Molecular Biology - Scripps Research Institute · Neel Krishna, Ph.D.** ... 156 MOLECULAR BIOLOGY 2004 THE SCRIPPS RESEARCH INSTITUTE ... Palo Alto Research Center Palo Alto, California

The Scripps Research Institute

Molecular Biology

Published by TSRI Press®. © Copyright 2004, The Scripps Research Institute. All rights reserved.


Elizabeth Thomas, Ph.D., Assistant Professor, Department of Molecular Biology

D E P A R T M E N T O F

M O L E C U L A R B I O L O G Y

S T A F F

Peter E. Wright, Ph.D.*Professor and ChairmanCecil H. and Ida M. Green

Investigator in MedicalResearch

Ruben Abagyan, Ph.D.Professor

Carlos F. Barbas III, Ph.D.*ProfessorJanet and W. Keith Kellogg II

Chair, Molecular Biology

Donald E. Bashford, Ph.D.**Associate ProfessorSt. Jude’s Children’s HospitalMemphis, Tennessee

Michael N. Boddy, Ph.D.Assistant Professor

Charles L. Brooks III, Ph.D.Professor

Monica J. Carson, Ph.D.Associate Professor

David A. Case, Ph.D.Professor

Geoffrey Chang, Ph.D.*Assistant Professor

Jerold Chun, M.D., Ph.D.Professor

Lisa Craig, Ph.D.Assistant Professor

Valerie De Crécy-Lagard,Ph.D.

Assistant Professor

Luis De Lecea, Ph.D.***Associate Professor

Lluis Ribas De Pouplana,Ph.D.

Adjunct Assistant Professor

Ashok Deniz, Ph.D.Assistant Professor

H. Jane Dyson, Ph.D.Professor

John H. Elder, Ph.D.Professor

Martha J. Fedor, Ph.D.*Associate Professor

James Arthur Fee, Ph.D.Professor of Research

Elizabeth D. Getzoff,Ph.D.****

Professor

David B. Goodin, Ph.D.Associate Professor

David S. Goodsell, Jr., Ph.D.Associate Professor

Joel M. Gottesfeld, Ph.D.Professor

Robert Hallewell, D.Phil.Adjunct Associate Professor

Jennifer Harris, Ph.D.Assistant Professor of

Biochemistry

Christian A. Hassig, Ph.D.Adjunct Assistant Professor

Mirko Hennig, Ph.D.Assistant Professor

John E. Johnson, Ph.D.Professor

Gerald F. Joyce, M.D.,Ph.D.*****

Professor

Ehud Keinan, Ph.D.*Adjunct Professor

Richard A. Lerner, M.D.,Ph.D.*****

President, The ScrippsResearch Institute

Lita Annenberg HazenProfessor ofImmunochemistry

Cecil H. and Ida M. GreenChair in Chemistry

Scott Lesley, Ph.D.Assistant Professor of

Biochemistry

Tianwei Lin, Ph.D.Assistant Professor

Clare McGowan, Ph.D.†

Associate Professor

Duncan E. McRee, Ph.D.Adjunct Associate Professor

David P. Millar, Ph.D.Associate Professor

Louis Noodleman, Ph.D.Associate Professor

Arthur J. Olson, Ph.D.Professor

James C. Paulson, Ph.D.††

Professor

Vijay Reddy, Ph.D.Assistant Professor

Steven I. Reed, Ph.D.Professor

Victoria A. Roberts, Ph.D.Associate Professor

Paul Russell, Ph.D.†

Professor

Michel Sanner, Ph.D.Associate Professor

Harold Scheraga, Ph.D.Adjunct Professor

Paul R. Schimmel,Ph.D.*****

Ernest and Jean HahnProfessor of MolecularBiology and Chemistry

Anette Schneemann, Ph.D.Associate Professor

Subhash C. Sinha, Ph.D.*Associate Professor

Gary Siuzdak, Ph.D.Adjunct Associate Professor

Robyn L. Stanfield, Ph.D.Assistant Professor

Raymond C. Stevens,Ph.D.†††

Professor

Charles D. Stout, Ph.D.Associate Professor

Peiqing Sun, Ph.D.Assistant Professor

J. Gregor Sutcliffe, Ph.D.Professor

John A. Tainer, Ph.D.*Professor

Fujie Tanaka, Ph.D.Assistant Professor

Elizabeth Anne Thomas,Ph.D.

Assistant Professor

James R. Williamson,Ph.D.*****

Professor

Ian A. Wilson, D.Phil.*Professor

Curt Wittenberg, Ph.D.†

Associate Professor

Kurt Wüthrich, Ph.D.Cecil H. and Ida M. Green

Professor of StructuralBiology

Todd O. Yeates, Ph.D.Adjunct Professor

T H E S C R I P P S R E S E A R C H I N S T I T U T E M O L E C U L A R B I O L O G Y 2 0 0 4 1 5 5


S E C T I O N C O V E R F O R T H E D E P A R T M E N T O F M O L E C U L A R B I O L O G Y : Structure

of the integral membrane protein fatty acid amide hydrolase modeled into a lipid bilayer (determined

by scientists in the laboratory of R.C. Stevens, Ph.D., Department of Molecular Biology). The enzyme

(discovered and characterized by B.F. Cravatt, Ph.D., and colleagues, Department of Cell Biology) is

a homodimer assembled from 63-kD subunits. This view of the enzyme highlights the central twisted

β-sheet that forms the core of the structure. The inhibitor adduct methoxy arachidonyl fluorophospho-

nate (MAP; designed and synthesized by researchers in the laboratory of D.L. Boger, Department of

Chemistry) is depicted in the active site with van der Waals surfaces.

Guo-Fu Zhong, Ph.D.Assistant Professor

S E R V I C E F A C I L I T I E S

Ola Blixt, Ph.D.Core Manager, Consortium

for Functional Glycomics

John Chung, Ph.D.Manager, Nuclear Magnetic

Resonance Facilities

Gerard KroonAssistant Manager, Nuclear

Magnetic ResonanceFacilities

Michael E. PiqueDirector, Graphics

Development

Nahid Razi, Ph.D.Assistant Core Manager,

Consortium for FunctionalGlycomics

Peter Sobieszcsuk, Ph.D.Core Manager, Consortium

for Functional Glycomics

S T A F F S C I E N T I S T S

Aymeric Pierre De Parseval,Ph.D.

Karla Ewalt, Ph.D.

Brian M. Lee, Ph.D.

Maria Martinez-Yamout,Ph.D.

Garrett M. Morris, Ph.D.

Chiaki Nishimura, Ph.D.

Jeffrey Speir, Ph.D.

Manal Swairjo, Ph.D.

Mutsuo Yamaguchi, Ph.D.

S E N I O R R E S E A R C H

A S S O C I A T E S

Sultan C. Agalarov, Ph.D.**Institute of Protein ResearchPuschino, Russia

David Barondeau, Ph.D.

Kirk Beebe, Ph.D.

Udayan Chatterji, Ph.D.**Department of Immunology,

Scripps Research

Brian Collins, Ph.D.

Peter B. Hedlund, M.D.,Ph.D.

Neel Krishna, Ph.D.**Children’s Hospital of the

King’s DaughtersNorfolk, Virginia

Ying Chuan Lin, Ph.D.

Rebecca Page, Ph.D.

Mikhail Popkov, Ph.D.

Richard R. Rivera, Ph.D.

Lincoln Scott, Ph.D.

James Stevens, Ph.D.

Koji Tamura, Ph.D.

Liang Tang, Ph.D.

Xiang-Lei Yang, Ph.D.

R E S E A R C H A S S O C I A T E S

Sunny Abraham, Ph.D.

Fabio Agnelli, Ph.D.

Marcius Da Silva Almeida,Ph.D.

Beatriz Gonzalelz Alonso,Ph.D.

Jianghong An, Ph.D.

Yu An, Ph.D.

Crystal Stacy Anglen, Ph.D.

Brigitte Anliker, Ph.D.

Joseph W. Arndt, Ph.D.

Mabelle Ashe, Ph.D.

Jamie Mitchell Bacher, Ph.D.

Michael F. Bailey, Ph.D.

Christopher Baskerville, Ph.D.

Amy Christine Beltran,Ph.D.**

Miramar CollegeSan Diego, California

Per Bengston, Ph.D.

Albert E. Beuscher, Ph.D.**Stockholm UniversityStockholm, Sweden

William Henry Bisson, Ph.D.

Pilar Blancafort, Ph.D.

Andrew Bordner, Ph.D.**Molsoft, L.L.C.La Jolla, California

Brian Bothner, Ph.D.**Montana State UniversityBozeman, Montana

Ronald M. Brudler, Ph.D.

Ryan Burnett, Ph.D.

Rosa Maria Cardoso, Ph.D.

Andrew Barry Carmel, Ph.D.

Jesus M. Castagnetto, Ph.D.**San Diego Supercomputer

CenterLa Jolla, California

Qing Chai, Ph.D.

Eli Chapman, Ph.D.

Anju Chatterji, Ph.D.

Anton Vladislavovich Cheltsov,Ph.D.

I-Ju Chen, Ph.D.**Department of Molecular and

Experimental Medicine,Scripps Research

Jianhan Chen, Ph.D.

Jaeyoung Cho, Ph.D.

Jungwoo Choe, Ph.D.

Li-Chiou Chuang, Ph.D.

Jean-Pierre Clamme, Ph.D.

Linda Maria Columbus, Ph.D.

Adam Corper, Ph.D.

Stephane Coulon, Ph.D.**ImmunotechMarseille, France

James Covalt, Ph.D.††††

Qizhi Cui, Ph.D.

Carla P. Da Costa, Ph.D.

Douglas Daniels, Ph.D.

Sanjib Das, Ph.D.

Paramita Dasgupta, Ph.D.

Robert De Bruin, Ph.D.

Roberto N. De Guzman, Ph.D.

Sohela De Rozieres, Ph.D.

Qingdong Deng, Ph.D.

Paula Desplats, Ph.D.

Buchi RamacharyDhevalapally, Ph.D.

Zhanna Druzina, Ph.D.

Li-Lin Du, Ph.D.

Theresia Dunzendorfer-Matt,Ph.D.

Scott Eberhardy, Ph.D.

Marc-Olivier Ebert, Ph.D.

Stephen Edgcomb, Ph.D.

Susanna V. Ekholm-Reed,Ph.D.

Reza Mobini Farahani, Ph.D.

Michael Feig, Ph.D.**Michigan State UniversityEast Landing, Michigan

Daniel Felitsky, Ph.D.

Philippe Ferrara, Ph.D.**Novartis International AGBasel, Switzerland

1 5 6 M O L E C U L A R B I O L O G Y 2 0 0 4 T H E S C R I P P S R E S E A R C H I N S T I T U T E


Gerhard Fuhrmann, Ph.D.††††

Pierre Henri Gaillard, Ph.D.

Maria Alejandra Gamez-Abascal, Ph.D.

Hui Gao, Ph.D.

Elsa D. Garcin, Ph.D.

Edith Caroline Glazer, Ph.D.

Holger Gohlke, Ph.D.**Johann Wolfgang Goethe-

UniversitätFrankfurt, Germany

Torbjorn Graslund, Ph.D.**Royal Institute of TechnologyStockholm, Sweden

Björn Grünenfelder, Ph.D.

Gye Won Han, Ph.D.

Hongna Han, Ph.D.

Shoufa Han, Ph.D.

Wenge Han, Ph.D.

Jason W. Harger, Ph.D.

Anna-Maria Hays, Ph.D.**Ambrx, Inc.San Diego, California

Torsten Herrmann, Ph.D.**ETHZürich, Switzerland

Yunfeng, Hu, Ph.D.

Joy Huffman, Ph.D.

Laura M. Hunsicker, Ph.D.

Yuzuru Ikehara, Ph.D.**Aichi Cancer Center

Research InstituteNagoya, Japan

Wonpil Im, Ph.D.

Tasneem Islam, Ph.D.

Shuichiro Ito, Ph.D.

Nina Jendreyko, M.D.**Humboldt-Universität-ChariteBerlin, Germany

Glenn C. Johns, Ph.D.

Eric C. Johnson, Ph.D.

Margaret Alice Johnson, Ph.D.

Hamid Reza Kalhor, Ph.D.

Christian Kannemeier, Ph.D.

Mili Kapoor, Ph.D.

Anebouselvy Kengadarane,Ph.D.††††

Arto Tapio Kesti, Ph.D.**University of OuluOulun Yliopisto, Finland

Yang Khandogin, Ph.D.

Iija V. Khavrutski, Ph.D.

Reza Khayat, Ph.D.

Min Ju Kim, Ph.D.

Marcy A. Kingsbury, Ph.D.

Elizabeth Kompfner, M.D.

Milka Kostic, Ph.D.

Julio Kovacs, Ph.D.

Iaroslav Kuzmine, Ph.D.

Hugo Alfredo Lago-Zarrilli,Ph.D.

Jason Lanman, Ph.D.

Jonathan C. Lansing, Ph.D.

June Hyung Lee, Ph.D.

Kelly Lee, Ph.D.

Katrina Lehmann, Ph.D.

Chenglong Li, Ph.D.

Lian-Sheng Li, Ph.D.**Vicuron Pharmaceuticals, Inc.Fremont, California

Vasco Liberal, Ph.D.

Michael Lietzow, Ph.D.**Allergan, Inc.Irvine, California

William M. Lindstrom, Ph.D.

Jianming Liu, Ph.D.**AstraZeneca, Inc.Mölndal, Sweden

Hui-Yue Christine Lo, Ph.D.

Timothy Lovell, Ph.D.**AstraZeneca, Inc.Mölndal, Sweden

Kunheng Luo, Ph.D.

John Gately Luz, Ph.D.

Che Ma, Ph.D.

Ann MacLaren, Ph.D.

Laurent Magnenat, Ph.D.

Angele Maki, Ph.D.**Medarex, Inc.Sunnyvale, California

Darly Joseph Manayani, Ph.D.

Maria Victoria Martin-Sanchez, Ph.D.

Beatriz Maroto, Ph.D.**Department of Immunology,

Scripps Research

Nobuyuki Mase, Ph.D.**Shizuoka UniversityShizuoka, Japan

Daniel McElheny, Ph.D.

Christian Corey Melander,Ph.D.**

North Carolina StateUniversity

Raleigh, North Carolina

Benoit Melchior, Ph.D.

David Metzgar, Ph.D.

Heiko Michael Moeller, Ph.D.

Seongho Moon, Ph.D.

Bettina Moser, Ph.D.

Christopher Myers, Ph.D.

Sreenivasa Chowdari Naidu,Ph.D.

Toru M. Nakamura, Ph.D.

Tadateru Nishikawa, Ph.D.

Eishi Noguchi, Ph.D.

Brian Nordin, Ph.D.

Karin E. Norgard-Sumnicht,Ph.D.

Brian V. Norledge, Ph.D.

Michael Oberhuber, Ph.D.

Wendy Fernandez Ochoa,Ph.D.

Yoshiaki Zenmei Ohkubo,Ph.D.

Brian L. Olson, Ph.D.

Alexey Onufriev, Ph.D.**Virginia Polytechnic Institute

and State UniversityBlacksburg, Virginia

Andrew Orry, Ph.D.**Molsoft, L.L.C.La Jolla, California

Brian Paegel, Ph.D.

Covadonga Paneda, Ph.D.

Sandeep Patel, Ph.D.

Natasha Paul, Ph.D.

Stephanie Pebernard, Ph.D.

Suzanne Peterson, Ph.D.

Wolfgang Stefan Peti, Ph.D.

Goran Pljevaljcic, Ph.D.

Corinne Chantal Ploix, Ph.D.

Stephanie Pond, Ph.D.

Owen Pornillos, Ph.D.

Daniel Joseph Price, Ph.D.

Plachikkat Krishnan Radha,Ph.D.

Grazia Daniela Raffa, Ph.D.

John Reader, Ph.D.



Michael Recht, Ph.D.**Palo Alto Research CenterPalo Alto, California

Stevens Kastrup Rehen, Ph.D.

Jean-Baptiste Reiser, Ph.D.

Thomas Holm Rod, Ph.D.**Lund UniversityLund, Sweden

Miguel A. Rodriguez-Gabriel,Ph.D.

Robin Rosenfeld, Ph.D.**ActiveSightSan Diego, California

Stanislav Rudyak, Ph.D.

Sean Ryder, Ph.D.

Sanjita Sasmal, Ph.D.

Mika Aoyagi Scharber, Ph.D.

Jennifer S. Scorah, Ph.D.

Craig McLean Shepherd,Ph.D.

William Shih, Ph.D.

Hyunbo Shim, Ph.D.**Xoma, L.L.C.Berkeley, California

David S. Shin, Ph.D.

Adrian Smith, Ph.D.**Mary Lyon CentreHarwell, England

Holly Heaslet Soutter, Ph.D.

Natalie Spielewoy, Ph.D.

Greg Springsteen, Ph.D.

Deborah J. Stauber, Ph.D.

Derek Steiner, Ph.D.

Gudrun Stengel, Ph.D.

Daniel Stoffler, Ph.D.

Antitsa Stoycheva, Ph.D.**Gilead SciencesFoster City, California

Kenji Sugase, Ph.D.

Vidyasankar Sundaresan,Ph.D.

Jeff Suri, Ph.D.

Blair R. Szymczyna, Ph.D.

Florence Muriel Tama, Ph.D.

Guo-Qing Tang, Ph.D.**Robert Wood Johnson

Medical SchoolPiscataway, New Jersey

Jinghua Tang, Ph.D.

Nardos Tassew, Ph.D.

Hiroaki Tateno, Ph.D.

Michela Taufer, Ph.D.

Ewan Richardson Taylor,Ph.D.

Donato Tedesco, Ph.D.

Rhonda Torres, Ph.D.

Megan Wright Trevathan,Ph.D.

Frank Van Drogen, Ph.D.

Rani ParvathyVenkitakrishnan, Ph.D.††††

Philip Arno Venter, Ph.D.

Petra Verdino, Ph.D.

Stefan Vetter, Ph.D.

William Frederick Waas, Ph.D.

Shun-ichi Wada, Ph.D.

Ross Walker, Ph.D.

Lin Wang, Ph.D.

Raphaelle Winsky-Sommerer,Ph.D.

Eric L. Wise, Ph.D.

Jonathan Wojciak, Ph.D.

Dennis Wolan, Ph.D.

Hyung Sik Won, Ph.D.

Timothy I. Wood, Ph.D.

Eugene Wu, Ph.D.

Lan Xu, Ph.D.

Ramesh Yadava, Ph.D.**University of VirginiaCharlottesville, Virginia

Atsushi Yamagata, Ph.D.

Qi Yan, Ph.D.

Yong Yao, Ph.D.

Xiaoqin Ye, M.D., Ph.D.

Yongjun Ye, Ph.D.

Yong Yin, Ph.D.

Minmin Yu, Ph.D.Advanced Light SourceBerkeley, California

Veronica Yu, Ph.D.

Yuan Yuan, Ph.D.

Dirk M. Zajonc, Ph.D.

Markus Zeeb, Ph.D.

Hui Zhao, Ph.D.**Pfizer Global Research and

DevelopmentLa Jolla, California

Yong Zhao, Ph.D.

Peizhi Zhu, Ph.D.

Xueyong Zhu, Ph.D.

S C I E N T I F I C A S S O C I A T E S

Andrew S. Arvai, M.S.

Eric Birgbauer, Ph.D.

Xiaoping Dai, Ph.D.

Liliane Dickinson, Ph.D.

Michael Allen Hanson, Ph.D.

Diane Marie Kubitz, B.A.

Padmaja Natarajan, Ph.D.

Marianne Patch, Ph.D.

Gabriela Perez-Alvarado,Ph.D.

Nicholas Preece, Ph.D.

V I S I T I N G

I N V E S T I G A T O R S

Stephen J. Benkovic, Ph.D.Pennsylvania State UniversityUniversity Park, Pennsylvania

Astrid Graslund, Ph.D.Stockholm UniversityStockholm, Sweden

Arne Holmgren, M.D., Ph.D.Karolinska InstitutetStockholm, Sweden

Barry Honig, Ph.D.Columbia UniversityNew York, New York

Arthur Horwich, M.D.Yale UniversityNew Haven, Connecticut

Tai-huang Huang, Ph.D.Academica SinicaTaipei, Taiwan

Robert D. Rosenstein, Ph.D.Lawrence Berkeley National

LaboratoryBerkeley, California

* Joint appointment in The SkaggsInstitute for Chemical Biology

** Appointment completed; newlocation shown

*** Joint appointment in theDepartment of Neuropharmacology

**** Joint appointments in theDepartment of Immunology andThe Skaggs Institute forChemical Biology

***** Joint appointments in theDepartment of Chemistry andthe Skaggs Institute forChemical Biology

† Joint appointment in theDepartment of Cell Biology

†† Joint appointment in theDepartment of Molecular andExperimental Medicine

††† Joint appointment in theDepartment of Chemistry

†††† Appointment completed



Chairman’s Overview

Molecular biology forms the cornerstone of bio-logical and biomedical research. Research inour department covers the entire spectrum of

modern molecular biology, from molecular genetics atone extreme to computational and structural biology atthe other. Our scientists are making rapid progress towarda deeper understanding of the fundamental processes ofliving organisms. These processes include the molecularevents involved in control of the cell cycle, developmentof tumors, induction of sleep, development of neuronsand of CNS disorders, regulation of transcription, anddecoding of genetic information in translation. Majoradvances have been made in elucidating the structuralbiology of signal transduction and viral assembly, deter-mining the molecular basis of nucleic acid recognitionby proteins, and understanding mechanisms of viral infec-tivity. Finally, new advances have been made in the areaof biomolecular engineering, building novel functions intoviruses, antibodies and other proteins, RNA, and DNA.Progress in these and other areas is described in detailon the following pages; only a few highlights are men-tioned here.

Structural biology continues to be a major activity inthe department, and many new x-ray and nuclear mag-netic resonance structures of major biomedical signifi-cance were completed during the past year. Among the

highlights was the determination, in Ian Wilson’s labora-tory, of the structure of the influenza virus hemaggluti-nin from the 1918 “Spanish flu”; this structure providedstrong evidence that this deadly influenza outbreak origi-nated from transmission of an avian virus directly tohumans. Structural studies of a shark antibody by RobinStanfield, Ian Wilson, and colleagues provided novelinsights into antibody evolution and mechanisms of anti-gen recognition by a primitive single-chain antibody andrevealed potential for engineering minimal antigen-bind-ing domains for biomedical applications.

The spectacular success of structural biology, aidedby development of high-throughput technologies, has ledto a dramatic increase in the number of available pro-tein structures in recent years, with a correspondingincrease in our understanding of the proteins’ biologicalfunctions and mechanisms. However, the structures ofseveral classes of important eukaryotic proteins, thosethat are intrinsically disordered or are associated withcell membranes, remain poorly understood. Even though30% or more of eukaryotic proteins are membrane pro-teins, knowledge of their structures lags far behind knowl-edge of the structures of soluble proteins. A group ofinvestigators in the Department of Molecular Biologyrecently received a major grant from the National Insti-tutes of Health Roadmap initiative to develop technolo-gies to rectify this situation. This funding will support amajor new initiative to develop novel methods for pro-duction of membrane proteins for structural analysis byx-ray crystallography, nuclear magnetic resonance, andelectron microscopy. Other research groups in the depart-ment are using nuclear magnetic resonance to charac-terize intrinsically disordered proteins and investigatethe mechanisms by which these proteins fold on bind-ing to their biological targets.

New advances have been made in understandingthe mechanisms of repair of damaged DNA, both at thecellular and the structural level. The structures of theproteins flap endonuclease-1 and O6-alkylguanine-DNAalkyltransferase have been determined by John Tainerand colleagues, providing novel insights into mechanismsof DNA repair and into the function of flap endonucle-ase-1 in DNA replication. The alkyltransferase efficientlyrepairs alkylated DNA and can therefore make tumor cellsresistant to chemotherapeutic agents that function byDNA alkylation; knowledge of the enzyme structure willbe useful for design of inhibitors that could potentiallybe used as adjuvants in chemotherapy. Research in thelaboratories of Clare McGowan and Michael Boddy is



Peter E. Wright, Ph.D.

leading to new understanding of the DNA damageresponse in human cells. McGowan and coworkers haveidentified human checkpoint kinases that limit cell-cycleprogression when DNA is damaged, and researchers inthe Boddy laboratory have uncovered new links betweenthe cell-cycle replication checkpoint and DNA repair. Inhuman cells, the retinoblastoma protein Rb, a tumorsuppressor, plays a critical role as a master regulator ofcell division. Research by Curt Wittenberg and colleagueshas now led to the identification of a protein in yeastcells that performs the same function as Rb. The dis-covery of this protein, named Whi5, is a major advancebecause it allows the power of yeast genetics to beapplied to elucidate the pathways of cell-cycle regula-tion. The spin-off will be a more detailed understand-ing of the homologous regulatory pathways in humans,together with novel insights into the processes that leadto cancer.

A focus of research in the Department of MolecularBiology remains the design and engineering of biomole-cules with novel properties for potential applications innanotechnology and medicine. A major advance was madelast year by Jerry Joyce and coworkers, who success-fully designed a single DNA molecule that folds into theshape of an octahedron. This molecule can be readilycloned and evolved and could function as a versatilemolecular building block with potential applications innanotechnology and biomedicine. Tianwei Lin and JohnJohnson and their colleagues are adopting an alternativeapproach; they are using structure-based engineering toadapt a harmless plant virus for potential applicationsin nanotechnology and as a nanocapsule for delivery oftherapeutic agents. Researchers in other laboratoriesare engineering zinc finger proteins, antibodies, andvarious metalloproteins to endow the molecules withnovel properties.

Molecular biology remains a field of enormous oppor-tunity and excitement. The scientists in this departmentare taking full advantage of powerful new technologiesto advance our understanding of fundamental biologicalprocesses at the molecular level. Their discoveries willultimately be translated into new advances in biotech-nology and in medicine.



INVESTIGATORS’ REPORTS

Crystallographic Studies of Immune Recognition, Molecular Assemblies, and Anticancer TargetsI.A. Wilson, R.L. Stanfield, Y. An, T.A. Bowden, D.A. Calarese,

R.M.F. Cardoso, J.-W. Choe, A.L. Corper, M.D.M. Crispin,

T.H. Cross, X. Dai, W.L. Densley, E.W. Debler, M.-A. Elsliger,

S. Ferguson, P.A. Horton, S. Ito, M.S. Kelker, J.G. Luz,

J.B. Reiser, E.B. Shillington, D.A. Shore, D.J. Stauber,

R.S. Stefanko, J. Stevens, P. Verdino, D.W. Wolan, L. Xu,

M. Yu, D.M. Zajonc, Y. Zhang, X. Zhu

We are investigating many different families ofimmune recognition receptors and anticancertargets. We use primarily x-ray crystallogra-

phy to determine structures of these biomedically rele-vant proteins in complex with their respective ligandsor inhibitors. Structural information is used in someprojects for design of novel compounds to bind or inhibitthe proteins of interest. Our overall goal is to under-stand how foreign pathogens are recognized by the hostadaptive and innate immune systems.

V I R A L C O A T P R O T E I N S

In 1918–1919, the great influenza pandemic (Span-ish flu) killed an estimated 40 million persons. In col-laboration with J. Taubenberger, Armed Forces Instituteof Pathology, Washington, DC, and P. Palese, A. Garcìa-Sastre, and C. Basler, Mount Sinai School of Medicine,New York, New York, we are carrying out structuralanalyses of the 1918 viral proteins to understand whythis strain was so pathogenic. Our first structure deter-mined was of hemagglutinin, the major surface glyco-protein that is involved in binding and fusion of influenzavirus to human respiratory cells (Fig. 1). The structurereveals features conserved within avian viruses that mayhave contributed to the increased virulence of this virus.

H I V T Y P E 1 N E U T R A L I Z I N G A N T I B O D I E S

The HIV type 1 (HIV-1) envelope glycoproteinsgp120 and gp41 mutate rapidly in response to antibodychallenge, thus allowing the virus to evade the immunesystem. However, a few rare antibodies can neutralizeprimary strains of HIV-1. The crystal structures of theFabs of the broadly neutralizing antibodies 4E10 with a

gp41 peptide (Fig. 2), 2G12 with its carbohydrate epi-tope, and 447-52D with the gp120 V3 loop haverevealed key epitope conformations that are being usedas templates for rational design of HIV-1 vaccines. Theresearch on HIV is done in collaboration with D. Burton,Department of Immunology; P. Dawson, Department ofCell Biology; C.-H. Wong, Department of Chemistry;



F i g 1 . Ribbon representation of the hemagglutinin HA0 trimer from

the 1918 influenza virus. Each monomer has 2 important sites: the

receptor-binding site for virus attachment to the host lung epithelial

cells via sialic acid–containing host cell receptors and the cleavage

site where for full infectivity, the single chain (HA0) is cut into 2

chains (HA1 and HA2). At the N terminus of the HA2 chain is the

fusion peptide that is critical for subsequent membrane fusion events

that lead to infection.

F i g 2 . Antigen-binding site of the Fab of 4E10, a broadly neu-

tralizing antibody to HIV-1. This Fab recognizes a helical epitope

from the gp41 glycoprotein. The conformations of 4E10 complemen-

tarity-determining regions L1, L2, L3, H1, H2, and H3 and the

bound helical gp41 epitope are shown.

S. Danishefsky, Sloan-Kettering Institute, New York, New York; J.K. Scott, Simon Fraser University, Burnaby,British Columbia; S. Zolla-Pazner, New York UniversitySchool of Medicine, New York, New York; J. Moore, Cor-nell University, Ithaca, New York; Repligen Corporation,Waltham, Massachusetts; H. Katinger, R. Kunert, and G. Stiegler, University für Bodenkultur, Vienna, Austria;R. Wyatt and P. Kwong, Vaccine Research Center,National Institutes of Health, Bethesda, Maryland; andthe International Aids Vaccine Initiative.P E P T I D O G L Y C A N R E C O G N I T I O N

Peptidoglycans, essential components of the cellwalls of all bacteria, are among the conserved motifsthat invoke the innate immune system to induce strongantibacterial responses. A family of pattern recognitionmolecules, peptidoglycan recognition proteins (PGRPs),which interact with peptidoglycans, can be functionallydivided into “catalytic” PGRPs that have amidase activ-ity and “recognition” PGRPs that bind peptidoglycans.We determined the 1.56-Å crystal structure of PGRP-SA (Fig. 3), which does not contain the canonical zincfound in catalytic PGRPs. Comparison of PGRP-SA withthe catalytic PGRP-LB indicated overall structural con-servation and a hydrophilic groove that most likely isthe peptidoglycan core binding site. These investigationswere done in collaboration with L. Teyton, Departmentof Immunology.T R I G G E R I N G R E C E P T O R F A M I L Y

The triggering receptor expressed on myeloid cells(TREM) family of extracellular immunoglobulin recep-tors includes both activating and inhibitory isoformswhose ligands are unknown. TREM-1 amplifies theinflammation induced by both bacteria and fungi andis a potential therapeutic target. The 1.47-Å structureof the extracellular domain of human TREM-1, cou-

pled with analytical ultracentrifugation and deuterium-hydrogen nuclear magnetic resonance spectroscopy ofboth human and mouse TREM-1, conclusively showedthe monomeric state of this extracellular ectodomainin solution. This research was also done collaborationwith Dr. Teyton.C A T A L Y T I C A N T I B O D I E S

Development of effective immunotherapy for cocaineabuse, addiction, and overdose is under way. In studiesdone in collaboration with P. Wirsching and K.D. Janda,Department of Chemistry, the crystal structures ofcocaine-hydrolyzing antibodies 7A1 (free, with cocaine,with transition-state analog, and with products) and3A6 (with cocaine and with products) delineated themajor steps along the reaction coordinate.

Antibodies can catalyze the generation of hydrogenperoxide from singlet dioxygen and water via the postu-lated intermediate dihydrogen trioxide and other trioxygenspecies. In studies with R.A. Lerner and P. Wentworth,Department of Chemistry, we used x-ray analyses ofcatalytic antibodies 4C6 and 13G5 to elucidate thechemical consequences to the antibody molecule ofexposure to such reactive intermediates. The resultssuggested locations on the antibody where these inter-mediate species could be generated.

Antibody 34E4 catalyzes the base-promoted E2elimination of substituted benzisoxazoles and is one ofthe most efficient abzymes characterized to date. Struc-tures of the Fab of 34E4 reveal a deep binding pocketwith high shape complementarity to the transition-stateanalog and strong hydrophobicity provided by aromaticresidues. GluH50 interrupts this hydrophobic belt andcould act as a general base to abstract a proton fromthe substrate, triggering elimination of the phenoxide.This research is being done in collaboration with D. Hilvert, ETH Zürich, Zürich, Switzerland.C L A S S I C A L A N D N O N C L A S S I C A L M H C M O L E C U L E S

CD1 molecules, nonclassical homologs of class IMHC molecules, present lipid antigens to CD1-restrictedT-cell receptors (TCRs). Recently, mycobacterial lipo-peptide intermediates of the siderophore biosyntheticpathway were identified as CD1a antigens. The 2.8-Åstructure of CD1a in complex with a synthetic lipopep-tide derivative (Fig. 4) revealed that the lipid part ofthe ligand is buried deep within the binding groove,whereas the peptidic headgroup is exposed at the sur-face for recognition by specific CD8+ TCRs. Ligandsare provided by our collaborators D.B. Moody andM.B. Brenner, Harvard Medical School, Boston, Mass-achusetts, and C.-H. Wong, Department of Chemistry.



F i g . 3 . The 3-dimensional ribbon structure of PGRP-SA from

Drosophila melanogaster. The residues and atoms that line the bind-

ing groove and that may be involved in the interactions with the bac-

terial peptidoglycan core are shown in a ball-and-stick representation.

Collaborators in the research on CD1 and TCRs includeM. Kronenberg, La Jolla Institute for Allergy and Immu-nology, San Diego, California; V. Kumar, Torrey PinesInstitute for Molecular Studies, San Diego, California;Wayne Severn, AgResearch, Upper Hutt, New Zealand;and R. Dwek, P. Rudd, and S. Davis, University of Oxford,Oxford, England.

MUC1, a cancer mucin, is a promising target forthe development of an anticancer vaccine. In collabo-ration with V. Apostolopoulos, Austin Research Institute,Heidelberg, Australia, we are determining structuresfor a number of murine H2-Kb class I MHC moleculeswith MUC1-derived peptides to explain how noncanoni-cal, low-affinity peptides can stimulate cytotoxic T lym-phocytes. Structural comparisons between glycosylatedand nonglycosylated peptides will facilitate design ofnovel peptide mimics as tumor vaccines.

The TCR coreceptor CD8 is expressed on the sur-face of leukocyte cells as an αβ heterodimer and anαα homodimer; the former is the dominant isotype oncirculatory CD8+ cytotoxic T cells. Large quantities ofsoluble CD8 αα and αβ have been produced for crys-tallization, and CD8 αα has been crystallized in com-plex with the Fab YTS in collaboration with Dr. Teyton.C Y T O K I N E R E C E P T O R S

IL-2, a class I cytokine, functions as a growth fac-tor in the immune system, causing proliferation and

cytokine production in T cells, proliferation and anti-body production in B cells, activation of the cytotoxicactivity of natural killer cells, and proliferation andclonal expansion of tumor-specific T cells. IL-2 signal-ing occurs through a ligand-induced activation of thehigh-affinity heterotrimeric IL-2 receptor (α-, β-, andγ-chains) complex. The structure of this complex willprovide insight into the recognition, assembly, and sig-naling properties of the IL-2 receptor and will aid indesign of novel ligands to modulate function and activ-ity of the receptor. Our collaborator in this research isK. Smith, Weil Medical College of Cornell University,New York, New York.E N Z Y M A T I C C A N C E R T A R G E T S

Rapidly dividing cells are critically dependent onde novo nucleotide synthesis pathways, essential forDNA synthesis and repair. This vulnerability has longbeen exploited in the area of anticancer research, andantifolates are one of the most extensively investigatedclasses of antineoplastic agents. Our focus involves 2 enzymes in the de novo purine biosynthesis pathway:glycinamide ribonulceotide transformylase (Fig. 5) andthe bifunctional aminoimidazole carboxamide ribonu-cleotide transformylase inosine monophosphate cyclohy-



F i g . 4 . Antigen-binding groove of CD1a. The molecular surface

is shown as a transparent binding pocket with the bound lipopep-

tide ligand. The side view (B) shows both the deeply buried lipid

part and the peptidic headgroup, which is more exposed and

accessible by the TCR (top view, A).

F i g . 5 . Stereoview of the active binding site of human glycinamide

ribonucleotide (GAR) transformylase in complex with a folate ana-

log, 10-trifluoroacetyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic

acid (10-CF3CO-DDACTHF), and substrate β-GAR. The final refined

structure of 10-CF3CO-DDACTHF is superimposed on the 2Fo-Fc

electron density contoured at 2 σ, and the substrate β-GAR structure

is shown within the 2Fo-Fc electron density contoured at 1.5 σ.

drolase. The structures of these 2 enzymes in complexwith several different compounds revealed the mecha-nism of the formyl transfer reaction and provided aplatform for the design of inhibitors to develop antineo-plastic agents. These investigations are being done incollaboration with D. Boger, Department of Chemistry;G.P. Beardsley, Yale University, New Haven, Connecti-cut; and S.J. Benkovic, Pennsylvania State University,University Park, Pennsylvania.

PUBLICATIONSBurton, D.R., Desrosiers, R.C., Doms, R.W., Koff, W.C., Kwong, P.D., Moore, J.P.,Nabel, G.J., Sodroski, J., Wilson, I.A., Wyatt, R.T. HIV vaccine design and theneutralizing antibody problem. Nat. Immunol. 5:233, 2004.

Cheong, C.G., Wolan, D.W., Greasley, S.E., Horton, P.A., Beardsley, G.P., Wilson,I.A. Crystal structures of human bifunctional enzyme aminoimidazole-4-carboxam-ide ribonucleotide transformylase/IMP cyclohydrolase in complex with potent sul-fonyl-containing antifolates. J. Biol. Chem. 279:18034, 2004.

Desharnais, J., Hwang, I., Zhang, Y., Tavassoli, A., Baboval, J., Benkovic, S.J.,Wilson, I.A., Boger, D.L. Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novopurine biosynthetic pathway. Bioorg. Med. Chem. 11:4511, 2003.

Erlandsen, H., Canaves, J.M., Elsliger, M.A., et al. Crystal structure of an HEPNdomain protein (TM0613) from Thermotoga maritima at 1.75 Å resolution. Pro-teins 54:806, 2004.

Kuhmann, S.E., Pugach, P., Kunstman, K.J., Taylor, J., Stanfield, R.L., Snyder,A., Strizki, J.M., Riley, J., Baroudy, B.M., Wilson, I.A., Korber, B.T., Wolinsky,S.M., Moore, J.P. Genetic and phenotypic analyses of human immunodeficiencyvirus type 1 escape from a small-molecule CCR5 inhibitor [published correctionappears in J. Virol. 78:6706, 2004]. J. Virol. 78:2790, 2004.

Lee, H.K., Scanlan, C.N., Huang, C.Y., Chang, A.Y., Calarese, D.A., Dwek, R.A.,Rudd, P.M., Burton, D.R., Wilson, I.A., Wong, C.H. Reactivity-based one-pot syn-thesis of oligomannoses: defining antigens recognized by 2G12, a broadly neutral-izing anti-HIV-1 antibody. Angew. Chem. Int. Ed. 43:1000, 2004.

Marsilje, T.H., Hedrick, M.P., Desharnais, J., Capps, K., Tavassoli, A., Zhang, Y.,Wilson, I.A., Benkovic, S.J., Boger, D.L. 10-(2-benzoxazolcarbonyl)-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid: a potential inhibitor of GAR transformylase andAICAR transformylase. Bioorg. Med. Chem. 11:4503, 2003.

Marsilje, T.H., Hedrick, M.P., Desharnais, J., Tavassoli, A., Zhang, Y., Wilson,I.A., Benkovic, S.J., Boger, D.L. Design, synthesis, and biological evaluation ofsimplified α-keto heterocycle, trifluoromethyl ketone, and formyl substituted folateanalogues as potential inhibitors of GAR transformylase and AICAR transformylase.Bioorg. Med. Chem. 11:4487, 2003.

Mitra, A.K., Celia, H., Ren, G., Luz, J.G., Wilson, I.A., Teyton, L. Supine orientation ofa murine MHC class I molecule on the membrane bilayer. Curr. Biol. 14:718, 2004.

Moody, D.B., Young, D.C., Cheng, T.Y., Rosat, J.P., Roura-Mir, C., O’Connor, P.B.,Zajonc, D.M., Walz, A., Miller, M.J., Levery, S.B., Wilson, I.A., Costello, C.E.,Brenner, M.B. T cell activation by lipopeptide antigens [published correctionappears in Science 304:211, 2004]. Science 303:527, 2004.

Page, R., Nelson, M.S., von Delft, F., et al. Crystal structure of -glutamyl phos-phate reductase (TM0293) from Thermotoga maritima at 2.0 Å resolution. Pro-teins 54:157, 2004.

Pantazatos, D., Kim, J.S., Klock, H.E., Stevens, R.C., Wilson, I.A., Lesley, S.A.,Woods, V.L., Jr. Rapid refinement of crystallographic protein construct definitionemploying enhanced hydrogen/deuterium exchange MS. Proc. Natl. Acad. Sci. U. S. A. 101:751, 2004.

Pantophlet, R., Wilson, I.A., Burton, D.R. Hyperglycosylated mutants of humanimmunodeficiency virus (HIV) type 1 monomeric gp120 as novel antigens for HIVvaccine design. J. Virol. 77:5889, 2003.

Rader, C., Turner, J.M., Heine, A., Shabat, D., Sinha, S.C., Wilson, I.A., Lerner,R.A., Barbas, C.F. A humanized aldolase antibody for selective chemotherapy andadaptor immunotherapy. J. Mol. Biol. 332:889, 2003.

Rudolph, M.G., Shen, L.Q., Lamontagne, S.A., Luz, J.G., Delaney, J.R., Ge, Q.,Cho, B.K., Palliser, D., McKinley, C.A., Chen, J., Wilson, I.A., Eisen, H.N. A pep-tide that antagonizes TCR-mediated reactions with both syngeneic and allogeneicagonists: functional and structural aspects. J. Immunol. 172:2994, 2004.

Rudolph, M.G., Wingren, C., Crowley, M.P., Chien, Y.H., Wilson, I.A. Combinedpseudo-merohedral twinning, non-crystallographic symmetry and pseudo-transla-tion in a monoclinic crystal form of the γδ T-cell ligand T10. Acta Crystallogr. DBiol. Crystallogr. 60(Pt. 4):656, 2004.

Schwarzenbacher, R., Canaves, J.M., Brinen, L.S., et al. Crystal structure of uronateisomerase (TM0064) from Thermotoga maritima at 2.85 Å resolution. Proteins53:142, 2003.

Schwarzenbacher, R., Deacon, A.M., Jaroszewski, L., et al. Crystal structure of aputative glutamine amido transferase (TM1158) from Thermotoga maritima at 1.7Å resolution. Proteins 54:801, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of aphosphoribosylaminoimidazole mutase PurE (TM0446) from Thermotoga maritimaat 1.77-Å resolution. Proteins 55:474, 2004.

Schwarzenbacher, R., von Delft, F., Abdubek, P., et al. Crystal structure of a puta-tive PII-like signaling protein (TM0021) from Thermotoga maritima at 2.5 Å reso-lution. Proteins 54:810, 2004.

Schwarzenbacher, R., von Delft, F., Canaves, J.M., et al. Crystal structure of aniron-containing 1,3-propanediol dehydrogenase (TM0920) from Thermotoga mar-itima at 1.3 Å resolution. Proteins 54:174, 2004.

