SIMULACIONS COMPUTACIONALS PER A ENTENDRE COM FUNCIONEN ELS ENZIMS

Carme RoviraICREA― Universitat de Barcelona 

Institut de Química Teòrica i Computacional (IQTCUB)

Què és un enzim?

Adaptat de https://www.ebi.ac.uk/training/online/course/introduction‐protein‐classification‐ebi/protein‐classification

• Els enzims són molècules essencials

• Els enzims són proteïnes

2016

Carme Rovira

Què és un enzim?

• Els enzims són proteïnes

• Els enzims actúen de catalitzadors per a accelerar les reaccions bioquímiques

• Els enzims no es modifiquen durant aquest procés

2016

Carme Rovira

Saliva: amilasa

Estómac:proteases

Budell prim:lipases,amilasa, lactasa

Dibuix dewww.divavillage.com

ENZIMS DIGESTIUS

Unitats de sucre(monosacàrids)

Molècula de carbohidrat

digestió

GLICOSIDASES 2016

Carme Rovira

Saliva: amilasa

Estómac:proteases

Budell prim:lipases,amilasa, lactasa

Dibuix dewww.divavillage.com

ENZIMS DIGESTIUS

Unitats de sucre(monosacàrids)

digestió

Enllaços glicosídics

GLICOSIDASES 2016

Carme Rovira

R. Wolfenden & M. J. Snider.Acc. Chem. Res. 2001, 34, 938‐945

The power of enzymes as catalysts

t1/2 = 1 billion years

t1/2 = 1 million years

t1/2 = 1 thousand years

t1/2 = 1 year

t1/2 = 1 day

t1/2 = 1 minute

age of the earth

O‐glycoside hydrolysis 5‐8 million years

2016

Carme Rovira

How enzymes work

PASS

AB

© 2010 Nature Education All rights reserved. 

Without enzyme With enzyme

Energy

Reaction pathway

Activationenergy

A

B

A

B

Enzymes lower the activation energyof the biochemical reaction

2016

Carme Rovira

How enzymes work

PASS

AB

GLYCOSIDASE

2016

Carme Rovira

Enzymes are very specific

Example: α‐1,4‐glucosidase “cuts” glycosidic bondsbetween atoms 1 and 4 of the α‐glucose units of glycogen

n

1 4

...OH OH OH OH

2016

Carme Rovira

Lack of lactase causeslactose intolerance

GALACTOSE GLUCOSE

Enzymes and disease

Lack of α‐1,4‐glucosidase causes accumulation of glycogen in the liver, leading to the Pompe disease

GLYCOGEN

LACTOSE

GLUCOSE UNITS

2016

Carme Rovira

Knowing how enzymes work isimportant for drug design

Relenza Tamiflu

Synthesis of molecules that inhibit a disease‐related enzyme

2016

Carme Rovira

Sugars and common flu

Influenza virus

NeuraminidaseRelenza Tamiflu

N‐acetylneuraminic acid(sialic acid)

Approved since 1999

2016

Carme Rovira

Computer simulation to understandhow enzymes work

• Computer simulation provide molecular details of enzyme action

• Short‐lived states (e.g. transition states) can be identified

• Important for inhibitor (drug) design

Reaction pathway

A

B

2016

Carme Rovira

Impact of supercomputing

• The large increase of computer power in recent years has contributed enormously to the progress of computer simulation of enzymes (computational enzymology) 

• Better models and precise methods can be used

• Meaningful predictions can be made

2016

Carme Rovira

Clarifying mechanisms of enzymesthat process carbohydrates

Gas2 glucosidase, an antifungal target

2016

Carme Rovira

Raich et al. J. Am. Chem. Soc. 138, 3325 (2016) 

Clarifying mechanisms of enzymesthat process carbohydrates

2016

Carme Rovira

(Newton’s equations)

Molecular dynamics

Initial conditions:

• Atomic positions (e.g. crystal structure): {ri}• Atomic velocities (e.g. random velocities scaled to desired T): iv

Motion of atoms from

ri (t)i

2i

2

rr

dEd

tddmi

2016

Carme Rovira

Classical MD

E = Ebond + Eang + Edih + EvdW + Eelec

Energy from a force-field

I(r)ρ

Rρ(r),EminE Density Functional Theory

Molecular dynamics

Computer codes: GROMACS, NAMD, AMBER, …

Energy from quantum mechanics

Ab initio MD

Computer codes: CPMD, CP2K, QUANTUM EXPRESSO, …

C. RoviraMethods Mol. Biol. 305, 527 (2005)

Tobacco mosaic virusShulten & col.Structure 14, 437-449, 2006

2016

Carme Rovira

Classical MD

E = Ebond + Eang + Edih + EvdW + Eelec

Energy from a force-field

I(r)ρ

Rρ(r),EminE

Energy from quantum mechanics

Density Functional Theory

Molecular dynamics

Ab initio MD

2016

Carme Rovira

Active center

(quantum mechanics)Rest of the system

(molecular mechanics)

Multiscale methods

“for thedevelopment of multiscale modelsfor complexchemical systems”

The quantum mechanics / molecular mechanics (QM/MM) approach

ab initio MD classical MD

2016Carme Rovira

Carbohydrate degradation by glycosidases

glycosidic bondcleavage

sugar units(monosaccharides)

2016Carme Rovira

Carbohydrates are very flexible

Slide from Lluís Raich(University of Barcelona)

Sugar shapes

Chair

Most abundantfor a free sugar

Envelope Boat

2016

Carme Rovira

Boat‐likeconformation

O

Sugars change shape when they bindto enzymes

O

WHY?