Stanfield, R.L., Gorny, M.K., Williams, C., Zolla-Pazner, S., Wilson, I.A. Structuralrationale for the broad neutralization of HIV-1 by human monoclonal antibody 447-52D. Structure (Camb.) 12:193, 2004.

Stevens, J., Corper, A.L., Basler, C.F., Taubenberger, J.K., Palese, P., Wilson, I.A.Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenzavirus. Science 303:1866, 2004.

Wolan, D.W., Cheong, C.G., Greasley, S.E., Wilson, I.A. Structural insights intothe human and avian IMP cyclohydrolase mechanism via crystal structures withthe bound XMP inhibitor. Biochemistry 43:1171, 2004.

Wolan, D.W., Greasley, S.E., Wall, M.J., Benkovic, S.J., Wilson, I.A. Structure ofavian AICAR transformylase with a multisubstrate adduct inhibitor β-DADF identi-fies the folate binding site. Biochemistry 42:10904, 2003.

Zajonc, D.M., Elsliger, M.A., Teyton, L., Wilson, I.A. Crystal structure of CD1a incomplex with a sulfatide self antigen at a resolution of 2.15 Å. Nat. Immunol.4:808, 2003.

Zhu, X., Wentworth, P., Jr., Wentworth, A.D., Eschenmoser, A., Lerner, R.A., Wil-son, I.A. Probing the antibody-catalyzed water-oxidation pathway at atomic resolu-tion. Proc. Natl. Acad. Sci. U. S. A. 101:2247, 2004.

Zwick, M.B., Komori, H.K., Stanfield, R.L., Church, S., Wang, M., Parren, P.W.,Kunert, R., Katinger, H., Wilson, I.A., Burton, D.R. The long third complementar-ity-determining region of the heavy chain is important in the activity of the broadlyneutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J. Virol.78:3155, 2004.

Zwick, M.B., Parren, P.W., Saphire, E.O., Church, S., Wang, M., Scott, J.K.,Dawson, P.E., Wilson, I.A., Burton, D.R. Molecular features of the broadly neutral-izing immunoglobulin G1 b12 required for recognition of human immunodeficiencyvirus type 1 gp120. J. Virol. 77:5863, 2003.



Principles of Protein Structurefor Recognition, Interaction,Catalysis, and DesignE.D. Getzoff, M. Aoyagi, A.S. Arvai, D.P. Barondeau, R.M. Brudler, L. Craig, T.H. Cross, M. DiDonato, E.D. Garcin,U.K. Genick, S.W. Hennessy, C. Hitomi, K. Hitomi, C.J. Kassmann, I. Li, S.J. Lloyd, M.E. Pique, R.J. Rosenfeld, M.E. Stroupe, M.J. Thompson, J.L. Tubbs, T.I. Wood

P H O T O A C T I V E P R O T E I N S

To understand in atomic detail how proteins trans-late sunlight into defined conformational changesfor biological functions, we are exploring the reac-

tion mechanisms of the blue-light receptors photoactiveyellow protein and cryptochrome. We combined ourultra-high-resolution and time-resolved crystallographicstructures of the dark state and 2 photocycle intermedi-ates of photoactive yellow protein with site-directedmutagenesis, ultraviolet-visible spectroscopy, time-resolved Fourier transform infrared spectroscopy, andquantum mechanical and electrostatic computationalmethods to understand the protein photocycle. We foundthat the chromophore undergoes trans-to-cis isomer-ization after light absorption by first flipping its thioesterlinkage to the protein, and we analyzed how the dark-state protein anticipates motions required for this isomer-ization (Fig. 1). With L. Noodleman and D. Bashford,Department of Molecular Biology, we defined electronicchanges that occur upon photoexcitation of the pho-toactive yellow protein chromophore and determinedtheir subsequent effects on its isomerization.

Cryptochrome flavoproteins are homologs of light-dependent DNA repair photolyases that function asblue-light receptors in plants and as components ofcircadian clocks in animals. We identified the newcryptochrome DASH family in plants and bacteria andsolved the first crystallographic structure of a cryp-tochrome. Our structure reveals commonalities withphotolyases in DNA binding and redox-dependentfunction but shows distinct active-site and interaction-surface features. New structures of photolyases from 2 other branches of the photolyase/cryptochrome familythat repair cyclobutane pyrimidine dimers and photo-products help us decipher the cryptic structure, func-tion, and evolutionary relationships of these fascinatingredox-active proteins. We are also studying clock pro-teins, including those with PAS domains, which struc-turally resemble photoactive yellow protein.

M E T A L L O E N Z Y M E S T R U C T U R E A N D F U N C T I O N

Superoxide dismutases (SODs) act as master regu-lators of intracellular free radicals and reactive oxygenspecies by transforming superoxide to oxygen andhydrogen peroxide. Our crystallographic structures ofcopper-zinc SODs from mammals, bacterial symbionts,and pathogens revealed striking differences in theenzyme assembly and in the loops flanking the active-site channel, despite the shared β-barrel subunit fold,catalytic metal center, and electrostatic enhancementof activity. With J.A. Tainer, Department of MolecularBiology, we determined structures of mutant humanSODs found in patients with the disease amyotrophiclateral sclerosis (Lou Gehrig disease), and proposed ahypothesis for how single-site mutations cause thisfatal neurodegenerative disease.

Novel nickel SODs assemble into a hollow spheres(Fig. 2, top left), composed of six 4-helix bundle sub-units (Fig. 2, top right). After cleavage of a leadersequence, the 9 N-terminal residues fold into a uniquenickel hook motif that chelates the redox-active nickelion for catalysis (Fig. 2, bottom). This motif shows



F i g . 1 . Intermediates in the light cycle of photoactive yellow

protein. The cryo-trapped ultra-high-resolution (0.85 Å) protein

structure (bottom) shows the chromophore in its protein environ-

ment for the light-activated early intermediates highlighted in the

photocycle (top).

promise as a detectable metal ion–binding tag in pro-tein purification and structure determination.

To synthesize the cellular signal and defensive cyto-toxin nitric oxide, nitric oxide synthases (NOSs) requirecalmodulin-orchestrated interactions between their cata-lytic, heme-containing oxygenase module and theirelectron-supplying reductase module. Crystallographicstructures of wild-type and mutant NOS oxygenasedimers with substrate, intermediate, inhibitors, cofac-tors, and cofactor analogs, determined in collaborationwith D. Stuehr, the Cleveland Clinic, Cleveland, Ohio,and J. Tainer, provided insights into the catalytic mecha-nism and dimer stability.

Our structure-based drug design projects are aimedat selectively inhibiting either inducible NOS, to preventinflammatory disorders, or neuronal NOS, to preventmigraines, while maintaining blood pressure regulationby endothelial NOS. We integrated biochemical datawith our structures of NOS oxygenase, NOS reductase,and calmodulin in complex with peptides derived fromNOS to propose a model for the assembled holoenzymethat provides a moving-domain mechanism for electronflow from NADPH through 2 flavin cofactors to the heme.

Sulfite reductase catalyzes the 6-electron reductionof sulfite and nitrite required for the biogeochemicalcycling of sulfur and nitrogen. Our high-resolution struc-tures of the catalytic hemoprotein subunit of sulfitereductase characterize the cysteine-linked siroheme andFe4S4 cluster in 3 different states of oxidation and with

bound ligands to provide insights into the catalyticmechanism. Siroheme synthase completes the synthe-sis of the siroheme cofactor by catalyzing methylation,reduction, and metallation reactions. Our coupled struc-tural and functional studies define its 2 active sites andreveal a potential regulatory function for the unexpectedphosphoserine posttranslational modification.M E T A L L O P R O T E I N D E S I G N

An ultimate goal for protein engineers is to designand construct new protein variants with desirable cat-alytic or physical properties. As members of the ScrippsResearch Metalloprotein Structure and Design Group,we are testing our understanding of metal-ion affinity,selectivity, and activity by transplanting metal sites fromstructurally characterized metalloproteins into newprotein scaffolds. To aid our design efforts, at theMetalloprotein Database and Browser (available athttp://metallo.scripps.edu), we have organized quanti-tative information and interactive viewing of proteinmetal sites.

For green fluorescent protein and the homologousred fluorescent protein, we designed, constructed, andcharacterized metal-ion biosensors in which binding ofmetal ions is signaled by changes in the spectroscopicproperties of the naturally occurring fluorophores. Thegreen fluorescent protein scaffold provides advantagesover existing probes by allowing optimization with ran-dom mutagenesis, noninvasive expression in living cells,and targeting to specific cellular locations. By complet-ing the metalloprotein design cycle from prediction tohighly accurate structures, we can rigorously evaluateand improve our algorithms for the design of metal sites.Our related structural studies of green and red fluores-cent protein intermediates in chromophore cyclizationand oxidation provide a novel mechanism for the spon-taneous synthesis of these tripeptide fluorophores withinthe protein scaffold.

PUBLICATIONSBarondeau, D.P., Kassmann, C.J., Bruns, C.K., Tainer, J.A., Getzoff, E.D. Nickelsuperoxide dismutase structure and mechanism. Biochemistry 43:8038, 2004.

Garcin, E.D., Bruns, C.M., Lloyd, S.J., Hosfield, D.J., Tiso, M., Gachhui, R., Stuehr,D.J., Tainer, J.A., Getzoff, E.D. Structural basis for isozyme-specific regulation of elec-tron transfer in nitric-oxide synthase. J. Biol. Chem. 279:37918, 2004.

Stroupe, M.E., Leech, H.K., Daniels, D.S., Warren, M.J., Getzoff, E.D. CysGstructure reveals tetrapyrrole-binding features and novel regulation of sirohemebiosynthesis. Nat. Struct. Biol. 10:1064, 2003.



F i g . 2 . Structure of nickel SOD. A hollow sphere (top left) is

formed from six 4-helix bundle protein subunits (top right). The

N terminus of each subunit forms the nickel hook motif (bottom).

Nickel ions are shown as large spheres.

Structural Molecular Biology ofInteractions and Protein DesignJ.A. Tainer, A.S. Arvai, D.P. Barondeau, M. Bjoras,

B.R. Chapados, L. Craig, T.H. Cross, D.S. Daniels,

M. DiDonato, G. DiVita, L. Fan, C. Hitomi, K. Hitomi,

J.L. Huffman, C.J. Kassmann, I. Li, G. Moncalian,

M.E. Pique, D.S. Shin, O. Sundheim, T.I. Wood, A. Yamagata

At the level of basic research, we combine struc-ture and design projects with biochemistry andmutational analysis to characterize keystone

complexes, conformational changes, interfaces betweenmacromolecular machines, and the dynamics of poly-meric assemblies. We probe both active-site and sur-face chemistries to relate phenotypic and physiologicoutcomes to genomic polymorphisms. We are examiningprotein complexes involved in control of reactive oxygenspecies, DNA repair and genomic integrity, control ofthe cell cycle, bacterial pathogenesis, and cell death.

A second goal in research is advancement of thetechniques used to structurally characterize and visual-ize these systems. We focus on 3 major areas. First,we hope to close the resolution gaps between electronmicroscopy, small-angle x-ray scattering, and x-raycrystallography by merging these techniques computa-tionally. Second, we wish to accelerate structure deter-mination by improving synchrotron radiation facilities;in these studies, we are using combinatorial and sparsematrix approaches to isolate relevant protein complexesand are testing and validating model organisms. Third,we are using the synergy between basic research andadvancement in techniques to contribute to the basicunderstanding and treatment of degenerative and infec-tious diseases and cancer.R E A C T I V E O X Y G E N A N D X E N O B I O T I C

C O N T R O L E N Z Y M E S

Superoxide dismutases (SODs) are master regulatorsfor reactive oxygen species involved in injury, patho-genesis, aging, and degenerative diseases. For copper,zinc SOD, we defined the active-site structural chem-istry responsible for rapid catalysis and examined howsingle-site mutations cause the neurodegenerative dis-ease familial amyotrophic lateral sclerosis (FALS; alsoknown as Lou Gehrig disease). We used crystallogra-phy, atomic force, and electron microscopy to showthat point mutations destabilize the copper, zinc SODdimer and dramatically increase its propensity to aggre-gate and form filaments that resemble those seen in

motor neurons of patients with FALS. These filamentsbind both Congo red and thioflavin T, suggesting thepresence of amyloid-like stacked β-sheet interactions.These findings provide a molecular basis for the notionthat a single FALS phenotype arises from diverse pointmutations throughout the protein that reduce the struc-tural integrity of copper, zinc SOD and lower the energybarrier for fibrous aggregation.

D N A R E P A I R A N D G E N E T I C E V O L U T I O N

Life is impossible without constant repair of DNA.Structural and mutational analyses of DNA repairenzymes provide a framework for understanding themolecular basis for genetic integrity and its loss in can-cer and degenerative diseases. We are interested inhow specific types of damage are detected, how repairenzymes are coordinated within different pathways, andthe nature and role of conformational change in proteinsand DNA in repair pathways. We use a variety of tech-niques to probe the molecular machines involved inDNA repair.

To understand how flap endonuclease-1 and prolif-erating cell nuclear antigen interact in conjunction withDNA, we combined x-ray crystal structures with fluo-rescence techniques and mutational assays; the resultsled to a new kinked DNA model for the coordination ofDNA replication and repair. We used electron micros-copy, x-ray crystallography, small-angle x-ray scattering,and complementary in vitro and in vivo mutational analy-sis with the archaeal protein Rad51 to support a modelfor coordination and control of homologous recombina-tion (Fig. 1). Our results imply a mechanism for the lossof effective targeting of Rad51 to sites of DNA damageby BRCA2, a Rad51 recruitment protein whose defectsare associated with a hereditary type of breast cancer,leading to potential carcinogenesis. By combining crys-tal structures with computational modeling studies,we examined the mechanisms for controlling levels ofdeoxycytidine triphosphate relative to deoxythymidinetriphosphate and for removing harmful deoxyuridinetriphosphate from the nucleotide pool within cells.Understanding the structural chemistry and cell biol-ogy of DNA repair is critical for designing specificinhibitors to increase the effectiveness of chemother-apy and also for assessing how polymorphisms inDNA repair enzymes may affect diseases in humans.B A C T E R I A L P I L I A N D I N F E C T I O U S D I S E A S E S

Type IV pili are essential virulence factors for manygram-negative bacteria, playing key roles in surfacemotility, adhesion, formation of microcolonies and bio-



films, natural transformation, and signaling. Thesemultifunctional hairlike filaments are remarkably stronggiven their narrow dimensions (50–80 Å in diameterand several microns in length). Type IV pili and theirassembly machinery bear a strong resemblance to 2 other protein export systems: the type II secretionsystem, which exports bacterial toxins and hydrolyticenzymes, and the archaeal flagella, which are requiredfor motility in archaea. We derived computational mod-els for the type IV pili of Pseudomonas aeruginosa andVibrio cholerae by combining our atomic structures ofpilin subunits and information on helical symmetryderived from electron microscopy and fiber diffractionanalyses (Fig. 2). These models reveal a commonassembly mechanism that accounts for both the extrememechanical strength and the multifunctionality of typeIV pili. We are working to solve electron microscopyand x-ray structures of protein components and com-plexes common to all 3 protein export systems in aneffort to understand their overall architecture and assem-bly mechanism for use in the design of antibacterial vac-cines and therapeutic agents.

PUBLICATIONSArthur, L.M., Gustausson, K., Hopfner, K.P., Carson, C.T., Stracker, T.H., Karcher, A.,Felton, D., Weitzman, M.D., Tainer, J., Carney, J.P. Structural and functional analysisof Mre11-3. Nucleic Acids Res. 32:1886, 2004.

Barondeau, D.P., Kassmann, C.J., Bruns C.K., Tainer, J.A., Getzoff, E.D. Nickelsuperoxide dismutase structure and mechanism. Biochemistry 43:8038, 2004.

Barondeau, D.P., Putnam, C.D., Kassmann, C.J., Tainer, J.A., Getzoff, E.D. Mecha-nism and energetics of green fluorescent protein chromophore synthesis revealed bytrapped intermediate structures. Proc. Natl. Acad. Sci. U. S. A. 100:12111, 2003.

Chapados, B.R., Hosfield, D.J., Han, S., Qiu, J., Yelent, B., Shen, B., Tainer, J.A.Structural basis for FEN-1 substrate specificity and PCNA-mediated activation inDNA replication and repair. Cell 116:39, 2004.

Craig, L., Pique, M.E., Tainer, J.A. Type IV pilus structure and bacterial patho-genicity. Nat. Rev. Microbiol. 2:363, 2004.

Daniels, D.S., Woo, T.T., Luu, K.X., Noll, D.M., Clarke, N.D., Pegg, A.E., Tainer,J.A. DNA binding and nucleotide flipping by the human DNA repair protein AGT.Nat. Struct. Mol. Biol. 11:714, 2004.

DiDonato, M., Craig, L., Huff, M.E., Thayer, M.M., Cardoso, R.M., Kassmann,C.J., Lo, T.P., Bruns, C.K., Powers, E.T., Kelly, J.W., Getzoff, E.D., Tainer, J.A.ALS mutants of human superoxide dismutase form fibrous aggregates via frameworkdestabilization [published correction appears in J. Mol. Biol. 334:175, 2003]. J. Mol. Biol. 332:601, 2003.

Greenleaf, W.B., Perry, J., Hearn, A.S., Cabelli, D.E., Lepock, J.R., Stroupe, M.E.,Tainer, J.A., Nick, H.S., Silverman, D.N. Role of hydrogen bonding in the active site ofhuman manganese superoxide dismutase. Biochemistry 43:7038, 2004.

Hearn, A.S., Fan, L., Lepock, J.R., Luba, J.P., Greenleaf, W.B., Cabelli, D.E., Tainer,J.A., Nick, H.S., Silverman, D.N. Amino acid substitution at the dimeric interface ofhuman manganese superoxide dismutase. J. Biol. Chem. 279:5861, 2004.

Hencrickson, E.A., Huffman, J.L., Tainer, J.A. Structural aspects of Ku and theDNA-dependent protein kinase complex. In: DNA Damage Recognition. Seide, W.,Kow, Y.W., Doetsch, P.W. (Eds.). Marcel-Dekker, New York, in press.

Huffman, J.L., Li, H., White, R.H., Tainer, J.A. Structural basis for recognition andcatalysis by the bifunctional dCTP deaminase and dUTPase from Methanococcus jan-naschii. J. Mol. Biol. 331:885, 2003.

Huffman, J.L., Sundheim, O., Tainer, J.A. Structural features of DNA glycosylasesand AP endonucleases. In: DNA Damage Recognition. Seide, W., Kow, Y.W.,Doetsch, P.W. (Eds.). Marcel-Dekker, New York, in press.



F i g . 2 . Models of the type IV pilus filaments from V cholerae

toxin-coregulated pilus (A) and P aeruginosa PAK pilus (B). Filaments

assemble via interactions among the conserved hydrophobic N-termi-

nal α-helices and interactions between the structurally variable

αβ-loop and D-region (boxes).

F i g . 1 . Models of the active DNA-bound Rad51 nucleoprotein

filament. A, Computational docking of our Pyrococcus furiosus

Rad51 x-ray crystal structure into Sulfolobus solfataricus Rad51-

DNA electron microscopy 3-dimensional reconstructions. A polymer-

ization motif that tethers Rad51 subunits together in the ring form

was retained in this docking experiment, providing evidence that this

motif is necessary for dynamic ring-to-filament transitions. B, A con-

served helix-hairpin-helix (HhH) motif was used to identify a double-

stranded DNA (dsDNA) binding site for the filament. Channels allow

contact between single-stranded DNA within the interior and the dou-

ble-stranded DNA.

Manuel, R.C., Hitomi, K., Arvai, A.S., House, P.G., Kurtz, A.J., Dodson, M.L.,McCullough, A.K., Tainer, J.A., Lloyd, R.S. Reaction intermediates in the catalyticmechanism of Escherichia coli MutY DNA glycosylase. J. Biol. Chem., in press.

McMurray, C.T., Tainer, J.A. Cancer, cadmium and genome integrity. Nat. Genet.34:239, 2003.

Moncalian, G., Lengsfeld, B., Bhaskara, V., Hopfner, K.P., Karcher, A., Alden, E.,Tainer, J.A., Paull, T.T. The Rad50 signature motif: essential to ATP binding andbiological function. J. Mol. Biol. 335:937, 2004.

Qiu, J., Liu, R., Chapados, B.R., Sherman, M., Tainer, J.A., Shen, B. Interactioninterface of human flap endonuclease-1 with its DNA substrates. J. Biol. Chem.279:24394, 2004.

Shin, D.S., Chahwan, C., Huffman, J.L., Tainer, J.A. Structure and function of thedouble-strand break repair machinery. DNA Repair (Amst.) 3:863, 2004.

Shin, D.S., Pellegrini, L., Daniels, D.S., Yelent, B., Craig, L., Bates, D., Yu, D.S.,Shivji, M.K., Hitomi, C., Arvai, A.S., Volkmann, N., Tsuruta, H., Blundell, T.L., Venki-taraman, A.R., Tainer, J.A. Full-length archaeal Rad51 structure and mutants: mecha-nisms for RAD51 assembly and control by BRCA2. Embo J. 22:4566, 2003.

Wang, M., Howell, J.M., Libbey, J.E., Tainer, J.A., Fujinami, R.S. Manganese superox-ide dismutase induction during measles virus infection. J. Med. Virol. 70:470, 2003.

Structural Biology of IntegralMembrane ProteinsG. Chang, X. He, A. Ma, T. Nguyen, O. Pornillos,

C.R. Reyes, P. Szewczk, Y. Yin

Determination of the structure of integral mem-brane proteins is an exciting frontier in molec-ular structural biology. We are interested in 3

areas: the structural basis for substrate transport acrossthe cell membrane by transporters, signal transductionby receptors, and the structure of mammalian mem-brane-bound transporters. We use several experimentalmethods, including detergent/lipid protein biochemistry,3-dimensional crystallization of integral membrane pro-teins, and x-ray crystallography.

We are addressing the molecular basis of multidrugresistance, a significant challenge in the treatment ofdisease and the development of therapeutic agents.The x-ray structures of MsbA, a multidrug resistanceATP-binding cassette transporter, and the H+/drugantiporter EmrE reveal the molecular structural basisof lipid and drug transport. MsbA is organized as adimer; each subunit contains 6 transmembrane α-helicesand a nucleotide-binding domain. The asymmetric dis-tribution of charged residues lining a central chamberin the cell membrane suggests a general mechanismfor the translocation of hydrophobic substrate by MsbAand other multidrug resistance ATP-binding cassettetransporters. The open and closed conformations ofMsbA reveal the structural changes that are possibleby ATP-binding cassette exporters.

The x-ray structure of EmrE reveals a transportercomposed of identical dimers that assemble indepen-dently and associate to form a tetramer. Each half ofthe transporter is composed of a structural heterodimerwith 2 monomer protein chains that come together indifferent conformations. Because of this asymmetry,the positions of 2 pairs of important glutamate residuesare nonequivalent in the transporter and have differenttasks coupling proton translocation with drug transportthrough the cell membrane. The x-ray structures of MsbAand EmrE are excellent models for drug efflux systemsthat confer multidrug resistance to cancer cells andinfectious microorganisms.

A major part of our research is determining thestructure of receptors, the classic models for signaltransduction across the cell membrane. One of the mostwidely studied families of receptors is the family of G protein–coupled receptors. These receptors are phar-maceutically important drug targets because more than70% of all drugs on the market target them. All G pro-tein–coupled receptors are predicted to have 7 trans-membrane α-helices with domains that bind ligandand activate G proteins. How does a ligand or drugmolecule on the outside of the cell membrane transmita signal across the cell membrane to initiate a signalon the cytoplasmic side? A structure of these receptorswill shed light on this important biophysical question.

PUBLICATIONSChang, G. Multidrug resistance ABC transporters. FEBS Lett. 555:102, 2003.

Ma, C., Chang, G. Structure of the multidrug resistance efflux transporter EmrEfrom Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 101:2852, 2004.

Crystallography ofTranshydrogenaseC.D. Stout, H. Heaslet, M. Yamaguchi, V. Sundaresan

We use x-ray crystallography in combinationwith biophysical methods and focus on pro-teins and enzymes involved in fundamental

biological processes. The research involves close collab-oration with colleagues at Scripps. Our goal is to under-stand molecular structure and function; experimentsentail the design and preparation of specific complexesand enzymatic states, crystallization, structure determi-nation and analysis, and assay of biological function.Principal projects are studies of the enzymes cytochrome



P-450 and transhydrogenase and of electron-transferproteins that contain iron-sulfur clusters.

The mitochondrial enzyme transhydrogenase trans-ports protons across the membrane in concert withhydride exchange between NAD(H) and NADP(H), areaction that couples the proton motive force to theconcentration of reducing equivalents. Crystal struc-tures of the soluble NADP(H)- and NAD(H)-bindingdomains of transhydrogenase were determined. Essen-tial residues involved in proton translocation throughthe integral membrane domain were defined in muta-genesis and biochemical studies, identifying residuesand transmembrane helices involved in formation ofthe proton channel (Fig. 1). Recent crystal structuresof the NADP(H)-binding domain in both oxidized andreduced states revealed that extensive conformationalchange is required of this domain to transduce NADPH-binding energy into changes in pK associated with protontranslocation. A structure in progress, of the cocrystallizedsoluble domains in the presence of NADH and NADP,will provide a basis for studying the mechanism ofhydride transfer between the extramembranous domains.

In collaboration with M. Yeager and B. Carragher,Department of Cell Biology, we are using electron micros-copy of single particles and 2-dimensional crystals inlipid bilayers to visualize the intact 200-kD holoenzyme.

In addition, crystallization experiments are in progresswith purified, detergent-solubilized and monodispersedtranshydrogenase to obtain 3-dimensional crystals forx-ray diffraction experiments. The structural resultswill be correlated with ongoing mutagenesis and kinet-ics studies to establish the basis of proton pumping inthis essential enzyme of respiration.

PUBLICATIONSCamba, R., Jung, Y.S., Hunsicker-Wang, L.M., Burgess, B.K., Stout, C.D., Hirst, J.,Armstrong, F.A. Mechanisms of redox-coupled proton transfer in proteins: role ofthe proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandiiferredoxin I. Biochemistry 42:10589, 2003.

Hunsicker-Wang, L.M., Heine, A., Chen, Y., Luna, E.P., Todaro, T., Zhang, Y.M.,Williams, P.A., McRee, D.E., Hirst, J., Stout, C.D., Fee, J.A. High-resolution struc-ture of the soluble, respiratory-type Rieske protein from Thermus themophilus:analysis and comparison. Biochemistry 42:7303, 2003.

Schoch, G.A., Yano, J.K., Wester, M.R., Griffin, K.J., Stout, C.D., Johnson, E.F.Structure of human microsomal cytochrome P450 2C8: evidence for a peripheralfatty acid binding site. J. Biol. Chem. 279:9497, 2004.

Scott, E.E., He, Y.A., Wester, M.R., White, M.A., Chin, C.C., Halpert, J.R., John-son, E.F., Stout, C.D. An open conformation of mammalian cytochrome P450 2B4at 1.6-Å resolution. Proc. Natl. Acad. Sci. U. S. A. 100:13196, 2003.

Scott, E.E., White, M.A., He, Y.A., Johnson, E.F., Stout, C.D., Halpert, J.R. Struc-ture of mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imid-azole at 1.9 Å resolution: insight into the range of P450 conformations and coordinationof redox partner binding. J. Biol. Chem. 279:27294, 2004.

Stout, C.D. Induced fit, drug design and dUTPase. Structure (Camb.) 12:2, 2004.

Sundaresan, V., Yamaguchi, M., Chartron, J., Stout, C.D. Conformational changein the NADP(H) binding domain of transhydrogenase defines four states. Biochem-istry 42:12143, 2003.

Wester, M.R., Johnson, E.F., Marques-Soares, C., Dijols, S., Dansette, P.,Mansuy, D., Stout, C.D. Structure of mammalian cytochrome P450 2C5 com-plexed with diclofenac at 2.1 Å resolution: evidence for an induced fit model ofsubstrate binding. Biochemistry 42:9335, 2003.

Yamaguchi, M., Stout, C.D. Essential glycine in the proton channel of Escherichiacoli transhydrogenase. J. Biol. Chem. 278:45333, 2003.

High-Throughput Structure-Based Drug Discovery andStructural NeurobiologyR.C. Stevens, E.A. Abola, J. Arndt, S.E. Andryski, G. Asmar-

Rovira, R. Benoit, M.H. Bracey, Q. Chai, J. Chappie,

T. Clayton, B. Collins, A. Gámez, J. Godzik, C. Grittini,

M.A. Hanson, J. Joseph, W. Kim, K. Masuda, B. McManus,

K. Moy, M. Nelson, R. Page, M.G. Patch, E.C. Sims, V. Sridhar,

M. Straub, V. Subramanian, J. Velasquez, L. Wang

H I G H - T H R O U G H P U T S T R U C T U R A L B I O L O G Y

For the past several years, we have focused ondeveloping tools to change the field of structuralbiology by accelerating the rate of determination

of protein structures, an endeavor that includes pioneer-



F i g . 1 . The 4-helix bundle model for the proton channel in the

β-subunit of Escherichia coli transhydrogenase consists of α-helices

H9, H10, H13, and H14 containing the essential residues βHis91,

βSer139, βAsn222, and βGly252. βAsp213 and βGlu85 participate

in H+ conductance between the channel and βAsp392 and/or βHis345

in extramembranous domain III. An orthogonal view of the 4 helices

is shown on the right.

ing microliter expression/purification for structural stud-ies, nanovolume crystallization, and automated imagecollection. This effort, which now encompasses theJoint Center for Structural Genomics (JCSG), is a col-laboration between our laboratory and laboratories atScripps Research, the Genomics Institute of the Novar-tis Research Foundation, the San Diego Supercomput-ing Center, and the Stanford Synchrotron RadiationLaboratory. To demonstrate the power of the technology,members of the JCGS are trying to determine all of thenovel protein structures of a prokaryotic genome (Ther-motoga maritima) and signaling proteins of a eukary-otic genome (mouse). These experimental goals areambitious, and the bioinformatics challenge to effectivelymanage and mine the wealth of information that is beinggenerated is enormous. More recently, we started theJCSG Center for Innovative Membrane Protein Tech-nologies, where in collaboration with G. Chang and K. Wüthrich, Department of Molecular Biology, P. Kuhnand M. Yeager, Department of Cell Biology, and M.G.Finn, Department of Chemistry, we will pursue tech-nology developments for more challenging membraneproteins, such as G protein–coupled receptors, ion chan-nels, and drug transporters.S T R U C T U R A L N E U R O B I O L O G Y

Although we have developed high-throughput meth-ods to accelerate the determination of protein structures,our primary interest is using these tools to study thechemistry and biology of neurotransmission and ofdiseases that affect neurons. Our goals are to under-stand how neuronal cells function on a molecular leveland, on the basis of that understanding, create newmolecules and materials that mimic neuronal signaltransduction and recognition. We use high-throughputprotein crystallography and biochemical methods toprobe the structure and function of molecules involvedin neurotransmission and neurochemistry.F A T T Y A C I D A M I D E H Y D R O L A S E

In collaboration with B.F. Cravatt, Department ofCell Biology, we solved the structure of fatty acid amidehydrolase (FAAH), a degradative integral membraneenzyme responsible for setting intracellular levels ofendocannabinoids, to 2.8 Å (Figs. 1 and 2). FAAH isintimately associated with CNS signaling processes suchas retrograde synaptic transmission, a process that isalso modulated by the illicit substance δ9-tetrahydro-cannabinol. FAAH is a dimer capable of monotopicmembrane insertion; it has an active-site structure con-sistent with the capacity for hydrolysis of hydrophobic

fatty acid amides and structural features amenable tostructure-based drug design. With our knowledge of the3-dimensional structure, we are trying to understandhow the enzyme works at a basic level and how it mightbe the basis for potential drug discovery.



F i g . 1 . Structure of the integral membrane protein FAAH modeled

into a lipid bilayer. The enzyme is a homodimer assembled from

63-kD subunits. This view of the enzyme highlights the central twisted

β-sheet that forms the core of the structure. The inhibitor adduct

methoxy arachidonyl fluorophosphonate (MAP) is depicted in the

active site with van der Waals surfaces.

F i g . 2 . Active site of FAAH in complex with the arachidonyl inhib-

itor methoxy arachidonyl fluorophosphonate (MAP). The FAAH dimer

is shown with part of the protein surface removed to highlight the

continuous internal channel leading from the membrane binding face

at Arg486 and Asp403 to the active site and on to the cytosolic

port. Electron density corresponding to the arachidonyl inhibitor is

shown and lies in the hydrophobic substrate-binding pocket. Trp445

from the dimer mate is rendered in van der Waals surface to demon-

strate the effective plugging of this potential port.

B I O S Y N T H E S I S O F N E U R O T R A N S M I T T E R S

For neuronal signal transduction, the presynapticcell synthesizes neurotransmitters that then traversethe synaptic cleft. We are using the high-throughputmethods to determine the inclusive structures of com-plete biochemical pathways. Specifically, we are inter-ested in determining the structures of all the enzymesin the biosynthesis pathways of neurotransmitters inorder to understand the mechanistic details of eachindividual enzymatic reaction at the atomic level. Thisapproach also allows us to determine the best path fordrug discovery in the areas of neurotransmitter biosyn-thesis and catabolism.

Phenylalanine hydroxylase and tyrosine hydroxylaseinitiate the first committed steps in the biosynthesis ofthe neurotransmitters dopamine, adrenaline, and nor-adrenaline, and tryptophan hydroxylase catalyzes therate-determining step in the biosynthesis of serotonin.Because of the importance of these neurotransmittersin the proper functioning of the CNS, understandingthe molecular details involved in the catalysis and reg-ulation of these biosynthetic enzymes is crucial. Werecently determined the 3-dimensional structures fortyrosine hydroxylase, tryptophan hydroxylase, andphenylalanine hydroxylase, and we are now uncover-ing specific mechanistic details for these enzymes. Ourultimate goal is to selectively modulate serotonergic,noradrenergic, or dopaminergic function.

In addition to the basic enzymology questions underinvestigation, recent clinical studies suggest that somepatients with the metabolic disease phenylketonuria areresponsive to (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin(BH4), the natural cofactor of phenylalanine hydroxy-lase. We are doing studies to correlate how structurecan be used to predict which patients with phenylke-tonuria most likely will respond to treatment with thiscofactor. Currently, BH4 is entering phase 2 clinical tri-als. In addition, we have created an enzyme replacementtherapeutic agent that is being tested in animal models.The therapy is based on administration of a minimizedform of the phenylalanine hydroxylase discovered inour structural studies. Last, we are determining thestructural basis of diseases caused by several otherenzymes involved in the biosynthesis of neurotrans-mitters. Many of these diseases are rare or occur dur-ing childhood.N E U R O T O X I N S

The clostridial neurotoxins include tetanus toxin andthe 7 serotypes of botulinum toxin. We are determin-

ing the molecular events involved in the binding, poreformation, translocation, and catalysis of botulinumneurotoxin. Although botulinum toxin is most knownfor its deadly effects, it is now being used therapeuti-cally to treat involuntary muscle disorders. Recently,we determined the structure of the 900-kD complexform of the toxin, 150-kD holotoxin form, the catalyticdomain, and the catalytic domain bound to substratesand inhibitors. These structures are being used tounderstand the toxin’s mechanism of action and maybe useful in determining additional therapeutic appli-cations of the toxin.

PUBLICATIONSBakolitsa, C., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of anorphan protein (TM0875) from Thermotoga maritima at 2.00 Å resolution revealsa new fold. Proteins 56:607, 2004.

Bern, M., Goldberg, D., Stevens, R.C., Kuhn, P. Automatic classification of protein crys-tallization images using a curve-tracking algorithm. J. Appl. Crystallogr. 37:279, 2004.

Bracey, M.H., Cravatt, B.F., Stevens, R.C. Structural commonalities among inte-gral membrane enzymes. FEBS Lett. 567:159, 2004.

Canaves, J.M., Page, R., Stevens, R.C. Protein biophysical properties that corre-late with crystallization success in Thermotoga maritima: maximum clusteringstrategy for structural genomics. J. Mol. Biol., in press.

Erlandsen, H., Canaves, J.M., Elsliger, M.-A., et al. Crystal structure of an HEPNdomain protein (TM0613) from Thermotoga maritima at 1.75 Å resolution. Pro-teins 54:806, 2004.

Erlandsen, H., Patch, M.G., Gámez, A., Straub, M., Stevens, R.C. Structural stud-ies on phenylalanine hydroxylase and implications toward understanding and treat-ing phenylketonuria. Pediatrics 112(6 Pt. 2):1557, 2003.

Erlandsen, H., Pey, A.L., Gámez, A., Pérez, B., Desviat, L.R., Aguado, C., Koch,R., Surendrran, S., Tyring, T., Matalon, R., Scriver, C.R., Ugarte, M., Martínez,A., Stevens, R.C. Correction of kinetic and stability defects by the cofactor BH4 inPKU patients with certain phenylalanine hydroxylase mutations. Proc. Natl. Acad.Sci. U. S. A., in press.

Gámez, A., Wang, L., Straub, M., Patch, M.G., Stevens, R.C. Toward PKU enzymereplacement therapy: PEGylation with activity retention for three forms of recombinantphenylalanine hydroxylase. Mol. Ther. 9:124, 2004.

Heine, A., Canaves, J.M., von Delft, F., et al. Crystal structure of O-acetylserinesulfhydrylase (TM0665) from Thermotoga maritima at 1.8 Å resolution. Proteins56:387, 2004.

Jaroszewski, L., Schwarzenbacher, R., von Delft, F., et al. Crystal structure of anovel manganese-counting cupin (TM1459) from Thermotoga maritima at 1.65 Åresolution. Proteins 56:611, 2004.

Kim, W., Erlandsen, H., Surendran, S., Stevens, R.C., Gámez, A., Michols-Mat-alon, K., Tyring, S.K., Matalon, R. Trends in enzyme replacement therapy forphenylketonuria. Mol. Ther. 10:220, 2004.

Levin, I., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a puta-tive NADPH-dependent oxidoreductase (GI: 18204011) from mouse at 2.10 Å res-olution. Proteins 56:629, 2004.

Levin, I., Schwarzenbacher, R., Page, R., et al. Crystal structure of a PIN (PilT N-terminus) domain (AF0591) from Archeoglobus fulgidus at 1.90 Å resolution. Pro-teins 56:404, 2004.

Matalon, R., Koch, R., Michals-Matalon, K., Moseley, K., Surendran, S., Tyring, S.,Erlandsen, H., Gámez, A., Stevens, R.C., Romstad, A., Moller, L.B., Guttler, F.Biopterin responsive phenylalanine hydroxylase deficiency. Genet. Med. 6:27, 2004.



McMullan, D., Schwarzenbacher, R., Jaroszewski, L., et al. Crystal structure of anovel Thermotoga maritima enzyme (TM1112) from the cupin family at 1.83 Åresolution. Proteins 56:615, 2004.

Miller, M.D., Schwarzenbacher, R., von Delft, F., et al. Crystal structure of a tan-dem cystathionine-β-synthase (CBS) domain protein (TM0935) from Thermotogamaritima at 1.87 Å resolution. Proteins 57:213, 2004.

Page, R., Moy, K., Sims, E.C., Velasquez, J., McManus, B., Grittini, C., Clayton,T.L., Stevens, R.C. Scalable high-throughput micro-expression device for recombi-nant proteins. Biotechniques 37:364, 2004.

Page, R., Nelson, M.S., von Delft, F., et al. Crystal structure of γ-glutamyl phos-phate reductase (TM0293) from Thermotoga maritima at 2.0 Å resolution. Pro-teins 54:157, 2004.

Page, R., Stevens, R.C. Crystallization data mining in structural genomics: usingpositive and negative results to optimize protein crystallization screens. Methods34:373, 2004.

Pantazatos, D., Kim, J.S., Klock, H.E., Stevens, R.C., Wilson, I.A., Lesley, S.A.,Woods, V.L., Jr. Rapid refinement of crystallographic protein construct definitionemploying enhanced hydrogen/deuterium exchange MS. Proc. Natl. Acad. Sci. U. S. A. 101:757, 2004.

Pey, A.L., Pérez, B., Desviat, L.R., Erlandsen, H., Gámez, A., Stevens, R.C.,Ugarte, M., Martínez, A. Mechanisms underlying responsiveness to tetrahydro-biopterin in mild phenylketonuria mutations. Hum. Mutat., in press.

Pey, A.L., Thórólfsson, M., Erlandsen, H., Stevens, R.C., Ugarte, M., Desviat,L.D., Pérez, B., Martínez, A. Thermodynamic parameters for the binding oftetrahydrobiopterin to human phenylalanine hydroxylase. In: Pterins, Folates andNeurotransmitters in Molecular Medicine. Blau, N., Thöny, B. (Eds.) SPS Publica-tions, Heilbronn, Germany, 2004, p.155.

Santelli, E., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima(TM1621) at 1.60 Å resolution. Proteins 56:167, 2004.

Scriver, C.R., Hurtubise, M., Prevost, L., Phommarinh, M., Konecki, D., Erland-sen, H., Stevens, R.C., Waters, P.J., Ryan, S., McDonald, D., Sarkissan, C. APAH gene knowledge base: content, informatics, utilization. In: PKU and BH4:Advances in Phenylketonuria and Tetrahydrobiopterin Research. Blau, N. (Ed.).SPS Publications, Heilbronn, Germany, in press.

Schwarzenbacher, R., Canaves, J.M., Brinen, L.S., et al. Crystal structure of uronateisomerase (TM0064) from Thermotoga maritima at 2.85 Å resolution. Proteins53:142, 2003.

Schwarzenbacher, R., Deacon, A.M., Jaroszewski, L., et al. Crystal structure of aputative glutamine amino transferase (TM1158) from Thermotoga maritima at 1.7Å resolution. Proteins 54:801, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of anaspartate aminotransferase (TM1255) from Thermotoga maritima at 1.90 Å reso-lution. Proteins 55:759, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of aphosphoribosylaminoimidazole mutase PurE (TM0446) from Thermotoga maritimaat 1.77 Å resolution. Proteins 55:474, 2004.

Schwarzenbacher, R., Jaroszewski, L., von Delft, F., et al. Crystal structure of atype II quinolic acid phosphoribosyltransferase (TM1645) from Thermotoga mar-itima at 2.50 Å resolution. Proteins 55:768, 2004.

Schwarzenbacher, R., von Delft, F., Abdubek, P., et al. Crystal structure of a puta-tive PII-like signaling protein (TM0021) from Thermotoga maritima at 2.5 Å reso-lution. Proteins 54:810, 2004.

Schwarzenbacher, R., von Delft, F., Canaves, J.M., et al. Crystal structure of aniron-containing 1,3-propanediol dehydrogenase (TM0920) from Thermotoga mar-itima at 1.3 Å resolution. Proteins 54:174, 2004.

Schwarzenbacher, R., von Delft, F., Jaroszewski, L., et al. Crystal structure of aputative oxalate decarboxylase (TM1287) from Thermotoga maritima at 1.95 Åresolution. Proteins 56:392, 2004.

Shen, Z., Go, E.P., Gámez, A., Apon, J.V., Fokin, V., Greig, M., Ventura, M.,Crowell, J.E., Blixt, O., Paulson, J.C., Stevens, R.C., Finn, M.G., Siuzdak, G. Amass spectrometry plate reader: monitoring enzyme activity and inhibition with adesorption/ionization on silicon (DIOS) platform. Chembiochem 5:921, 2004.

Snell, G., Cork, C., Nordmeyer, R., Cornell, E., Meigs, G., Yegian, D., Jaklevic, J.,Jin, J., Stevens, R.C., Earnest, T. Automated sample mounting and alignment sys-tem for biological crystallography at a synchrotron source. Structure (Camb.)12:537, 2004.

Song, J., Stevens, R.C., Cheng, Q. Functional amphiphilic and bolaamphiphilicpoly(diacetylene) assemblies with controlled optical and morphological properties.In: Chromogenic Phenomena in Polymers: Tunable Optical Properties. Jenekhe,S.A., Kiserow, D.J. (Eds.). Oxford University Press, New York, 2004.

Spraggon, G., Schwarzenbacher, R., Kreusch, A., et al. Crystal structure of amethionine aminopeptidase (TM1478) from Thermotoga maritima at 1.9 Å resolu-tion. Proteins 56:396, 2004.

Spraggon, G., Schwarzenbacher, R., Kreusch, A., et al. Crystal structure of anUdp-n-acetylmuramate-alanine ligase MurC (TM0231) from Thermotoga maritimaat 2.3 Å resolution. Proteins 55:1078, 2004.

Stevens, R.C. Long live structural biology. Nat. Struct. Mol. Biol. 11:293, 2004.

Xu, Q., Schwarzenbacher, R., McMullan, D., et al. Crystal structure of a ribose-5-phosphate isomerase RpiB (TM1080) from Thermotoga maritima at 1.90 Å resolu-tion. Proteins 56:171, 2004.

Xu, Q., Schwarzenbacher, R., Page, R., et al. Crystal structure of an allantoicase(YIR029W) from Saccharomyces cerevisiae at 2.4 Å resolution. Proteins 56:619, 2004.

Nuclear Magnetic Resonance of3-Dimensional Structure andDynamics of Proteins in SolutionP.E. Wright, H.J. Dyson, A. Atkins, J. Covalt, R. De Guzman,

T. Dunzendorfer-Matt, T.-H. Huang, B. Hudson, J. Lansing,

B. Lee, G. Perez-Alvarado, M. Landes, M. Martinez-Yamout,

T. Nishikawa, J. Wojciak, C. Reyes, L.L. Tennant, J. Chung,

D.A. Case, J. Gottesfeld, B. Cunningham, R. Evans,*

M. Montminy*

*Salk Institute, La Jolla, California

We use multidimensional nuclear magneticresonance (NMR) spectroscopy to investi-gate the structures, dynamics, and interac-

tions of proteins in solution. Such studies are essentialfor understanding the mechanisms of action of theseproteins and for elucidating structure-function relation-ships. The focus of our current research is protein-pro-tein and protein–nucleic acid interactions involved inthe regulation of gene expression.T R A N S C R I P T I O N F A C T O R – N U C L E I C

A C I D C O M P L E X E S

NMR methods are being used to determine the 3-dimensional structures and intramolecular dynamics ofzinc finger motifs from several eukaryotic transcriptional



regulatory proteins, both free and complexed with tar-get nucleic acid. Zinc fingers are among the most abun-dant domains in eukaryotic genomes. They play a centralrole in the regulation of gene expression at both thetranscriptional and the posttranscriptional levels, medi-ated through their interactions with DNA, RNA, or pro-tein components of the transcriptional machinery. TheC2H2 zinc finger, first identified in transcription factorIIIA (TFIIIA), is used by numerous transcription factorsto achieve sequence-specific recognition of DNA. Thereis growing evidence, however, that some C2H2 zincfinger proteins control gene expression both throughtheir interactions with DNA regulatory elements and,at the posttranscriptional level, by binding to RNA.

The best-characterized example of a C2H2 zinc fin-ger protein that binds specifically to both DNA and toRNA is TFIIIA, which contains 9 zinc fingers. Weshowed previously that different subsets of zinc fingersare responsible for high-affinity binding of TFIIIA to DNA(fingers 1–3) and to 5S RNA (fingers 4–6). To obtaininsights into the mechanism by which the TFIIA zinc fin-gers recognize both DNA and RNA, we are using NMRmethods to determine the structures of the complexesformed by zf1-3 (a protein containing fingers 1–3) withDNA and by zf4-6 (a protein consisting of fingers 4–6)with a fragment of 5S RNA.

Three-dimensional structures were determined pre-viously for the complex of zf1-3 with the cognate 15-bpoligonucleotide duplex. The structures contain severalnovel features and reveal that prevailing models of DNArecognition, which assume that zinc fingers are inde-pendent modules that contact bases through a limitedset of amino acids, are outmoded.

In addition to its role in binding to and regulatingthe 5S RNA gene, TFIIIA also forms a complex withthe 5S RNA transcript. NMR studies of the complexformed by zinc fingers 4–6 with a truncated form of5S RNA are in progress and should give importantinsights into the structural basis for 5S RNA recogni-tion. At this stage, the interactions involving finger 4have been fully delineated, showing that the proteinrecognizes both the structure of the RNA backboneand the specific bases in the loop E motif.

NMR studies of 2 alternate splice variants of theWilms tumor zinc finger protein are in progress. Theseproteins differ only through insertion of 3 additionalamino acids (the tripeptide lysine-threonine-serine) inthe linker between fingers 3 and 4, yet have markeddifferences in their DNA-binding properties and sub-

cellular localization. 15N relaxation measurementsindicate that the insertion increases the flexibility ofthe linker between fingers 3 and 4 and abrogatesbinding of the fourth zinc finger to its cognate site inthe DNA major groove, thereby modulating DNA-bind-ing activity. The x-ray structure of the DNA complexhas now been determined, and NMR studies of RNAbinding are in progress.

Several novel zinc binding motifs have recentlybeen identified that mediate gene expression at theposttranscriptional level by regulating mRNA process-ing and metabolism. Regulatory proteins of the TIS11family bind specifically, through a pair of novel CCCHzinc fingers, to the adenosine-uridine–rich element inthe 3′ untranslated region of short-lived cytokine, growthfactor, and proto-oncogene mRNAs and control expres-sion by promoting rapid degradation of the message.We determined the NMR structure of the complexformed between the tandem zinc finger domain ofTIS11d and its binding site on the adenosine-uridine–rich element (Fig. 1). This structure provides novelinsights into mechanisms of sequence-specific recogni-tion of single-stranded RNA through formation of anetwork of hydrogen bonds between the polypeptidebackbone and the Watson-Crick edges of the bases.

P R O T E I N - P R O T E I N I N T E R A C T I O N S I N

T R A N S C R I P T I O N A L R E G U L A T I O N

Transcriptional regulation in eukaryotes relies onprotein-protein interactions between DNA-bound fac-



F i g . 1 . Structure of the TIS11d complex with its cognate adeno-

sine-uridine–rich RNA recognition element. The protein backbone is

shown as a light gray ribbon, the zinc atoms are shown as spheres,

and the RNA backbone and bases are dark gray.

tors and coactivators that, in turn, interact with thebasal transcription machinery. The transcriptional coac-tivator CREB-binding protein (CBP) and its homologp300 play an essential role in cell growth, differentia-tion, and development. Understanding the molecularmechanisms by which CBP and p300 recognize theirvarious target proteins is of fundamental biomedicalimportance. CBP and p300 have been implicated indiseases such as leukemia, cancer, and mental retarda-tion and are novel targets for therapeutic intervention.

We previously determined the structure of thekinase-inducible activation domain of the transcriptionfactor CREB bound to its target domain (the KIXdomain) in CBP. Ongoing work is directed toward map-ping the interactions between KIX and the transcrip-tional activation domains of the proto-oncogene c-Myband of the mixed-lineage leukemia protein. The solu-tion structure of the complex between KIX and c-Mybhas been completed (Fig. 2) and provides insights intothe factors that determine constitutive vs inducibleactivation of transcription. Our work has also providednew understanding of the thermodynamics of the cou-pled folding and binding processes involved in interac-tion of KIX with transcriptional activation domains.

Recently, we determined the structure of the com-plex between the hypoxia-inducible factor Hif-1α andthe CH1 domain of CBP. The interaction between Hif-1αand CBP/p300 is of major therapeutic interest becauseof the central role Hif-1α plays in tumor progressionand metastasis; disruption of this interaction leads toattenuation of tumor growth. A protein named CITED2

functions as a negative feedback regulator of the hypoxicresponse by competing with Hif-1α for binding to theCH1 domain of CBP. We determined the structure ofthe complex formed between CITED2 and CBP (Fig. 3).The intrinsically unstructured Hif-1α and CITED2domains use partly overlapping surfaces of the CH1motif to achieve high-affinity binding and competeeffectively with each other for CBP/p300. We are con-tinuing to map the multiplicity of interactions betweenCBP/p300 domains and their numerous biological tar-gets to understand the complex interplay of interac-tions that mediate key biological processes in healthand disease.

PUBLICATIONSChen, J., Brooks, C.L. III, Wright, P.E. Model-free analysis of protein dynamics:assessment of accuracy and model selection protocols based on molecular dynam-ics simulation. J. Biomol. NMR 29:243, 2004.

De Guzman, R.N., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Interaction ofthe TAZ1 domain of the CREB-binding protein with the activation domain ofCITED2: regulation by competition between intrinsically unstructured ligands fornon-identical binding sites. J. Biol. Chem. 279:3042, 2004.

De Guzman, R.N., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Structureand function of TAZ domains. In: Zinc Finger Proteins: From Atomic Contact to Cel-lular Function. Iuchi, S., Kuldell, N. (Eds.). Landes Bioscience, Georgetown, TX,2004, Chap. 17.

Demarest, S.J., Deechongkit, S., Dyson, H.J., Evans, R.M., Wright, P.E. Packing,specificity and mutability at the binding interface between the p160 coactivatorand CREB-binding protein. Protein Sci. 13:203, 2004.

Hudson, B.P., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. Recognition of themRNA AU-rich element by the zinc finger domain of TIS11d. Nat. Struct. Biol.11:257, 2004.

Love, J.J., Li, X., Chung, J., Dyson H.J., Wright, P.E. The LEF-1 high-mobilitygroup domain undergoes a disorder-to-order transition upon formation of a complexwith cognate DNA. Biochemistry 43:8725, 2004.



F i g . 2 . Surface representation of the KIX domain of CBP show-

ing the Myb transactivation domain bound in a hydrophobic groove.

F i g . 3 . Structure of a complex between the transactivation

domain of CITED2 (dark gray) and the CH1 domain of CBP.

Matt, T., Martinez-Yamout, M.A., Dyson, H.J., Wright, P.E. The CBP/p300 TAZ1domain in its native state is not a binding partner of MDM2. Biochem. J. 381(Pt.3):685, 2004.

Zor, T., De Guzman, R.N., Dyson, H.J., Wright, P.E. Solution structure of the KIX domainof CBP bound to the transactivation domain of c-Myb. J. Mol. Biol. 337:521, 2004.

Folding of Proteins and Protein FragmentsP.E. Wright, H.J. Dyson, M. Lietzow, R. Mohana-Borges,

C. Nishimura, D. Felitsky, J. Chung, L.L. Tennant,

V. Bychkova** Institute of Protein Research, Puschino, Russia

The molecular mechanism by which proteins foldinto their 3-dimensional structures remains oneof the most important unsolved problems in struc-

tural biology. Nuclear magnetic resonance (NMR) spec-troscopy is uniquely suited to provide information onthe structure of transient intermediates formed duringprotein folding. Previously, we used NMR methods toshow that many peptide fragments of proteins have atendency to adopt folded conformations in water solu-tion. The presence of transiently populated folded struc-tures, including reverse turns, helices, nascent helices,and hydrophobic clusters, in water solutions of shortpeptides has important implications for initiation ofprotein folding. Formation of elements of secondarystructure probably plays an important role in the initi-ation of protein folding by reducing the number of con-formations that must be explored by the polypeptidechain and by directing subsequent folding pathways.

A major program in our laboratory is directedtoward a structural and mechanistic description of theapomyoglobin folding pathway. Previously, we usedquenched-flow pulse labeling methods in conjunctionwith 2-dimensional NMR spectroscopy to map thekinetic folding pathway of the wild-type protein. Withthese methods, we showed that an intermediate inwhich the A, G, and H helices adopt hydrogen-bondedsecondary structure is formed within 6 ms of the initi-ation of refolding. Folding then proceeds by stabiliza-tion of structure in the B helix and then in the C andE helices. We are using carefully selected myoglobinmutants and both optical stopped-flow spectroscopyand NMR methods to further probe the kinetic foldingpathway. For some of the mutants studied, the changesin amino acid sequence resulted in changes in the fold-ing pathway of the protein. These experiments are pro-

viding novel insights into both the local and the long-range interactions that stabilize the kinetic foldingintermediate. Of particular importance, long-rangeinteractions have been observed that indicate native-like packing of some of the helices in the kinetic mat-ter globule intermediate (Fig. 1).

Apomyoglobin provides a unique opportunity fordetailed characterization of the structure and dynamicsof a protein-folding intermediate. Conditions were pre-viously identified under which the apomyoglobin moltenglobule intermediate is sufficiently stable for acquisi-tion of multidimensional heteronuclear NMR spectra.Analysis of 13C and other chemical shifts and measure-ments of polypeptide dynamics provided unprecedentedinsights into the structure of this state.

The A, G, and H helices and part of the B helix arefolded and form the core of the molten globule. Thiscore is stabilized by relatively nonspecific hydrophobicinteractions that restrict the motions of the polypeptidechain. Fluctuating helical structure is formed in regionsoutside the core, although the population of helix is lowand the chain retains considerable flexibility. The F helixacts as a gate for heme binding and only adopts stablestructure in the fully folded holoprotein.

The acid-denatured (unfolded) state of apomyoglo-bin is an excellent model for the fluctuating local inter-actions that lead to the transient formation of unstableelements of secondary structure and local hydrophobicclusters during the earliest stages of folding. NMR dataindicated substantial formation of helical secondarystructure in the acid-denatured state in regions thatform the A and H helices in the folded protein and alsorevealed nonnative structure in the D and E helix region.



F i g . 1 . Regions of the apomyoglobin kinetic folding intermediate

(indicated by the black spheres) that are destabilized by mutation

of leucine 32 to alanine. The observed destabilization of the G

helix is evidence of nativelike long-range interactions between the

B and G helices in the molten globule folding intermediate.

Because the A and H regions adopt stabilized heli-cal structure in the earliest detectable folding interme-diate, these results lend strong support to folding modelsin which spontaneous formation of local elements ofsecondary structure plays a role in initiating formationof the A-[B]-G-H molten globule folding intermediate. Inaddition to formation of transient helical structure, for-mation of local hydrophobic clusters has been detectedby using 15N relaxation measurements. Significantly,these clusters are formed in regions where the averagesurface area buried upon folding is large. In contrastto acid-denatured unfolded apomyoglobin, the urea-denatured state is largely devoid of structure, althoughresidual hydrophobic interactions have been detectedby using relaxation measurements.

We measured residual dipolar couplings for unfoldedstates of apomyoglobin by using partial alignment instrained polyacrylamide gels. These data provide novelinsights into the structure and dynamics of the unfoldedpolypeptide chain. We have shown that the residualdipolar couplings arise from the well-known statisticalproperties of flexible polypeptide chains (Fig. 2), notfrom the persistence of native structure under denatur-ing conditions as has been claimed by other workers.Residual dipolar couplings provide valuable insightsinto the dynamic and conformational propensities ofunfolded and partially folded states of proteins andhold great promise for charting the upper reaches ofprotein-folding landscapes.

To probe long-range interactions in unfolded andpartially folded states of apomyoglobin, we introducedspin-label probes at several sites throughout the poly-peptide chain. These experiments led to the surprisingdiscovery that structures with nativelike topology exist

within the ensemble of conformations formed by theacid-denatured state of apomyoglobin. They also indi-cated that the packing of helices in the molten globulestate is similar to that in the native folded protein.

The view of protein folding that results from ourwork on apomyoglobin is one in which collapse of thepolypeptide chain to form increasingly compact statesleads to progressive accumulation of secondary struc-ture and increasing restriction of fluctuations in thepolypeptide backbone. Chain flexibility is greatest atthe earliest stages of folding, in which transient ele-ments of secondary structure and local hydrophobicclusters are formed. As the folding protein becomesincreasingly compact, backbone motions become morerestricted, the hydrophobic core is formed and extended,and nascent elements of secondary structure are pro-gressively stabilized. The ordered tertiary structurecharacteristic of the native protein, with well-packedside chains and relatively low-amplitude local dynam-ics, appears to form rather late in folding.

PUBLICATIONSDyson, H.J., Wright, P.E. Elucidation of the protein folding landscape by NMR.Methods Enzymol., in press.

Dyson, H.J., Wright, P.E. Unfolded proteins and protein folding studied by NMR.Chem. Rev. 104:3607, 2004.

Mohana-Borges, R., Goto, N.K., Kroon, G.J., Dyson, H.J., Wright, P.E. Structuralcharacterization of unfolded states of apomyoglobin using residual dipolar cou-plings. J. Mol. Biol. 340:1131, 2004.

Nishimura, C., Wright, P.E., Dyson, H.J. Role of the B helix in early folding eventsin apomyoglobin: evidence from site-directed mutagenesis for native-like long-rangeinteractions. J. Mol. Biol. 334:293, 2003.

Nuclear Magnetic ResonanceStudies of the Structure andDynamics of EnzymesH.J. Dyson, P.E. Wright, M.O. Ebert, D. Groff, G. Kroon,

J. Lansing, T. Lehmann,* M. Martinez-Yamout, D. McElheny,

K. Sugase, R.P. Venkitakrishnan, H.S. Won, Y. Yao,

L.L. Tennant, J. Chung, S.J. Benkovic,** A. Holmgren***

* Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela

** Pennsylvania State University, University Park, Pennsylvania

*** Karolinska Institutet, Stockholm, Sweden

We use site-specific information from nuclearmagnetic resonance (NMR) to further theunderstanding of enzyme function through

study of structure and dynamics. We focus on the



F i g . 2 . Principal axes of the alignment tensors for statistical seg-

ments of an unfolded polypeptide that preferentially populate the

β-region (A) and the α-region (B) of the Ramachandran map.

mechanisms of enzymes and catalytic antibodies andthe relationship between dynamics and function.D Y N A M I C S I N E N Z Y M E A C T I O N

Dynamic processes are implicit in the catalyticfunction of all enzymes. We use state-of-the-art NMRmethods to elucidate the dynamic properties of severalenzymes. New methods were developed for analysis ofNMR relaxation data for proteins that tumble aniso-tropically and for analysis of slow time scale motions.

Dihydrofolate reductase plays a central role in folatemetabolism and is the target enzyme for a number ofanticancer agents. 15N relaxation experiments on dihy-drofolate reductase from Escherichia coli revealed arich diversity of backbone dynamics for a broad rangeof time scales (picoseconds to milliseconds). Thesestudies were extended to additional intermediates inthe reaction cycle and to forms of the enzyme withmutations at various motional “hot spots.”

In addition, we are using 2H relaxation measure-ments in triple-labeled dihydrofolate reductase to elu-cidate the dynamics of critical active-site side chains.So far, we have identified functionally important motionsin loops that control access to the active site of thereductase on the same time scale as the hydride trans-fer chemistry. These motions become attenuated oncethe NADPH cofactor is bound in the active site, lock-ing the nicotinamide ring in a geometry conducive tohydride transfer to substrate. We also found evidenceof motion of active-site side chains that are implicatedin the catalytic process.

Dynamics measurements on inhibitor complexes ofa metallo-β-lactamase, one of the most important agentsof antibiotic resistance in bacteria, provided novelinsights into the role of dynamics in catalysis by anenzyme that has evolved rapidly to catalyze the hydroly-sis of a wide variety of substrates. Using NMR, wedetected motions on a wide range of time scales thatcan be correlated with enzyme function.

These studies are being extended into other classesof enzymes, particularly those such as kinases, thatcatalyze bimolecular reactions.D Y N A M I C S O F A C A T A L Y T I C A N T I B O D Y

Because they have evolved over millions of years,enzymes are exquisitely tuned to the reactions they cat-alyze and may also be tolerant of mutations. In con-trast, catalytic antibodies have much lower efficiencyand specificity. Knowledge of the local structure anddynamics of the catalytic site will allow novel insightsinto the mechanisms of antibody catalysis, provide

insight into the structural and functional evolution ofenzymes, and guide future work aimed at enhancingthe catalytic efficiency.

The Fv fragment of the catalytic antibody 43C9 wasexpressed and labeled uniformly with 2H, 13C, and 15N,and backbone resonance assignments were completedfor both unliganded antibody and the product complex.We found that binding of product caused markedchanges in the backbone dynamics in the complemen-tarity-determining regions. In addition, we are doingNMR studies of a family of aldolase catalytic antibod-ies of exceptionally broad scope to determine the struc-tural and dynamic bases of differences in substratespecificity and catalytic efficiency.R E D O X C O N T R O L B Y T H I O L - D I S U L F I D E C H E M I S T R Y

Many cellular functions are regulated by thiol-disul-fide chemistry. We did an extensive study of the struc-tural basis for the activity of several thiol-disulfideenzymes. Thioredoxin, a small, 108-residue thiol-disul-fide oxidoreductase, has many functions in the cell,including reduction of ribonucleotides to form deoxyri-bonucleotides for DNA synthesis. A primary function ofthioredoxin in the cell is as a protein disulfide reductase,a function vital for the prevention of misfolded proteinsin vivo. The E coli thioredoxin system has been fullycharacterized by using NMR, including the calculation ofhigh-resolution structures for both the oxidized (disul-fide) and the reduced (dithiol) forms of the protein.

Using backbone dynamics and amide proton-hydro-gen exchange, we found that functional differences inphage systems between oxidized and reduced thiore-doxin were due to differences in the flexibility of themolecules rather than to structural differences. Wealso delineated of the mechanism of E coli thioredoxin.We found that the reduction reaction of thioredoxindepends critically on the movement of protons, duringthe 2-electron–2-proton transfer reaction, as a sub-strate disulfide is reduced.

Glutaredoxins are another major class of thiol-disul-fide regulatory proteins. We recently determined thestructure of glutaredoxin-2 from E coli. This proteinappears to be a link between the glutaredoxin-thioredoxinclass of small thiol-active proteins and the extensive glu-tathione-S-transferase class of detoxification enzymes.

In other projects, we are focusing on the control ofcellular processes by thiol-disulfide reactions. We areusing NMR to investigate the molecular basis of theredox switch reactions in the chaperone Hsp33 and inthe transcription factor OxyR.



PUBLICATIONSChen, J., Won, H.-S., Im, W., Dyson, H.J., Brooks, C.L. III. Generation of native-like models from limited NMR data. J. Biomol. NMR, in press.

Graf, P.C.F., Martinez-Yamout, M.A., VanHaerents, S., Lilie H., Dyson, H.J.,Jakob, U. Activation of the redox-regulated chaperone Hsp33 by domain unfold-ing. J. Biol. Chem. 279:20529, 2004.

Kamikubo, Y., De Guzman, R., Kroon G., Curriden, S., Neels, J., Churchill, M.J.,Dawson, P., Oldziej, S., Jagielska, A., Scheraga, H.A., Loskutoff, D.J., Dyson,H.J. Disulfide bonding arrangements in active forms of the somatomedin B domainof human vitronectin. Biochemistry 43:6519, 2004.

Osborne, M.J., Venkitakrishnan, R.P., Dyson, H.J., Wright, P.E. Diagnostic chemi-cal shift markers for loop conformation and substrate and cofactor binding in dihy-drofolate reductase complexes. Protein Sci. 12:2230, 2003.

Schnell, J.R., Dyson, H.J., Wright, P.E. Effects of cofactor binding and loop confor-mation on side chain methyl dynamics in dihydrofolate reductase. Biochemistry43:374, 2004.

Schnell, J.R., Dyson, H.J., Wright, P.E. Structure, dynamics, and catalytic functionof dihydrofolate reductase. Annu. Rev. Biophys. Biomol. Struct. 33:119, 2004.

Venkitakrishnan, R.P., Zaborowski, E., McElheny, D., Benkovic, S.J., Dyson, H.J.,Wright, P.E. Conformational changes in the active-site loops of dihydrofolate reduc-tase during the catalytic cycle. Biochemistry, in press.

Won, H.-S., Low, L.Y., De Guzman, R.N., Martinez-Yamout, M., Jakob, U., Dyson,H.J. The zinc-dependent redox switch domain of the chaperone Hsp33 has a novelfold. J. Mol. Biol. 341:893, 2004.

Transcription Regulation WithSmall MoleculesJ.M. Gottesfeld, R. Burnett, L.A. Dickinson, C. Melander,

M.R. Wood, P.B. Dervan,* K. Luger**

* California Institute of Technology, Pasadena, California

** Colorado State University, Fort Collins, Colorado

C H E M I C A L R E G U L A T I O N O F G E N E T R A N S C R I P T I O N

Pyrrole-imidazole polyamides are the only availableclass of synthetic small molecules that can bedesigned to bind predetermined DNA sequences

with affinities comparable to those of cellular gene reg-ulatory proteins. In collaboration with P.B. Dervan andcolleagues at the California Institute of Technology, Pasa-dena, California, we showed that polyamides inhibit theDNA-binding activities of various transcriptional regula-tory proteins and can be used to inhibit transcription incell culture experiments. For these molecules to be use-ful as modulators of endogenous cellular gene expres-sion, they must be able to access their target sites inthe context of cellular chromatin. To address this issue,we used polyamides conjugated to a fluorescent dye anddeconvolution microscopy to determine the kinetics ofuptake and the subcellular distribution of polyamides incell culture. To show ligand binding at its target site innuclear DNA, we used a polyamide–DNA alkylator conju-

gate (chlorambucil) and ligation-mediated polymerasechain reaction to directly monitor polyamide binding inthe cell nucleus. DNA microarray experiments werecarried out to examine the specificity of polyamides incell culture. In contrast with the large number of poten-tial binding sites for polyamides in the human genome,the transcription patterns of only a limited number ofgenes were affected by polyamides designed to target7-bp sequences.

Site-specific alkylation by polyamide-chlorambucilconjugates within a coding region of a gene resulted ina strong block to transcription elongation by mamma-lian RNA polymerase II, both in vitro and in reportergene transfection experiments in cell culture. A seriesof polyamide-chlorambucil conjugates with differentDNA sequence specificities was screened for the effectsof the conjugates on morphology and growth charac-teristics of human colon carcinoma cell lines. We identi-fied one compound that caused cells to arrest in theG2/M stage of the cell cycle, without any apparent cyto-toxic effects. This change in growth properties requiredboth the DNA-binding specificity of the polyamide andthe alkylator moiety, suggesting that growth arrest isdue to the silencing of a set of specific genes by site-specific alkylation.

Surprisingly, DNA microarray analysis indicated thatonly 1 gene of approximately 18,000 probed genes wassignificantly downregulated by the polyamide, andreverse transcription–polymerase chain reaction andWestern blotting confirmed that histone H4c mRNAand total H4 protein levels were reduced in treated cells.Downregulation of H4c mRNA by small interfering RNAyielded the same cellular response, providing targetvalidation. The gene for histone H4c contains bindingsites for the active polyamide, and DNA alkylationwithin the coding region of the H4c gene was confirmedin cell culture by using ligation-mediated polymerasechain reaction. Cells treated with this compound didnot grow in soft agar and did not form tumors in nudemice, indicating that polyamide-treated cells are nolonger tumorigenic. The compound is active in vivo,blocking tumor growth in mice, without any obvioustoxic effects. Our results suggest that polyamide-DNAalkylators may lead to a new class of cancerchemotherapeutic agents.P O L Y A M I D E S A S A C T I V A T O R S O F G E N E E X P R E S S I O N

In several human diseases, activation of a repressedgene might be useful as a therapeutic approach. Oneexample is the neurodegenerative disease Friedreich’s



ataxia in which gene silencing caused by an unusualDNA structure is the primary cause of the disease. TheDNA abnormality found in 98% of patients with Friedre-ich’s ataxia is the unstable hyperexpansion of a GAAtriplet repeat in the first intron of the frataxin X25 gene,which adopts a triplex DNA structure, resulting indecreased transcription and reduced levels of frataxinprotein. We designed pyrrole-imidazole polyamides totarget GAA repeats with high affinity, and we foundthat these molecules relieved transcription inhibitionof the frataxin gene in cell lines derived from affectedindividuals, most likely by stabilizing canonical Watson-Crick B-type DNA. Changing the sequence specificityof the active polyamide abolished its ability to inducefrataxin expression. The results of deconvolution micros-copy established that fluorescent dye–polyamide con-jugates localize in the cell nucleus in these cultured cells.The GAA-specific polyamides are a first step towardtherapeutic agents for this neurodegenerative disease.D N A R E C O G N I T I O N W I T H I N C H R O M A T I N

Biochemical and x-ray crystallography studies indi-cate that nucleosomal DNA is largely available formolecular recognition by pyrrole-imidazole polyamides.Polyamide binding sites that are located 80 bp apart onlinear DNA lie across the 2 gyres of the DNA superhelixin the nucleosome, forming a supergroove that isunique to the nucleosome. On the basis of this observa-tion, we developed bivalent pyrrole-imidazole polyamideclamps that bind with high specificity across the nucle-osomal supergroove. X-ray crystallography studies per-formed in the laboratory of our collaborator, K. Luger,Colorado State University, Fort Collins, Colorado, indi-cated that the clamps bind as designed and effectivelycross-link the 2 genes of the DNA superhelix in thenucleosome. These molecules stabilize nucleosomalDNA from dissociation and may be useful as molecu-lar probes for dynamics and structural studies, as wellas transcription inhibitors with chromatin templates.

PUBLICATIONSDervan, P.B., Fechter, E.J., Edelson, B.S., Gottesfeld, J.M. Regulation of geneexpression with pyrrole-imidazole polyamides. In: Pseudo-Peptides in Drug Discov-ery. Nielsen, P.E. (Ed.). Wiley-VCH, New York, 2004, p. 121.

Dudouet, B., Burnett, R., Dickinson, L.A., Wood, M.R., Melander, C., Belitsky, J.M.,Edelson, B., Wurtz, N., Briehn, C., Dervan, P.B., Gottesfeld, J.M. Accessibility ofnuclear chromatin by DNA binding polyamides. Chem. Biol. 10:859, 2003.

Edayathumangalam, R.S., Weyerman, P., Gottesfeld, J.M., Dervan, P.B., Luger,K. Molecular recognition of the nucleosomal “supergroove.” Proc. Natl. Acad. Sci.U. S. A. 101:6864, 2004.

Melander, C., Burnett, R., Gottesfeld, J.M. Regulation of gene expression with pyr-role-imidazole polyamides. J. Biotechnol. 112:195, 2004.

Thastrom, A., Gottesfeld, J.M., Luger, K., Widom, J. Histone-DNA binding freeenergy cannot be measured in dilution-driven dissociation experiments. Biochem-istry 43:736, 2004.

Single-Molecule Conformational Dynamics ofNucleic Acid EnzymesD.P. Millar, M.F. Bailey, G. Pljevaljcic, S. Pond, G. Stengel,N. Tassew, E.J.C. Van der Schans

The focus of our research is the assembly andconformational dynamics of nucleic acid–basedmacromolecular machines. We use single-mole-

cule fluorescence methods to investigate a range ofsystems, including ribozymes, DNA polymerases, andtopoisomerases. Our studies reveal the large structuralrearrangements that occur as an integral component ofthe catalytic mechanism of these enzymes.R I B O Z Y M E S

RNA conformation plays a central role in the mech-anism of ribozyme catalysis. The hairpin ribozyme is asmall nucleolytic ribozyme that serves as a model sys-tem for detailed biophysical studies of RNA folding andcatalysis. The essential components of the hairpin ribo-zyme are 2 loop-containing duplexes, 1 of which con-tains the scissile phosphodiester bond. In a minimalform of the ribozyme, the 2 duplexes are connectedby a single-stranded hinge; in the natural form of theribozyme, the duplexes are arranged as 2 arms of a 4-way helical junction.

To attain catalytic activity, the ribozyme must foldinto a specific conformation in which the 2 loops aredocked with each other, forming a network of tertiaryhydrogen bonds. We monitor the formation of thisdocked structure by using fluorescence resonanceenergy transfer (FRET) and ribozyme constructs labeledwithin donor and acceptor dyes. By measuring FRETat the level of single ribozyme molecules, we reveal sub-populations of docked and extended conformers that arehidden in conventional experiments. Using this approach,we found that the docking of the 2 loops is stronglydependent on the nature of the intervening helicaljunction. Our results indicate that the role of the 4-wayjunction in the natural ribozyme is to facilitate dockingof the loops, by populating an intermediate state inwhich the loop-carrying arms are brought into proxim-ity and the 2 loops are juxtaposed for the subsequentformation of tertiary interactions.



D N A P O L Y M E R A S E S

DNA polymerases are remarkable for their abilityto synthesize DNA at rates close to several hundredbase pairs per second while maintaining an extremelylow frequency of errors. To elucidate the origin of poly-merase fidelity, we are using single-molecule fluores-cence methods to examine the dynamic interactionsthat occur between a DNA polymerase and its DNAand nucleotide substrates. The FRET method is beingused to observe conformational transitions of theenzyme-DNA complex that occur during selection andincorporation of an incoming nucleotide substrate. Ourresults reveal that binding of a correct nucleotide sub-strate triggers the translocation of the DNA primer ter-minus from the insertion site to the postinsertion siteof the polymerase, allowing the incoming nucleotide toenter the insertion site in readiness for the next roundof nucleotide incorporation. Single-pair FRET is alsobeing used to monitor the movement of the DNA primer-template between the separate polymerizing and edit-ing sites of the enzyme. This active-site switching ofDNA plays a key role in the proofreading process usedto remove misincorporated nucleotides from the DNA.The advantage of single-molecule observations is thatthey eliminate the need to synchronize a population ofmolecules, allowing these dynamic processes to beobserved directly.T O P O I S O M E R A S E S

Topoisomerases are enzymes that control the stateof DNA supercoiling in the cell. Type I topoisomerasesintroduce a nick into a strand of DNA and becomecovalently joined to the cleaved strand. This processallows the other strand to freely swivel around the first,resulting in the relaxation of supercoils within the DNA.The enzyme-DNA connection is then reversed, and thebroken strand is rejoined, completing the process ofsupercoil removal. We are using single-pair FRET meth-ods to observe the DNA-unwinding activity of singletype I topoisomerases in real time. The purpose ofthese studies is to directly observe DNA rotationalmotions during supercoil relaxation and to determinewhether the same number of supercoils is removedduring each enzyme-DNA encounter.

PUBLICATIONSBailey, M.F., Van der Schans, E.J.C., Millar, D.P. Thermodynamic dissection of thepolymerizing and editing modes of a DNA polymerase. J. Mol. Biol. 336:673, 2004.

Klostermeier, D., Sears, P., Wong, C.H., Millar, D.P., Williamson, J.R. A three-flu-orophore FRET assay for high-throughput screening of small-molecule inhibitors ofribosome assembly. Nucleic Acids Res. 32:2707, 2004.

Millar, D.P., Traskelis, M., Benkovic, S.J. On the solution structure of the T4 slid-ing clamp (gp45). Biochemistry, in press.

Pljevaljcic, G., Millar, D.P., Deniz, A.A. Freely diffusing single hairpin ribozymesprovide insights into the role of secondary structure and partially folded states inRNA folding. Biophys. J. 87:457, 2004.

Single-Molecule BiophysicsA.A. Deniz, S.Y. Berezhna, J.P. Clamme, A. Maharaj, M.M. Sandy, P. Zhu

We develop and use state-of-the-art single-molecule fluorescence methods to addresskey biological questions. Single-molecule and

small-ensemble methods offer several key advantagesover traditional measurements, such as the ability todirectly observe the behavior of subpopulations in mix-tures of molecules and to measure kinetics of structuraltransitions under equilibrium conditions. These meth-ods are particularly well suited for the study of complexbiological systems and processes involving stochas-tic dynamics and multiple structural states or reactionpathways. Our research involves a diverse array of meth-ods from biology, chemistry, and physics.

One major goal is to apply single-molecule methodsto studies of protein and RNA folding. Using relativelysimple model systems such as the protein barnase, weare addressing several fundamental questions aboutfolding mechanisms, such as the nature of partiallyfolded structures, the connectivity between differentfolding states, and the cooperativity of structural transi-tions, thereby exploring folding energy landscapes. Par-tially folded or misfolded protein structures are thoughtto play important cellular roles, and these states couldbe studied by using single-molecule methods.

In collaboration with D.P. Millar, Department ofMolecular Biology, we carried out a single-moleculefluorescence resonance energy transfer study on thefolding of RNA hairpin ribozymes. Using several ribo-zyme molecules with different combinations of helicaljunction and loop elements, we probed the contribu-tions of these secondary structural features to the fold-ing and stability of these molecules. We found that thenatural 4-way junction ribozyme can uniquely populatean undocked state with nativelike structure, most likelycontributing to the higher stability of the native structure.

In collaboration with J.R. Williamson, Departmentof Molecular Biology, we are using single-moleculemethods to study the detailed mechanisms of assem-bly of the bacterial ribosome. The small 30S subunitof the ribosome is assembled from a large RNA and21 small proteins through a complex process that



involves several steps of binding and conformationalchanges. Initially, we are focusing on the conformationalproperties of small RNA fragments from the 30S sub-unit and on the interactions of the fragments with theirprotein partners. These studies are also being extendedto the assembly of entire domains of the 30S subunit.

Finally, we are developing new multicolor single-molecule fluorescence resonance energy transfer meth-ods to enhance our capabilities to monitor multipleconformational changes or binding events in biologicalfolding, assembly, or activity. We are beginning to applythese methods to studies of folding and assembly.

PUBLICATIONSPljevaljcic, G., Millar, D.P., Deniz, A.A. Freely diffusing single hairpin ribozymesprovide insights into the role of secondary structure and partially folded states inRNA folding. Biophys. J. 87:457, 2004.

Computer Modeling of Proteinsand Nucleic AcidsD.A. Case, S. Brozell, J. Carlsson, M. Crowley, Q. Cui,

P. Dasgupta, F. Dupradeau,* T. Dwyer,** H. Gohlke,

D. Groff, D. Mathews, S. Moon, D. Nguyen, A. Onufriev,

R. Torres, R.C. Walker

* Université Jules Verne, Amiens, France

** University of San Diego, San Diego, California

Computer simulations offer an exciting approachto the study of many aspects of biochemicalinteractions. We focus primarily on molecular

dynamics simulations (in which Newton’s equations ofmotions are solved numerically) to model the solutionbehavior of biomacromolecules. Recent applicationsinclude detailed analyses of electrostatic interactionsin short peptides (folded and unfolded), proteins, andoligonucleotides in solution.

In addition, molecular dynamics methods are use-ful in refining solution structures of proteins by usingconstraints derived from nuclear magnetic resonance(NMR) spectroscopy, and we continue to explore newmethods in this area. Our developments are incorpo-rated into the Amber molecular modeling package,designed for large-scale biomolecular simulations, andinto other software, including Nucleic Acid Builder, fordeveloping 3-dimensional models of unusual nucleicacid structures; SHIFTS, for analyzing chemical shiftsin proteins and nucleic acids; and RNAMotif, for find-ing structural motifs in genomic sequence databases.

Additional studies on active sites of nitrogenaseand other metalloenzymes are described in the reportof L. Noodleman, Department of Molecular Biology.N M R A N D T H E S T R U C T U R E A N D D Y N A M I C S O F

P R O T E I N S A N D N U C L E I C A C I D S

Our overall goal is to extract the maximum amountof information on biomolecular structure and dynamicsfrom NMR experiments. To this end, we are studyingthe use of direct refinement methods for determiningbiomolecular structures in solution, going beyond dis-tance constraints to generate closer connections betweencalculated and observed spectra. We are also usingquantum chemistry to study chemical shifts and spin-spin coupling constants. As an example, Figure 1 showsa fragment taken from a molecular dynamics simula-tion of thioredoxin. Quantum mechanical calculationsof chemical shifts on fragments of this size can provideimportant information about the connections betweenprotein structure and NMR parameters. Other types ofdata, such as chemical shift anisotropies, direct dipo-lar couplings in partially oriented samples, and analy-sis of cross-correlated relaxation, are also being usedto guide structure refinement. In recent structural stud-ies, we focused on minor groove–binding drugs in com-plex with DNA and on complexes of zinc finger proteinswith RNA.M O D E L I N G O F N U C L E I C A C I D S

Another project centers on the development of novelcomputer methods to construct models of “unusual”nucleic acids that go beyond traditional helical motifs.We are using these methods to study circular DNA,small RNA fragments, and 3- and 4-stranded DNA com-



F i g . 1 . Part of the protein thioredoxin shows some of the impor-

tant hydrogen-bonding interactions that influence nitrogen and pro-

ton chemical shifts.

plexes, including models for recombination sites. Wecontinue to develop efficient computer implementationsof continuum solvent methods to allow simplified simu-lations that do not require a detailed description of thesolvent (water) molecules; this approach also provides auseful way to study salt effects.

This research is part of a larger effort to develop“low resolution” models for nucleic acids that can beextended to much larger structures such as circularDNA, viruses, or models of ribosomal particles. Figure 2gives an example of the reduction in the number ofatoms that would be achieved for a small piece of theribosome consisting of a stretch of RNA and an asso-ciated ribosomal protein. A computer language, NAB,was developed to make it easier to construct and sim-ulate molecular models for complex and often low-res-olution problems. The language is being used to studycompact and swollen viruses, to analyze curved andcircular DNA, and to simulate ribosomal assembly.D Y N A M I C S A N D E N E R G E T I C S O F N A T I V E A N D

N O N N A T I V E S T A T E S O F P R O T E I N S

Analysis methods similar to those described fornucleic acids are also being used to estimate thermo-dynamic properties of “molten globules” and unfoldedstates of proteins. These studies are an extension of ourearlier work on the folding of peptide fragments of pro-teins. A key feature is the development of computa-

tional methods that can be used to model pH and saltdependence of complex conformational transitions, suchas unfolding events.

A second aspect of this research is a detailed inter-pretation of NMR results for protein nonnative statesthrough molecular dynamics simulations and the con-struction of models for molecular motion and disorder.In a parallel effort, we are studying correlated fluctua-tions about native conformations in a variety of proteins,including dihydrofolate reductase, metallo-β-lactamase,binase, and cyclic-dependent kinase, in an effort tomake more secure connections between the motionsof proteins and the activities of enzymes.

All of these modeling activities are based on molec-ular mechanics force fields, which provide estimatesof energies as a function of conformation. We continueto work on improvements in force fields; recently, wefocused on adding aspects of electronic polarizability,going beyond the usual fixed-charge models, and onmethods for handling arbitrary organic molecules thatmight be considered potential inhibitors in drug dis-covery efforts. Overall, the new models should providea better picture of the noncovalent interactions betweenpeptide groups and their surroundings, leading ultimatelyto more faithful simulations.

PUBLICATIONSCase, D.A. NMR parameters in proteins and nucleic acids. In: Calculation of NMRand EPR Parameters. Kaupp, M., Bühl, M., Malkin, V.G. (Eds.). Wiley-VCH, NewYork, 2004, p. 341.

Cui, J., Crich, D., Wink, D., Lam, M., Rheingold, A.L., Case, D.A., Fu, W., Zhou, Y.,Rao, M., Olson, A.J., Johnson, M.E. Design and synthesis of highly constrained fac-tor Xa inhibitors: amidine-substituted bis(benzoyl)-[1,3]-diazepan-2-ones and bis(ben-zylidene)-bis(gem-dimethyl)cycloketones. Bioorg. Med. Chem. 11:3379, 2003.

Feig, M., Onufriev, A., Lee, M.S., Im, W., Case, D.A., Brooks, C.L. III. Performancecomparison of generalized Born and Poisson methods in the calculation of electrostaticsolvation energies for protein structures. J. Comput. Chem. 25:265, 2004.