2016

Carme Rovira

Biarnés et al. J. Biol. Chem. 2006 Biarnés et al.  J. Am. Chem. Soc. 2011

Sugar distortion facilitates thebiochemical reaction

Sugars change shape when they bindto enzymes

twistedboat

O

Collaboration withAntoni Planas(IQS‐URL)

2016

Carme Rovira

Biarnés et al. J. Biol. Chem. 2006 Biarnés et al.  J. Am. Chem. Soc. 2011

Sugars change shape when they bindto enzymes

Sugar distortion facilitates thebiochemical reaction

Energy

Reaction pathway

ActivationenergyA

B

2016

Carme Rovira

http://biology.tutorvista.com

Example: Golgi α‐mannosidase II 

Golgi complex

Cell

mannose2

Anticancer target

2016

Carme Rovira

Mechanism of action

Golgi α‐mannosidase II

Petersen et al.  J. Am. Chem. Soc. 132, 8291 (2010)

boat

Initialcomplex

O

Collaboration withPeter Reilly(Iowa State University)

2016

Carme Rovira

Petersen et al.  J. Am. Chem. Soc. 132, 8291 (2010)Williams et al. Angew. Chem. Int. Ed. 53, 1087 (2014)

Golgi α‐mannosidase IIMechanism of action

anticancer inhibitors

transitionstate

2016

Carme Rovira

Privateer software: conformational validation of carbohydrate structures Agirre, Iglesias‐Fernández, Rovira, Davies, Wilson, Cowtan. Nat. Struct. Mol. Biol. 22, 833 (2015)

Computer simulation for structural validation

wrong conformation correct conformation

(high energy)

β α

(low energy)

Collaboration withGideon J. Davies(Universityof York)

Jon Agirre(Universityof York)

2016

Carbohydrate synthesis

glycosidic bondformation

2016

Carme Rovira

© 2010 Nature Education All rights reserved. http://commonfund.nih.gov/Singlecell/index

Sugars on the cell surface

cytoplasm

cell surface carbohydrates (glycans)

2016

Carme Rovira

Outsideof cell

Cellmembrane

Insideof cell

(cytoplasm)

virusbacteria

Sugars on the cell surface

Special Issue.March 2001: Vol. 291. no. 5512. 

Carbohydrates and Glycobiology

2016

Carme Rovira

Problems associated to a failure of cell‐cell communication

Outsideof cell

Cellmembrane

Insideof cell

(cytoplasm)

virusbacteria

Inflammation

Autoinmune

diseases: 

asthma 

diabetes

allergies 

multiple sclerosis

Infection

2016

Carme Rovira

Abnormal glycosilation: cancer

cancer cells

2016

Carme Rovira

• GalNAc‐T2 attaches one sugar (GalNAc, a galactose derivative) to proteins

• It initiates protein glycosylation

N‐acetylgalactosaminyltransferase 2 (GalNAc‐T2)

GalNAc‐T2

2016

Carme Rovira

Structure of GalNAc‐T2

Catalyticdomain

Lectin domain

Linker

enzymeactive site

Collaboration withRamón Hurtado‐Guerrero(BIFI‐Zaragoza)

2016

Carme Rovira

Structure of GalNAc‐T2

enzymeactive site

proteinfragment

2016

Carme Rovira

Metadynamics simulation

The lectin domain “catches” the peptide and brings it to the catalytic site. 

Lira‐Navarrete et al. Nat. Commun. 2015, 6, 6937.

Peptide binding to GalNAc‐T2 2016

Carme Rovira

Computer simulation

Atomic Force Microscopy

Lira‐Navarrete et al. Nat. Commun. 2015, 6, 6937.

Computationalmicroscope

Adapted from:DOI: 10.1021/acs.jcim.5b00249(A. Magistrato)

2016Carme Rovira

GalNAc‐T2 biochemical reaction

PEPTIDE

UDP

SUGAR

Angew. Chem. Int. Ed. 2014, 53, 8206

SUGAR TRANSFER

2016

Carme Rovira

Agraïments

Col∙laboradors teòrics:

Xevi Biarnés (IQS, Barcelona)

Víctor Rojas-Cervellera (Barcelona)

Albert Ardèvol (MPI, Frankfurt)

Javier Iglesias-Fernández (KCL, London)

Lluís Raich (UB, Barcelona)

Col∙laboradors experimentals:

Antoni Planas (IQS, Barcelona)

Peter Reilly (Iowa State U., USA)

Gideon J. Davies (U. York, UK)

Spencer Williams (Melbourne U., Australia)

Ramón Hurtado-Guerrero (BIFI, Zaragoza)

2016

Carme Rovira