Gohlke, H., Case, D.A. Converging free energy estimates: MM-PB(GB)SA studieson the protein-protein complex Ras-Raf. J. Comput. Chem. 25:238, 2004.

Gohlke, H., Kiel, C., Case, D.A. Insights into protein-protein binding by binding freeenergy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDScomplexes. J. Mol. Biol. 330:891, 2003.

Gohlke, H., Kuhn, L.A., Case, D.A. Change in protein flexibility upon complex for-mation: analysis of Ras-Raf using molecular dynamics and a molecular frameworkapproach. Proteins 56:322, 2004.

Lovell, T., Liu, T., Case, D.A., Noodleman, L. Structural, spectroscopic, and redoxconsequences of a central ligand in the FeMoco of nitrogenase: a density functionaltheoretical study. J. Am. Chem. Soc. 125:8377, 2003.

Moulnier, L., Case, D.A., Simonson, T. Reintroducing electrostatics into protein x-ray structure refinement: bulk solvent treated as a dielectric continuum. ActaCrystallogr. D Biol. Crystallogr. 59(Pt. 12):2094, 2003.

Onufriev, A., Bashford, D., Case, D.A. Exploring protein native states and large-scale conformational changes with a modified generalized Born model. Proteins55:383, 2004.



F i g . 2 . An example of a low-resolution description of a piece of

the central domain of the small subunit of the ribosome shows the

decrease in complexity compared with an all-atom model.

Roberts, M.F., Cui, Q., Turner, C.J., Case, D.A., Redfield, A.G. High-resolutionfield-cycling NMR studies of a DNA octamer as a probe of phosphodiester dynam-ics and comparison with computer simulation. Biochemistry 43:3637, 2004.

Roberts, V.A., Case., D.A., Tsui, V. Predicting interactions of winged-helix tran-scription factors with DNA. Proteins 57:172, 2004.

Simonson, T., Carlsson, J., Case, D.A. Proton binding to proteins: pKa calculationswith explicit and implicit solvent models. J. Am. Chem. Soc. 126:4167, 2004.

Wang, J., Wolf, R.M., Caldwell, J.W., Kollman, P.A., Case, D.A. Development andtesting of a general Amber force field. J. Comput. Chem. 25:1157, 2004.

Quantum BioinorganicChemistry and PhotochemistryL. Noodleman, D.A. Case, W.-G. Han, T. Lovell,* T. Liu,**

M.J. Thompson,*** R.A. Torres

* AstraZeneca R&D, Mölndal, Sweden

** Montana State University, Bozeman, Montana

*** Boston University, Boston, Massachusetts

We use a combination of modern quantumchemistry (density functional methods) andclassical electrostatics to describe the ener-

getics, reaction pathways, and spectroscopic propertiesof enzymes and to analyze systems with novel catalytic,photochemical, or photophysical properties.

Our major efforts are still directed toward under-standing the intermediates and transition states forreactions of redox-active metalloenzymes. The iron-molybdenum cofactor center of nitrogenase catalyzesthe multielectron reduction of molecular nitrogen toammonia and molecular hydrogen. This complex cofac-tor contains a MoFe7S9X prismane active site. Theultra-high-resolution x-ray structure (1.16 Å) and ourdensity functional analysis of redox potentials, struc-tures, and Mössbauer isomer shifts indicate that thestable endogenous central ligand X (Fig. 1) most likelyis nitride (N3–). We also predicted the resting oxidationand protonation state on the basis of these calculations.

Class I ribonucleotide reductases are aerobic enzymesthat catalyze the reduction of ribonucleotides to deoxy-ribonucleotides, providing the required building blocksfor DNA replication and repair. These ribonucleotide-to-deoxyribonucleotide reactions occur by a long-rangeradical (or proton-coupled electron transfer) propaga-tion mechanism initiated by a fairly stable tyrosine rad-ical, “the pilot light.” When this pilot light goes out,the tyrosine radical is regenerated by a high-oxidation-state Fe(III)-Fe(IV)-oxo enzyme intermediate, calledintermediate X. We are using density functional and

electrostatics calculations in combination with analy-sis of Mössbauer and electron nuclear double reso-nance spectroscopies to search for a proper structuraland electronic model for intermediate X (Fig. 2).

In a collaboration with F. Neese, W. Lubitz, and S. Sinnecker, Max-Planck-Institut für Strahlenchemie,Mülheim an der Ruhr, Germany, we calculated electron



F i g . 1 . The FeMoco of nitrogenase with an unknown ligand (X)

sitting in the center.

F i g . 2 . Selected core structures of our initial tested models for

ribonucleotide reductase intermediate state X.

paramagnetic resonance and electron nuclear doubleresonance parameters for a well-defined exchange cou-pled Mn(III)Mn(IV)-di-µ-oxo complex for which carefulstudies with these experimental spectroscopies havebeen done. These methods will be useful for spin-cou-pled manganese complexes and related enzymes (e.g.,manganese catalases).

In studies with E. Getzoff and M.J. Thompson,Department of Molecular Biology, and D. Bashford,St. Jude Children’s Research Hospital, Memphis, Ten-nessee, we are examining the basis for the spectraltuning of the chromophore at the active site of photoac-tive yellow protein as an example of a light-activatedsignal-transducing protein. This research combines theexpertise of our different groups in x-ray structure andspectroscopy, electrostatics, and quantum chemistryfor ground and excited states.

In collaboration with K. Hahn, A. Toutchkine, andD. Gremiachinsky, Department of Cell Biology; F. Himo,Royal Institute of Technology, Stockholm, Sweden; andM. Ullmann, Universität Bayreuth, Bayreuth, Germany,we examined the optical properties of solvent-dependentfluorescent dyes as prototypes for fluorescent tags thatcan act as reporters of protein conformational changedue to ligand binding. These detailed calculations willbe used to deduce general principles to improve designstrategies for stable and optically useful dyes.

Also, with Dr. Bashford’s group, we are studyingreaction pathways for the catalytic dephosphorylationof a tyrosine side chain by a low molecular weightprotein tyrosine phosphatase. The reaction occurs in 2 distinct steps: first, formation and then hydrolysis ofa phosphocysteine intermediate. The related reactionpathways have different proton-transfer characteristics.

In collaboration with T.C. Bruice, University ofCalifornia, Santa Barbara, we used density functionaltheory to study the magnesium-catalyzed hydrolysis ofthe phosphodiester bond in the hammerhead ribozymein order to generate a 2′,3′ cyclic phosphate ester withelimination of the adjacent 5′ alkoxy group. The reac-tion pathway shows an associative mechanism with 2 structurally distinct transition states (Fig. 3) and 1 intermediate, but all are close in energy. Two differ-ent proton transfers within the inner magnesium ionhydration sphere are involved in the reaction.

We are continuing our collaboration with K.B. Sharp-less, V. Fokin, R. Hilgraf, and V. Rostovtsev, Depart-ment of Chemistry, on the catalytic mechanisms usedby transition metal ions in click chemistry, in which

metal centers catalyze ring formation from multiplybonded precursors. Our current focus is copper(I)reactions, because copper(I) in water shows great ver-satility in ligating organic azides and alkynes to form5-membered heterocycles (triazoles) with wide molec-ular diversity.

PUBLICATIONSHan, W.-G., Liu, T., Himo, F., Toutchkine, A., Bashford, D., Hahn, K., Noodle-man, L. A theoretical study of the UV/visible absorption and emission solvato-chromic properties of solvent-sensitive dyes. Chemphyschem 4:1084, 2003.

Han, W.-G., Lovell, T., Liu, T., Noodleman, L. Density functional study of a µ-1,1-carboxylate bridged Fe(III)-O-Fe(IV) model complex, 2: comparison with ribonu-cleotide reductase intermediate X. Inorg. Chem. 43:613, 2004.

Himo, F., Demko, Z.P., Noodleman, L. Density functional study of the intramolecu-lar [2 + 3] cycloaddition of azide to nitriles. J. Org. Chem. 68:9076, 2003.

Liu, T., Han, W.-G., Himo, F., Ullmann, G.M., Bashford, D., Toutchkine, A.,Hahn, K., Noodleman, L. Density functional vertical self-consistent reaction fieldtheory for solvatochromism studies of solvent-sensitive dyes. J. Phys. Chem. A108:3545, 2004.

Liu, T., Lovell, T., Han, W.-G., Noodleman, L. DFT calculations of isomer shifts andquadrupole splitting parameters in synthetic iron-oxo complexes: applications tomethane monooxygenase and ribonucleotide reductase. Inorg. Chem. 42:5244, 2003.

Noodleman, L., Lovell, T., Han, W.-G., Li, J., Himo, F. Quantum chemical studiesof intermediates and reaction pathways in selected enzymes and catalytic syntheticsystems. Chem. Rev. 104:459, 2004.

Noodleman, L., Lovell, T., Han, W.-G., Liu, T., Torres, R.A., Himo, F. Densityfunctional theory. In: Fundamentals: Physical Methods, Theoretical Analysis, andCase Studies. Lever, A.B.P. (Ed.). Philadelphia, Elsevier, 2003, p. 491. Compre-hensive Coordination Chemistry II: From Biology to Nanotechnology, Vol. 2.McCleverty, J.A., Meyer, T.J. (Eds.).

Sinnecker, S., Neese, F., Noodleman, L., Lubitz, W. Calculating the electron para-magnetic resonance parameters of exchange coupled transition metal complexesusing broken symmetry density functional theory: application to a MnIII/MnIVmodel compound. J. Am. Chem. Soc. 126:2613, 2004.

Torres, R.A., Himo, F., Bruice, T.C., Noodleman, L., Lovell, T. Theoretical exami-nation of Mg2+-mediated hydrolysis of a phosphodiester linkage as proposed forthe hammerhead ribozyme. J. Am. Chem. Soc. 125:9861, 2003.



F i g . 3 . The quantum mechanically optimized structure corre-

sponding to the first transition state (TS1) in our examination of

magnesium-mediated phosphodiester hydrolysis in the hammer-

head ribozyme. Atom names reflect RNA nomenclature.

Theoretical and ComputationalMolecular BiophysicsC.L. Brooks III, C. An, S.R. Brozell, J. Chen, P. Cicotti, M.F. Crowley, M. Feig,* P. Ferrara,** O. Guvench, R. Hills,W. Im, J. Karanicolas,*** J. Khandogin, I. Khavrutzki, M. Michino, Y.Z. Ohkubo, M. Olson,**** S. Patel, D.J. Price, T.H. Rod,***** V. Reddy, H.A. Scheraga,†

C. Shepard, A. Stoycheva, F.M. Tama, M. Taufer, K.A. Taylor,†† I.F. Thorpe, C. Wildman* Michigan State University, East Lansing, Michigan

** Novartis Pharma AG, Basel, Switzerland

*** University of Washington, Seattle, Washington

**** U.S. Army Medical Research Institute of Infectious Diseases, Fort

Detrick, Maryland

***** Lund University, Lund, Sweden

† Cornell University, Ithaca, New York

†† Florida State University, Tallahassee, Florida

Understanding the forces that determine the struc-ture of proteins, peptides, nucleic acids, andcomplexes containing these molecules and the

processes by which the structures are adopted is essen-tial to complete our knowledge of the molecular natureof structure and function. To address such questions, weuse statistical mechanics, molecular simulation, statis-tical modeling, and quantum chemistry.

Creating atomic-level models to simulate biophysi-cal processes (e.g., folding of a protein or binding of aligand to a biological receptor) requires (1) the devel-opment of potential energy functions that accuratelyrepresent the atomic interactions and (2) the use ofquantum chemistry to aid in parameterizing these mod-els. Calculation of thermodynamic properties requiresthe development and implementation of new theoreti-cal and computational approaches that connect aver-ages over atomistic descriptions to experimentallymeasurable thermodynamic and kinetic properties.

Interpreting experimental results at more micro-scopic levels is fueled by the development and investi-gation of theoretical models for the processes of interest.Massive computational resources are needed to realizethese objectives, and this need motivates our effortsaimed at the efficient use of new computer architec-tures, including large supercomputers, Linux Beowulfclusters, and computational grids. Each of the objec-tives and techniques mentioned represents an ongoingarea of development within our research program incomputational biophysics. The following are highlightsof a few specific projects.

F O L D I N G , S T R U C T U R E , A N D F U N C T I O N O F

M E M B R A N E - B O U N D P R O T E I N S

Folding and stability of membrane proteins aredirectly governed by the unique hydrophilic and hydro-phobic environment provided by biological membranes.Modeling this heterogeneous environment is both anobstacle and an essential requisite to experimental andcomputational studies of the structure and function ofmembrane proteins. The experimental difficulties indetermining the structure of these proteins are quiteevident; compared with soluble proteins, only a hand-ful of membrane-associated protein structures are foundin the Protein Data Bank. Because of the biologicalimportance and significant presence of membrane pro-teins in known genomes (i.e., they account for about30% of all proteins), one aim of modern computationalbiology should be the development of methods thatcan be used in experimental studies to understand thestructure and function of these systems. We recentlydeveloped theoretical methods that enable the explo-ration of protein insertion and folding in membranes.These developments combine the modern samplingmethods of replica-exchange molecular dynamics withnovel generalized Born implicit solvent/implicit mem-brane continuum electrostatic theories to study thefolding of integral membrane peptides and proteins.

We recently completed de novo folding simulationsof the major pVIII coat protein from the filamentous fdbacteriophage (Fig. 1). The quality of the predicted



F i g . 1 . Folding progress of the major pVIII coat protein from fila-

mentous fd bacteriophage (a 56-residue integral membrane pro-

tein) and comparison of computed and experimentally determined

solid-state NMR measurements of the 15N chemical shift.

structures, judged by comparison with experimentalstructures from both solid-state nuclear magnetic reso-nance (NMR) in lipid bilayers and solution-phase NMRon the protein in micelles, was excellent. The trans-membrane helical segment of the protein was welldefined in the membrane environment; the amphipathichelical fragment remained at the membrane–aqueousphase boundary while undergoing significant confor-mational flexibility due to the loop connecting the 2 helical segments of the protein. From our simula-tions of the protein at room temperature, we com-puted solid-state NMR properties, the 15N chemicalshift and 15N-1H dipolar coupling constants, whichindicated nearly quantitative agreement with the cor-responding measurements. These findings suggest anemerging potential for the de novo investigation ofintegral membrane peptides and proteins and a mech-anism to assist in experimental approaches to thecharacterization and structure determination of theseimportant systems.L A R G E - S C A L E F U N C T I O N A L D Y N A M I C S I N

M O L E C U L A R A S S E M B L I E S

Many naturally occurring “machines,” such as ribo-somes, which process mRNA for protein synthesis, ormyosin, which produces the force needed for the normalcontraction of muscle, require large-scale dynamicalmotions as a component of their normal functioning.These motions often involve the “mechanical” reorgani-zation of major parts of the structure of the machine inresponse to binding of effectors or to the addition ofenergy in the form of thermal fluctuations or provided bychemical catalysis. Exploring and understanding thecharacter and nature of such large-scale reorganizationof biological machines are an ongoing in our laboratory.Using theoretical approaches derived from the treatmentof mechanoelastic materials, we are constructing theo-retical models for the motions of large molecular assem-blies, including viral capsids, ribosomes, and myosin.

Ribosomes undergo a range of structural changesduring protein synthesis. Using the lower-resolutionstructural methods of electron cryomicroscopy, ourcollaborator J. Frank, Wadsworth Center, Albany, NewYork, characterized 2 key motions: the ratchetlike dis-placement of the major ribosomal domains (30S and50S subunits) with respect to each other and large-scale displacement of the protein L1. The rachetlikedisplacement occurs during the translocation of tRNAfrom the A and P binding sites to the P and E sites(Fig. 2); the large-scale displacement of the protein

L1 may facilitate the dissociation of amino acid–exhausted tRNA from the E site.

Using mechanoelastic models based on the crys-tallographic structure of the 70S ribosome, we showedthat these functionally critical motions arise as naturaldisplacements of “elastic bodies” with the shape of theribosome. Emerging from these calculations are atomic-level pathways for these steps in translocation. In par-ticular, our calculations suggest that the ratchetlikerotation of the 50S subunit relative to the 30S subunitleads to initial displacement of the tRNA molecules inA and P sites toward the P and E sites. Also prevalentas a “normal mode” of displacement of the complexis the “reaching” of the L1 protein “arm” to possiblyfacilitate the removal of spent tRNA in the E site. Wehypothesize that the strength and power of simple shape-dependent dynamics for functional motions critical forparticular biological processing are exploited in natu-rally occurring machines.

We are also exploiting shape-dependent dynamicsto develop structural models consistent with experimen-tal data from electron microscopy and tomography. Incollaboration with K.A. Taylor, Florida State University,Tallahassee, Florida, we are developing models for thestructural rearrangement associated with the transitionfrom the activated state of smooth muscle heavy mero-myosin to its inhibited state that forms upon regulatory



F i g . 2 . Normal mode analysis of a mechanoelastic model of

the ribosome. Left, Ratchetlike reorganization of the ribosome, as

required for tRNA translocation, is accomplished by rotation of the

30S domain relative to the 50S domain. The relative motion of the

2 major domains facilitates the motion of the tRNA molecules from

the A and P sites toward the P and E sites. Right, Also evident is

the opening and closing dynamics of the L1 domain (from position

I to position III). This domain may be involved in assisting the exit-

ing of tRNA from the E site of the ribosome. The pivot point (white

circle) for the rearrangement is near the base of the L1 stalk.

dephosphorylation of the light chain of the protein(Fig. 3). Beginning with a homology model of heavymeromyosin, including a hypothetical S2 (dimeriza-tion) domain, we computed mechanoelastic normalmodes of displacement of the molecule and exploredthe ability of the modes to account for the large reor-ganization of this structure required for consistencywith low-resolution structural data from electron cryo-microscopy. Construction and exploration of the dynam-ical motions led to new interpretation of the electroncryomicroscopy data and to an interpretation of therole of flexibility of the S2/S1 junction region in facili-tating the conformational change.

PUBLICATIONSFeig, M., Karanicolas, J., Brooks, C.L. III. MMTSB Tool Set: enhanced samplingand multiscale modeling methods for applications in structural biology. J. Mol.Graph. Model. 22:377, 2004.

Im, W., Brooks, C.L. III. De novo folding of membrane proteins: an exploration of thestructure and NMR properties of the fd coat protein. J. Mol. Biol. 337:513, 2004.

Im, W., Feig, M., Brooks, C.L. III. An implicit membrane generalized Born theoryfor the study of structure, stability, and interactions of membrane proteins. Bio-phys. J. 85:2900, 2003.

Karanicolas, J., Brooks, C.L. III. Integrating folding kinetics and protein function:biphasic kinetics and dual binding specificity in a WW domain. Proc. Natl. Acad.Sci. U. S. A. 101:3432, 2004.

Lee, M.S., Salsbury, F.R., Jr., Brooks, C.L. III. Constant-pH molecular dynamicsusing continuous titration coordinates. Proteins 56:738, 2004.

MacKerell, A.D., Jr., Feig, M., Brooks, C.L. III. Improved treatment of the proteinbackbone in empirical force fields. J. Am. Chem. Soc. 126:698, 2004.

Patel, S., Brooks, C.L. III. CHARMM fluctuating charge force field for proteins, I:parameterization and application to bulk organic liquid simulations. J. Comput.Chem. 25:1, 2004.

Stoycheva, A.D., Onuchic, J.N., Brooks, C.L. III. Effects of gatekeepers on the earlyfolding kinetics of a model β-barrel protein. J. Chem. Phys. 119:5722, 2003.

Tama, F., Miyashita, O., Brooks, C.L. III. Flexible multi-scale fitting of atomicstructures into low-resolution electron density maps with elastic network normalmode analysis. J. Mol. Biol. 337:985, 2004.

Tama, F., Miyashita, O., Brooks, C.L. III. NMFF: normal mode based flexible fittingof high-resolution structure into low-resolution experimental data from cryo-EM. J.Struct. Biol., in press.

Thorpe, I.F., Brooks, C.L. III. Barriers to hydride transfer in wild type and mutantdihydrofolate reductase from E. coli. J. Phys. Chem. B 107:14042, 2003.

Thorpe, I.F., Brooks, C.L. III. The coupling of structural fluctuations to hydridetransfer in dihydrofolate reductase. Proteins, in press.

Trylska, J., Konecny, R., Tama, F., Brooks, C.L. III, McCammon, J.A. Ribosomemotions modulate electrostatic properties. Biopolymers 74:423, 2004.

Computational Structural BiologyA.J. Olson, B.S. Duncan, D.S. Goodsell, M.F. Sanner, A. Beuscher, S. Coon, A. Gillet, Y. Hu, R. Huey, C. Li, W. Lindstrom, G.M. Morris, A. Omelchenko, M. Pique, B. Norledge, R. Rosenstein, D. Stoffler, Y. Zhao

In the molecular graphics laboratory, we develop novelcomputational methods to analyze, understand, andcommunicate the structure and interactions of com-

plex biomolecular systems. This past year, we fabricatedhundreds of 3-dimensional molecular models and usedthem as a tangible human-computer interface for explo-ration in research and education. Within our respon-sive, modular software environment, we continue todevelop methods for predicting biomolecular interac-tions, analyzing biomolecular structure and function,and presenting the biomolecular world in educationand outreach.

We have applied these methods to several importantareas in human health and welfare. We continue thesearch for inhibitors of HIV protease that are active withdrug-resistant strains of the virus. We are exploring themechanisms of protein interaction in the blood coagula-tion cascade. We are also pursuing several diverse tar-gets for the development of new anticancer drugs.N O V E L H U M A N - C O M P U T E R I N T E R F A C E S

We combined 2 cutting-edge research technologies,3-dimensional printing and augmented reality, to cre-ate an environment for teaching, collaboration, andresearch. We used 3-dimensional printing technology(rapid prototyping or layered fabrication) to createphysical, 3-dimensional models of molecular struc-tures and complexes derived from crystallography,nuclear magnetic resonance, and electron microscopystudies. Augmented reality (or “mixed” reality) links



F i g . 3 . A progression of structural reorganization of smooth mus-

cle heavy meromyosin during the dynamical transition from a model

activated state (top left) to the inhibited form (bottom right). From

our normal mode analysis of models with various degrees of non–

coiled coil helices at the S1/S2 junction, we found optimal agreement

with data from electron microscopy for uncoiling of 2 or 3 heptad

repeats (14–21 residues), consistent with experimental observations.

these tangible models to the computational models,tracking the models as they are manipulated and link-ing the motion to a computer graphics representationof the molecule (Fig. 1). The environment allows stu-dents and researchers to access diverse molecular data,to guide exploration of the molecule, and to presentdynamic characteristics of the molecule. We are usingcost-effective consumer laptops and video cameras. Wedemonstrated the approach at workshops and confer-ences, and in collaboration with the Human InterfacesTechnology Laboratory at the University of Washing-ton, Seattle, Washington, we are testing these meth-ods in molecular biology classrooms.

During the past year, we developed an articulated3-dimensional model of protein folding. In the model,magnets are used to simulate hydrogen bonds, androtatable connections along the backbone are designedto favor specified ϕ-ψ torsion angles such as those incommon α-helix and β-strand conformations. The modelwas integrated into the augmented-reality environmentby placing generic markers at side-chain positions, allow-ing users to display and manipulate virtual amino acidsattached to the physical backbone.A M O D U L A R S O F T W A R E E N V I R O N M E N T F O R

B I O M O L E C U L A R C O M P U T I N G

During the past decade, we showed the usefulnessof the Python programming language as a platform for

creating a modular, programmable, dynamic environmentto solve the challenges of new biomolecular applications.In the past year, we continued work on 2 central compo-nents of this environment: the Python Molecular Viewer(PMV) and Vision, a visual programming environment.We also developed Python-based tools for the aug-mented-reality environment described earlier.

We improved the stability, usability, and perfor-mance of PMV and Vision and the software componentsthese applications are built on. We also added Macin-tosh OS X to the list of supported operating systems.We made 2 releases of PMV and its companion pro-gram AutoDockTools, which have been downloaded bymore than 7250 scientists around the world; 3400downloaded our software during the past 12 months.To support this rapidly growing user community, weestablished various mailing lists, and we taught sev-eral tutorials at conferences and at Scripps Research.

Vision (formerly known as ViPEr) has undergoneprofound refactoring; we changed the underlying soft-ware architecture while retaining the functionality.Vision is an interactive visual programming environ-ment that allows users to build networks with novelcombinations of computing methods. Enhancementsinclude added support for discovering computationalnodes by searching their documentation, fail-safe sav-ing and restoring of networks, scheduling networkscontaining cyclic execution dependencies (i.e., loops),and a package for working with volumetric data.

During the past year, we improved and extendedPyARTK, the augmented-reality tools integrated intoPMV and Vision. The environment now allows auto-matic registration of the virtual computer graphics rep-resentation with the tangible model, on the basis ofthe files used to fabricate the tangible model. We nowcan display the dynamic properties of a molecule byplaying an animation when a marker is detected.

We also began work on a modular flexibility treefor the study of biomolecular dynamics. Abstractinginformation on macromolecular flexibility and represent-ing the flexibility in an intuitive and high-level form aredifficult problems. However, such a high-level under-standing of the flexibility of biomolecules and thecomputational encoding of that flexibility are of funda-mental importance for the design of new methods thatbetter represent molecular flexibility in simulations ofmacromolecular interactions. The goal of the flexibilitytree is to develop the computational infrastructure toencode a hierarchy of motions.



F i g . 1 . This augmented-reality environment, which uses off-

the-shelf consumer hardware, tracks markers on the surface of an

autofabricated tangible model and overlaps a representation of the

electrostatic field on the video image.

The Python programming language gained accep-tance rapidly over the past few years, and as a resultmany laboratories in all areas of science use this lan-guage as a development platform. To provide a forumfor researchers who use Python for scientific comput-ing, we coorganized the SciPy ’03 workshop (http://www.scipy.org/documentation/Workshops/SciPy03) atCalifornia Institute of Technology, Pasadena, California,and a workshop for the Computational Representation ofBiological Molecules (http://mgldev.scripps.edu/CRBM/)at the University of California, San Diego. A substan-tial amount of overlap of the different software presentedat these workshops was detected, and a clear will tocollaborate on an open source, common implementa-tion of a molecular representation was established.D O C K I N G O F B I O M O L E C U L A R C O M P L E X E S

AutoDock is a suite of programs for the predictionof bound conformations and binding energies for bio-molecular complexes. AutoDock is currently in use in2600 academic and commercial laboratories worldwide.The advances we made in the usability of AutoDockduring recent years helped open up the suite to abroader community of chemists and biologists. In thepast year, we continued to support the use of AutoDockwith the hands-on tutorial Using AutoDock With Auto-DockTools (available at http://www.scripps.edu/pub/olson-web/doc/autodock/index.html) and with an activeAutoDock mailing list with more than 600 subscribers.

We also continued to improve AutoDock, makingspecific modifications to improve computational speed,updating the force field, and incorporating methods forprotein motion. The new linear free-energy force fieldincludes an improved hydrogen-bonding potential thatcorrectly treats groups with multiple hydrogen bonds,such as the pairing of bases in DNA (Fig. 2). We alsoimproved our pairwise desolvation potential. We arecurrently calibrating the force field by using a large setof complexes taken from the Ligand-Protein Databasefrom the laboratory of C.L. Brooks, Department of Molec-ular Biology, and we are testing methods for localizedmotion of side chains.

Looking to the future, we are restructuring AutoDockby using models from object-oriented technology todesign a new architecture that promises great flexibil-ity and innovations in the science of docking. Initiallywe are focusing on the design of a general-purposemodular scorer that evaluates the interaction energyon the basis of physicochemical or knowledge-basedpotentials. The scorer is being used in several different

applications, including a support vector machine anda statistical decision tree under development by R. Belew, University of California, San Diego, for thestudy of HIV protease inhibitors.F I G H T I N G A I D S W I T H H I V P R O T E A S E I N H I B I T O R S

As part of an ongoing project, we are continuingwork on inhibitors to fight drug resistance in HIV dis-ease. Using click chemistry, in collaboration with K.B.Sharpless and C-H. Wong, Department of Chemistry,we focused on the design of inhibitors that assemblewithin the active site of HIV protease. We performedmultiple cycles of drug design, identifying the optimalplacement of the triazole formed in this reaction andoptimizing the binding of the 2 molecular fragments inthe active site. Using this approach, we identified andtested a series of potent nanomolar inhibitors (Fig. 3).

Using the FightAIDS@Home system, we performeda virtual screening of compounds in the National Can-



F i g . 2 . Improved hydrogen-bonding potentials model the direc-

tionality of hydrogen bonds in molecules that form multiple interac-

tions, such as the cytosine shown here.

F i g . 3 . Predicted (light bonds) and crystallographic (dark bonds)

structures of a nanomolar HIV protease inhibitor synthesized by using

click chemistry. The pentagonal triazole, shown in black, forms a

hydrogen bond with a structural water, shown as a large sphere.

cer Institute Diversity Set against a panel of HIV pro-tease mutants. FightAIDS@Home enlists the worldwidecommunity in a large computational effort to designeffective therapeutic agents to fight AIDS; personalcomputers are used in the program when the comput-ers are not in use by their owners. The goal of thecurrent study is to find novel molecules with strongaffinity for HIV protease. By evaluating the binding of the compounds against a panel of viral mutants, weestimate the potential of the compound to remain effec-tive against the protease and any resistant strains thatmight develop.S P E C I F I C I T Y I N C A T A L Y T I C A N T I B O D I E S

Catalytic antibodies are among the best syntheticcatalysts, excelling in enantioselectivity and enantio-specificity. We used computational modeling to study2 different systems with unusual specificities. In ourcollaboration with C.F. Barbas, I.A. Wilson, and R.A.Lerner, Department of Molecular Biology, we usedAutoDock to analyze 2 separate groups of aldolaseantibodies that catalyze the same aldol reactions withantipodal selectivity. Mutations based on these com-putational studies confirmed that the 2 antibodiesbind transition states selectively by forming differenthydrogen bonds between substrate and antibody in awide range of reactions.

We also studied, in collaboration with K.D. Janda,Department of Chemistry, and I.A. Wilson, a periph-eral-blockade approach to cocaine addiction in whichcatalytic antibodies break down cocaine in the blood.In our predicted transition state (Fig. 4), a hydroxideion that is held in place by 2 tyrosines conducts a nucle-

ophilic attack on cocaine carbonyl to form a methylester and benzoic acid.M U L T I P L E T A R G E T S F O R C A N C E R C H E M O T H E R A P Y

We continued to characterize and design inhibitorsfor several targets in cancer chemotherapy. In collabo-ration with Dr. Wilson and D.L. Boger, Department ofChemistry, we continued the search for inhibitors of 5-aminoimidazole-4-carboxamide ribonucleotide trans-formylase. Rapidly dividing cells rely more on the denovo purine biosynthetic pathway than on the usualsalvage pathway for their purine sources, so theseinhibitors are attractive potential antineoplastic drugs.

After a successful pilot study, we screened the wholeNational Cancer Institute Diversity Set of compoundsand selected 138 structurally similar compounds. Thesecond-round AutoDock screening of these compoundssuggested 14 possible inhibitors. Enzyme assays con-firmed that 11 of the 14 are inhibitors; 1 of the 11 hasa submicromolar inhibition constant. So far, we havediscovered inhibitors capable of binding to the sub-strate site, others that bind to the cofactor site, and“spanners” that cover both sites. Further analysis ofthese compounds revealed that electrostatic interac-tions play a large role in the binding process and inthe formation of an “oxyanion hole” similar to thatfound in the serine proteases.

Pleiotrophin is a heparin-binding cytokine that isstrongly expressed in a high proportion of human tumorcells. Pleiotrophin initiates the dimerization of a recep-tor protein tyrosine phosphatase, and the dimerizationinhibits the phosphorylation of catenin and the associ-ation of catenin with cadherin inside the cell, which canlead to uncontrolled cell growth. We modeled pleiotro-phin dimerization by using Surfdock, which allows thestudy of protein-protein interactions at different levelsof detail in order to explore the primary factors for pro-tein recognition such as shape complementarity, elec-trostatics, or individual salt bridges. We found thatpleiotrophin always forms a dimer with C-2 symmetryat various resolutions, in agreement with our proposedpleiotrophin signaling mechanism. Mutation experimentson the pleiotrophin dimer interface suggested by Surf-dock models are currently being done by T.F. Deuel,Department of Molecular and Experimental Medicine.

The protein Myc is a cell-cycle checkpoint presentat abnormally high concentrations in many tumor celltypes. Myc functions as a transcriptional activator whenbound to the protein Max, so small molecules that inhibitthe formation of the Myc-Max complex would be ideal



F i g . 4 . Two tyrosines (at the top) from a catalytic antibody posi-

tion a hydroxyl group to attack a cocaine molecule (at the bottom).

candidates for anticancer therapeutic agents. We usedAutoDock to model the binding of several inhibitorsdiscovered by P.K. Vogt, Department of Molecular andExperimental Medicine, and we are currently searchingthe National Cancer Institute Diversity Set for addi-tional leads.P R O T E I N I N T E R A C T I O N I N T H E

C O A G U L A T I O N C A S C A D E

Tissue factor (TF) is a membrane-associated proteinthat initiates coagulation. The extracellular domain of TF,in complex with coagulation factor VIIa, triggers thethrombogenic cascade by activating coagulation factorsIX and X. In collaboration with T.S. Edgington, Depart-ment of Immunology, we developed a model of the inter-action between TF and a peptide that inhibits activationof factor X but not factor IX (Fig. 5). Molecules such asthis one may be valuable tools in isolating the signalingproperties of TF from its activities in coagulation.

In collaboration with W. Ruf, Department of Immu-nology, and G. Legg, University of Texas, Austin, weare combining nuclear magnetic resonance with com-puter modeling to investigate interactions that involvethe cytoplasmic domain of TF. The cytoplasmic domaincontains the phosphoserine-proline motif associatedwith WW-domain binding. We used AutoDock to modelthe interaction of a peptide from the cytoplasmic tailof TF (which contains this motif) with the WW domainof proline isomerase 1. Binding of substrate to the WWdomain of proline isomerase 1 occurs in a cleft betweenthe WW domain and the proline isomerase domains ofproline isomerase 1 and is thought to have an allosteric

effect on the activity of the isomerase. These interac-tions modulate the signaling events arising from acti-vation of members of the family of protease-activatedreceptors, which lead to inflammatory responses andalso play an important role in sepsis.E D U C A T I O N A N D O U T R E A C H

Understanding structural molecular biology is essen-tial in our society to foster progress and critical deci-sion making among students, policy makers, and thegeneral public. We continued our longstanding work inscience outreach with a variety of projects, includingposters, presentations, textbook and popular illustra-tions, sites on the World Wide Web, scientific artshows, and a new book on bionanotechnology for thelay audience.

We continued 2 regular features that informally pres-ent molecular structure and function. The “Molecule ofthe Month” at the Protein Data Bank (http://www.pdb.org)provides an accessible introduction to the database.Each month, a new molecule is presented, and theplace of the molecule in human health and welfare isdescribed. Visitors are given suggestions as to how tobegin their own exploration of these molecules. Anexample is shown in Figure 6. The “Molecular Perspec-tive” is presented in The Oncologist, a journal for clini-cal oncologists. Each short article presents a singlemolecule and describes the place of the molecule in thedevelopment and treatment of cancer. The column is asource of continuing medical education for physicians.



F i g . 5 . Molecular docking of the inhibitory Hlp611 peptide

(shown in ball-and-stick representation) to the complex of TF and

factor VIIa.

F i g . 6 . An illustration showing the action of DNA tweezers.

Reprinted from Bionanotechnology by D.S. Goodesll, copyright ©

2004. This material is used by permission of John Wiley & Sons, Inc.

PUBLICATIONSBrik, A., Muldoon, J., Lin, Y.-C., Elder, J.H., Goodsell, D.S., Olson, A.J., Fokin, V.V.,Sharpless, K.B., Wong, C.-H. Rapid diversity-oriented synthesis in microtiter plates forin situ screening of HIV protease inhibitors. Chembiochem 4:1246, 2003.

Goodsell, D.S. Bionanotechnology: Lessons From Nature. Wiley-Liss, Hoboken, NJ,2004.

Goodsell, D.S. Looking at molecules: an essay on art and science. Chembiochem4:1293, 2003.

Goodsell, D.S. The molecular perspective: DNA polymerase. Oncologist 9:108,2004; Stem Cells 22:236, 2004.

Goodsell, D.S. The molecular perspective: epidermal growth factor. Oncologist8:496, 2003; Stem Cells 21:702, 2003.

Goodsell, D.S. The molecular perspective: histone deacetylase. Oncologist 8:389,2003; Stem Cells 21:620, 2003.

Goodsell, D.S. The molecular perspective: protein farnesyltransferase. Oncologist8:597, 2003; Stem Cells 22:119, 2004.

Goodsell, D.S. The molecular perspective: ubiquitin and the proteosome. Oncolo-gist 8:293, 2003; Stem Cells 21:509, 2003.

Huang, H., Norledge, B.V., Liu, C., Olson, A.J., Edgington, T.S. Selective attenua-tion of the extrinsic limb of the tissue factor-driven coagulation protease cascade byoccupancy of a novel peptidyl docking site on tissue factor. Biochemistry42:10619, 2003.

Huey, R., Goodsell, D.S., Morris, G.M., Olson, A.J. Grid-based hydrogen bondpotentials with improved directionality. Lett. Drug Des. Discov. 1:178, 2004.

Lin, Y.-C., Beck, Z., Morris, G.M., Olson, A.J., Elder, J.H. Structural basis for dis-tinctions between substrate and inhibitor specificities for feline immunodeficiencyvirus and human immunodeficiency virus proteases. J. Virol. 77:6589, 2003.

Norledge, B.V., Petrovan, R.J., Ruf, W., Olson, A.J. Tissue factor/factor VIIa/factorXa complex: a model built by docking and site-directed mutagenesis. Proteins53:640, 2003.

Rosenfeld, R.J., Goodsell, D.S., Musah, R.A., Morris, G.M., Goodin, D.B., Olson, A.J.Automated docking of ligands to an artificial active site: augmenting crystallographicanalysis with computer modeling. J. Comput. Aided Mol. Des. 17:525, 2003.

Zhang, Y., Desharnais, J., Marsilje, T.H., Li, C., Hedrick, M.P., Gooljarsingh, L.T.,Tavassoli, A., Benkovic, S.J., Olson, A.J., Boger, D.L., Wilson, I.A. Rationaldesign, synthesis, evaluation and crystal structure of a potent inhibitor of humanGAR tfase: 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid.Biochemistry 42:6043, 2003.

Computational ProteomicsR. Abagyan, J. An, A. Cheltsov, J. Kovacs, A. Orry, C. Smith,

V. Tseitin, A. Bordner,* C. Cavasotto,* J. Fernandez-Recio,**

M. Schapira,*** M. Totrov,* Y. Zhou****

* Molsoft L.L.C., La Jolla, California

** Cambridge University, Cambridge, England

*** Aptanomics SA, Lyon, France

**** Genomics Institute of the Novartis Research Foundation, San Diego,

California

B I O I N F O R M A T I C S A N D M O D E L I N G

We continue to do sequence-structure analysisand modeling of homologous proteins and usethe results to formulate or test specific bio-

logical hypotheses. Sequence-structure alignment is akey component of homology modeling. We generated adatabase of 1927 pairs of homologous proteins withknown 3-dimensional structures and then generated astructural alignment database that we used to optimizeparameters of sequence-structure alignments.

General informatics and data analysis are integralparts of computational proteomics. We compared andreviewed our previously developed match-only integraldistribution algorithm for a more accurate analysis ofthe expression data obtained by using high-density oli-gonucleotide chips. We also developed the informaticsenvironment for the mass spectrometry/metabolomics(a quantitative and dynamic description of all low molec-ular weight metabolites in an organism) project of G. Siuzdak, Department of Molecular Biology.

F O R C E F I E L D D E V E L O P M E N T

A major obstacle in predicting molecular structuresis insufficient accuracy of the force field. We showedthat the calculation of van der Waals energy can beimproved if the force field parameters are free fromunphysical combination rules. Furthermore, we showedthat accurate van der Waals parameters can be deriveddirectly from the calculated quantum mechanical ener-gies. The derived parameters were close to experimen-tal values from a validation data set, and this fit wassignificantly better than the MMFF94 force field. Thisresult can provide a platform for derivation of a newgeneral-purpose force field because the benchmark forthe force field is no longer limited by scope or accu-racy to the experimentally characterized molecules orexperimental measurements.

L I G A N D D O C K I N G

In the past 5 years, the potential of using of flexi-ble docking of millions of available or virtual com-pounds to design inhibitors on the basis of proteinstructure became apparent. Using the Internal Coordi-nates Mechanics platform, we developed and createdbenchmarks for the docking and scoring technologyand identified new inhibitors based on the 3-dimen-sional models of proteins of interest. This past yearwe published studies on 4 aspects of ligand docking:benchmarking, predicting the specificity of ligand bind-ing, applying our methods to the discovery of ligands,and dealing with the problem of induced fit.

We studied the use of multiple receptors and homol-ogy models on a benchmark of nuclear receptors. Morethan 5000 structurally diverse compounds, including78 known ligands for nuclear receptors, were screened



against 18 crystal structures and 1 computer model of10 ligand-binding domains of nuclear receptors in activeor inactive states of the receptors. We were able togenerate highly focused subsets of the input chemicallibrary that were enriched 33- to 100-fold for all but1 receptor studied. For a particular ligand, virtual ligandscreening can often be used to identify the correct tar-get within the receptor family, although reliable discrimi-nations cannot be made between the closely relatedreceptor isoforms. Additionally, our results suggest thatwhen an experimental structure of the receptor is notavailable, virtual ligand screening can be applied suc-cessfully by using a homology model.

The main complicating factor in structure-baseddrug design is receptor rearrangement upon ligand bind-ing, also termed induced fit. It is the induced fit thatcomplicates cross-docking of ligands from differentligand-receptor complexes. Few docking methods areavailable that can be used to predict the induced fit reli-ably and, at the same time, improve on discriminatingbetween binders and nonbinders in the virtual screeningprocess. We compiled a benchmark of 33 cocrystalstructures of 4 kinases and showed that the inducedconformations can be generated by our procedure bycodocking of the ligand with side-chain and backboneoptimization (Fig. 1). These conformations improve theefficiency of virtual ligand screening by a factor of 1.89compared with docking to multiple experimental confor-mations. A review of methods to account for receptorflexibility and induced fit in ligand docks is in press.

P R O T E I N - P R O T E I N D O C K I N G

Association of 2 biological macromolecules is afundamental biological phenomenon and an unsolvedtheoretical problem. We optimized and applied a 2-stepdocking procedure for protein-protein docking of uncom-

plexed structures for the Critical Assessment of PRe-diction of Interactions competition (CAPRI; http://capri.ebi.ac.uk) with good results (Fig. 2). Subsequently, we

used ensembles of the rigid-body docking solutionsgenerated by the simulations to project the dockingenergy landscapes onto the protein surfaces. We foundthat highly populated low-energy regions consistentlycorresponded to actual binding sites. The procedurewas validated on a test set of 21 known protein-proteincomplexes not used in the training set. As much as 81%of the predicted high-propensity patch residues werelocated correctly in the native interfaces. This approachcan be used to guide the design of mutations on the sur-faces of proteins, provide geometrical details of a pos-sible interaction, and help annotate protein surfaces instructural proteomics.

Only about one third of the protein complexes canbe docked without serious considerations for the inducedconformational changes that occur upon docking. Previ-ously, we successfully used side-chain refinement ofthe interfaces to improve the ranking of the near-nativedocking geometries. This year we analyzed how differ-ential geometry measures and low-resolution modelscan be used to analyze the backbone flexibility and togenerate alternative conformations.

PUBLICATIONSBordner, A.J., Cavasotto, C.N., Abagyan, R.A. Direct derivation of van der Waalsforce field parameters from quantum mechanical interaction energies. J. Phys.Chem. B 107:9601, 2003.

Bursulaya, B.D., Totrov, M., Abagyan, R., Brooks, C.L. III. Comparative study of sev-eral algorithms for flexible ligand docking. J. Comput. Aided Mol. Des. 17:755, 2003.

Cavasotto, C.N., Abagyan, R.A. Protein flexibility in ligand docking and virtualscreening to protein kinases. J. Mol. Biol. 337:209, 2004.

Cavasotto, C.N., Orry, A.J.W., Abagyan R. Receptor flexibility in ligand docking.In: Handbook of Theoretical and Computational Nanotechnology. Rieth, M., Schom-mers, W. (Eds.). American Scientific Publishers, Stevenson Ranch, Calif, in press.

Fernandez-Recio, J., Totrov, M., Abagyan R. Identification of protein-protein inter-action sites from docking energy landscapes. J. Mol. Biol. 335:843, 2004.



F i g . 1 . Docking of a kinase inhibitor to the flexible kinase pocket.

F i g . 2 . A, Three protein-protein complexes predicted for the CAPRI

competition. B, Refinement of target 6 dramatically improved the

near-native solution.

Kovacs, J., Chacon, P., Abagyan, R. Predictions of protein flexibility: first ordermeasures. Proteins 56:661, 2004.

Marsden, B., Abagyan, R. SAD—a normalized structural alignment database:improving sequence-structure alignments. Bioinformatics, in press.

Schapira, M., Abagyan, R., Totrov, M. Nuclear hormone receptor targeted virtualscreening. J. Med. Chem. 46:3045, 2003.

Schapira, M., Raaka, B.M., Das, S., Fan, L., Totrov, M., Zhou, Z., Wilson, S.R.,Abagyan, R., Samuels, H.H. Discovery of diverse thyroid hormone receptor antago-nists by high-throughput docking. Proc. Natl. Acad. Sci. U. S. A. 100:7354, 2003.

Computer Modeling of Protein Structure and Intermolecular InteractionsV.A. Roberts, L. Fan, S. Hennessy, J.-L. Pellequer,

M.E. Pique, M.M. Thayer, A.E. Karu,* J.C. Mitchell,**

L.F. Ten Eyck*** University of California, Berkeley, California

** San Diego Supercomputing Center, La Jolla, California

The rapid increase in the number of known pro-tein sequences and structures is fueling theneed for methods to predict protein structure

and intermolecular interactions. We use computationaland computer graphics techniques in conjunction withprotein crystallography and site-directed mutagenesisto develop testable hypotheses and to direct proteinengineering experiments.P R E D I C T I N G M A C R O M O L E C U L A R C O M P L E X E S

We developed the computer program DOT to pre-dict macromolecular interactions. DOT rapidly performsa complete, 6-degrees-of-freedom search of all config-urations between 2 molecules. Intermolecular energies,consisting of van der Waals and electrostatic terms,are evaluated as correlation functions, which are effi-ciently computed with fast Fourier transforms. Theelectrostatic term takes into account the effects of sol-vent and counterions that surround the macromolecules.Currently, the energies of more than 60 billion config-urations between 2 molecules can be calculated in4 hours with 25 desktop workstations.

We applied DOT to a wide range of macromolecu-lar systems, from highly stable interactions with large,precisely fit contact regions to transient interactionsthat bring together highly charged surfaces. Calculatedenergies are sufficiently accurate to reflect the variedcontribution of electrostatic and van der Waals termsto protein association and to reproduce the complexesdetermined by x-ray crystallography. DOT has been

particularly useful for predicting protein-DNA interac-tions. Because the DOT search is over all space, wecan identify multiple DNA-binding sites on a proteinwith just a single calculation.

For example, we examined HIV type I integrase,which inserts viral DNA into the host genome. Integraseis a target for therapeutic inhibitors to treat AIDS, butthe search for potential drugs is impeded by the lackof a DNA-integrase structure. With DOT, we found2 DNA-binding sites that meet over the catalytic site(Fig. 1), providing a clue for how integrase incorpo-rates the viral DNA into host DNA.

E N H A N C I N G V I S U A L I Z A T I O N O F D N A

In collaboration with E.D. Getzoff and J.A. Tainer,Department of Molecular Biology, we created the Mod-elzilla graphics program, which combines molecularvisualization with solid geometry manipulation. Weconstructed simplified models of DNA by twisting andbending cylindrical geometric solids. We applied thereverse transformations to the molecular surfaces ofB-form DNA upon which molecular properties had beenmapped. Analysis of the resulting cylindrical objectsmakes it easier to correlate specific molecular proper-ties with DNA sequence and to compare the propertiesamong a series of DNA fragments.H I G H - R E S O L U T I O N A N T I B O D Y M O D E L S

Our database of superimposed crystallographicantibody structures reveals the structural conservation



F i g . 1 . Computational model of the interaction of the catalytic

domain of HIV type 1 integrase (gray ribbons) with bound viral

(white) and host (black) DNA fragments. The 2 DNA molecules

meet over the catalytic site, which includes 2 metal ions (dark gray

spheres). The terminal hydroxyl group of the conserved 3′-adeno-

sine (shown as white spheres) of the viral DNA contacts 1 of the

metal ions and also is near 1 of the strands of the host DNA.

of both the antibody backbone fold and the side chainsthat shape the antigen-binding pocket. Using the data-base, we constructed 3-dimensional models of anti-bodies to investigate antigen-antibody interactions,metal-site design, and the mechanism of catalysis.These models provide a structural basis for directingmutagenesis experiments to enhance binding, selectiv-ity, and catalysis.

One focus of this research is to assist developmentof antibodies that can be used to detect environmentalcontaminants. For example, the antibody S2B1 bindspolychlorinated biphenyls (PCBs) that lack chlorineatoms at the ortho positions. The toxicity of PCBs isthought to be due to their ability to bind proteins suchas the estrogen receptor. Our model of S2B1 with abound PCB (Fig. 2) reveals the structural basis for selec-tivity and suggests mutagenesis experiments for develop-ing antibodies selective for other groups of PCBs.

PUBLICATIONSAdesokan, A.A., Roberts, V.A., Lee, K.W., Lins, R.D., Briggs, J.M. Prediction ofHIV-1 integrase/viral DNA interactions in the catalytic domain by fast moleculardocking. J. Med. Chem. 47:821, 2004.

Roberts, V.A., Case, D.A., Tsui, V. Predicting interactions of winged-helix transcrip-tion factors with DNA. Proteins 57:172, 2004.

Mass SpectrometryG. Siuzdak, J. Apon, T. Brandon, E. Go, K. Harris, R. Lowe,

A. Meyers, N. Reisdorph, Z. Shen, C. Smith, G. Tong,

S. Trauger, W. Uritboonthai, E. Want, W. Webb, C. Wranik

H U M A N M E T A B O L I T E S

Small molecules ubiquitous in biofluids are nowwidely used to predict disease states. The inher-ent advantage of monitoring small molecules

rather than proteins is the relative ease of quantitativeanalysis with mass spectrometry. We are implement-ing novel mass spectrometry (Fig. 1) and bioinformat-ics techniques to investigate the metabolite profile ofsmall molecules as a diagnostic indicator of disease.The ultimate goal is to develop analytical and chemi-cal technologies and a data management system toidentify and structurally characterize metabolites ofphysiologic importance.

V I R A L C H A R A C T E R I Z A T I O N

We developed novel methods for characterizingviruses that have applications to whole viruses and viralproteins. Our results enabled us to examine both localand global viral structure, gaining insight into the dynamicchanges of proteins on the viral surface.

M A S S S P E C T R O M E T R Y I N S I L I C O

We are also developing ultra-high-sensitivityapproaches in mass spectrometry with a new strategythat involves pulsed laser desorption/ionization from asilylated silicon surface. In desorption/ionization on sili-con, silicon is used to capture analytes, and laser radia-tion is used to vaporize and ionize the analytes. Usingthis technology, we can analyze a wide range of mole-cules with unprecedented sensitivity, in the yoctomolerange (Fig. 2).



F i g . 2 . Structural model of the antibody S2B1 (gray tubes repre-

sent Cα backbone) with bound 3,4,3′,4′-PCB (ball-and-stick figure

with chlorine atoms in black). The S2B1 binding pocket (white sur-

face) is cut away to show the tight fit around the PCB ring carbon

atoms at the ortho positions (gray). This fit results in the observed

binding selectivity of S2B1.

F i g . 1 . A metabolite identified from plasma by using high-resolu-

tion mass analysis.

PUBLICATIONSBorman, S., Russell, H., Siuzdak, G. A mass spec timeline. Todays Chem. Sep-tember 2003, p. 47.

Bothner, B., Siuzdak, G. Electrospray ionization of a whole virus: analyzing mass,structure, and viability. Chembiochem 5:258, 2004.

Compton, B.J., Siuzdak, G. Mass spectrometry in nucleic acid, carbohydrate, andsteroid analysis. Spectroscopy 17:699, 2003.

Go, E.P., Shen, Z., Harris, K., Siuzdak, G. Quantitative analysis withdesorption/ionization on silicon mass spectrometry using electrospray deposition.Anal. Chem. 75:5475, 2003.

Kriwacki, R., Reisdorph, N., Siuzdak, G. Protein structure characterization withmass spectrometry. Spectroscopy 18:37, 2004.

Meng, J.-C., Averbuj, C., Lewis, W.G., Siuzdak, G., Finn, M.G. Cleavable linkers forporous silicon-based mass spectrometry. Angew. Chem. Int. Ed. 43:1255, 2004.

Prenni, J.E., Shen, Z., Trauger, S., Chen, W., Siuzdak, G. Protein characterizationusing liquid chromatography desorption ionization on silicon mass spectrometry(LC-DIOS-MS). Spectroscopy 17:693, 2003.

Reisdorph, N., Thomas, J.J., Katpally, U., Chase, E., Harris, K., Siuzdak, G.,Smith, T.J. Human rhinovirus capsid dynamics is controlled by canyon flexibility.Virology 314:34, 2003.

Shen, Z., Go, E.P., Gámez, A., Apon, J.V., Fokin, V., Greig, M., Ventura, M.,Crowell, J.E., Blixt, O., Paulson, J.C., Stevens, R.C., Finn, M.G., Siuzdak, G. Amass spectrometry plate reader: monitoring enzyme activity and inhibition with adesorption/ionization on silicon (DIOS) platform. Chembiochem 5:921, 2004.

Siuzdak, G. An introduction to mass spectrometry ionization. J. Assoc. Lab.Autom. 9:50, 2004.

Thomas, J.J., Bothner, B., Traina, J., Benner, W.H., Siuzdak, G. Electrospray ionmobility spectrometry of intact viruses. Spectroscopy 18:31, 2004.

Trauger, S.A., Junker, T., Siuzdak, G. Investigating viral proteins and intact viruseswith mass spectrometry. Top. Curr. Chem. 225:265, 2003.

Trauger, S.A., Wu, E., Bark, S.B., Nemerow, G.R., Siuzdak, G. The identificationof a unique adenovirus receptor by using affinity capture and mass spectrometry.Chembiochem 5:1095, 2004.

Want, E., Compton, B.J., Hollenbeck, T., Siuzdak, G. The application of massspectrometry in pharmacokinetics studies. Spectroscopy 17:681, 2003.

Wu, E., Trauger, S.A., Pache, L., Mullen, T.-M., von Seggern, D.J., Siuzdak, G.,Nemerow, G.R. Membrane cofactor protein is a receptor for adenoviruses associ-ated with epidemic keratoconjunctivitis. J. Virol. 78:3897, 2004.

Assembly of the 30S Ribosomal SubunitJ.R. Williamson, F. Agnelli, A. Beck, A. Bunner, A. Carmel,

J. Chao, S. Edgcomb, M. Hennig, E. Johnson, E. Kompfner,

K. Lehmann, P.K. Radha, M.I. Recht, W. Ridgeway,

S.P. Ryder, L.G. Scott, E. Sperling, B. Szymczyna,

M. Trevathan

The ribosome, the macromolecular machineresponsible for all protein synthesis in all cells,is composed of 3 large RNA molecules and more

than 50 small protein molecules. The process by whichthis machine is assembled is carefully orchestrated andhighly efficient, but the details of the process are poorlyunderstood. We are using a wide variety of biochemicaland biophysical methods to study the assembly of thesmall ribosomal 30S subunit. Understanding howassembly is efficiently carried out will lead to insightsinto assembly of RNA-protein complexes in general.

The 30S subunit is composed of a large 16S ribo-somal RNA molecule and 20 small proteins (S2–S21).The RNA is divided into 3 domains, 5′, central, and3′, and each domain binds to a subset of the proteins.In the past year, we made significant progress in under-standing the assembly of the 30S subunit in 2 areas.First, focusing on the central domain, we did a detailedthermodynamic analysis of the cooperative binding ofthe first 3 proteins of this domain. Second, we devel-oped methods that allow us to measure the bindingrates of all 20 ribosomal proteins simultaneously.T H E R M O D Y N A M I C A N A L Y S I S O F T H E C O O P E R A T I V E

B I N D I N G O F C E N T R A L D O M A I N P R O T E I N S

The central domain constitutes about one third ofthe 30S subunit, making up what is termed the plat-form region, which is near the mRNA decoding sitewhere the genetic code is read out. Six proteins, S6,S8, S11, S15, S18, and S21, bind specifically to thisregion of the RNA. During assembly of the centraldomain (Fig. 1A), S15 binds first and then a cascadeof other proteins binds, including S6 and S18. We arefocusing on the binding of S15, S6, and S18. Our goalis to understand why S6 and S18 do not bind to RNAuntil after S15 is bound.

We used isothermal titration calorimetry to studythe thermodynamics of binding of S15, S6, and S18to the central domain RNA. Interestingly, we found thatthe free energies of binding are closely correlated tothe enthalpies of binding, but the enthalpies can be



F i g . 2 . Laser desorption/ionization mass spectrometry on struc-

tured silylated silicon has sensitivity rivaling that of fluorescence.

measured more accurately (Fig. 1B). S15 binds tightlyto the central domain RNA, with an enthalpy of –14.5kcal/mol, and then S6 and S18 bind as a heterodimer,with an enthalpy of –32.5 kcal/mol. This ordered path-way is the one observed in the assembly map (Fig. 1A).

To understand why the assembly occurs in thisorder, we measured the binding enthalpy for additionof S6 and S18 in the absence of S15. The result was–12.7 kcal/mol, almost 20 kcal/mol less enthalpy thanis observed for binding of S6 and S18 to the preformedS15-RNA complex. From additional experiments withvarious RNA fragments, we were able to determinethat this difference corresponds to RNA tertiary inter-actions that are formed after S15 binds. This findingprovides a structural explanation for why S15 bindingoccurs before assembly of S6 and S18. We are extend-ing this analysis to include the remaining proteins in thecentral domain and the proteins in the other 2 domains.S I M U L T A N E O U S M E A S U R E M E N T O F T H E B I N D I N G

O F A L L 2 0 R I B O S O M A L P R O T E I N S

Although the study of domains and smaller riboso-mal fragments permits detailed mechanistic studies,using these approaches for studies of the entire 30Ssubunit is very difficult. We developed an isotope pulse-chase assay that allows measurement of the bindingrate of all 20 ribosomal proteins simultaneously. Weused this assay to learn about the kinetic mechanismof assembly of the 30S subunit.

The assembly of the 30S subunit is initiated bycombining 16S ribosomal RNA with a mixture of all 20proteins that are uniformly labeled with nitrogen 15.

After an assembly time, the binding of the labeled pro-teins is chased with an excess of unlabeled proteinscontaining the naturally occurring nitrogen 14. Theassembly of the 30S subunits is allowed to proceed,and the subunits are purified. Analysis of the proteinsby mass spectrometry determines the fraction of eachprotein that is labeled with nitrogen 15 as a functionof the incubation time. In this way, the time course ofbinding for all 20 proteins can be determined.

Most of the proteins in the 5′ and central domainsbind in less than 10 seconds, another set binds within1 minute, and a third set binds during a period of sev-eral minutes (labeled fast, medium, and slow, respec-tively, in Fig. 2). In general, the proteins that bind atthe 5′ end of the RNA bind more quickly than do thoseat the 3′ end. This finding is consistent with the mecha-nism of ribosome biogenesis in vivo, which occurscotranscriptionally from the 5′ end to the 3′ end as theRNA is synthesized by RNA polymerase. Further experi-ments under various conditions will reveal more detailin the kinetic mechanism as we continue to learn moreabout the assembly landscape for the 30S subunit.

PUBLICATIONSChao, J.A., Williamson, J.R. Joint x-ray and NMR refinement of the yeast L30e-mRNA complex. Structure (Camb.) 12:1165, 2004.

Gonsalvez, G.B., Lehmann, K.A., Ho, D.K., Stanitsa, E.S., Williamson, J.R.,Long, R.M. RNA-protein interactions promote asymmetric sorting of the ASH1mRNA ribonucleoprotein complex. RNA 9:1383, 2003.

Johnson, E.C., Feher, V.A., Peng, J.W., Moore, J.M., Williamson, J.R. Application ofSHAPES NMR screening to an RNA target. J. Am. Chem. Soc. 125:15724, 2003.



F i g . 1 . Cooperativity in the assembly of the central domain of

the 30S ribosomal subunit. A, The assembly map shows the observed

binding dependencies of the proteins in the central domain. Binding

of S15 precedes binding of S6 and S18. B, Measured enthalpies

for binding of ribosomal proteins to 16S rRNA. Binding of S6 and

S18 is enhanced by prior binding of S15; this enhanced binding is

due to the formation of additional RNA structural contacts near the

protein-binding sites.

F i g . 2 . Binding kinetics for 20 proteins binding to 16S ribosomal

RNA. The assembly map of the 30S subunit is annotated with the

binding rate as measured by a pulse-chase isotope mass spectrom-

etry method developed for this project. Proteins that bind at the 5′end generally bind more quickly than do proteins at the 3′ end, a

finding consistent with the order of events that occur inside cells.

Klostermeier, D., Sears, P., Wong, C.H., Millar, D.P., Williamson, J.R. A three-flu-orophore FRET assay for high-throughput screening of small-molecule inhibitors ofribosome assembly. Nucleic Acids Res. 32:2707, 2004.

Ryder, S.P., Frater, L.A., Abramowitz, D.L., Goodwin, E.B., Williamson, J.R. RNAtarget specificity of the STAR/GSG domain post-transcriptional regulatory proteinGLD-1. Nat. Struct. Mol. Biol. 11:20, 2004.

Nuclear Magnetic ResonanceStudies of RNA and RNA-LigandComplexes in SolutionM. Hennig, L.G. Scott, N. Kirchner, G.C. Pérez-Alvarado

During the early stages of HIV replication, thedefault pathway for viral RNA processing is tofully splice both introns of the RNA genome

before nuclear export. This splicing produces a num-ber of regulatory proteins, including Tat and Rev, thatsubsequently interact with the retroviral RNA genome.The formation of a complex consisting of the trans-activating response element, Tat, RNA, and proteinprovides an essential gene regulatory function for HIV.The protein Tat binds the trans-activating responseelement, an RNA hairpin located at the 5′ end ofnascent viral transcripts, and thereby enhances theinefficient elongation of transcription complexes initi-ated at the HIV promoter. The nuclear export of singlyspliced and unspliced HIV type 1 viral mRNA is medi-ated by the activity of Rev. Translation of these longermRNAs late in the replication phase of HIV is requiredto produce viral protein components that are needed toassemble infectious virions. The Rev response elementserves as the structured viral RNA target for multipleRev proteins.

Our goals are to identify specific, high-affinity RNA-binding ligands and to understand and, ultimately,manipulate the essential structural features of the trans-activating response element–Tat and the Rev responseelement–Rev recognition processes. We establishedefficient and economical enzymatic syntheses of thefluorinated nucleotide analogs 5F-UTP, 5F-CTP, and2F-ATP and showed that the analogs can be specifi-cally incorporated into RNA in template-directed tran-scription reactions by using T7 RNA polymerase. Theintroduction of fluorine 19 (19F) substitutions into theheterocyclic bases is nonperturbing and provides usuniquely positioned, sensitive reporter groups that canbe used for nuclear magnetic resonance monitoring ofconformational changes that occur upon binding.

RNA with its limited diversity of chemical groupsand its high, uniformly distributed negative chargeappears to be prone to bind small molecules non-specifically. Therefore, we use a target-based methodfor screening because the localization of binding siteson the isotopically labeled RNA distinctly distinguishesbetween specific and nonspecific binding. We use RNAcontaining 5-fluoropyrimidine (major groove reporter)or 2-fluoroadenine (minor groove reporter) in nuclearmagnetic resonance screening assays. By identifyingperturbations of assigned 19F resonances, we can notonly identify ligands but also localize their binding sites.The 19F chemical shift of a covalently bound fluorineatom is extraordinarily sensitive to changes in the micro-environment. Furthermore, because naturally occurringRNA lacks 19F probes, the specifically fluorinated RNAcounterparts are particularly well suited for monitoringligand-binding interactions without any interfering back-ground signals from the perspective of the RNA.

PUBLICATIONSScott, L.G., Geierstanger, B.H., Williamson, J.R., Hennig, M. Enzymatic synthesisand 19F NMR studies of 2-fluoroadenine-substituted RNA. J. Am. Chem. Soc.126:11776, 2004.

Genetic Code Components inTranslation and Cell BiologyP. Schimmel, J. Bacher, A. Bates, K. Beebe, V. de Crécy-Lagard,

Z. Druzina, K. Ewalt, M. Kapoor, E. Merriman, D. Metzgar,

C. Myers, L. Nangle, B. Nordin, F. Otero, J. Reader, M. Swairjo,

K. Tamura, W. Waas, X.-L. Yang

The genetic code is an algorithm that defines howcodons, nucleotide triplets, specify amino acids,thus providing the instructions for the synthesis

of proteins. The code was established by aminoacyl-tRNA synthetases more than 2 billion years ago, duringthe transition from a putative RNA world to the theaterof proteins. The synthetases are enzymes that carry outaminoacylation reactions that match each of the 20amino acids with the appropriate cognate tRNA. EachtRNA in turn has an anticodon nucleotide triplet that,in the algorithm of the code, corresponds to the aminoacid covalently joined to the tRNA by the synthetase.

The recognition system used by aminoacyl-tRNAsynthetases to correctly join an amino acid to its cog-nate tRNA can depend on direct recognition of theanticodon nucleotides, but the enzymes also use anindependent system that is sometimes called the “sec-



ond genetic code.” This second code is made up ofnucleotides in the acceptor stems of tRNAs, a regionthat is well removed from the anticodon triplet. Plausi-bly, this second genetic code was the original system forpairing amino acids with tRNAs or tRNA-like molecules.

Alanyl-tRNA synthetase is a prime example in whichthe anticodon has no role in determining aminoacyla-tion specificity. Rather, the enzyme recognizes a singleacceptor stem guanine-uridine wobble base pair throughan interaction that is largely conserved throughoutevolution. We recently solved the crystal structure ofan alanyl-tRNA synthetase (Fig. 1) and generated amodel of the synthetase-tRNA complex. This modelalso provided a mechanism for how the enzyme canadapt its structure to accommodate small shifts in thepositional location of the guanine-uridine wobble pairwithin the tRNA acceptor stem.

The precision of the modern genetic code isenhanced by proofreading, or editing, reactions bysynthetases for alanine, threonine, isoleucine, leucine,phenylalanine, and valine. Editing clears via hydrolysisan amino acid that has been joined to the wrong tRNA.The reaction takes place at a specific active site onthe synthetase. This site is separate from the activesite for aminoacylation. For instance, correct aminoa-cylation by alanyl-tRNA synthetase produces Ala-tRNAAla.The closely related amino acid glycine can becomemisacylated to produce Gly-tRNAAla. Through editing,the glycyl moiety is translocated from the aminoacyla-tion active site to the editing site and hydrolyzed.

Currently, we are focusing on how mischargedtRNAs are recognized and cleared at the editing sites,

including the role of specific nucleotides in the tRNA.Because these editing reactions are essential for cellviability, most likely small defects in editing can leadto specific diseases in humans, possibly autoimmunedisorders. Recent experiments in mammalian cellsestablished a link between cell morphology and partialdefects in editing and have encouraged the study of ani-mal models that incorporate partial defects in editing.

Throughout evolution, proteins have occurred thatare homologous to pieces of synthetases. For example,homologs of the editing domain of alanyl-tRNA syn-thetase exist as independent proteins or as fusionswith other proteins. Recently, researchers found thattryptophanyl-tRNA synthetase from Plasmodium falci-parum contains a domain related to the editing domainof alanyl-tRNA synthetase. Although its function is notknown, the domain is an unusual example of an editing-like domain from one synthetase fused to an entirelyunrelated synthetase like the one for tryptophan.

Before the development of the modern tRNA struc-ture, smaller RNAs most likely implemented RNA-dependent protein synthesis. Recently, we showedtemplate-dependent peptide synthesis by highly reac-tive aminoacyl-oligonucleotides. These results are con-sistent with the idea that small RNA oligonucleotideshad a role in the development of coded peptide synthesis.

Most modern tRNAs have posttranscriptional nucle-otide modifications that range from reductions to methy-lations to hypermodifications. Modifications are foundin the tRNAs of organisms in all 3 kingdoms of life.The biosynthetic pathways for these modifications arecomplex, and the genes that code for the enzymes thatmake up the pathways have, in many instances, beendifficult to identify. Using comparative genomics, wefirst identified potential genes for biosynthesis of dihy-rouridine (produced by reduction of uridine) and queu-osine (a hypermodified base). Biochemical experimentsthen provided definitive evidence for the modificationactivity encoded by these genes.

In mammals, tRNA synthetases are linked to cellsignaling and to disease. Biological fragments of tryp-tophanyl-tRNA synthetase and tyrosyl-tRNA synthetaseinhibit and stimulate angiogenesis, respectively (Fig. 2).These opposing angiogenic functions are regulated bythe presence of extra domains that must be cleaved toactivate cell signaling. An N-terminal domain is cleavedfrom human tryptophanyl-tRNA synthetase (by alterna-tive splicing or proteolysis) to generate forms that inhibitangiogenesis through receptor signaling pathways. A



F i g . 1 . Crystal structure of alanyl-tRNA synthetase (1–453) from

Aquifex aeolicus modeled with tRNA (L-shaped continuous thin tube)

to explain positional recognition of identity nucleotides in the accep-

tor stem. The identity nucleotides (small black and small gray spheres)

mark, or identify, the particular kind of tRNA.

C-terminal domain similar to the cytokine endothelialmonocyte-activating polypeptide II is cleaved fromhuman tyrosyl-tRNA synthetase to liberate a proangio-genic factor. The 2 opposing activities suggest thatthese tRNA synthetase fragments coordinately regulatethe growth of blood vessels.

Our studies of crystal structures of active synthetasefragments and the inactive (for angiogenesis) full-lengthenzymes provided a model for how the full-lengthenzymes are activated by removal of the extra domains.The structures also help us understand of how similar-ities and differences between the 2 enzymes explaintheir opposing angiogenic activities.

PUBLICATIONSBeebe, K., Merriman, E., Ribas de Pouplana, L., Schimmel, P. A domain for edit-ing by an archaebacterial tRNA synthetase. Proc. Natl. Acad. Sci. U. S. A.101:5958, 2004.

Beebe, K., Merriman, E., Schimmel, P. Structure-specific tRNA determinants forediting a mischarged amino acid. J. Biol. Chem. 278:45056, 2003.

De Pereda, J.M., Waas, W.F., Jan, Y., Ruoslahti, E., Schimmel, P., Pascual, J.Crystal structure of a human peptidyl-tRNA hydrolase reveals a new fold and sug-gests basis for a bifunctional activity. J. Biol. Chem. 279:8111, 2004.

Hendrickson, T.L., de Crécy-Lagard, V., Schimmel, P. Incorporation of nonnaturalamino acids into proteins. Annu. Rev. Biochem. 73:147, 2004.

Liu, J., Shue, E., Ewalt, K.L., Schimmel, P. A new γ-interferon-inducible promoterand splice variants of an anti-angiogenic human tRNA synthetase. Nucleic AcidsRes. 32:719, 2004.

Lovato, M.A., Swairjo, M.A., Schimmel, P. Positional recognition of a tRNA deter-minant dependent on a peptide insertion. Mol. Cell 13:843, 2004.

Nordin, B.E., Schimmel, P. Transiently misacylated tRNA is a primer for editing ofmisactivated adenylates by class I aminoacyl-tRNA synthetases. Biochemistry42:12989, 2003.

Pezo, V., Metzgar, D., Hendrickson, T.L., Waas, W.F., Hazebrouck, S., Döring, V.,Marlière, P., Schimmel, P., de Crécy-Lagard, V. Artificially ambiguous genetic codeconfers growth yield advantage. Proc. Natl. Acad. Sci. U. S. A. 101:8593, 2004.

Reader, J.S., Metzgar, D., Schimmel, P., de Crécy-Lagard, V. Identification of fourgenes necessary for biosynthesis of the modified nucleoside queuosine. J. Biol.Chem., 279:6280, 2004.

Ribas de Pouplana, L., Schimmel, P. Aminoacyl tRNA synthetases as potentialmarkers for the development of the genetic code. In: Life Sciences for the 21st Cen-tury. Keinan, E., Schechter, I., Sela, M. (Eds.). Wiley-VCH, New York, 2004, p. 81.

Schimmel, P., Beebe, K. Molecular biology: genetic code seizes pyrrolysine. Nature431:257, 2004.

Schimmel, P., Söll, D. The world of tRNA synthetases. In: Aminoacyl tRNA Syn-thetases. Ibba, M., Francklyn, C., Cusack, S. (Eds.). Landes Bioscience/Eurekah.com,Georgetown, TX, in press.

Schimmel, P., Yang, X.-L. Two classes give lessons about CCA. Nat. Struct. Mol.Biol. 11:807, 2004.

Swairjo, M.A., Otero, F.J., Yang, X.-L., Lovato, M.A., Skene, R.J., McRee, D.E.,Ribas de Pouplana, L., Schimmel, P. Alanyl-tRNA synthetase crystal structure anddesign for acceptor-stem recognition. Mol. Cell 13:829, 2004.

Tamura, K., Schimmel, P. Chiral-selective aminoacylation of an RNA minihelix. Sci-ence 305:1253, 2004.

Tang, H.-L., Yeh, L.-S., Chen, N.-K., Ripmaster, T., Schimmel, P., Wang, C.-C.Translation of a yeast mitochondrial tRNA synthetase initiated at redundant non-AUG codons. J. Biol. Chem., in press.

Tzima, E., Reader, J.S., Irani-Tehrani, M., Ewalt, K.L., Schwartz, M.A., Schim-mel, P. Biologically active fragment of a human tRNA synthetase inhibits fluidshear stress-activated responses of endothelial cells. Proc. Natl. Acad. Sci. U. S. A.100:14903, 2003.

Yang, X.-L., Otero, F.J., Skene, R.J., McRee, D.E., Schimmel, P., Ribas de Pou-plana, L. Crystal structures that suggest late development of genetic code compo-nents for differentiating aromatic amino acids. Proc. Natl. Acad. Sci. U. S. A.100:15376, 2003.

Yang, X.-L., Schimmel, P., Ewalt, K.L. Relationship of two human tRNA syn-thetases used in cell signaling. Trends Biochem. Sci. 29:250, 2004.

Mechanisms of RNA Assemblyand CatalysisM.J. Fedor, E.M. Calderon, J.W. Cottrell, C.P. Da Costa,

J.W. Harger, Y.I. Kuzmin, E.M. Mahen, R.S. Yadava

The ways that RNA enzymes accomplish catalysisare of considerable interest, particularly becauseof compelling evidence that protein synthesis

itself is an RNA-catalyzed reaction. Like proteins, RNAsassemble into ordered structures that can facilitatecatalysis by positioning reactive groups in an optimalorientation. However, ribonucleosides lack the chemi-



F i g . 2 . Domain cleavage in human tyrosyl- and tryptophanyl-tRNA

synthetases (TyrRS and TrpRS, respectively) activates the 2 enzymes

for the regulation of angiogenesis. EMAP II indicates endothelial

monocyte-activating polypeptide II.

cal versatility of amino acids. At first, it seemed thatall RNA enzymes compensated for this lack of chemi-cal versatility by recruiting metal cation cofactors. Thenresearchers found that a catalytic RNA, the hairpin ribo-zyme, catalyzes a reversible phosphodiester cleavagereaction in the absence of metal cations (Fig. 1). Theability of the hairpin ribozyme to accomplish catalysiswithout metal cations raised the possibility that RNAfunctional groups might participate directly in catalyticchemistry. Our goals are to determine which parts ofthe hairpin ribozyme contribute to catalytic activityand to understand the chemical basis of this activity.

Biochemical and structural studies implicate 2 active-site nucleobases, G8 and A38, in the catalytic mecha-nism. High-resolution structures of the active site ledto a general acid-base model in which G8 removes aproton to activate the attacking 2′ hydroxyl nucleophile,and A38 protonates the 5′ oxygen-leaving group. Wedeveloped an approach involving exogenous nucleobaserescue of abasic ribozymes to probe potential catalyticroles of G8 and A38. In this approach, abasic phospho-diester linkages replace individual active-site nucleo-bases. Characterization of the residual activity of abasicvariants provides valuable clues about the function ofthe missing nucleobase.

We found that activity increases with increasing pHin reactions with unmodified ribozymes, consistent withthe idea that the ionization state of a general base cata-lyst could be important for activity. Abasic ribozymeslacking G8 are much less active than are unmodifiedribozymes, as expected if G8 participates in catalysis.However, the residual activity of a variant lacking G8has the same pH dependence as reactions of unmodi-fied ribozymes. This similarity in pH dependence forunmodified and abasic ribozyme reactions argues againsta role for G8 as a general base catalyst because depro-

tonation of G8 could not account for the pH transitionin the abasic variant. On the other hand, loss of A38shifts this pH transition by several pH units, evidencethat a deprotonation event associated with A38 couldbe important for activity.

The activity that is lost when G8 is replaced by anabasic residue can be rescued by providing certainnucleobases in solution. Rescue of activity by exoge-nous nucleobases increases sharply with decreasingpH, indicating that a step that requires protonationbecomes rate determining for rescue. Several modelsof the rescue mechanism could explain this pH depen-dence. In a general acid catalysis model, the rescuingnucleobase protonates the 5′ oxygen-leaving group. Inan electrostatic stabilization model, interactions withthe rescuing nucleobase counter negative charge thatdevelops in the transition state. Alternatively, protona-tion might simply facilitate bindings of exogenousnucleobases to restore active-site geometry.

Detailed analyses of exogenous nucleobase rescueallow us to begin to distinguish among these models.Rescue increased with decreasing pH for both cleav-age and the reverse reaction of ligation, arguing againsta role for the rescuing nucleobase as a general acidbecause a nucleobase that contributes general acidcatalysis in the cleavage pathway should provide gen-eral base catalysis in ligation. Analysis of the concen-tration dependence of nucleobase rescue at high andlow pH indicated that protonation promotes catalysiswithin the nucleobase-bound ribozyme complex butdoes not stabilize nucleobase binding in the groundstate. Taken together, these results support a novelelectrostatic stabilization mechanism in which exoge-nous nucleobase binding counters negative charge thatdevelops in the transition state (Fig. 2).



F i g . 1 . Chemical mechanism of RNA cleavage mediated by the

family of small catalytic RNAs that includes the hairpin ribozyme.

Cleavage proceeds through an SN2-type mechanism that involves

in-line attack of the 2′ oxygen nucleophile on the adjacent phospho-

rus. Breaking of the 5′ oxygen-phosphorus bond generates products

with 5′ hydroxyl and 2′,3′-cyclic phosphate termini.

F i g . 2 . Model of electrostatic stabilization by a cationic nucleo-

base. The exogenous nucleobase stacks between A7 and A9 in the

pocket left by the G8 deletion, restoring active-site architecture. The

amidine group of the rescuing nucleobase forms hydrogen bonds

with 2′ hydroxyl and phosphoryl oxygens. These interactions stabi-

lize negative charge that develops in the transition state and posi-

tion reactive groups in the orientation appropriate for an SN2 in-

line nucleophilic attack.

PUBLICATIONSFedor, M.J. Determination of kinetic parameters for hammerhead and hairpinribozymes. Methods Mol. Biol. 252:19, 2004.

Kuzmin, Y.I., Da Costa, C.P., Fedor, M.J. Role of an active site guanine in hairpin ribo-zyme catalysis probed by exogenous nucleobase rescue. J. Mol. Biol. 340:233, 2004.

Yadava, R.S., Mahen, E.M., Fedor, M.J. Kinetic analysis of ribozyme-substratecomplex formation in yeast. RNA 10:863, 2004.

Directed Evolution of NucleicAcid EnzymesG.F. Joyce, J.T. Hillman, T.A. Jackson, G.C. Johns,

H.R. Kalhor, M. Oberhuber, B.M. Paegel, N. Paul,

W.M. Shih, G.G. Springsteen, S.B. Voytek

It is generally accepted that RNA-based life precededDNA- and protein-based life during the early his-tory of life on Earth. This earlier era is referred to

as the “RNA world.” During that time, genetic infor-mation resided in the sequence of RNA molecules,and phenotype was derived from the catalytic proper-ties of RNA. By studying the properties of RNA in thelaboratory, especially with regard to the evolution ofcatalytic function, we can gain insight into the RNAworld. In addition, we can develop novel nucleic acidenzymes that have applications in biology and medicine.S Y N T H E S I S A N D D E R I V A T I Z A T I O N O F R I B O S E

Ribose, the sugar in nucleic acids, is a minor com-ponent among the many products that result from thecondensation of formaldehyde. In addition, ribose ismore reactive than most other sugars and degradesmore rapidly than they do. Thus, it is difficult to under-stand why ribose is included in genetic material.

We exploited the greater reactivity of ribose by allow-ing it to react preferentially with cyanamide to form astable bicyclic product (Fig. 1). This product crystallizedspontaneously in aqueous solution under a broad range

of conditions; the corresponding products derived fromother sugars did not. Furthermore, the ribose-cyanamidecrystals gave the same diffraction pattern when derivedfrom D-ribose, L-ribose, or a racemic mixture of D- andL-ribose. The crystals derived from racemic ribose areenantiomorphously twinned, containing separate domainswith either pure D or pure L stereochemistry.

In a related study, we examined the ability of anucleic acid template to control the aldol condensa-tion between glycoaldehyde and glyceraldehyde to formribose and other pentose sugars. We prepared oligonu-cleotides that were derivatized at either the 5′ or the3′ end with either glycoaldehyde (the aldol donor) orglyceraldehyde (the aldol acceptor), respectively. The2 oligonucleotide-linked aldehydes were allowed tobind at adjacent positions along a complementarytemplate, resulting in an aldol reaction that gave riseto pentose sugars. No reaction was detected in theabsence of the template. This reaction is now beingused as the basis for in vitro evolution experiments,selecting RNAs that catalyze the template-dependentaldol condensation and, ultimately, aldol condensationreactions in a template-independent manner.E V O L U T I O N O F A D N A E N Z Y M E F R O M A N

R N A E N Z Y M E

The ability of RNA to catalyze the replication ofRNA molecules is an activity that would have beennecessary for RNA-based evolution in the RNA world.We previously developed an RNA enzyme, termed theR3C ligase, that catalyzes the template-directed join-ing of 2 RNA molecules. This enzyme was convertedto a format that allows it to produce additional copiesof itself through the joining of 2 component subunits.The copies in turn give rise to additional copies, result-ing in an exponential increase in the number of enzymemolecules over time. During the past year, using invitro evolution, we sought to convert this self-replicat-ing RNA enzyme to a self-replicating DNA enzyme.

The 54 ribonucleotides within the catalytic domainof the R3C ligase were replaced by deoxynucleotides,and random mutations were introduced at a frequencyof 12% per position. The resulting population wassubjected to 10 rounds of in vitro evolution, culminat-ing in a catalytic DNA with a kcat of 0.030 min–1 anda Km of 2.5 µM in the RNA ligation reaction. Theevolved DNA enzyme contains 11 mutations relativeto the starting RNA enzyme (Fig. 2). We are continu-ing the evolution, replacing the RNA substrates withDNA substrates to develop a system for the DNA-cat-alyzed ligation of DNA.



F i g . 1 . Ribose quickly decomposes in aqueous solution to form a

tarlike mixture of compounds, but in the presence of cyanamide, it

forms a bicyclic product that crystallizes spontaneously, protecting

ribose from further degradation.

C O N T I N U O U S E V O L U T I O N O F R N A E N Z Y M E S

We developed methods to carry out the in vitroevolution of RNA enzymes in a continuous manner.During continuous evolution, RNA molecules that per-form RNA ligation give rise to “progeny” molecules,which in turn are eligible to perform another reaction.Evolution takes place within a single reaction mixture,without intervention by the experimenter, until the sup-ply of substrate is exhausted. At that point, a smallaliquot of the mixture can be transferred to a fresh reac-tion vessel, allowing evolution to continue indefinitely.

We used continuous in vitro evolution to examinethe evolutionary response of an RNA enzyme to condi-tions of extreme pH. The RNA enzyme, which originallywas evolved to operate at pH 8.5, was exposed to pro-gressively more extreme environmental conditions. Theresults were 2 evolved populations that had adapted toeither acidic (pH 5.8) or alkaline (pH 9.8) conditions.Individual molecules were clonally isolated from each ofthe 2 evolved populations and characterized. Those thathad adapted to the low pH condition had a 10-foldincrease in catalytic rate compared with the parent mol-ecule. Those that had adapted to the high pH conditionhad substantially increased structural stability, leadingto measurable catalytic rates at conditions under whichthe parent RNA enzyme simply denatures.

PUBLICATIONSCadwell, R.C., Joyce, G.F. Mutagenic PCR. In: PCR Primer: A Laboratory Manual,2nd ed. Dieffenbach, C.W., Dveksler, G.S. (Eds.). Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, 2003, p. 453.

Joyce, G.F. Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem.73:791, 2004.

Kumar, R.M., Joyce, G.F. A modular, bifunctional RNA that integrates itself into atarget RNA. Proc. Natl. Acad. Sci. U. S. A. 100:9738, 2003.

Shih, W.M., Quispe, J.D., Joyce, G.F. A 1.7-kilobase single-stranded DNA thatfolds into a nanoscale octahedron. Nature 427:618, 2004.

Studies at the Interface ofMolecular Biology, Chemistry,and MedicineC.F. Barbas III, L. Asawapornmongkul, P. Blancafort,

S. Eberhardy, B. Dhevalapally, R. Fuller, R. Gordley, J. Guo,

N. Jendreyko, R.A. Lerner, C. Lund, L. Magnenat, A. Maki,

N. Mase, R. Mobini, S. Naidu, M. Popkov, D. Steiner,

J. Suri, F. Tanaka, R. Thayumanavan, Y. Ye, Y. Yuan,

G. Zhong

We are concerned with problems in molecularbiology, chemistry, and medicine. Many ofour studies involve learning or improving on

Nature’s strategies to prepare novel molecules thatperform specific functional tasks, such as regulating agene, destroying cancer, or catalyzing a reaction withenzymelike efficiency. We hope to apply these novelinsights, technologies, methods, and products to pro-vide solutions to human diseases, including cancer,HIV disease, and genetic diseases.D I R E C T I N G T H E E V O L U T I O N O F C A T A L Y T I C

F U N C T I O N

Using our concept of reactive immunization, wehave developed antibodies that catalyze aldol as wellas retro-aldol reactions of a wide variety of molecules.The catalytic proficiency of the best of these antibodiesis almost 1014, a value 1000 times that of the bestcatalytic antibodies reported to date and overall thebest of any synthetic protein catalyst. We have shownthe efficient asymmetric synthesis and resolution of avariety of molecules, including tertiary and fluorinatedaldols, and have applied these chiral synthons to thesynthesis of natural products (Fig. 1). The resultshighlight the potential synthetic usefulness of catalyticantibodies as artificial enzymes in addressing problemsin organic chemistry that are not solved by using nat-ural enzymes or more traditional synthetic methods.

To further evolve these catalytic antibodies, we aredeveloping genetic selection methods. Other advancesin this area include the development of the first peptidealdolase enzymes. By using both design and selection,we created small peptide catalysts that recapitulatemany of the kinetic features of large protein catalysts.With these smaller enzymes, we can address how thesize of natural proteins is related to catalytic efficiency.



F i g . 2 . Sequence and secondary structure of a DNA enzyme with

RNA ligase activity that was evolved from a corresponding RNA

enzyme. Mutations relative to the starting RNA enzyme are circled;

RNA residues are indicted by lowercase letters.

O R G A N O C A T A L Y S I S : A B I O O R G A N I C A P P R O A C H T O

C A T A L Y T I C A S Y M M E T R I C C A R B O N - C A R B O N

B O N D – F O R M I N G R E A C T I O N S

To further explore the principles of catalysis, weare studying amine catalysis as a function of catalyticscaffold. Using insights garnered from our studies ofaldolase antibodies, we determined the efficacy of sim-ple chiral amines and amino acids for catalysis of aldoland related imine and enamine chemistries such asMichael, Mannich, Knoevenagel, and Diels-Alder reac-tions (Fig. 2). Although aldolase antibodies are supe-rior in terms of the kinetic parameters, these moresimple catalysts are enabling us quantify the impor-tance of pocket sequestration in catalysis.

Furthermore, many of these catalysts are cheap,environmentally friendly, and practical for large-scalesynthesis. With this approach, we showed the scopeand usefulness of the first efficient amine catalysts of

direct asymmetric aldol, Mannich, Diels-Alder, andMichael reactions. The organocatalyst approach is adirect outcome of our studies of catalytic antibodiesand provides an effective alternative to organometallicreactions that use severe reaction conditions and oftentoxic catalysts.

We think that our discovery that the simple natu-rally occurring amino acids such as L-proline and otheramines can effectively catalyze a variety of enantiose-lective intermolecular reactions will change the waymany reactions will be performed. Furthermore, thesecatalysts are functional in related ketone addition reac-tions such as Mannich- and Michael-type reactions. Asa testament to the mild nature of this approach, wedeveloped the first catalytic asymmetric aldol, Man-nich, and Michael reactions involving aldehydes asnucleophiles. Previously, such reactions were consid-ered out of the reach of traditional synthetic methods.

In an extension of these concepts, we invented avariety of novel multicomponent or asymmetric assem-bly reactions (Fig. 3). Our finding that a variety ofoptically active amino acids can be synthesized withproline catalysis in which an L-amino acid begets other



F i g . 1 . A variety of compounds synthesized with the world’s

first commercially available catalytic antibody, 38C2, produced at

Scripps Research.

F i g . 2 . L-Proline and other organocatalysts developed for a vari-

ety of catalytic asymmetric syntheses via aldol, Michael, Mannich,

Diels-Alder, and Knoevenagel reactions provide access to important

classes of compounds. These catalysts make reactions that were

once complex multistep reactions, simple 1-step reactions. A wide

variety of medicinally important products can be assembled by

using the Mannich reaction manifold alone.

F i g . 3 . A few recently developed catalytic asymmetric assembly

reactions. In these reactions, designed small organic molecules are

used to synthesize complex molecules.

L-amino acids suggests that this route may have beenused in prebiotic syntheses of optically active aminoacids. In addition, we showed that our strategy can beused to synthesize carbohydrates directly, thereby pro-viding a provocative prebiotic route to the sugars essen-tial for life.

Unlike most catalysts obtained via traditionalapproaches, our catalysts are environmentally safe andare available in both enantiomeric forms. The reactionsdo not require inert conditions or heavy metals and canbe performed at room temperature without preactivationof the donor substrates. Because amines can act as cat-alysts via both nucleophilic (enamine based) and elec-trophilic (iminium based) activation, they have greatpotential in catalytic asymmetric synthesis.T H E R A P E U T I C A N T I B O D I E S , I N A N D O U T O F C E L L S

We developed the first human antibody phage dis-play libraries and the first synthetic antibodies and meth-ods for the in vitro evolution of antibody affinity. Theability to manipulate large libraries of human antibodiesand to evolve such antibodies in the laboratory providestremendous opportunities to develop new medicines.Laboratories and pharmaceutical companies around theworld now apply the phage display technology that wedeveloped for antibody Fab fragments. In our laboratory,we are targeting cancer and HIV disease. One of ourantibodies, IgG1-b12, protects animals against primarychallenge with HIV type 1 (HIV-1) and has been furtherstudied by many other researchers. We improved thisantibody by developing in vitro evolution strategies thatenhanced its neutralization activity. By coupling labo-ratory-evolved antibodies with potent toxins, we showedthat immunotoxins can effectively kill infected cells.

We are also developing methods to halt HIV byusing gene therapy. We created unique human antibod-ies that can be expressed inside human cells to makethe cells resistant to HIV infection. In the future, theseantibodies might be delivered to the stem cells ofpatients infected with HIV-1, allowing the developmentof a disease-free immune system that would precludethe intense regimen of antiviral drugs now required totreat HIV disease.

Using our increased understanding of antibody-anti-gen interactions, we extended our efforts in cancer ther-apy and developed rapid methods for creating humanantibodies from antibodies derived from other species.We produced human antibodies that should enable usto selectively starve a variety of cancers by inhibitingangiogenesis and antibodies that will be used to deliver

radionuclides to colon cancers to destroy the tumors.We hope that some of these antibodies will be used inclinical trials done by our collaborators at the Sloan-Kettering Cancer Center in New York City.

On the basis of our studies on HIV-1, we used intra-cellular expression of antibodies directed against angio-genic receptors to create a new gene-based approachto cancer. We are determining if this new approachcan be applied in vivo to halt tumor growth. Our pre-liminary studies indicate that this type of gene therapycan be successfully applied to the treatment of cancer.T H E R A P E U T I C A P P L I C A T I O N S O F

C A T A L Y T I C A N T I B O D I E S

The development of highly efficient catalytic anti-bodies opens the door to many practical applications.One of the most fascinating is the use of such antibod-ies in human therapy. We think that use of this strat-egy can improve chemotherapeutic approaches todiseases such as cancer and AIDS. Chemotherapeuticregimens are typically limited by nonspecific toxic effects.To address this problem, we developed a novel andbroadly applicable drug-masking chemistry that oper-ates in conjunction with our unique broad-scope cata-lytic antibodies. This masking chemistry is applicableto a wide range of drugs because it is compatible withvirtually any heteroatom. We showed that generic drug-masking groups can be selectively removed by sequen-tial retro-aldol–retro-Michael reactions catalyzed byantibody 38C2 (Fig. 4). This reaction cascade is notcatalyzed by any known natural enzyme.

Application of this masking chemistry to the anti-cancer drugs doxorubicin, camptothecin, and etopo-side produced prodrugs with substantially reducedtoxicity. These prodrugs are selectively unmasked by



F i g . 4 . Targeting cancer and HIV with prodrugs activated by catalytic

antibodies. A bifunctional antibody is shown targeting a cancer cell

for destruction. A nontoxic analog of doxorubicin, pro-doxorubicin, is

being activated by an aldolase antibody to the toxic form of the drug.

the catalytic antibody when the antibody is applied attherapeutically relevant concentrations. The efficacy ofthis approach has been shown in in vivo models ofcancer. Currently, we are developing more potent drugsand novel antibodies that will allow us to target breast,colon, and prostate cancer as well as cells infectedwith HIV-1. On the basis of our preliminary findings,we think that our approach can become a key tool inselective chemotherapeutic strategies. To see a movieillustrating this approach, visit http://www.scripps.edu/mb/barbas/index.html.A D A P T O R I M M U N O T H E R A P Y : T H E A D V E N T

O F C H E M O B O D I E S

We think that combining the chemical diversity ofsmall synthetic molecules with the immunologic charac-teristics of antibody molecules can lead to therapeuticagents with superior properties. Therefore, we devel-oped a conceptually new device that equips small syn-thetic molecules with both the immunologic effectorfunctions and the long serum half-life of a generic anti-body molecule. As a prototype, we developed a target-ing device based on the formation of a covalent bondof defined stoichiometry between (1) a 1,3-diketonederivative of an arginine–glycine–aspartic acid peptido-mimetic that targets the integrins αvβ3 and αvβ5 and(2) the reactive lysine of aldolase antibody 38C2. Theresulting complex spontaneously assembled in vitro andin vivo, selectively retargeted antibody 38C2 to thesurface of cells expressing integrins αvβ3 and αvβ5,dramatically increased the circulatory half-life of thepeptidomimetic, and effectively reduced tumor growthin animal models of human Kaposi sarcoma and coloncancer (Fig. 5). These studies have been extended tomelanoma therapy. Z I N C F I N G E R G E N E S W I T C H E S

The solutions to many diseases might be simplyturning genes on or off in a selective way. In order toproduce switches that can turn genes or off, we arestudying molecular recognition of DNA by zinc fingerproteins and methods of creating novel zinc fingerDNA-binding proteins (Fig. 6). Because of their modu-larity and well-defined structural features, zinc fingerproteins are particularly well suited for use as DNA-binding proteins. Each finger forms an independentlyfolded domain that typically recognizes 3 nucleotidesof DNA. We showed that proteins can be selected ordesigned that contain zinc fingers that recognize novelDNA sequences. These studies are aiding the elucida-tion of rules for sequence-specific recognition within

this family of proteins. We selected and designed spe-cific zinc finger domains that will constitute an alpha-bet of 64 domains that will allow any DNA sequenceto be bound selectively. The prospects for this “secondgenetic code” are fascinating and should have a majorimpact on basic and applied biology.

We showed the potential of this approach in multi-ple mammalian and plant cell lines and in whole organ-isms. With the use of characterized modular zinc fingerdomains, polydactyl proteins capable of recognizing an18-nucleotide site can be rapidly constructed. Our



F i g . 5 . Adaptor immunotherapy dramatically slows tumor growth.

A variety of cancer xenografts have been effectively treated with

chemobodies, a combination of small-molecule drugs and antibod-

ies. Chemobodies have characteristics that can be superior to those

of either the small molecule or the antibody alone.

F i g . 6 . A designed polydactyl zinc finger binds 18 bp of DNA. A

single zinc finger domain is highlighted. With this direct approach, we

can construct more than a billion gene switches and use the switches

to specifically turn genes on or off in multiple organisms. With further

elaboration of the approach, every gene in the genome can be either

upregulated or downregulated, providing a new approach to probe

gene function across the genome.

results suggest that zinc finger proteins might be use-ful as genetic regulators for a variety of human alimentsand provide the basis for a new strategy in gene ther-apy. Our goal is to develop this class of therapeuticproteins to inhibit or enhance the synthesis of pro-teins, providing a direct strategy for fighting diseasesof either somatic or viral origin.

We are also developing proteins that will inhibit thegrowth of tumors and others that will inhibit the expres-sion of the protein CCR5, which is a key to infection ofhuman cells by HIV-1. This past year, we reported thedevelopment of an HIV-1–targeting transcription factorthat strongly suppresses HIV-1 replication. Geneticdiseases such as sickle cell anemia are also being tar-geted. Using a library of transcription factors, we devel-oped a strategy that allows us to turn on and turn offevery gene in the genome. With this powerful new strat-egy, we can quickly regulate a target gene or discoverother genes key in disease. In the future, we hope touse novel DNA-modifying enzymes directed by zincfingers to manipulate chromosomes themselves.

PUBLICATIONSBetancort, J.M., Kandasamy, S., Thayumanavan, R., Tanaka, F., Barbas, C.F. III.Catalytic direct asymmetric Michael reactions: addition of unmodified ketone andaldehyde donors to alkylidene malonates and nitro olefins. Synthesis (Mass), in press.

Chowdari, N.S., Ramachary, D.B., Barbas, C.F. III. Organocatalysis in ionic liq-uids: highly efficient L-proline-catalyzed direct asymmetric Mannich reactionsinvolving ketone and aldehyde nucleophiles. Synlett 12:1906, 2003.

Chung, J., Rader, C., Popkov, M., Hur, Y.-M., Kim, H.-K., Lee, Y.-J., Barbas, C.F.III. Integrin αIIbβ3-specific synthetic human monoclonal antibodies and HCDR3peptides that potently inhibit platelet aggregation. FASEB J. 18:361, 2004.

Gottesfeld, J.M., Barbas, C.F. III. RNA as a transcriptional activator. Chem. Biol.10:584, 2003.

Holbro, T., Beerli, R.R., Maurer, F., Koziczak, M., Barbas, C.F. III, Hynes, N.E.The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2 requiresErbB3 to drive breast tumor cell proliferation. Proc. Natl. Acad. Sci. U. S. A.100:8933, 2003.

Jendreyko, N., Popkov, M., Beerli, R.R., Chung, J., McGavern, D.B., Rader, C.,Bar-bas, C.F. III. Intradiabodies: bispecific, tetravalent antibodies for the simultaneous func-tional knockout of two cell surface receptors. J. Biol. Chem. 278:47812, 2003.

Lund, C.V., Blancafort, P., Popkov, M., Barbas, C.F. III. Promoter-targeted phagedisplay selections with preassembled synthetic zinc finger libraries for endogenousgene regulation. J. Mol. Biol. 340:599, 2004.

Mase, N., Tanaka, F., Barbas, C.F. III. Rapid fluorescent screening for bifunctionalamine-acid catalysts: efficient syntheses of quaternary carbon-containing aldolsunder organocatalysis. Org. Lett. 5:4369, 2003.

Mase, N., Tanaka, F., Barbas, C.F. III. Synthesis of β-hydroxyaldehydes with stere-ogenic quarternary carbon centers by direct organocatalytic asymmetric aldol reac-tions. Angew. Chem. Int. Ed. 43:2420, 2004.

Notz, W., Tanaka, F., Watanabe, S.-I., Chowdari, N.S., Turner, J.M., Thayumana-van, R., Barbas, C.F. III. The direct organocatalytic asymmetric Mannich reaction:unmodified aldehydes as nucleophiles. J. Org. Chem. 68:9624, 2003.

Popkov, M., Jendreyko, N., Gonzalez-Sapienza, G., Mage, R.G., Rader, C., Bar-bas, C.F. III. Human/mouse cross-reactive anti-VEGF receptor 2 recombinant anti-bodies selected from an immune b9 allotype rabbit antibody library. J. Immunol.Methods 288:149, 2004.

Rader, C., Turner, J.M., Heine, A., Shabat, D., Sinha, S.C., Wilson, I.A., Lerner,R.A., Barbas, C.F. III. A humanized aldolase antibody for selective chemotherapyand adaptor immunotherapy. J. Mol. Biol. 332:889, 2003.

Ramachary, D.B., Anebouselvy, K., Chowdari, N.S., Barbas, C.F. III. Direct organo-catalytic asymmetric heterodomino reactions: the Knoevenagel/Diels-Alder/epimer-ization sequence for the highly diastereoselective synthesis of symmetrical andnonsymmetrical synthons of benzoannelated centropolyquinanes. J. Org. Chem.69:5838, 2004.

Ramachary, D.B., Chowdari, N.S., Barbas, C.F. III. The first organocatalytic het-ero-domino Knoevenagel-Diels-Alder-epimerization reactions: diastereoselective syn-thesis of highly substituted spiro[cyclohexane-1,2′-indan]-1,3′,4-triones. Synlett12:1910, 2003.

Ramachary, D.B., Chowdari, N.S., Barbas, C.F. III. Organocatalytic asymmetricdomino Knoevenagel/Diels-Alder reactions: a bioorganic approach to the diastere-ospecific and enantioselective construction of highly substituted spiro-[5,5]-unde-cane-1,5,9-triones. Angew. Chem. Int. Ed. 42:4233, 2003.

Segal, D.J., Gonclaves, J., Eberhardy, S., Swan, C.H., Torbett, B.E., Barbas, C.F.III, Li, X. Attenuation of HIV-1 replication in primary human cells with a designedzinc finger transcription factor. J. Biol. Chem. 279:14509, 2004.

Shim, H., Karlström, A., Touami, S.M., Fuller, R.P., Barbas, C.F. III. Flow cytometricscreening of aldolase catalytic antibodies. Bioorg. Med. Chem. Lett. 14:4065, 2004.

Tan, W., Zhu, K., Segal, D.J., Barbas, C.F. III, Chow, S.A. Fusion proteins consist-ing of human immunodeficiency virus type 1 integrase and the designed polydactylzinc finger protein E2C direct integration of viral DNA into specific sites. J. Virol.78:1301, 2004.

Tanaka, F., Barbas, C.F. III. Antibody-catalyzed aldol reactions. In: Enolates, Bio-catalysis and Natural Product Synthesis. Mahrwald, R. (Ed.). Wiley-VCH, NewYork, 2004, p. 273. Modern Aldol Reactions, Vol. 1.

Tanaka, F., Barbas, C.F. III. Organocatalytic approaches to enantioenriched β-amino acids. In: Enantioselective Synthesis of β-Amino Acids, 2nd ed. Juaristi, E.(Ed.). Wiley-VCH, New York, in press.

Tanaka, F., Barbas, C.F. III. Reactive immunization: a unique approach to aldolaseantibodies. In: Catalytic Antibodies. Keinan. E. (Ed.). Wiley-VCH, New York, inpress.

Tanaka, F., Flores, F., Kubitz, D., Lerner, R.A., Barbas, C.F. III. Antibody-catalyzedaminolysis of a chloropyrimidine derivative. Chem. Commun., in press.

Tanaka, F., Fuller, R., Shim, H., Lerner, R.A., Barbas, C.F. III. Evolution of aldol-ase antibodies in vitro: correlation of catalytic activity and reaction-based selection.J. Mol. Biol. 335:1007, 2004.

Tanaka, F., Mase, N., Barbas, C.F. III. Design and use of fluorogenic aldehydes for moni-toring the progress of aldehyde transformations. J. Am. Chem. Soc. 126:3692, 2004.

Tanaka, F., Thayumanavan, R., Barbas, C.F. III. Fluorescent detection of carbon-carbon bond foundation. J. Am. Chem. Soc. 125:8523, 2003.

Tanaka, F., Thayumanavan, R., Mase, N., Barbas, C.F. III. Rapid analysis of sol-vent effects on enamine formation by fluorescence: how might enzymes facilitateenamine chemistry with primary amines? Tetrahedron Lett. 45:325, 2004.



Catalytic Antibodies, SyntheticEnzymes, Organic Synthesis,and Biomolecular ComputingE. Keinan, C.H. Lo, H. Han, S. Sasmal, M. Arifuddin,

S. Ledoux, S. Saphier, A. Brik, N. Metanis, G. Sklute,

E. Kossoy, M. Soreni, D. Vebenov, R. Piran, M. Sinha,

A. Alt, I. Ben-Shir

We focus on antibody-catalyzed photochemicalreactions, oxidation reactions, and organo-metallic reactions, as illustrated in the fol-

lowing examples.

C A T A L Y T I C A N T I B O D I E S

Although the solution photochemical reaction ofthe ketone 1 (in Fig. 1) yields only the cleavage prod-ucts 2 and 3, in the presence of 20F10, an antibodyto 5a and 5b, this Norrish type II reaction results inthe selective formation of cis-cyclobutanol (compound4 in Fig. 1). Furthermore, the fact that compound 4,which consists of 2 asymmetric centers, is obtainedas a single diastereomer makes this photoproduct avaluable building block for the synthesis of naturalproducts. Another reaction that is exclusively cat-alyzed by 20F10 is the photochemical formation ofcyclopropanol products.

An aldolase antibody, 24H6, obtained from immu-nization with large diketone haptens has an active-sitelysine residue with a perturbed pKa of 7.0. This antibodycatalyzes both the aldol addition and the retrograde aldolfragmentation with a broad range of substrates thatdiffer structurally from the hapten. This observationsuggests that in reactive immunization with 1,3-dike-

tones, the hapten structure governs the chemistry butnot the overall organization of the active site. Antibody24H6 also catalyzes the oxidation of α-hydroxyketonesto α-diketones. The deuterium exchange at the α posi-tion of many ketones and aldehydes is also efficientlycatalyzed by aldolase antibodies 38C2 and 24H6. Allreactions were carried out in deuterium oxide under neu-tral conditions and showed regioselectivity, chemoselec-tivity, and high catalytic rates.

During our ongoing efforts to develop catalytic anti-bodies for organometallic reactions, we discovered anew aqueous chemistry of platinum. A novel, air-sta-ble dihydrido(methyl)platinum(IV) complex (compound7 in Fig. 2) was formed by the reaction of compound6 with water. When the reaction is done with deuteriumoxide, compound 6 undergoes a surprising hydrogen-deu-terium exchange to produce 6-d3 before the completeformation of compound 7, probably via reversible for-mation of a sticky σ-methane ligand. Orchestrating therelative orientation and cooperativity of several func-tional groups, as occurs naturally in the catalytic triad,to organometallic chemistry could lead to catalysis ofdifficult chemical transformations.

S Y N T H E T I C E N Z Y M E S

Efforts to generate new enzymatic activities fromexisting protein scaffolds may not only provide bio-technologically useful catalysts but also lead to a bet-ter understanding of the natural process of evolution.Enzymes are usually characterized as catalyzing a spe-cific reaction by a unique chemical mechanism. How-ever, small changes in the amino acid sequence of someenzymes can significantly alter the catalytic properties



F i g . 1 . The photochemical Norrish type II reaction of ketone 1produces in solution the cleavage products 2 and 3. Antibody

20F10, which was elicited against a mixture of 5a and 5b, cat-

alyzes enantioselective formation of cis-cyclobutanol (4).

F i g . 2 . The hydrogen-deuterium exchange in complex 7 reflects

a 3-station proton walk mechanism, which is an organometallic

version of the catalytic triad that exists in serine proteases and

other natural enzymes. The efficiency of this process relies on the

spatial organization of functional groups that operate in concert to

activate a water molecule by the bidentate-tris-pyrazolylborate 6′′,as shown in A, and then to achieve a multistep proton walk from

water to the methyl ligand via an agostic-type σ-bond metathesis

mechanism, as shown in D, leading to the σ-methane intermediate E.

of the enzymes, affecting the substrate selectivity andsubtle aspects of the catalytic mechanism. The cata-lytic promiscuity displayed in these enzymes may bean important factor in the natural evolution of newcatalytic activities and in the development of new cat-alysts through protein engineering methods.

We profoundly changed the catalytic activity andmechanism of 4-oxalocrotonate tautomerase by creat-ing a rationally designed single amino acid substitutionthat corresponds to a mutation in a single base pair.Although the wild-type enzyme catalyzes only the tau-tomerization of oxalocrotonate to 2-oxo-3E-hexenedioate,the mutant P1A catalyzes 2 reactions: the original tau-tomerization reaction via a general acid-base mechanismand the decarboxylation of oxaloacetate via a nucle-ophilic mechanism. The observation that a single cat-alytic group in an enzyme can catalyze 2 reactions by2 different mechanisms supports the theory that newenzymatic activity can evolve in a continuous manner.

Highly evolved enzymes are optimized not only tocatalyze a desired reaction but also to avoid undesiredprocesses. Mutation of active-site residues designed todecrease the optimized catalytic activity may alsoenhance alternative reaction pathways. Thus, even aminor change in the active-site residues could resultin a dramatic change in the delicately optimized bal-ance of the enzymes’ chemical reactivities. We showedthat the mutant P1A, which catalyzes the isomeriza-tion of the double bond in 4-oxalocrotonate, also under-goes specific 1,4-addition to the tautomerization productto form a stable covalent adduct. This research on syn-thetic enzymes is being done in collaboration with P.E.Dawson, Department of Cell Biology.O R G A N I C S Y N T H E S I S

Annonaceous acetogenins, particularly those withadjacent bis-tetrahydrofuran rings, have remarkablecytotoxic, antitumor, antimalarial, immunosuppressive,pesticidal, and antifeedant activities. More than 350different acetogenins have been isolated from only 35of 2300 plants of the family Annonaceae. We devel-oped synthetic approaches that can be used to gener-ate chemical libraries of stereoisomeric acetogenins.These efforts resulted in the total synthesis of severalnaturally occurring acetogenins, including asimicin,bullatacin, trilobacin, rolliniastatin, solamin, reticulat-acin, rollidecins C and D, goniocin, cyclogoniodeninand mucocin, and many nonnatural stereoisomers. Asubstituted photoactive derivative of asimicin has beenprepared for photoaffinity labeling of the target protein

subunit in the mitochondrial complex I. This researchis being done in collaboration with S.C. Sinha, Depart-ment of Molecular Biology.B I O M O L E C U L A R C O M P U T I N G D E V I C E S

In fully autonomous molecular computing devices,all components, including input, output, software, andhardware, are specific molecules that interact witheach other through a cascade of programmable chemi-cal events, progressing from the input molecule to themolecular output signal. DNA molecules and DNAenzymes have been used as convenient, readily avail-able components of such computing devices becausethe DNA materials have highly predictable recognitionpatterns, reactivity, and information-encoding features.Furthermore, DNA-based computers can become partof a biological system, generating outputs in the formof biomolecular structures and functions.

Our previously reported 2-symbol–2-state finiteautomata computed autonomously, and all of theircomponents were soluble biomolecules mixed in solu-tion. The hardware consisted of 2 enzymes, an endo-nuclease and a ligase, and the software and the inputwere double-stranded DNA oligomers (Fig. 3). Morerecently, we designed and created 3-symbol–3-stateautomata that can carry out more complex computa-tions. In addition, we found that immobilization of theinput molecules on chips allowed parallel computa-tion, a system that can be used for encryption of infor-mation. We also developed an advanced computingdevice in which the input is a molecule but the outputis a biological phenomenon.



F i g . 3 . A biomolecular computing machine made of molecules.

The hardware consists of a restriction nuclease and a ligase; the

input, transition molecules (software), and detection molecules are

all made of double-stranded DNA.

PUBLICATIONSDubnikova, F., Kosloff, R., Almog, J., Zeiri, Y., Boese, R., Itzhaky, H., Alt, A.,Keinan, E. Detonation of TATP is an entropic explosion. J. Am. Chem. Soc., in press.

Han, H., Sinha, M.K., D’Souza, L.J., Keinan, E., Sinha, S.C. Total synthesis of34-hydroxyasimicin and its photoactive derivative for affinity labeling of the mito-chondrial complex I. Chemistry 10:2149, 2004.

Keinan, E., Alt, A., Amir, G., Bentur, L., Bibi, H., Shoseyov, D. Natural ozonescavenger prevents asthma in sensitized rats. Bioorg. Med. Chem., in press.

Metanis, N., Brik, A., Dawson, P.E., Keinan, E. Electrostatic interactions dominatethe catalytic contribution of Arg39 in 4-oxalocrotonate tautomerase. J. Am. Chem.Soc. 126:12726, 2004.

Saphier, S., Hu, Y., Sinha, S.C., Houk, K.N., Keinan, E. The origin of selectivity inthe antibody 20F10-catalyzed Yang cyclization. J. Am. Chem. Soc., in press.

Sasmal, S., Sinha, M.K., Keinan, E. Facile purification of rare cucurbiturils byaffinity chromatography. Org. Lett. 6:1225, 2004.

Sklute, G., Oizerowich, R., Shulman, H., Keinan, E. Antibody-catalyzed benzoinoxidation as a mechanistic probe for nucleophilic catalysis by an active site lysine.Chemistry 10:2159, 2004.

Soreni, M., Yogev, S., Kossoy, E., Shoham, Y., Keinan, E. Parallel biomolecularcomputation on surfaces with advanced finite automata. J. Am. Chem. Soc., in press.

Antibody Catalysis, OrganicSynthesis, and Prodrug andTargeting TherapiesS.C. Sinha, R.A. Lerner, S. Das, L.-S. Li, S. Abraham

Our research interests involve synthetic methods,total synthesis of biologically important naturalproducts, antibody catalysis, and development

of prodrug and targeting approaches for cancer therapy.A L D O L - T Y P E A D D I T I O N O F C A R B O N Y L C O M P O U N D S

T O A C E T A L S A N D K E T A L S

Development of new stereoselective synthetic meth-ods holds a key to the efficient synthesis of biologicallyimportant natural products and their analogs. Recently,we developed a new method for an aldol-type additionof a methyl ketone with an acetal or ketal that producesthe corresponding β-alkoxy ketones. Our method is suit-able for both intramolecular and intermolecular aldol-type reactions, and it is an alternative approach to theMukaiyama reaction. The intramolecular reaction ishighly stereoselective although limited in scope, whereasthe intermolecular reaction is versatile but less stereo-selective (Fig. 1).S Y N T H E S I S O F N A T U R A L P R O D U C T S A N D

T H E I R A N A L O G S

For the past several years, we have focused on thesynthesis of a number of naturally occurring molecules,including analogs of sorangiolides A and B, epothilones,

and bis-tetrahydrofuran annonaceous acetogenins.Sorangiolides and epothilones (Fig. 2) are macrocycliclactones and have interesting biological properties.Sorangiolides are weakly active against gram-positivebacteria, and epothilones and their analogs are extremelyactive against many cancer cell lines. Bis-tetrahydro-furan acetogenins are also among the most active can-cer agents and compared with doxorubicin are toxic fora number of human cancer cell lines at much lowerconcentrations. In a joint effort with E. Keinan, Depart-ment of Molecular Biology, we synthesized an analogof asimicin, an annonaceous acetogenin, for photoaffinitylabeling studies of bovine mitochondrial NADH-ubiqui-none oxidoreductase (complex 1). These studies mayshed light on the structure and function of this intricateenzyme and on the origin of the high antitumor activityof annonaceous acetogenins.

P R O D R U G A N D T A R G E T I N G T H E R A P I E S

Prodrug therapy provides a unique approach thatcan be used to minimize the toxic effects of a drug.Ideally, a nontoxic prodrug selectively releases the cyto-toxic drug at an appropriate site. We are developingnew prodrugs of cytotoxic molecules, including pacli-taxel, doxorubicin analogs, enediynes, CBI analogs, andepothilones. Using an analog of dynemicin B, we syn-thesized and evaluated a number of new prodrugs(Fig. 3). We are also developing new linkers for theprodrugs, so that the activation of the prodrugs can beselectively achieved at high catalytic rate.

We synthesized a number of integrin-targeting com-pounds. These compounds are equipped with a linker,which allowed us to prepare the corresponding biotinand diketone derivatives. All these compounds bound



F i g . 1 . Dibutylboron triflate/i-Pr2NEt–mediated aldol-type reac-

tion of ketones with acetals.

F i g . 2 . Structure of sorangiolides A and B and epothilones A and B.

to cells expressing the integrins αvβ3 and αvβ5 andreacted covalently with the reactive lysine residues inthe binding site of an aldolase monoclonal antibody.The resultant conjugates have several merits, includ-ing the prolongation of half-life of the antagonist andin vivo assembly of the conjugate. We used a conjugatecomposed of the integrin-targeting compound SCS-873and the monoclonal antibody 38C2 to study the effectof the conjugate on the growth of Kaposi sarcoma andmelanoma, which express the integrins αvβ3 and αvβ5.In mice xenografted with the human cancers, comparedwith SCS-873 alone, the SCS-873–38C2 conjugatesignificantly inhibited tumor growth.

PUBLICATIONSDas, S., Li, L.-S., Sinha, S.C. Stereoselective aldol-type cyclization reaction medi-ated by dibutylboron triflate/diisopropylethylamine. Org. Lett. 6:123, 2004.

Han, H., Sinha, M.K., D’Souza, L., Keinan, E., Sinha, S.C. Total synthesis of 34-hydroxyasimicin and its photoactive derivative for affinity labeling of the mitochon-drial complex I. Chemistry 10:2149, 2004.

Li, L.-S., Das, S., Sinha, S.C. Efficient one-step aldol-type reaction of ketones withacetals and ketals mediated by dibutylboron triflate/diisopropylethylamine. Org.Lett. 6:127, 2004.

Li, L.-S., Rader, C., Matsushita, M., Das, S., Barbas C.F. III, Lerner, R.A., Sinha,S.C. Chemical adaptor immunotherapy: design, synthesis, and evaluation of novelintegrin-targeting devices. J. Med. Chem, in press.

Sinha, S.C., Li, L.-S., Dutta, S., Rader, C., Lerner, R.A. New prodrugs of dyne-micin analogs for selective chemotherapy mediated by an aldolase antibody. Proc.Natl. Acad. Sci. U. S. A. 101:3095, 2004.

Sinha, S.C., Li, L.-S., Watanabe, S.-I., Kaltgrad, E., Tanaka, F., Rader, C., Lerner,R.A., Barbas, C.F. III. Aldolase antibody activation of prodrugs of potent aldehyde-containing cytotoxics for selective chemotherapy. Chemistry, in press.

Structure, Function, andApplications of Virus ParticlesJ.E. Johnson, B. Bothner, A. Chatterji, W. Fernandez-Ochoa,

L. Gan, I. Gertsman, R. Khayat, M.-J. Kim, G. Lander,

J. Lanman, K. Lee, P. Natarajan, J. Speir, J. Tang, L. Tang,

V. Volpetti, H. Walukiewicz, E. Wu

For humans, viruses are two-edged swords. Manyof these microorganisms are dangerous pathogens(e.g., HIV and the coronavirus associated with

severe acute respiratory syndrome) that cause extraor-dinary suffering, mortality, and economic loss. How-ever, viruses benign to humans provide the means forunderstanding numerous biological processes that, byanalogy, lead to an understanding of cellular functionscrucial for the development of therapies for disease.

We investigate model virus systems that continueto provide insights for understanding the entry, local-ization, and replication of nonenveloped viruses. Morerecently, some of the benign viruses were recognizedas practical reagents for applications in nanotechnol-ogy, chemistry, and biology. Using viral capsids asreagents, we discovered novel and, in some instances,unique properties of macromolecular organizations anddynamics. We investigate viruses that infect bacteria,insects, fish, yeast, plants, and, recently, the extremethermophile Sulfolobus. These viruses have genomesof single-stranded RNA, double-stranded RNA, anddouble-stranded DNA. In many instances, we use virus-like particles that do not contain infectious genomes.

We use a variety of physical methods to investi-gate structure-function relationships, including single-crystal and static and time-resolved solution x-raydiffraction, electron cryomicroscopy and image recon-struction, mass spectrometry, structure-based compu-tational analyses, and methods associated withthermodynamic characterization of virus particles andtheir transitions. Biological methods we use includegenetic engineering of viral genes and their expressionin Escherichia coli, mammalian cells, insect cells, andyeast and the characterization of these gene productsby the physical methods mentioned previously. Forcytologic studies of viral entry and infection, we usefluorescence and electron microscopy and particlesassembled in heterologous expression systems. Ourstudies depend on extensive consultations and collab-orations with others at Scripps Research, including thegroups led by C.L. Brooks, D.A Case, B. Carragher,M.G. Finn, M.R. Ghadiri, T. Lin, M. Manchester, D.P.Millar, R.A. Milligan, C. Potter, V. Reddy, A. Schneemann,G. Siuzdak, K.F. Sullivan, and M. Yeager, and a varietyof groups outside Scripps.D O U B L E - S T R A N D E D D N A V I R U S E S

HK97 is a double-stranded DNA bacterial virussimilar to phage λ. It undergoes a remarkable morpho-genesis in its assembly and maturation, and this pro-cess can be recapitulated in vitro. The most dramaticevent is the expansion from prohead (~500 Å in size)to head (~650 Å) in which both particles have identi-



F i g . 3 . Structure of the prodrugs of an analog of dynemicin B.

cal protein composition. Four years ago, we determinedthe atomic resolution structure of the mature particleand discovered the mechanism used to concatenatethe subunits of the particle into a chain-mail fabricsimilar to that seen in the armor of medieval knights.Three years ago, we created a model of the procapsidon the basis of a 12-Å electron cryomicroscopy structureand the subunits defined in the head. We investigatedthe transition by using time-resolved intrinsic fluores-cence, circular dichroism, and solution scattering.

The data indicated that a highly pH-sensitive earlyevent (<10 minutes) is followed by a slow (hours)pH-independent late event. We also determined thekinetics of the subunit autoligation and the structureof a mutant that is not cross-linked. The structure andthe biochemical data indicate that the native quater-nary structure depends on the covalent cross-link forformation. DNA-packaging studies of bacteriophagehave progressed, with crystallographic and electroncryomicroscopy studies of the portal complex frombacteriophage P22 (Fig. 1).

Sulfolobus turreted icosahedral virus was isolatedfrom the extreme thermophile (90°C, pH 2) Sulfolobus,which grows in the sulfur springs of Yellowstone NationalPark. The virus was isolated and its double-strandedDNA genome was sequenced by our collaborator M. Young, Montana State University, Bozeman, Mon-tana. Using electron cryomicroscopy reconstruction of

the virus, we found that the capsid has pseudo T = 31quasi-symmetry and is 1000 Å in diameter, includingthe gold pentons. The trimeric nature of the hexonsstrongly indicates that this virus is related to the humanadenovirus, the PRD1 bacteriophage, and a virus thatinfects algae. Recently, we obtained crystals of theisolated subunit and a structure determination isunder way.S I N G L E - S T R A N D E D R N A V I R U S E S

Nudaurelia capensis ω virus and helicoverpa armi-gera stunt virus are single-stranded RNA viruses withT = 4 (240 subunits) capsids that infect Lepidoptera.We expressed the genes for both capsid proteins in thebaculovirus system and assembled large quantities ofparticles that exist in 2 pH-dependent morphologies.The diameter of the particle is 480 Å at pH 7 and410 Å at pH 5. Electron cryomicroscopy reconstruc-tions were done on both forms and on 2 intermediatestructures for nudaurelia capensis ω virus. We foundthat mutations in the internal helical domain dramat-ically affect assembly and morphology. A pH-depen-dent helix-coil transition may be the driving force forthe particle dynamics. The particles are nanomachineswith potential as sensors and as “engines” for drivingother nanodevices.

Cowpea mosaic virus is a pH-driven reagent forchemistry and nanotechnology. In collaboration with T. Lin, Department of Molecular Biology, and M.G. Finn,Department of Chemistry, we generated and produceda large variety of viable mutations of the virus in gramquantities for nanopatterning, molecular electronicscaffolds, and platforms for novel chemistry.

PUBLICATIONSBrumfield, S., Willits, D., Tang, L., Johnson, J.E., Douglas, T., Young, M.J. Het-erologous expression of the modified coat protein of cowpea chlorotic mottle bro-movirus results in the assembly of protein cages with altered architectures andfunction. J. Gen. Virol. 85(Pt. 4):1049, 2004.

Chatterji, A., Ochoa, W., Paine, M., Ratna, B., Johnson, J.E., Lin, T. Newaddresses on an addressable virus nanoblock: uniquely reactive Lys residues oncowpea mosaic virus. Chem. Biol., in press.

Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S.P., Wong, S.M., Clinton, G.,Ghosh, P., Lin, T., Johnson, J.E. Chemical conjugation of heterologous proteins onthe surface of cowpea mosaic virus. Bioconjug. Chem. 15:807, 2004.

Fourme, R., Ascone, I., Kahn, R., Girard, E., Mezouar, M., Lin, T., Johnson, J.E.New trends in macromolecular crystallography at high hydrostatic pressure. In:Advances in High Pressure Bioscience and Biotechnology II: Proceedings of the 2ndInternational Conference on High Pressure Bioscience and Biotechnology, Dortmund,September 16-19, 2002. Winter, R. (Ed.). Springer Verlag, New York, 2003, p. 161.

Fourme, R., Girard, E., Kahn, R., Ascone, I., Mezouar, M., Dhaussy, A.C., Lin, T.,Johnson, J.E. Using a quasi-parallel x-ray beam of ultrashort wavelength for highpressure virus crystallography: implications for standard macromolecular crystallog-raphy. Acta Crystallogr. D Biol. Crystallogr. 59(Pt.10):1767, 2003.



F i g . 1 . The structure of the portal complex from bacteriophage

P22 at 8-Å resolution by electron cryomicroscopy. Double-stranded

DNA bacteriophages and herpesviruses use similar mechanisms for

DNA packaging. Central to this packaging is the portal complex

located at a unique vertex of the virus, where it serves as a channel

through which DNA enters. The P22 portal complex is the largest one

reported so far, a dodecameric ring with a mass of approximately 1

million daltons.

Fourme, R., Girard, E., Kahn, R., Ascone, I., Mezouar, M., Lin, T., Johnson, J.E.State of the art and prospects of macromolecular crystallography at high hydrosta-tic pressure. In: High Pressure Crystallography. Katrusiak, A., McMillan, P. (Eds.).Kluwer Academic, Dordrecht, the Netherlands, 2004, p. 527. NATO ScienceSeries, II: Mathematics, Physics, and Chemistry, Vol. 140.

Gan, L., Conway, J.F., Firek, B.A., Cheng, N., Hendrix, R.W., Steven, A.C., John-son, J.E., Duda, R.L. Control of crosslinking by quaternary structure changes dur-ing bacteriophage HK97 maturation. Mol. Cell 14:559, 2004.

Helgstrand, C., Munshi, S., Johnson, J.E., Liljas, L. The refined structure ofnudaurelia capensis ω virus reveals control elements for a T = 4 capsid matura-tion. Virology 318:192, 2004.

Helgstrand, C., Wikoff, W.R., Duda, R.L., Hendrix, R.W., Johnson, J.E., Liljas, L.The refined structure of a protein catenane: the HK97 bacteriophage capsid at3.44 Å resolution. J. Mol. Biol. 334:885, 2003.

Johnson, J.E. An atomic model of a plant reovirus: rice dwarf virus. Structure(Camb.) 11:1193, 2003.

Johnson, J.E. Virus assembly and maturation. In: Folding and Self-Assembly ofBiological Macromolecules: Proceedings of the Deuxièmes Entretiens de Bures,Bures-sur-Yvette, France, 27 November-1 December 2001. Westhoff, E., Hardy, N. (Eds.). World Scientific, Hackensack, NJ, 2003, p. 349.

Johnson, J.E. Virus particle dynamics. Adv. Protein Chem. 64:197, 2003.

Johnson, J.E. Virus structure analysis with synchrotron radiation: methods andresults. J. Synchrotron Radiat. 11(Pt. 1):89, 2004.

Lee, K.K., Gan, L., Tsuruta, H., Hendrix, R.W., Duda, R.L., Johnson, J.E. Evi-dence that a local refolding event triggers maturation of HK97 bacteriophage cap-sid. J. Mol. Biol. 340:419, 2004.

Lee, K.K., Johnson, J.E. Complementary approaches to structure determination oficosahedral viruses. Curr. Opin. Struct. Biol. 13:558, 2003.

Lee, K.K., Tang, J., Taylor, D., Bothner, B., Johnson, J.E. Small compounds tar-geted to subunit interfaces arrest maturation in a nonenveloped, icosahedral animalvirus. J. Virol. 78:7208, 2004.

Lin, T., Cavarelli, J., Johnson, J.E. Evidence for assembly-dependent folding ofprotein and RNA in an icosahedral virus. Virology 314:26, 2003.

Lin, T., Johnson, J.E. Structures of picorna-like plant viruses: implications andapplications. Adv. Virus Res. 62:167, 2003.

Raja, K.S., Wang, Q., Gonzalez, M.J., Manchester, M., Johnson, J.E., Finn, M.G.Hybrid virus-polymer materials, 1: synthesis and properties of PEG-decorated cow-pea mosaic virus. Biomacromolecules 4:472, 2003.

Reddy, V., Schneemann, A., Johnson, J.E. Nodavirus endopeptidase. In: Hand-book of Proteolytic Enzymes, 2nd ed. Barret, A., Rawlings, N., Woessner, J. (Eds.).Academic Press, San Diego, in press.

Rice, G., Tang, L., Stedman, K., Roberto, F., Spuhler, J., Gillitzer, E., Johnson,J.E., Douglas, T., Young, M.J. The structure of a thermophilic archeal virus showsa doubled-stranded DNA viral capsid type that spans all domains of life. Proc.Natl. Acad. Sci. U. S. A. 101:7716, 2004.

Taylor, D.J., Wang, Q., Bothner, B., Natarajan, P., Finn, M.G., Johnson, J.E. Cor-relation of chemical reactivity of nudaurelia capensis ω virus with a pH-inducedconformational change. Chem. Commun. (Camb.) 2770, 2003, Issue 22.

Tihova, M., Dryden, K.A., Le, T.V., Harvey, S.C., Johnson, J.E., Yeager, M.,Schneemann, A. Nodavirus coat protein imposes dodecahedral RNA structureindependent of nucleotide sequence and length. J. Virol. 78:2897, 2004.

Wikoff, W.R., Che, Z., Duda, R.L., Hendrix, R.W., Johnson, J.E. Crystallizationand preliminary analysis of a dsDNA bacteriophage capsid intermediate: prohead IIof HK97. Acta Crystallogr. D Biol. Crystallogr. 59(Pt. 12):2060, 2003.

Yin, Z., Zheng, Y., Doerschuk, P.C., Natarajan, P., Johnson, J.E. A statistical approachto computer processing of cryo-electron microscope images: virion classificationand 3-D reconstruction. J. Struct. Biol. 144:24, 2003.

Structure-Based Engineering of an Icosahedral Virus forNanomedicine andNanotechnologyT. Lin, J.E. Johnson, A. Chatterji, W.F. Ochoa, V. Volpetti, T. Ueno

Cowpea mosaic virus (CPMV) is a picorna-like plantvirus with a diameter of 30 nm. For structuralstudies of the virus, we use x-ray crystallography,

electron microscopy, and molecular virology. Because ofits exceptional stability, high yield, ease of production,and accessible genetic programmability, CPMV can beengineered for biotechnology and nanotechnology.F U N C T I O N A L I Z A T I O N O F C P M V F O R

N A N O T E C H N O L O G Y

Previously, we showed that CPMV can be used asa template for nanochemistry and nanomaterials byintroducing unique cysteine residues and exploitingthe native lysine residues. We showed the specificityof the reactivity by attaching nano-sized gold parti-cles. Structural characterization of the gold conjugatesindicated that the environment dictated the mode ofligand presentation. Gold particles attached in the val-ley-like structure on the viral surface were associatedwith refined electron density, whereas those in openand flat areas adopted more flexible structures. Pat-terning of CPMV-gold conjugates on a mica surface bydrop-and-dry methods and imaging via atomic forcemicroscopy revealed that the geometric features of thevirus particles dominated the formation of arrays andthat the individual particles interacted with the sur-face along either the 5- or the 3-fold axis.D E V E L O P M E N T O F A P L A N T V I R U S A S A D E L I V E R Y

V E H I C L E F O R T H E R A P E U T I C A G E N T S

We are using polyvalent presentation of exogenousproteins on the surface of CPMV to enhance biologicalfunctions. Conjugation of the viral capsid with inter-nalin B, a surface protein on bacteria that facilitatesentry of the microorganisms into mammalian cells, andherstatin, a cancer inhibitor that recognizes HER2, acancer-related cell-surface receptor, led to the targetingof CPMV to mammalian cells. This targeting illustratesthe feasibility of using a plant virus as a nanocapsulefor delivery of therapeutic agents. Efforts are beingmade to load different cargos in the capsid interior fortargeted delivery.



To increase the loading capacity, we excavated thenative viral RNA molecules packaged in the capsid inte-rior by using an alkaline treatment. The synthetic emptyparticles were associated with an enhanced capacityin loading exogenous materials. Characterization of theempty particles by electron microscopy revealed notonly the hollow interior due to the degradation of thepackaged RNA but also the opening of a port alongthe capsid 5-fold axis, through which fragmented RNAmight exit (Fig. 1). Studies are under way to investi-gate the control of the opening and the type of cargothat can be loaded.

PUBLICATIONSBlum, A.S., Soto, C.M., Wilson, C.D., Cole, J.D., Kim, M., Gnade, B., Chatterji,A., Ochoa, W.F., Lin, T., Johnson, J.E., Ratna, B.R. Cowpea mosaic virus as ascaffold for 3-D patterning of gold nanoparticles. Nano Lett. 4:867, 2004.

Fourme, R., Girard, E., Kahn, R., Ascone, I., Mezouar, M., Dhaussy, A.-C., Lin,T., Johnson, J.E. Using a quasi-parallel x-ray beam of ultrashort wavelength forhigh-pressure virus crystallography: implications for standard macromolecular crys-tallography. Acta Crystallogr. D Biol. Crystallogr. 59(Pt. 10):1767, 2003.

Lin, T., Cavarelii, J., Johnson, J.E. Evidence for assembly-dependent folding ofprotein and RNA in an icosahedral virus. Virology 314:26, 2003.

Lin, T., Johnson, J.E. Structures of picorna-like plant viruses: implications andapplications. Adv. Virus Res. 62:167, 2003.

Protein Design and Informaticsin Structural VirologyV.S. Reddy, C.M. Shepherd, C.L. Brooks, III, J.E. Johnson,

M. Manchester, A. Schneemann

Viruses are highly evolved macromolecularmachines that perform a variety of functionsduring their life cycle. Simple viruses, such as

nonenveloped viruses, form closed protein shells orcapsids of uniform size and character by the self-asso-ciation of structural and functional components. How-ever, under different physicochemical conditions (e.g.,pH and ionic strength), these capsids undergo con-certed structural changes that may be suggestive ofcapsid dynamics that occur during the viral life cycle:assembly, maturation, and infection.

We use structural, computational, and geneticapproaches to understand the structure-function rela-tionships of viral capsids and to design new and novelcapsids. To reach this goal, we develop structure-basedcomputational tools to analyze the existing capsid struc-tures in terms of protein-protein interactions and sur-face characteristics. With these tools and geneticapproaches, we can use viral capsids as the platformsto display foreign epitopes of interest. Such reagentsare being used as potential vaccines and antitoxins.

To facilitate the systematic and global analysis ofvirus structures, we continue to maintain the VirusParticle Explorer Web site (http://mmtsb.scripps.edu/viper) as a repository of viral structures. Currently, weare using MySQL software to develop a virus structuredatabase that would, in future, act as the engine behindthe Web site. Such a database would also provide quickaccess to and comparison of properties between groupsof viruses.

Recently, we developed a new measure of quanti-fying protein-protein interactions, the protein-proteininteraction (PPI) index, as a ratio of solvent-accessiblesurface area of a subunit in the context of a capsidassembly to the isolated subunit. Calculation of thisindex for all the quasi-equivalent capids (T ≥ 3) sug-gested that the normalized extent of protein-proteininteractions is constant (PPI = ~0.45) across all thecapsids, independent of the subunit molecular weightand capsid diameter. However, there is a linear depen-dence of the PPI index on the subunit molecular weightin T = 1 capsids. These results suggest that althoughthe information to form a particular type of capsid isbuilt into the coat protein subunit, polymorphism ofcapsids could be achieved by altering the interactionbetween these subunit building blocks.

We are also involved in designing suitable viralplatforms for the display of complete or partial proteindomains from pathogenic organisms that potentially canbe used as vaccines and antitoxins. Currently, we areusing tomato bushy stunt virus as a display platformto create decoys of pathogenic agents.



F i g . 1 . Top, Surface renderings of the native CPMV (left) and the

empty CPMV (right) show that a pentameric port is open. Bottom,

Schematic diagram shows the opening of the port along the viral

5-fold axis.

PUBLICATIONSRao, A.L.N., Reddy V.S. Architecture of plant viruses. In: Handbook of Plant Virol-ogy. Khan, J.D., Dijkstra, J. (Eds.). Haworth Press, Binghamton, NY, in press.

Reddy, V.S., Johnson J.E. Structure derived insights into virus assembly. Adv. VirusRes., in press.

Reddy, V.S., Natarajan, P., Lander, G., Qu, C., Brooks, C.L. III, Johnson, J.E.Virus Particle Explorer (VIPER): a repository of virus capsid structures. In: Confor-mational Proteomics of Macromolecular Architecture: Approaching the Structure ofLarge Molecular Assemblies and Their Mechanisms of Action. Cheng, R.H., Ham-mer, L. (Eds.). World Scientific, Hackensack, NJ, 2004, p. 403.

Capsid Biology of Icosahedral Viruses

A. Schneemann, A. Venter, H. Lago, J. Lee, D.J. Manayani

Coat proteins of nonenveloped, icosahedral virusesperform multiple functions during the course ofviral infection, including assembly of the viral

capsid, specific encapsidation of the viral genome, bind-ing to a cellular receptor, and uncoating. In some viruses,a single type of protein is sufficient to carry out thesefunctions; we are interested in the determinants thatendow a polypeptide chain with such versatility. Wefocus on a structurally and genetically well-character-ized model system, the T = 3 nodaviruses.

Nodaviruses are composed of 180 copies of a sin-gle coat protein and 2 strands of positive-sense RNA.To acquire infectivity, particles must first mature byautocatalytic cleavage of the coat subunits into 2 poly-peptides. We are investigating the precise pathway ofviral assembly and the mechanism of the associatedcleavage reaction. Our studies are guided by the high-resolution structures of several nodaviruses that weredetermined in the laboratory of J.E. Johnson, Depart-ment of Molecular Biology.

Analysis of the atomic structures of these virusesenabled us to identify regions in the coat protein thatappear to be critical in regulating viral assembly, sta-bility, and maturation. Using genetic, biochemical, andbiophysical approaches, we showed that the N-termi-nal part of the coat protein plays a role in regulatingthe geometry of virus particles and in recognizing theviral RNAs for encapsidation. Another region requiredfor specific encapsidation of nodaviral RNAs is locatedat the C terminus of the capsid protein.

Currently, we are elucidating the mechanism bywhich the 2 genomic RNAs are packaged into a singlevirion. Our data indicate that the 2 RNAs are recog-nized separately, but it is not yet known whether pack-

aging occurs sequentially and whether one or more coatprotein subunits are involved in this process.

Another project we are involved in is a multiscaleeffort by several collaborators at Scripps Research todevelop nonenveloped, icosahedral plant and insectviruses as biomolecular sensors for targeting cancer.The high-resolution x-ray structure of the nodavirusflock house virus (FHV) revealed the potential for usingchemical approaches to attach ligands to the viral sur-face and genetic strategies to modify the capsids. Theparticles are stable, easily manipulated, biocompati-ble, and nontoxic in vivo and can be produced easilyand in high quantities. The project is divided into sev-eral parts.

The first part is display or attachment of differenttumor-targeting moieties on the surface of the virion,for example, a tumor-binding peptide, a tumor-specificantibody, or a tumor antigen–specific T-cell receptor.These moieties will be displayed by incorporating theircoding sequences into the coding sequence of the FHVcoat protein or by direct chemical conjugation that takesadvantage of strategically placed reactive lysine and/orcysteine residues on the viral surface. Direct chemicalconjugation has many advantages because it circum-vents genetic instability of viral clones, inability ofmodified genomes to replicate efficiently, and interfer-ence with viral assembly due to insertion of foreignsequences. The modified particles are characterizedstructurally, and their ability to bind to cells displayingthe respective tumor antigen is tested. In a parallelapproach, FHV is being developed as a gene deliveryvehicle. Because of our knowledge of FHV assemblyand RNA encapsidation, we are in a powerful positionto engineer the capsid protein so that packaging ofnucleic acid can be manipulated in a systematic manner.

Last, we are developing FHV as a tumor-imagingagent. Virus particles are loaded with gadolinium ionsby using certain chelators, the particles are injectedinto small animals with tumors, and then magneticresonance imaging is done at Loma Linda University,Loma Linda, California. Using virus particles that tar-get cells of interest, we hope to be able to image tumorswith greater specificity than is currently possible.

PUBLICATIONSTihova, M., Dryden, K., Le, T.L., Harvey, S.C., Johnson, J.E., Yeager, M., Schnee-mann, A. Nodavirus coat protein imposes dodecahedral RNA structure independentof nucleotide sequence and length. J. Virol. 78:2897, 2004.



Molecular Biology of RetrovirusesJ.H. Elder, A.P. de Parseval, U. Chatterji, Y.-C. Lin, S. de Rozieres, M.D. Carlson, B.E. Torbett*

* Department of Molecular and Experimental Medicine, Scripps Research

Our research centers on molecular characteriza-tion of retroviruses, with emphasis on felineimmunodeficiency virus (FIV). FIV causes an

AIDS-like syndrome in domestic cats, and although itdoes not infect humans, the feline retrovirus has manystructural and functional similarities to HIV, the causa-tive agent of AIDS in humans. Thus, study of FIV canyield insights into ways to interfere with the retroviruslife cycle that may ultimately result in the developmentof treatments for infections in both cats and humans.During the past year, we continued to focus on 2 majorareas: receptor studies and development of inhibitorsagainst the aspartic protease encoded by FIV.R E C E P T O R S T U D I E S

The surface glycoprotein (gp95) of FIV binds toseveral cell-surface molecules, including CXCR4; hep-aran sulfate proteoglycans; DC-SIGN, a specific C-typelectin expressed on dendritic cells; and a 43-kD cell-surface receptor on T cells recently identified as CD134.The first 3 receptor molecules are also used by HIV,but the human virus uses CD4 instead of CD134 as aprimary binding receptor. CXCR4 is the receptor thatactually facilitates FIV entry into the cell in all knowncases, and the other molecules act as binding receptors.

We confirmed and extended the finding that CD134is a primary receptor for FIV, and we showed that tem-perature critically influences the binding properties ofFIV gp95 to CXCR4. Our results indicate that factorsthat increase the effective concentration of CXCR4enhance FIV infectivity and may involve (1) tempera-ture- or ligand-induced conformational changes inCXCR4 that enhance gp95 binding, (2) interactionsbetween coreceptors and gp95 that either alter gp95conformation to enhance CXCR4 binding or increasethe localized concentration of receptor or ligand, or(3) a direct increase in the concentration of CXCR4via overexpression.

HIV uses CD4 as a primary binding receptor to infectand kill CD4+ T cells, eventually leading to immuno-deficiency and death of the host via opportunisticinfections. An apparent paradox of FIV infections isthat the feline retrovirus also primarily infects and killsCD4+ T cells, but unlike HIV, it does not bind to CD4.As indicated earlier, the FIV surface glycoprotein spe-

cifically binds to a 43-kD glycoprotein, now identifiedas CD134, on the surface of at least one subset of T cells in peripheral blood mononuclear cells. We showedthat the FIV glycoprotein binds to CD134 and thatthis receptor is specifically upregulated on activatedfeline CD4+ T cells. The results help explain the spe-cific depletion of the CD4+ T-cell population from theperipheral blood of infected animals, independent ofthe use of CD4 as a coreceptor.P R O T E A S E I N H I B I T O R S T U D I E S

We previously reported the development of a broad-based protease inhibitor, termed TL-3. This uniquemolecule inhibits replication of both FIV and HIV, aswell as replication of many HIV drug-resistant strainsthat have arisen as a consequence of treating patientswith other protease inhibitors. This inhibitor is the firstone that can effectively block FIV replication despite amarked similarity in the overall 3-dimensional struc-tures of HIV and FIV proteases. We thus wished todetermine if TL-3 would be effective in treating FIVinfections in cats.

In vivo tests were performed to assess the influenceof TL-3 on FIV-induced CNS deficits. A total of 20 catswere divided into 4 groups of 5 animals each. Group1 received no treatment; group 2 received TL-3 only;group 3 received the PPR strain of FIV only; and group 4received the PPR strain of FIV and TL-3. Animals weremonitored for immunologic and virologic status, andchanges in brain stem auditory evoked potentials (BAEPs)were measured.

Groups 1 and 2 remained FIV negative; groups 3and 4 became FIV positive and seroconverted by 3–5weeks after inoculation. No adverse effects were notedin the cats given TL-3 only. The mean peak viral loadswere 1.32 x 106 RNA copies/mL for the virus-onlygroup 3 animals and 6.9 x 104 copies/mL for TL-3–treated group 4 cats. Cats in group 3 had marked pro-gressive delays in BAEPs starting at 2 weeks afterexposure to FIV, typical of infection with the PPR strain.In contrast, the cats in group 4 (virus plus TL-3) hadBAEPs similar to the cats in groups 1 and 2.

At 97 days after infection, treatments were switched;treatment with TL-3 was stopped in the cats in group 4and started in the cats in group 3. BAEPs in the group 3animals returned to control levels, and BAEPs in group 4animals remained at control levels. After 70 days,treatment with TL-3 was stopped in group 3. Delays inBAEPs immediately increased to levels observed beforeTL-3 treatment.



The findings indicate that early treatment with TL-3can effectively eliminate FIV-induced changes in the CNS.Furthermore, TL-3 can counteract the effects of FIV onthe CNS of infected cats, although continued treatment isrequired to maintain unimpaired CNS function.

PUBLICATIONSBrik, A., Muldoon, J., Lin, Y.-C., Elder, J.H., Goodsell, D.S., Olson, A.J., Fokin, V.V.,Sharpless, K.B., Wong, C.-H. Rapid diversity-oriented synthesis in microtiter plates forin situ screening of HIV protease inhibitors. Chembiochem 4:1246, 2003.

de Parseval, A., Ngo, S., Sun, P., Elder, J.H. Factors that increase the effectiveconcentration of CXCR4 dictate feline immunodeficiency virus tropism and kineticsof replication. J. Virol. 78:9132, 2004.

de Parseval, A., Su, S.V., Elder, J.H., Lee, B. Specific interaction of feline immunode-ficiency virus surface glycoprotein with human DC-SIGN. J. Virol. 78:2597, 2004.

de Rozieres, S., Mathiason, C.K., Rolston, M.R., Chatterji, U., Hoover, E.A., Elder, J.H.Characterization of a highly pathogenic molecular clone of feline immunodeficiency virusclade C. J. Virol. 78:8971, 2004.

Huitrón-Reséndiz, S., de Rozieres, S., Sanchez-Alavez, M., Bühler, B., Lin, Y.-C.,Lerner, D.L., Henriksen, N.W., Burudi, M., Fox, H.S., Torbett, B.E., Henriksen, S.,Elder, J.H. Resolution and prevention of feline immunodeficiency virus-induced neuro-logical deficits by treatment with the protease inhibitor TL-3. J. Virol. 78:4525, 2004.

Kutilek, V.D., Sheeter, D.A., Elder, J.H., Torbett, B.E. Is resistance futile? Curr.Drug Targets Infect. Disord. 3:295, 2003.

Lin, Y.-C., Beck, Z., Morris, G.M., Olson, A.J., Elder, J.H. Structural basis for dis-tinctions between substrate and inhibitor specificities for feline immunodeficiencyvirus and human immunodeficiency virus proteases. J. Virol. 77:6589, 2003.

Mak, C.C., Brik, A., Lerner, D.L., Elder, J.H., Morris, G.M., Olson, A.J., Wong,C.H. Design and synthesis of broad-based mono- and bi-cyclic inhibitors of FIV andHIV proteases. Bioorg. Med. Chem. 11:2025, 2003.

Metalloenzyme EngineeringD.B. Goodin, C.D. Stout, A.J. Olson, J. Rebek, Jr.,

A.E. Pond, A.-M.A. Hays, S. Vetter, L. Kröck, H.B. Gray,*

J.R. Winkler,* J.H. Dawson,** T.L. Poulos***

* California Institute of Technology, Pasadena, California

** University of South Carolina, Columbia, South Carolina

*** University of California, Irvine, California

Our overall goals are to understand the funda-mental protein structural features of metalloen-zyme catalysts and to create catalysts for useful

chemical reactions. We use a number of techniques instructural biology and spectroscopy and strategies ofrational protein redesign and molecular evolution. In thepast year, we made progress in several of these areas.

The ubiquitous cytochrome P450 family catalyzesa vast range of biologically important reactions in mam-mals, plants, fungi, and bacteria. Despite several recentadvances in the structural characterization of thesesystems, the molecular mechanism by which theseenzymes control the promiscuity or specificity of sub-

strate binding and how they achieve regioselective andstereoselective catalysis remains elusive. In a collabo-ration with H.B. Gray, California Institute of Technology,Pasadena, California, we determined the crystal struc-tures of cytochrome P450cam complexed with 2 sensi-tizer-linked substrate probes (Fig. 1). These probesinduce specific conformational responses in the proteinstructure that suggest the nature of the dynamic inter-mediates that must exist transiently in solution duringsubstrate entry and product egress. They also providea view of the complementary nature of substrate inter-actions induced by pliability of the P450 active-sitestructure. These conformational variants show how sub-strate recognition, by elements of the F and G helixregions, is intimately coupled to the active site througha conformational switch. This coordinated proteinrearrangement creates new solvent sites within thesubstrate-binding channel, which may modulate thecatalytic activity and allow P450cam to bind a multi-tude of substrates, ligands, and inhibitors.

In a separate study, we developed a model systemfor using artificially created cavities in a protein struc-ture to introduce specific protein-ligand interactions. Aset of cationic heterocycles was previously shown tobind in the buried, solvent-filled pocket created by theW191G mutation of cytochrome c peroxidase. Wecompleted docking studies of these ligands into thiscavity. Predicted binding energies differed from mea-sured values by ±0.8 kcal/mol. For most ligands, thedocking simulation clearly predicted a single bindingmode that closely matched the crystallographic struc-ture. For 2 ligands, docking indicated multiple bindingmodes. This finding was consistent with ambiguities



F i g . 1 . Ribbon diagram of P450cam containing a sensitizer-

linked substrate. The ligand induces movements in the F, G, and

I helices. These changes reveal conformational states associated

with substrate promiscuity and a conformational modulation of

critical active-site residues.

in the observed electron density of the 2 ligands, sug-gesting disordered binding of these ligands. Ligandscould be distinguished, to some extent, from nonbindersby using a combination of predicted binding energiesand the clustering of predicted docking conformations.These studies and others now under way illustrate theapplication of these artificial ligand-receptor sites asuseful benchmarks for the calibration and refinementof ligand-protein docking tools used in drug discovery.

We made significant progress in our efforts toremove and replace the native electron-transfer path-way of cytochrome c peroxidase. Previous engineeringstudies resulted in the precise excision of the electron-transfer pathway. This past year we determined thecrystal structure of an artificial peptide within this chan-nel that accurately replaces the native electron-trans-fer pathway. Measurements of electron transfer of theseinstalled “molecular wires” are under way and will bedirected toward rapidly initiating reactions, trappingreactive intermediates, and answering fundamentalquestions about electron-transfer pathways in proteins.

Finally, we collaborated with J. Rebek, The SkaggsInstitute for Chemical Biology, in a study in which elec-tron paramagnetic resonance spectroscopy and spinlabeling were used to characterize weak host-guestinteractions in a class of ligand-binding resorcinarenes.Examples of these compounds were known to bindsmall ligands in the crystalline state, but no formationof complexes could be detected in solution by usingnuclear magnetic resonance. By placing 4 spin-labelgroups on the resorcinarene, we showed that electronparamagnetic resonance spectroscopy could be usedto monitor small changes in dipolar interactions asso-ciated with ligand binding.

PUBLICATIONSKröck, L., Shivanyuk, A., Goodin, D.B., Rebek, J., Jr. Spin labeling monitors weakhost-guest interactions. Chem. Commun. (Camb.) 272, 2004, Issue 3.

Rosenfeld, R.J., Goodsell, D.S., Musah, R.A., Morris, G.M., Goodin, D.B., Olson,A.J. Automated docking of ligands to an artificial active site: augmenting crystallo-graphic analysis with computer modeling. J. Comput. Aided Mol. Des. 17:525, 2003.

Control of Cell DivisionS.I. Reed, C. Baskerville, L.-C. Chuang, S. Ekholm-Reed,

B. Grunenfelder, M. Henze, J. Keck, V. Liberal, K. Luo,

B. Olson, S. Rudyak, O. Sangfelt, A. Smith, C. Spruck,

D. Tedesco, F. van Drogen, J. Wohlschlegel, V. Yu

Biological processes of great complexity can beapproached by beginning with a systematicgenetic analysis in which the relevant compo-

nents are first identified and the consequences of theirselective elimination by mutation are investigated. Weuse yeast, which is uniquely tractable to this type ofanalysis, to investigate control of cell division. In recentyears, it has become apparent that the most centralcellular processes throughout the eukaryotic phylogenyare highly conserved in terms of both the regulatorymechanisms used and the proteins involved. Thus, ithas been possible in many instances to generalize fromyeast cells to human cells.C O N T R O L I N Y E A S T

Recently, we focused on the role and regulation ofthe Cdc28 protein kinase (Cdk1). Initially identified bymeans of a mutational analysis of the yeast cell cycle,this protein kinase and its analogs are ubiquitous ineukaryotic cells and are central to a number of aspectsof control of cell-cycle progression.

One current area of interest is regulation of cellularmorphogenesis by Cdk1. The activity of Cdk1 drivenby mitotic cyclins modulates polarized growth in yeastcells. Specifically, these activities depolarize growth byaltering the actin cytoskeleton. Because many of theproteins required for polarized growth have been iden-tified in yeast, these microorganisms are an ideal sys-tem for investigating the role of Cdks in morphogenesis.

A second major area of interest is the regulation ofmitosis. A key aspect of mitotic regulation in yeast isthe accumulation of Cdc20, which triggers the transi-tion from metaphase to anaphase. Cdc20 is an essen-tial cofactor of the protein-ubiquitin ligase known asthe anaphase-promoting complex or APC/C. It is throughthe ubiquitin-mediated proteolysis of a specific ana-phase inhibitor, securin (Pds1 in yeast), that anaphaseis initiated. We are investigating the role of Cdk1 andassociated cyclins in the transcription of CDC20, thegene for Cdc20, and the regulation of Cdc20 by prote-olysis. Previously, we showed that cells are preventedfrom entering mitosis when DNA replication is blockedby the drug hydroxyurea, which causes the destabi-lization and resultant downregulation of Cdc20.



While investigating mitosis, we found that Cks1, asmall Cdk1-associated protein, appears to regulate pro-teasomes. Proteasomes are complex proteases thattarget ubiquitylated proteins, including important cell-cycle regulatory proteins. Surprisingly, we found thatCks1 regulates a nonproteolytic function of proteasomes,the transcriptional activation of Cdc20. Specifically,Cks1 is required to recruit proteasomes to CDC20 forefficient transcriptional elongation. Our investigationsof CDC20 led to the conclusion that Cks1 is requiredfor recruitment of proteasomes to and transcriptionalelongation of many other genes as well. Currently, weare elucidating the mechanism whereby Cks1 recruitsproteasomes and facilitates transcriptional elongation.C O N T R O L I N M A M M A L I A N C E L L S

We showed previously that the human homologsof the Cdc28 protein kinase are so highly conserved,structurally and functionally, relative to the yeast pro-tein kinase that they can function and be regulatedproperly in a yeast cell. Analyzing control of the cellcycle in mammalian cells, we produced evidence forthe existence of regulatory schemes, similar to thoseelucidated in yeast, that use networks of both positiveand negative regulators.

A principal research focus is the positive regulatorof Cdk2, cyclin E. Cyclin E is often overexpressed and/orderegulated in human cancers. Using a tissue culturemodel, we showed that deregulation of cyclin E con-fers genomic instability, probably explaining the link tocarcinogenesis. The observation that deregulation ofcyclin E confers genomic instability led us to hypothe-size a mechanism of cyclin E–mediated carcinogenesisbased on accelerated loss of heterozygosity at tumorsuppressor loci. We are testing this hypothesis in trans-genic mouse models. We showed previously that acyclin E transgene expressed in the mammary epithe-lium significantly increases loss of heterozygosity atthe p53 locus, leading to enhanced mammary carcino-genesis. We are extending these investigations byusing mouse prostate, testis, and skin models.

In an attempt to understand cyclin E–mediated geno-mic instability, we are investigating how deregulationof cyclin E affects both S phase and mitosis. Our recentdata suggest that deregulation of cyclin E impairs DNAreplication by interfering with assembly of the prerepli-cation complex. Cyclin E deregulation also impairs thetransition from metaphase to anaphase.

Our interest in cyclin E deregulation in cancer ledus to examine the pathway for turnover of cyclin E. We

showed that phosphorylation-dependent proteolysis ofcyclin E depends on a protein-ubiquitin ligase knownas SCFhCdc4. The F-box protein hCdc4 is the specific-ity factor that targets phosphorylated cyclin E.

Because of its functional relationship to cyclin E,we are studying the role of mutations in hCDC4, thegene that encodes hCdc4, in carcinogenesis. We foundthat hCDC4 is mutated and most likely is a tumor sup-pressor in endometrial cancer and breast cancer. Inendometrial cancer, tumors with mutations in hCDC4are more aggressive than those without such mutations.Because we showed that loss of hCdc4 leads to dereg-ulation of cyclin E through the cell cycle, these resultsconfirm the observation that in some cancers at leastderegulation of cyclin E is associated with aggressivedisease and poor outcome.

Another area of interest is the regulation and func-tion of the pRb-related protein p130. This protein is atranscriptional repressor as well as a Cdk2 inhibitor.Unlike pRb, p130 undergoes cell cycle–dependent pro-teolysis, important in regulation of Cdk2 activity. Weshowed that turnover of p130 is mediated by phos-phorylation by Cdk4 and subsequent ubiquitylationby SCFSkp2.

A major area of interest is the role of Cks proteinsin mammals, complementing our research in yeast.Mammals express 2 orthologs of yeast Cks1, knownas Cks1 and Cks2. Experiments in mice lacking thegene for Cks1 and Cks2 revealed that each orthologhas a specialized function. Cks1 is required as a cofac-tor for Skp2-mediated ubiquitylation and turnover ofinhibitors p27 and p130. Cks2 is required for thetransition from metaphase to anaphase in both maleand female meiosis I. Nevertheless, mice nullizygousat the individual loci are viable. However, doubly nul-lizygous mice have not been observed, consistent withan essential redundant function. We are using bothmouse genetic approaches and interfering RNA to elu-cidate this function.

PUBLICATIONSDonovan, P.J., Reed, S.I. Germline exclusion of Cks1 in the mouse reveals a metaphaseI role for Cks proteins in male and female meiosis. Cell Cycle 2:275, 2003.

Ekholm-Reed, S., Mendez, J., Tedesco, D., Zetterberg, A., Stillman, B., Reed,S.I. Deregulation of cyclin E in human cells interferes with prereplication complexassembly. J. Cell Biol. 165:789, 2004.

Ekholm-Reed, S., Spruck, C.H., Sangfelt. O., van Drogen, F., Mueller-Holzner, E.,Widschwendter, M., Zetterberg, A., Reed, S.I. Mutation of hCDC4 leads to cellcycle deregulation of cyclin E in cancer [published correction appears in CancerRes. 64:2939, 2004]. Cancer Res. 64:795, 2004.



Hubalek, M.M., Widschwendter, A., Erdel, M., Gschwendtner, A., Fiegl, H.M.,Muller, H.M., Goebel, G., Mueller-Holzner, E., Marth, C., Spruck, C.H., Reed,S.I., Widschwendter, M. Cyclin E dysregulation and chromosomal instability inendometrial cancer. Oncogene 23:4187, 2004.

Reed, S.I. Ratchets and clocks: the cell cycle, ubiquitylation and protein turnover,Nat. Rev. Mol. Cell Biol. 4:855, 2003.

Regulating Cell Proliferation:Flipping Transcriptional andProteolytic SwitchesC. Wittenberg, M. Ashe, R. de Bruin, M. Guaderrama,

T. Kalashnikova, T. Kesti, N. Spielewoy

Cell proliferation is governed primarily by control-ling the activities of positive and negative regu-lators of cell-cycle transitions. Inhibitors of

cyclin-dependent protein kinases (CDKs) and the posi-tive regulatory subunits, cyclins, are critical in estab-lishing the proper timing of cell-cycle transitions andin imposing cell-cycle checkpoints. The activities ofthose proteins are largely regulated via periodic tran-scriptional activation coupled with regulated proteoly-sis. We focus primarily on those regulatory mechanisms.

As in animal cells, initiation of the cell cycle inthe budding yeast Saccharomyces cerevisiae occursduring late G1 phase and is governed by the controlledaccumulation of G1 CDK activity. A large family of G1-specific genes, including those for the G1 cyclins Cln1and Cln2, are coordinately regulated by the SBF andMBF transcription factors. As in metazoans, the tran-scriptional activation of those genes depends on theactivity of a distinct G1 cyclin, Cln3, that acts on pro-moter-bound transcription factors to promote recruit-ment of components of the RNA polymerase II complex.

By analogy with metazoan Rb, an inhibitor of theE2F transcription factor that is antagonized by cyclinD/CDK, we predicted the existence of a G1-specifictranscriptional repressor that is inactivated by Cln3/CDK.Using the combined application of molecular geneticsand the mass spectrometry–based multidimensionalprotein identification technology, we identified an SBF-specific transcriptional repressor, Whi5, that is inacti-vated via phosphorylation by Cln3/CDK. This discoveryprovides a unifying mechanism for initiation of the cellcycle in yeast and metazoans (Fig. 1). Further analysisof Whi5 and other regulators identified in that studyshould facilitate our understanding of this regulationin eukaryotes.

One of the primary roles of G1 cyclin–associatedCDKs is to promote the ubiquitin-dependent proteoly-sis of cell-cycle regulators, including the G1 cyclinsthemselves. CDK-dependent phosphorylation of a num-ber of proteins targets the proteins for recognition bythe Cdc34-SCF ubiquitin ligase complex. Grr1, one ofseveral distinct F box proteins that associate with thecomplex, confers recognition of specific phosphorylatedtargets. We are interested in the molecular basis of thatrecognition. Previously, we showed that the interactionbetween Grr1 and Cln2 requires basic residues resid-ing in the pocket of the leucine-rich repeat of Grr1 anddefined a transferable “degron” in the C terminus ofCln2 that is phosphorylated by the CDK.

In addition to its role in cell-cycle control, SCFGrr1

plays a central role in regulating the expression of genesinduced by glucose and amino acids. We showed thatthe glucose signal promotes phosphorylation-dependent,ubiquitin-mediated proteolysis of Mth1, which is requiredfor maintenance of transcriptional repression of glu-cose-inducible genes. Surprisingly, recognition of phos-phorylated Mth1 requires properties of Grr1 distinctfrom those required for recognition of phosphorylatedG1 cyclins. Some of the properties are also importantfor Grr1-dependent recognition of an as yet unknowntarget required for the activation of amino acid–regu-lated genes via SPS signaling. Efforts are under way toidentify that target and to investigate the possibilitythat Grr1 mediates the coordination of cell-cycle pro-gression with the availability of environmental nutrients.



F i g . 1 . Unification of G1/S-phase control in eukaryotes. In yeast,

Cln3-associated CDK1 phosphorylates Whi5, promoting release of

Whi5 from SBF and leading to derepression of SBF-dependent tran-

scription. Activation of SBF-dependent genes leads to accumulation

of G1 and S-phase cyclins, which promote entry into S phase. Accu-

mulation of Cln1- and Cln2-associated CDK1 may provide additional

regulation of Whi5 and SBF. In metazoans, cyclin D/CDK4/6 phosphor-

ylates Rb, relieving Rb inhibition of E2F/DP1 and leading to derepres-

sion of G1 and S-phase gene expression. Activation of E2F-responsive

genes leads to the accumulation of cyclins E and A, which promote

entry into S phase. Accumulation of cyclin E/CDK2 further phosphory-

lates Rb, contributing to the activation of E2F-responsive genes.

CDKs also regulate DNA replication at the transi-tion from the G1 phase to the S phase. Our interest inthe essential noncatalytic function of DNA polymeraseε has led us to study Dbp2, a component of the poly-merase ε holoenzyme. Using mass spectrometry andmutational analysis, we identified a number of CDK-dependent phosphorylation sites in Dbp2 that arephosphorylated during late G1 phase. Genetic analysissuggests that phosphorylation facilitates assembly andactivity of DNA polymerase ε, one of the major replica-tive DNA polymerases.

PUBLICATIONSde Bruin, R., McDonald, W.H., Kalashnikova, T., Yates, J. III, Wittenberg, C.Cln3 activates G1-specific transcription via phosphorylation of the SBF boundrepressor Whi5. Cell 117:887, 2004.

Kesti, T., McDonald, W.H., Yates, J.R. III, Wittenberg, C. Cell cycle-dependentphosphorylation of the DNA polymerase ε subunit, Dbp2, by the Cdc28 cyclin-dependent protein kinase. J. Biol. Chem. 279: 14245, 2004.

Reed, S.I., Wittenberg, C. Cell cycle-dependent transcription in yeast: promoters,transcription factors, and transcriptomes. Oncogene, in press.

Spielewoy, N., Flick, K., Kalashnikova, T.I., Walker, J.R., Wittenberg, C. Regula-tion and recognition of SCFGrr1 targets in the glucose and amino acid signalingpathways. Mol. Cell. Biol. 24:8994, 2004.

Cell-Cycle Checkpoints, DNA Repair, and OxidativeStress ResponseP. Russell, C. Chahwan, S. Coulon, L.-L. Du, P.-H. Gaillard,

V. Martin, T. Nakamura, C. Noguchi, E. Noguchi,

M. Rodriguez, P. Shanahan, K. Tanaka, H. Zhao

Genome maintenance and stress response mech-anisms are highly conserved through evolution,reflecting their crucial role in the reproductive

success of all organisms. A fortunate byproduct of thisfact is that “simple” eukaryotes such as the fission yeastSchizosaccharomyces pombe can be used as relevantmodels for more complex multicellular organisms. Weuse S pombe to study cell-cycle checkpoints, DNA repair,and stress response mechanisms. Defects in these 3 areas underlie a number of human diseases, not theleast of which is cancer.D N A R E P L I C A T I O N C H E C K P O I N T

The difficult task of replicating a eukaryotic genomeis made even more challenging by intrinsic and extrinsicagents that interrupt DNA replication. Protein complexesbound to DNA can impede replication forks, as willchemical adducts in DNA or deoxyribonucleotide starva-

tion. To cope with these difficulties, cells have a DNAreplication monitoring system that senses stalled repli-cation forks and directs various responses. One of theseresponses is the checkpoint that delays the onset of mito-sis (M phase) while DNA synthesis (S phase) is underway. This “mitotic arrest” response buys time to recoverfrom stalled replication forks. Interestingly, the samecheckpoint also controls how damaged DNA is replicated.

The keystone of the replication checkpoint is theDNA-dependent protein kinase–like family that includesthe kinases ATM and ATR in humans and Rad3 in fis-sion yeast. In conjunction with regulatory subunits(e.g., Rad26 in fission yeast) and 2 other proteincomplexes, these kinases activate effector kinases viadirect phosphorylation. Cds1 (Chk2) is the effector ofthe replication checkpoint in fission yeast. We areinvestigating the regulation and function of Cds1. Afew years ago, we discovered mediator of replicationcheckpoint-1 (Mrc1), an adaptor or mediator proteinthat directs the replication checkpoint signal fromRad3 to Cds1. We recently discovered that Mrc1 is asubstrate of Rad3. The phosphorylated form of Mrc1is thought to recruit Cds1 to Rad3.

Cds1 controls repair systems that are required totolerate stalled replication forks. To understand thesesystems, we identified proteins that associate with theforkhead-associated protein-docking domain of Cds1.One of the proteins identified in this screen was Mus81,a novel protein related to the XPF nucleotide excisionrepair protein. Subsequent studies revealed that Mus81associates with the protein Eme1 to form a structure-specific endonuclease. We found that Mus81-Eme1 isa Holliday junction resolvase, an enzyme the makessymmetric cuts in X-shaped Holliday junctions that ariseduring certain types of genetic recombination. Werecently discovered that meiotic intergenic recombi-nation is abolished in mus81 mutants, showing thatMus81-Eme1 is essential for resolution of Hollidayjunctions. We also found that Mus81-Eme1 cleavesHolliday junctions by a nick-and-counternick mecha-nism, with a large rate enhancement of the secondcut because of the flexible nature of the nicked Holli-day junction intermediate.

Stalled replication forks are unstable structures proneto rearrangement and collapse. We have expandedour efforts to understand how stalled forks are stabi-lized. We investigated Swi1, a protein required for aprogrammed fork-pausing event that initiates a generearrangement program that switches mating type. We



found that Swi1 prevents collapse of replication forksand controls activation of Cds1. Swi1 is a nuclear pro-tein that associates with chromatin specifically in S phase, indicating that Swi1 is a component of thereplisome that replicates DNA (Fig. 1). Swi1 has 2 homologs in humans, one of which is the Timeless pro-tein that is involved in the control of circadian rhythms.

D N A D A M A G E C H E C K P O I N T

The DNA damage checkpoint prevents the onset of mitosis when DNA is damaged. This checkpoint isenforced by the protein kinase Chk1, which is itselfactivated by Rad3. Chk1 activation requires the adap-tor protein Crb2. Crb2 is rapidly recruited to double-stranded breaks in DNA. We recently found that Rad3and Tel1 (the ATM homolog in fission yeast) stimulateCrb2 recruitment by phosphorylating histone H2A atthe DNA break site (Fig. 2). Large-scale recruitment ofCrb2 to DNA damage appears to control the mode ofDNA repair.

O X I D A T I V E S T R E S S R E S P O N S E

Oxidative stress caused by reactive oxygen speciescan be highly toxic, causing damage to proteins, lipids,and nucleic acids. Oxidative stress elicits a complexgene expression response that is orchestrated in largepart by MAP kinase cascades. The fission yeast Spc1MAP kinase pathway is homologous to the p38 path-way in humans. We recently discovered Csx1, a proteinthat collaborates with Spc1 to control gene expressionin response to oxidative stress. Csx1 is an RNA-bindingprotein that mediates global control of gene expressionin response to oxidative stress by binding and stabiliz-ing mRNA that encodes Atf1, a transcription factorthat is also regulated by Spc1. These findings reveal anovel mechanism of controlling MAP kinase–regulatedtranscription factors and suggest how gene expressionpatterns can be customized to specific forms of stress.

PUBLICATIONSBlais, V., Gao, H., Elwell, C.A., Boddy, M.N., Gaillard, P.-H.L., Russell, P., McGowan,C.H. RNA interference inhibition of Mus81 reduces mitotic recombination in humancells. Mol. Biol. Cell 15:552, 2004.



F i g . 1 . Model of the involvement of Swi1 in the response to

replication fork arrest. Swi1 is recruited to chromatin during S phase,

suggesting that it might be an ancillary component of the replisome.

Swi1 is required for proficient activation of Cds1 in response to

replication arrest, suggesting that it stabilizes the stalled fork in a

conformation that is recognized by the checkpoint sensor proteins

(Rad3-Rad26 and Rad9-Rad1-Hus1 complexes) and Mrc1. Mrc1

facilities Cds1 activation by recruiting it to Rad3 and perhaps by

mediating Cds1 autophosphorylation. Cds1 enhances fork and repli-

some stabilization while the damaged area is removed.

F i g . 2 . Phosphorylation of histone H2A by the protein kinases

Rad3 and Tel1 controls recruitment of the checkpoint adaptor protein

Crb2 to sites of DNA damage. Wild-type (wt) and mutant strains

were exposed to 36 Gy of ionizing radiation (+IR) or were mock irra-

diated (–IR). Fluorescent micrographs show that a fusion protein

consisting of Crb2 and yellow fluorescent protein is recruited to dou-

ble-stranded breaks in DNA, forming distinct nuclear foci. Foci form

in wild-type cells and in rad3∆ and tel1∆ mutants but do not appear

in rad3∆ tel1∆ double-mutant cells. Foci also do not form in hta1

hta2 cells that express mutant versions of histone H2A that lack a

site that is phosphorylated by Rad3 and Tel1. Scale bar = 5 µm.

Coulon, S., Gaillard, P.-H., Chahwan, C., McDonald, W.H., Yates, J.R. III, Rus-sell, P. Slx1-Slx4 are subunits of a structure-specific endonuclease that maintainsribosomal DNA in fission yeast. Mol. Biol. Cell 15:71, 2003.

Nakamura, T.M., Du, L.L., Redon, C., Russell, P. Histone H2A phosphorylationcontrols Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influ-ences DNA repair in fission yeast. Mol. Cell. Biol. 24:6215, 2004.

Rodriguez-Gabriel, M.A., Burns, G., McDonald, W.H., Martin, V., Yates, J.R. III,Bahler, J., Russell, P. RNA-binding protein Csx1 mediates global control of geneexpression in response to oxidative stress. EMBO J. 22:6256, 2003.

Smith, G.R., Boddy, M.N., Shanahan, P., Russell, P. Fission yeast Mus81-Eme1Holliday junction resolvase is required for meiotic crossing over but not for geneconversion. Genetics 165:2289, 2003.

DNA Damage Responses inHuman CellsC.H. McGowan, V. Blais, H. Gao, A. MacLaren, J. Scorah,

E. Taylor

Together, DNA repair and checkpoint responsesensure the integrity of the genome and thus sur-vival. Coordination of cell-cycle checkpoints and

DNA repair is especially important when unusually highamounts of DNA damage occur after radiation therapyor genotoxic chemotherapy. Cellular survival requiresnot only extreme accuracy in the replication and seg-regation of chromosomal DNA but also repair of spon-taneous and induced mutations with minimal changes(mutations). Thus, surveillance mechanisms that mon-itor the structure of chromosomes and coordinate repairand cell-cycle progression have evolved.

Discovering the molecular mechanisms that regu-late cellular responses to damaged DNA is a majorgoal in cell biology. Anticancer therapy is largely basedon the use of genotoxic agents that damage DNA andthus kill dividing cells. Hence, a detailed understand-ing of cellular responses to DNA damage is also essen-tial to understanding the development and treatmentof disease in humans.

The mammalian cell cycle is driven by the sequen-tial activation of a number of cyclin-dependent kinases.Each kinase phosphorylates and alters proteins requiredfor the execution of specific cell-cycle events. The activ-ities of cyclin-dependent kinases are regulated by changesin the abundance of the cyclin subunit, by inhibitoryphosphorylation, and by association of the kinases withinhibitory proteins. Each of these processes is subject tonegative regulation by checkpoint pathways. Checkpointscontrol the order and timing of cell-cycle events, ensur-ing that biochemically independent processes are cou-

pled so that a delay in a critical cell-cycle process willcause a delay in all other aspects of cell-cycle progres-sion. Checkpoints also coordinate processes of repairwith delays in progression of the cell cycle.

We identified 2 checkpoint kinases in humans thatlimit progression of the cell cycle when DNA is dam-aged. One of these kinases, Chk2, is activated by theprotein ATM in response to DNA damage. The gene forATM is mutated in the cancer-prone disorder ataxiatelangiectasia, and cells that lack ATM have diminishedresponses to DNA damage. The role of Chk2 as a check-point kinase depends on its ability to phosphorylatecell-cycle regulators such p53 and Cdc25. In addition,we recently found evidence of a role for Chk2 in pro-moting repair.

Chk2 physically interacts with Mus81, a conservedDNA damage and replication stress-tolerance proteinthat has homology to the xeroderma pigmentosum Ffamily of endonucleases. Xeroderma pigmentosum is adisorder that results from a failure to appropriately repairdamaged DNA, and persons with the disease are proneto cancer. Biochemical analysis indicates that Mus81has associated endonuclease activity against structure-specific DNA substrates, including Holliday junctions.

Holliday junctions are 4-stranded DNA crossoverstructures that form when 2 strands of duplex DNAswitch binding partners. The junctions can be thoughtof as knots that form between 2 separate duplex DNAstructures. Because the junctions link 2 DNA duplexes,the knot must be resolved before DNA can be separatedand division can occur. Enzymatic analysis, immuno-fluorescence studies, and the use of interference RNAindicate that Mus81 is required for recombination repairin human cells. As a point of interaction between check-point control and DNA repair, the relationship betweenMus81 and Chk2 most likely provides information crit-ical to understanding the response to DNA damage asa whole and thus will aid in the rational developmentof novel and improved anticancer therapies.

PUBLICATIONSBlais, V., Gao, H., Elwell, C.A., Boddy, M.N., Gaillard, P.-H., Russell, P., McGowan,C.H. RNA interference inhibition of Mus81 reduces mitotic recombination in humancells. Mol. Biol. Cell 15:552, 2004.

Gao, H., Chen, X.-B., McGowan, C.H. Mus81 endonuclease localizes to nucleoliand to sites of DNA damage in S-phase cells. Mol. Biol. Cell 14:4826, 2003.

McGowan, C.H. Regulation of cell cycle progression. In: Handbook of Cell Signaling,Vol. 3. Bradshaw, R.A., Dennis, E. (Eds.). Academic Press, San Diego, 2003, p. 401.

McGowan, C.H. Running into problems: how cells cope with replicating damagedDNA. Mutat. Res. 532:75, 2003.



DNA Repair and the Maintenanceof Genomic StabilityM.N. Boddy, Y. Pavlova, S. Pebenard, G. Raffa

Multiple DNA repair pathways have evolved tomaintain genomic integrity in response to aconstant barrage of genotoxic agents. It is now

well established that defects in various DNA repairmechanisms strongly predispose to cancer. In additionto DNA repair pathways, the replication (S phase)checkpoint plays an important role in maintaininggenomic stability. In response to perturbations in the S phase, the replication checkpoint coordinates cellu-lar responses such as cell-cycle arrest, fork stabiliza-tion, DNA repair, and transcription. In humans, defectsin proteins required for the replication checkpoint pre-dispose to the development of tumors, in addition toneurologic and developmental disorders.

Despite its clear importance, the interface betweenthe replication checkpoint and the DNA repair machin-ery remains poorly defined. We identified a physicalinteraction between the replication checkpoint kinaseCds1 and Rad60, an essential DNA repair factor requiredfor the homologous recombination repair of DNA. Wethus have an ideal opportunity to study the functionalinterplay between DNA repair and checkpoints. Inresponse to arrest of replication, Rad60 is hyperphos-phorylated and delocalizes from the nucleus in a Cds1-dependent manner. We made mutants of Rad60 andfound that the gene for one, rad60-4, is refractory toregulation by Cds1 and makes cells hypersensitive toreplication arrest. We are determining the molecularfunction of Rad60 so that we can understand how itsregulation by Cds1 mitigates genomic instability.

In collaboration with J. Yates, Department of CellBiology, we used mass spectrometry to identify proteinsthat interact with Rad60. Intriguingly, this approachresulted in the identification of components of theessential structural maintenance of chromosomes(SMC) complex Smc5-Smc6. The Smc5-Smc6 com-plex is related to the SMC complexes that hold repli-cated sister chromatids together (cohesin) and condensechromatin before its segregation at mitosis (condensin).The molecular functions of Smc5-Smc6, however, remainenigmatic. We purified the Smc5-Smc6 complex andin collaboration with Dr. Yates, determined the identityof the hitherto unknown core components. The evolu-tionarily conserved core complex consists of the Smc5-

Smc6 heterodimer and 4 additional non-SMC subunits,which we call non-SMC elements or Nse1–Nse4. Muta-tions in Nse1–Nse4 cause extreme cellular sensitivityto many genotoxic agents.

Nse1 and Nse2 contain certain zinc finger domainsthat implicate these 2 subunits in the modification oftarget proteins with ubiquitin and the small ubiquitin-like protein SUMO. We are therefore working to identifytargets of Nse1 and Nse2; this information will lead toa better understanding of how the Smc5-Smc6 holo-complex functions in DNA repair and the maintenanceof chromatin structure.

PUBLICATIONSBlais, V., Gao, H., Elwell, C.A., Boddy, M.N., Gaillard, P.H., Russell, P., McGowan,C.H. RNA interference inhibition of Mus81 reduces mitotic recombination in humancells. Mol. Biol. Cell 15:552, 2004.

McDonald, W.H., Pavlova, Y., Yates, J.R. III, Boddy, M.N. Novel essential DNArepair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 com-plex. J. Biol. Chem. 278:45460, 2003.

Smith, G.R., Boddy, M.N., Shanahan, P., Russell, P. Fission yeast Mus81.Eme1Holliday junction resolvase is required for meiotic crossing over but not for geneconversion. Genetics 165:2289, 2003.

Identification of OncogenicAlterations Via Genetic ApproachesP. Sun, Q. Deng, C. Kannemeier, R. Liao, B. Moser

Our major interest is the systematic identifica-tion of genetic alterations involved in tumori-genesis. Currently, we are using 2 approaches.

In the first approach, we try to define the geneticrequirements for the transformation of normal humancells. Our goal is to determine the cellular pathwaysthat must be disrupted during oncogenic transforma-tion. Specifically, we analyze the behaviors of primarycultures of normal human cells after stable transduc-tion of known oncogenes via recombinant retroviruses.We focus mainly on 2 oncogenes: ras and MDM2.

Members of the ras family of oncogenes encodesmall GTP-binding proteins that transduce growth sig-nals from the cell surface. Often, ras is activated intumors, leading to constitutive cell proliferation. Innormal cells, activation of ras triggers an antioncogenicresponse called premature senescence, a permanentgrowth arrest morphologically similar to replicativesenescence. We showed that ras induces senescencethrough sequential activation of 2 MAP kinase path-ways. Initially, ras activates the MAP kinase kinase(MEK)–extracellular signal–regulated kinase (ERK)



pathway. Sustained activation of MEK-ERK turns onthe stress-induced p38 pathway, which subsequentlycauses senescence.

Experiments are under way to identify additionalsignaling components involved in the senescence-inducing pathway. Several proteins have been foundthat act either upstream or downstream of p38 tomediate senescence. Furthermore, in an attempt todetermine how premature senescence is bypassed inhuman tumors, we dissected the functions of an ade-novirus-encoded oncoprotein, E1A, that can rescuecells from ras-induced senescence. Multiple activitiesof E1A are essential for bypass of senescence. Cur-rently, we are searching for cellular proteins that mimicthese E1A activities.

MDM2 is a proto-oncogene that is often overex-pressed in cancers. The oncogenic activity of MDM2 isthought to be achieved primarily through inactivationof the p53 tumor suppressor. However, we found thatin epithelial cells, MDM2 confers resistance to trans-forming growth factor-β, a growth-inhibitory cytokine,through a p53-independent mechanism. Because ahigh percentage of human tumors have acquired resis-tance to growth inhibition induced by this cytokine,this finding suggests that the p53-independent activityof MDM2 plays an important role in oncogenesis. Weare delineating this p53-independent activity of MDM2.

In the second approach, we are screening cDNAlibraries to systematically search for genetic alterationsthat contribute to a specific tumor-associated pheno-type. We developed a retrovirus-based expression sys-tem that allows efficient phenotype-based genetic screensin cultured mammalian cells. Our major interests includetamoxifen resistance in breast cancer, cellular immor-talization, and metastasis.

PUBLICATIONSBian, D., Su, S., Mahanivong, C., Chen, R.K., Han, Q., Pan, Z.K., Sun, P., Huang, S.Lysophosphatidic acid stimulates ovarian cancer cell migration via a ras-MEK kinase 1pathway. Cancer Res. 64:4209, 2004.

Deng, Q., Liao, R., Wu, B.L., Sun, P. High intensity ras signaling induces prema-ture senescence by activating p38 pathway in primary human fibroblasts. J. Biol.Chem. 279:1050, 2004.

Li, X., Zhao, Q., Liao, R., Sun, P., Wu, X. The SCFSkp2 ubiquitin ligase complexinteracts with the human replication licensing factor Cdt1 and regulates Cdt1degradation. J. Biol. Chem. 278:30854, 2003.

The 5-HT7 Receptor as a Targetin DepressionP.B. Hedlund, P.E. Danielson, A.J. Roberts, T. Krucker, P. Bonaventure, T. Lovenberg, S. Huitrón-Reséndiz, S.J. Hendriksen, J.G. Sutcliffe

Serotonin (5-HT) is produced by a small group ofnuclei in the brain stem that send their projec-tions to a vast number of receptive fields. The

family of receptors for 5-HT is the most diverse familythat binds a single ligand, with at least 14 members.One of these is the 5-HT7 receptor, which we previ-ously discovered. In earlier studies, we showed thatthis receptor mediates resetting of circadian rhythmsby the hypothalamus. Despite vast differences in aminoacid sequence between the 5-HT7 receptor and the 5-HT1A receptor, the 2 share considerable pharmacol-ogy and have been implicated in some of the samefunctions. 5-HT1A is more abundant than 5-HT7, butthe areas of the brain that express the 2 receptorsoverlap considerably. Although 5-HT7-selective ago-nists have not been described, the drugs SB-266970and SB-656104 act as 5-HT7-selective antagonists.

We produced mutant mice in which the gene forthe 5-HT7 receptor was inactivated. In behavioral stud-ies, compared with normal mice, the mutant mice hadimpairment in contextual fear conditioning but not cuedfear conditioning. We found no difference between themutant mice and normal mice in other learning tasks.The mutant mice had normal basic synaptic excitabilityin the hippocampal CA1 region and unaltered short-termplasticity; however, they had reduced long-term plastic-ity, possibly contributing to the deficit in contextual fear.

We also studied hypothermia induced by nonselective5-HT7 agonists in normal and mutant mice. In normalmice, low concentrations of 5-HT, 5-carboxamidotryp-tamine, and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced hypothermia, which was reversedby the 5-HT7-selective antagonist SB-266970. Theagonists did not induce hypothermia in mutant mice.Higher concentrations of the agonists induced hypother-mia in both mutant mice and normal mice. This effectwas completely inhibited by a 5-HT1A-selective antagonistbut not completely by SB-266970. These results suggestthat both 5-HT1A and 5-HT7 receptors mediate serotonin-induced hypothermia. The 5-HT7 receptor is more impor-tant at low concentrations of agonist, consistent with finetuning of temperature homeostasis, whereas the 5-HT1A



receptor becomes activated only at higher concentra-tions, possibly as a defense against hyperthermia.

Sleep, circadian rhythm, and mood are related phe-nomena. 5-HT7-selective antagonists increase REMsleep latency and decrease the cumulative duration ofREM sleep, patterns the opposite of those found inpatients with clinical depression. Several antidepres-sants activate 5-HT7 neurons in the circadian controlarea of the hypothalamus, and chronic treatment withantidepressants diminishes both activation and 5-HT7binding there. We examined sleep parameters in themutant mice in which the gene for the 5-HT7 receptorwas inactivated. We found that they spent less timethen normal mice in REM sleep. This pattern is theopposite of that found in humans with depression.

Two models of behavioral despair, the forced swimtest and the tail suspension test, make rats and miceimmobile. This immobility, or helplessness, is likenedto depression in humans because a high correlationexists between the ability of antidepressant drugs toreverse immobility in rodents and to be effective clini-cally in humans. Furthermore, mice selectively bred tohave increased helplessness in these behavioral despairtests resemble patients with clinical depression. Themice have decreased REM latency and more cumula-tive REM sleep, elevated levels of corticosterone, adecreased 5-HT metabolism index, and greater hypo-thermia in response to 8-OH-DPAT. We examinedunmedicated 5-HT7 mutant mice in these tests andfound that they remained significantly more mobilethan did unmedicated normal mice during both theforced swim test and the tail suspension test. Normalmice treated with the 5-HT7-selective antagonist SB-266970 mimicked the mobility of unmedicated mutantmice, whereas the selective antagonist had no effecton the mobility of mutant mice. A selective serotoninreuptake inhibitor increased mobility in both types ofmice (albeit at a lower concentration in the mutantmice), suggesting that the inhibitor worked through anindependent mechanism.

These results are consistent with the notion thatthe 5-HT7 mutant mice have characteristics of a par-tially “antidepressed” state: they spend less time inREM sleep, have reduced immobility in the forced swimand tail suspension tests, and have decreases in 8-OH-DPAT–induced hypothermia. Normal mice medicatedwith 5-HT7-selective antagonists resemble unmedicated5-HT7 mutant mice in these measures. These findingssuggest that a 5-HT7-selective antagonist might be suffi-cient treatment for some aspects of clinical depression.

PUBLICATIONSBonaventure, P., Nepomuceno, D., Hein, L., Sutcliffe, J.G., Lovenberg, T., Hed-lund, P.B. Radioligand binding analysis of knockout mice reveals 5-hydroxytrypta-mine7 receptor distribution and uncovers 8-hydroxy-2-(di-n-propylamino)tetralininteraction with α2-adrenergic receptors. Neuroscience 124:901, 2004.

Hedlund, P.B., Kelly, L., Mazur, C., Lovenberg, T., Sutcliffe, J.G., Bonaventure, P.8-OH-DPAT acts on both 5-HT1A and 5-HT7 receptors to induce hypothermia inrodents. Eur. J. Pharmacol. 487:125, 2004.

Roberts, A.J., Krucker, T., Levy, C.L., Slanina, K.A., Sutcliffe, J.G., Hedlund, P.B.Mice lacking 5-HT7 receptors show specific impairments in contextual learning.Eur. J. Neurosci. 19:1913, 2004.

Thomas, E.A., Copolov, D.L., Sutcliffe, J.G. From pharmacotherapy to pathophysi-ology: emerging mechanisms of apolipoprotein D in psychiatric disorders. Curr.Mol. Med. 3:408, 2003.

Thomas, E.A., George, R.C., Danielson, P.E., Nelson, P.A., Warren, A.J., Lo, D.,Sutcliffe, J.G. Antipsychotic drug treatment alters expression of mRNAs encodinglipid metabolism-related proteins. Mol. Psychiatry 8:983, 2003.

Thomas, E.A., George, R.C., Sutcliffe, J.G. Apolipoprotein D modulates arachi-donic acid signaling in cultured cells: implications for psychiatric disorders.Prostaglandins Leukot. Essent. Fatty Acids 69:421, 2003.

Molecular Neurobiology of CNS DisordersE.A. Thomas, J.G. Sutcliffe, P.A. Desplats, K.E. Kass,

E.L. Woodward

M E C H A N I S M S O F S T R I A T A L D I S O R D E R S

The striatum is a brain region of interest becauseof its unique susceptibility to neurodegeneration,as occurs in Huntington’s disease and Parkinson’s

disease, and because of its association with movementdisorders, psychiatric disturbances, and addiction. Wecontinue to identify and catalog genes that have restrictedexpression in the striatum, because these most likelyencode proteins that contribute to the specific physio-logic and behavioral processes associated with thisregion. We recently focused on a subset of transcrip-tion factors that have restricted expression in thestriatum, including FoxP1, BTE-binding protein, andmRit1, and we are studying how these might regulategene expression in this region of the brain.

Using cDNA microarray technology, we are investi-gating how the expression of striatal genes is alteredin a transgenic mouse model of Huntington’s disease.Using in situ hybridization on individual striatal genes,we found that expression of mRit1 and the sodiumchannel subunit β4 was lower in the transgenic micethan in control mice. We are also using high-throughputmRNA display methods to identify differences in geneexpression between postmortem brain samples frompatients with different stages of schizophrenia and



postmortem samples from control subjects. These stud-ies should shed new light on the pathologic pathwaysinvolved in Huntington’s disease and schizophrenia.A P O L I P O P R O T E I N D

Apolipoprotein D is an atypical apolipoprotein thatis expressed by neurons and glia in the brain. Previ-ously, we found that levels of apolipoprotein D are ele-vated in several types of CNS abnormalities, includingschizophrenia, bipolar disorder, and Alzheimer’s dis-ease, and in response to treatment with antipsychoticdrugs. Although its mechanism of action is unknown,we showed that apolipoprotein D can modulate arachi-donic acid signaling in cultured cells, suggesting a rolefor this apolipoprotein in fatty acid transport and/orcellular membrane stability. Currently, to understandthe potential roles of in apolipoprotein D in humandisorders, we are investigating models of neuropatho-logic changes and behavior in mice that lack the genefor this apolipoprotein.

We found that mice lacking the gene for apolipo-protein D had both increases and decreases in theexpression of several myelin-associated genes andincreases in myelin markers. These changes may affectneuronal connectivity, which is disrupted in patientswith schizophrenia. We are also investigating the effectsof antipsychotic drugs on weight gain in these mice.Preliminary results indicated that unlike female controlmice that gained significant weight in response to cloza-pine, mice lacking the gene for apolipoprotein D didnot gain weight after drug treatment. These studiessuggest that apolipoprotein D plays diverse roles inneuronal homeostasis.

PUBLICATIONSThomas, E.A., George, R.C., Danielson, P.E., Nelson, P.A., Warren, A.J., Lo, D.,Sutcliffe, J.G. Antipsychotic drug treatment alters expression of mRNAs encodinglipid metabolism-related proteins. Mol. Psychiatry 8:983, 2003.

Thomas, E.A., George, R.C., Sutcliffe, J.G. Apolipoprotein D modulates arachi-donic acid signaling in cultured cells: implications for psychiatric disorders.Prostaglandins Leukot. Essent. Fatty Acids 69:421, 2003.

Thomas, E.A., Sutcliffe, J.G. The role of apolipoprotein D in the mechanism ofaction of clozapine and in schizophrenia. In: Phospholipid Spectrum Disorder inPsychiatry and Neurology, 2nd ed. Peet, M., Glen, I., Horrobin D.F. (Eds.). MariusPress, Carnforth, England, 2003, p. 257.

Yao, J.K., Thomas, E.A., Reddy, R.D., Kesharen, M.S. Association of plasma apo-lipoprotein D with RBC membrane arachidonic acid levels in schizophrenia. Schizo-phr. Res., in press.

Microglia and CNS InflammationM.J. Carson, C.S. Anglen, K. Masek, B. Melchior, C. Ploix,

J.C. Thrash, A. Rynko, L. Tran, J.G. Sutcliffe

Afunctional CNS is required for mammalian sur-vival, and therefore the CNS must be defendedfrom pathogens by the immune system. Although

the very molecules produced by the activated immunesystem can disrupt CNS function, activated immunecells can also infiltrate the CNS without inducing destruc-tive disease. Activated immune cells may provide neu-roprotection from neurodegenerative signals. Therefore,we did studies to identify interactions between the CNSand immune cells that are beneficial for maintenanceof CNS function.

First we sought to determine whether microglia,the tissue macrophages of the brain, play a uniquerole in regulating T-cell function in vivo. Previously,K.J. Jones, Loyola University, Chicago, Illinois, andV.M. Sanders, Ohio State University, Columbus, Ohio,showed that CD4+ T cells limit degeneration of motorneurons within the CNS after transection of the facialnerve. In collaboration with Drs. Jones and Sanders,we found that microglia and CNS-infiltrating macro-phages play distinct and nonoverlapping roles in gen-erating neuroprotective T-cell responses. In brief, wediscovered that although peripheral macrophages and/ordendritic cells were necessary to initiate the CNS-spe-cific T-cell response, microglia were necessary to evokeand/or sustain the neuroprotective effector function ofCD4+ T cells.

Second, we sought to identify CNS-specific mech-anisms that regulate T-cell function. We compared2 transgenic models of autoimmunity in which thetarget antigen, influenza virus hemagglutinin, andthe responding T-cell population, hemagglutinin-spe-cific CD4+ T cells, were identical. Only the site of anti-gen expression differed: hemagglutinin was expressedby pancreatic islet beta cells (Ins-HA mice) or by CNSastrocytes (GFAP-HA mice).

Hemagglutinin-specific T cells underwent antigen-independent (homeostatic) and antigen-induced prolif-eration and caused autoimmune destruction of islettissue when transferred into lymphopenic Ins-HA mice.In contrast, upon transfer into lymphopenic GFAP-HAmice, both homeostatic and antigen-induced T-cell pro-liferation and activation were reduced, and no CNS auto-



immunity occurred. Strikingly, upon transfer intoGFAP-HA–Ins-HA double transgenic mice, a 60% reduc-tion occurred in the incidence of T cell–induced dia-betes. Protection from autoimmunity correlated with anincreased ratio of IL-10 to IFN-γ produced by antigen-triggered T cells. These CNS protective mechanismswere overridden by treatment with pertussis toxin andrevealed that CNS integrity cannot be maintained dur-ing an effective autoimmune attack on astrocytes. Theseresults indicate that CNS immune privilege is in part aconsequence of continual presentation of CNS antigensto instruct the immune system of privileged targets.

PUBLICATIONSByram, S.C., Carson, M.J., DeBoy, C.A., Serpe, C.J., Sanders, V.M., Jones, K.J.CD4+ T cell-mediated neuroprotection requires dual compartment antigen presen-tation. J. Neurosci. 24:4333, 2004.

Carson, M.J. The two faces of CNS inflammation: can we tell Dr. Jekyll from Mr.Hyde? Brain Behav. Immun. 17:415, 2003.

Carson, M.J., Anglen, C.S., Ploix, C. Multiple sclerosis: a disease of miscommuni-cation between the CNS and immune systems? In: Inflammatory Disorders of theNervous System: Pathogenesis, Immunology and Clinical Management. Minagar,A., Alexander, J.S. (Eds.). Totowa, NJ, Humana Press, in press.

Carson, M.J., Thrash, J.C., Lo, D. Analysis of microglial gene expression: towardtargeted therapies for CNS neurodegenerative and autoimmune disease. Am. J.Pharmacogenomics, in press.

Molecular Biology of SleepL. de Lecea, R. Winsky-Sommerer, C. Suzuki, B. Boutrel,*

S. Huitrón-Reséndiz,* A. Roberts,* J.G. Sutcliffe,

S.J. Henriksen,* G.R. Siggins*

* Department of Neuropharmacology, Scripps Research

Our goal is to understand the cellular and molec-ular components that modulate cortical activityand sleep. In particular, we focus on the char-

acterization of neuropeptides first described by ourgroup: cortistatin and the hypocretins.C O R T I S T A T I N

Cortistatin is a neuropeptide expressed in the cere-bral cortex. Of its 14 residues, 11 also occur in theneuropeptide somatostatin. However, cortistatin andsomatostatin clearly have different physiologic func-tions. Electrophysiologic and behavioral data fromstudies in transgenic mice done in collaboration withM. Tallent, Drexel University, Philadelphia, Pennsylva-nia, and A. Roberts, Department of Neuropharmacology,indicated that overexpression of cortistatin dramaticallyimpairs hippocampal synaptic plasticity and spatiallearning. These results may have physiologic relevance,

because cortistatin mRNA is upregulated in a mousemodel of Alzheimer’s disease.T H E H Y P O C R E T I N S

The hypocretins, also called orexins, are 2 neuro-peptides expressed in a few thousand cells in the lat-eral part of the hypothalamus. Recent studies indicatedthat patients with narcolepsy lack hypocretin-express-ing cells, suggesting that narcolepsy is a neurodegen-erative disease of the hypocretinergic system. On thebasis of data from these and other studies, we thinkthat hypocretin neurons receive inputs from multiplephysiologic parameters, including metabolic state, cir-cadian time, motivation, mood, and stress, and inte-grate these signals into a coherent output that involvesmultiple components of arousal networks.

In anatomic and electrophysiologic experiments, wefound that hypocretin neurons are contacted by neu-rons expressing corticotrophin-releasing factor (CRF), amajor component of the stress response. Hypocretinneurons contain CRF receptors and are depolarized byCRF. Further, hypocretin neurons are not activated inresponse to stress in mice that lack a receptor for CRF.Our results suggest that the hypocretinergic system ispart of the stress response.

In collaboration with G.F. Koob and A. Markou,Department of Neuropharmacology, we analyzed therole of the hypocretinergic system in brain reward.Infusion of hypocretin into the brain ventricles in micereduced brain reward as measured by intracranial self-stimulation. Our data indicate a role of hypocretins inthe hyperaroused state associated with stress anddrug addiction.N E U R O P E P T I D E S

Neuropeptide S is a newly discovered neuropeptideexpressed prominently in a few hundred neurons in thelocus coeruleus. In collaboration with R.R. Reinscheidand O. Civelli, University of California, Irvine, Califor-nia, we investigated the role of neuropeptide S in sleepand wakefulness. Infusion of neuropeptide S into thebrain ventricles in mice dramatically enhanced wake-fulness. Neuropeptide S activated several brain nucleirelated to arousal, including neurons in the lateral partof the hypothalamus that express hypocretins. Ourdata strongly suggest that neuropeptide S is an impor-tant modulator of sleep and waking.

PUBLICATIONSde Lecea L. Reverse genetics and the study of sleep. In: Sleep: Circuits and Func-tions. Luppi, P.-H. (Ed.). CRC Press, Boca Raton, FL, in press.

García-Frigola, C., Burgaya, F., Calbet, M., Lopez-Domenech, G., de Lecea, L.,Soriano, E. A collection of cDNAs enriched in upper cortical layers of the embry-onic mouse brain. Brain Res. Mol. Brain Res. 122:133, 2004.



Guillén-Gómez, E., Calbet, M,, Casado, J., de Lecea, L., Soriano, E., Pastor-Anglada, M., Burgaya, F. Distribution of CNT2 and ENT1 transcripts in rat brain:selective decrease of CNT2 mRNA in the cerebral cortex of sleep-deprived rats. J.Neurochem. 90:883, 2004.

Martin, G., Ahmed, S., Guadaño-Ferraz, A., Morte, B., Koob, G.F., de Lecea, L.,Siggins, G. Chronic morphine treatment alters NMDA receptors in freshly isolatedneurons from nucleus accumbens. J. Pharmacol. Exp. Ther., in press.

Siggins, G.R., Martin, G., Roberto, M., Nie, Z., Madamba, S., de Lecea, L. Glutamater-gic transmission in opiate and alcohol dependence. N. Y. Acad. Sci. 1003:196, 2003.

Spier, A.D., Fabre, V., de Lecea, L. Autoradiographic localization of cortistatin bindingin the brain: evidence for cortistatin selective binding sites. Regul. Pept., in press.

Winsky-Sommerer, R., Boutrel, B., de Lecea, L. The role of the hypocretinergicsystem in the integration of networks that dictate the states of arousal. Drug NewsPerspect. 16:504, 2003.

Xu, Y., Reinscheid, R.R., Huitrón-Reséndiz, S., Clark, S.D., Wang, Z., Lin, S.H.,Brucher, F.A., Zeng, J., Ly, H.K., Henriksen, S.J., de Lecea, L., Civelli, O. Neu-ropeptide S: a novel neuropeptide promoting arousal and anxiolytic-like effects.Neuron, in press.

Molecular Neuroscience:Lysophospholipid Signaling,Neural AneuploidyJ. Chun, B. Almeida, B. Anliker, E. Birgbauer, K. Cabral,

J.Y. Cho,* M. Fontanoz, F. Gomes,** C. Higgins, D. Kaushal,

G. Kennedy, M. Kingsbury, M. McConnell, M. McCreight,

S. Peterson, S.K. Rehen, R. Rivera, P. Venkatesan,

A.H. Yang, X.Q. Ye

* Seoul National University, Seoul, Korea

** Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Understanding the brain—how it arises develop-mentally, how it carries out its myriad complextasks—is a major challenge. Two areas with both

basic and potentially therapeutic relevance drive ourcurrent studies: the role of lysophospholipid signalingand the role of genomic alterations within individualneurons as manifested by aneuploidy.

L Y S O P H O S P H O L I P I D S I G N A L I N G

Lysophospholipids are simple phospholipids con-taining a glycerophosphate or sphingoid backbone anda single acyl chain of varied length and saturation. Twomajor forms of lysophospholipids are lysophosphatidicacid and sphingosine-1-phosphate. It is now clear fromour research and that of many others that most impor-tant actions of lysophospholipids are mediated by cog-nate G protein–coupled receptors. A growing range ofneurobiological functions is being identified, particu-larly effects on Schwann cells and oligodendrocytes,which are involved in myelination, and on neuropro-

genitor cells of the cerebral cortex. To determinereceptor selectivity and actual neurobiological func-tion, we are producing mice that lack the genes forsingle and multiple receptors. In collaboration withother scientists at Scripps Research, we are develop-ing chemical tools to dissect the in vivo function oflysophosphatidic acid and sphingosine-1-phosphate.

During the past year, we found that lysophospha-tidic acid has a remarkable influence on the formationof the cerebral cortex. This area of the brain is normallysmooth in mice and is crenulated in higher mammals,the so-called sulci and gyri that correspond to valleysand hills on the surface of the brain. Embryonic expo-sure to lysophosphatidic acid produced marked changesin neural cell populations and created sulcilike andgyrilike structural changes of the brain’s surface inmice (Fig. 1). These findings indicate that lysophos-pholipids can have important effects on the organiza-tion of the brain. Studies are in progress to determinethe precise nature of these changes and to uncoverrelated mechanisms for other lipid molecules.

N O R M A L N E U R A L A N E U P L O I D Y

Are all neurons of the brain genetically identical,as is widely assumed, or are differences encoded withinindividual genomes? Using a combination of spectralkaryotyping, which “paints” chromosomes to allow theirunambiguous detection, and fluorescence in situ hybrid-ization, which uses labeled point-probes to identifydiscrete genetic loci in interphase cells, we havedetected a substantial degree of genomic variation inthe normal brain. During neurogenesis, approximatelyone third of all cells are aneuploid. In postmitotic neu-rons, in which spectral karyotyping cannot be usedbecause neurons are in interphase, fluorescence in situhybridization of sex chromosomes revealed a high per-centage of aneuploidy, and the total number of aneu-



F i g . 1 . Receptor-mediated lysophospholipid signaling can alter

cerebral cortical architecture during development. Left, Section of

brain from a control animal. Right, Section of brain from an animal

exposed to lysophospholipid.

ploid cells is almost certainly higher if the remainingautosomes are also considered.

During the past year, we examined basic mecha-nisms that might account for the presence of aneu-ploidy. We found chromosome missegregation defects(e.g., Fig. 2, nondisjunction) in normal developingbrain, indicating that aneuploidy in neural cells can,at least in part, be accounted for by known mecha-nisms. The results also provide a clear example inwhich mechanisms characteristic of cancer can alsooccur in normal cells. Currently, we are exploring thefunctional significance of neural aneuploidy duringbrain development and in disease processes.

PUBLICATIONSKaushal, D., Contos, J.J.A., Treuner, K., Yang, A.H., Kingsbury, M.A., Rehen, S.K.,McConnell, M.J., Okabe, M., Barlow, C., Chun, J. Alteration of gene expression bychromosome loss in the postnatal mouse brain. J. Neurosci. 23:5599, 2003.

Kingsbury, M.A., Rehen, S.K., Contos, J.J.A., Higgins, C., Chun, J. Nonprolifera-tive effects of lysophosphatidic acid enhance cortical growth and folding. Nat. Neu-rosci. 6:1292, 2003.

Liu, H., Toman, R.E., Goparaju, S., Maceyka, M., Nava, V.E., Sankala, H., Payne, S.G.,Bektas, M., Ishii, I., Chun, J., Milstien, S., Spiegel, S. Sphingosine kinase type 2 is aputative BH3-only protein that induces apoptosis. J. Biol. Chem. 278:40330, 2003.

Olivera, A., Rosenfeldt, H.M., Bektas, M., Wang, F., Ishii, I., Chun, J., Milstien,S., Spiegel, S. Sphingosine kinase type 1 induces G12/13-mediated stress fiber for-mation yet promotes growth and survival independent of G protein-coupled recep-tors. J. Biol. Chem. 278:46452, 2003.

Rao, T.S., Lariosa-Willingham, K.D., Lin, F.-F., Palfreyman, E.L., Yu, N., Chun, J.,Webb, M. Pharmacological characterization of lysophospholipid receptor signal trans-duction pathways in rat cerebrocortical astrocytes. Brain Res. 990:182, 2003.

Yang, A.H., Kaushal, D., Rehen, S.K., Kriedt, K., Kingsbury, M.A., McConnell,M.J., Chun, J. Chromosome segregation defects contribute to aneuploidy in normalneural progenitor cells. J. Neurosci. 23:10454, 2003.

Zhao, X., Ueba, T., Christie, B.R., Barkho, B., McConnell, M.J., Nakashima, K.,Lein, E.S., Eadie, B.D., Willhoite, A.R., Muotri, A.R., Summers, R.G., Chun, J.,Lee, K.-F., Gage, F.H. Mice lacking methyl-CpG binding protein 1 have deficits inadult neurogenesis and hippocampal function. Proc. Natl. Acad. Sci. U. S. A.100:6777, 2003.

Roles of Carbohydrate-BindingProteins in Immune Functionand Human DiseaseJ.C. Paulson, P. Bengtson, O. Blixt, B.E. Collins, T. Islam,

K. Norgard-Sumnicht, H. Tateno, Q. Yan

We investigate the roles of carbohydrate-bindingproteins that mediate cellular processes cen-tral to immune regulation and human disease.

All projects are interrelated and fall into 3 main areas:(1) functions of carbohydrate-binding proteins expressedon leukocytes, (2) regulation of the synthesis of thecarbohydrate ligands of the proteins during leukocyteactivation and differentiation, and (3) development ofglycosylation inhibitors that modulate immune func-tion. Our multidisciplinary approach is complementedby a diverse group of chemists, biochemists, cell biol-ogists, and molecular biologists.S I G L E C F A M I L Y O F C E L L A D H E S I O N P R O T E I N S

Of the 11 known Siglecs, 10 are expressed on whiteblood cells and tissue macrophages. The Siglecs are asubfamily of the immunoglobulin superfamily. They have2–17 extracellular Ig domains, including a unique,homologous N-terminal Ig domain that confers the abil-ity to bind to sialic acid–containing carbohydrate groups(sialosides) of glycoproteins and glycolipids. The cyto-plasmic domains of the Siglecs typically contain one ormore immunoreceptor tyrosine-based inhibitory motifsthat are often dynamically phosphorylated and are char-acteristic of accessory proteins that negatively regulatetransmembrane signaling of cell-surface receptor proteins.

To dissect the biology of the Siglecs, we create novelcarbohydrate probes of their function. We use chemoen-zymatic approaches to synthesize sialosides recognizedby Siglecs, and we are developing potent and specificinhibitors for each Siglec to explore the biological rolesof these proteins. Projects on several members of theSiglec family are ongoing.

CD22 (Siglec-2) is an important accessory mole-cule of the B-cell receptor complex; it has both positive



F i g . 2 . Fluorescence in situ hybridization shows chromosome

missegregation defects in the nuclei of neural progenitor cells. A,

Normal cells. B, Cells with 3:1 nondisjunction for chromosome X

(Chr. X). C, Cells with 3:1 nondisjunction for both chromosome X

and chromosome 4 (Chr. 4).

and negative effects on receptor signaling. The carbohy-drate ligand recognized by CD22 is the sequence sialicacid α-2-6-galactose, which commonly terminates N-linked carbohydrate groups of glycoproteins.

The enzyme responsible for synthesis of this carbo-hydrate in mice is a specific sialyltransferase, ST6Gal I.Significantly, mice that lack the gene for ST6Gal I havea marked deficiency in antibody production in responseto vaccination with T cell–dependent or T cell–indepen-dent antigens, establishing the importance of the ligandin CD22 function. We are identifying the functionalglycoprotein ligands of CD22 and are establishingtheir mechanisms for regulation of B-cell receptor sig-naling (Fig. 1).

Other members of the Siglec family differ from CD22both in cellular distribution and in specificity for recog-nition of sialic acid–containing oligosaccharides. Weare evaluating the roles of Siglec-7 and Siglec-9 inregulation of T-cell receptor signaling and the role ofmyelin-associated glycoprotein (Siglec-4) in regulationof neurite formation.R E G U L A T I O N O F L E U K O C Y T E G L Y C O S Y L A T I O N

Activation of lymphocytes and other leukocytesinduces programmed changes in glycosylation. Suchchanges regulate leukocyte trafficking and can modu-late the functions of carbohydrate-binding proteins. Weare systematically investigating the changes in glyco-sylation that occur in B and T lymphocytes after acti-vation in order to determine the underlying molecularmechanisms for these changes and establish their bio-logical relevance. To this end, in collaboration withS. Head and the Consortium for Functional Glycomics(http://www.functionalglycomics.org), we participatedin the development and use of a custom microarray ofglycosyltransferase genes and correlated changes ingene expression with changes in the glycan profiles ofthe resting and activated cells in collaboration with A. Dell, Imperial College London, London, England.

I N H I B I T O R S O F G L Y C O S Y L T R A N S F E R A S E S

In principle, the glycosyltransferases responsiblefor the synthesis of carbohydrates that play key rolesin immune function and human disease are targets fordevelopment of novel therapeutic agents. For example,in mice deficient in ST6Gal I, the absence of the car-bohydrate ligand for CD22 resulted in an immunosup-pressed phenotype. Therefore, an inhibitor of ST6Gal Imight be a novel immunosuppressive agent. Similarly,an inhibitor of the fucosyltransferase TVII might be aneffective anti-inflammatory agent, because this enzymeelaborates the carbohydrate sialyl-LewisX, which par-ticipates in selectin-dependent recruitment of leuko-cytes to sites of inflammation. With the long-term goalof developing potent glycosyltransferase inhibitors, weseek to determine the structures of key glycosyltrans-ferases and develop in vitro and in vivo models toevaluate the efficacy of inhibitors of these enzymes.

PUBLICATIONSCollins, B.E., Blixt, O., DeSieno, A.R., Bovin, N., Marth, J.D., Paulson, J.C.Masking of CD22 by cis ligands does not prevent redistribution of CD22 to sites ofcell contact. Proc. Natl. Acad. Sci. U. S. A. 101:6104, 2004.

Comelli, E.M., Amado, M., Lustig, S.R., Paulson, J.C. Identification and expres-sion of Neu4, a novel murine sialidase. Gene 321:155, 2003.

Danzer, C.P., Collins, B.E., Blixt, O., Paulson, J.C., Nitschke, L. Transitional andmarginal zone B cells have a high proportion of unmasked CD22: implications forBCR signaling. Int. Immunol. 15:1137, 2003.

Kalovidouris, S.A., Blixt, O., Nelson, A., Vidal, S., Turnbull, W.B., Paulson, J.C.,Stoddart, J.F. Chemically defined sialoside scaffolds for investigation of multivalentinteractions with sialic acid binding proteins. J. Org. Chem. 68:8485, 2003.

Shen, Z., Go, E.P., Gamez, A., Apon, J.V., Fokin, V., Greig, M., Ventura, M.,Crowell, J.E., Blixt, O., Paulson, J.C., Stevens, R.C., Finn, M.G., Siuzdak, G. Amass spectrometry plate reader: monitoring enzyme activity and inhibition with adesorption/ionization on silicon (DIOS) platform. Chembiochem 5:921, 2004.

Zuber, C., Paulson, J.C., Toma, V., Winter, H.C., Goldstein, I.J., Roth, J. Spatiotem-poral expression patterns of sialoglycoconjugates during nephron morphogenesis andtheir regional and cell type-specific distribution in adult rat kidney. Histochem. CellBiol. 120:143, 2003.



F i g . 1 . Redistribution of CD22 to sites of contact between B cells

is independent of the putative ligand CD45.

Documents

Molecular Biology - Scripps Research Institute · Neel Krishna, Ph.D.** ... 156 MOLECULAR BIOLOGY 2004 THE SCRIPPS RESEARCH INSTITUTE ... Palo Alto Research Center Palo Alto, California