Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases

doi:10.1016/j.jmb.2009.12.021 J. Mol. Biol. (2010) 396, 908–923

Available online at www.sciencedirect.com

Crystal Structure of the LasA Virulence Factor fromPseudomonas aeruginosa: Substrate Specificity andMechanism of M23 Metallopeptidases

James Spencer1,2⁎, Loretta M. Murphy3, Rebecca Conners4,Richard B. Sessions4 and Steven J. Gamblin5

1Department of Cellular andMolecular Medicine, Universityof Bristol School of MedicalSciences, University Walk,Bristol BS8 1TD, UK2Department of MolecularPhysiology and BiologicalPhysics, University of Virginia,Jordan Hall, 1300 JeffersonPark Avenue, Charlottesville,VA 22908, USA3Department of Chemistry,University of Wales Bangor,Bangor, Gwynedd LL57 2UW,UK4Department of Biochemistry,University of Bristol School ofMedical Sciences, UniversityWalk, Bristol BS8 1TD, UK5Division of Protein Structure,National Institute for MedicalResearch, The Ridgeway, MillHill, London NW7 1AA, UK

Received 31 August 2009;received in revised form8 December 2009;accepted 11 December 2009Available online21 December 2009

*Corresponding author. E-mail [email protected] address: J. Spencer, Depar

Molecular Medicine, University of BMedical Sciences, University Walk,Abbreviation used: CPA, carboxy

0022-2836/$ - see front matter © 2009 E

Pseudomonas aeruginosa is an opportunist Gram-negative bacterial pathogenresponsible for a wide range of infections in immunocompromizedindividuals and is a leading cause of mortality in cystic fibrosis patients. Anumber of secreted virulence factors, including various proteolytic enzymes,contribute to the establishment and maintenance of Pseudomonas infection.One such is LasA, an M23 metallopeptidase related to autolytic glycylgly-cine endopeptidases such as Staphylococcus aureus lysostaphin and LytM,and to DD-endopeptidases involved in entry of bacteriophage to hostbacteria. LasA is implicated in a range of processes related to Pseudomonasvirulence, including stimulating ectodomain shedding of the cell surfaceheparan sulphate proteoglycan syndecan-1 and elastin degradation inconnective tissue. Here we present crystal structures of active LasA as acomplex with tartrate and in the uncomplexed form. While the overall foldresembles that of the other M23 family members, the LasA active site is lessconstricted and utilizes a different set of metal ligands. The active site ofuncomplexed LasA contains a five-coordinate zinc ion with trigonalbipyramidal geometry and two metal-bound water molecules. Using thesestructures as a starting point, we propose a model for substrate binding byLasA that explains its activity against a wider range of substrates than thoseused by related lytic enzymes, and offer a catalytic mechanism for M23metallopeptidases consistent with available structural and mutagenesisdata. Our results highlight howLasA is a structurally distinctmember of thisendopeptidase family, consistent with its activity against a wider range ofsubstrates and with its multiple roles in Pseudomonas virulence.

© 2009 Elsevier Ltd. All rights reserved.

Keywords: M23 metallopeptidase; zinc hydrolase; Pseudomonas aeruginosa;structure mechanism
Edited by M. Guss
ress:

tment of Cellular andristol School ofBristol BS8 1TD, UK.peptidase A.

lsevier Ltd. All rights reserve

Introduction

The Gram-negative bacterium Pseudomonas aeru-ginosa is a versatile opportunist pathogen capable ofcausing a range of infections in humans, including anumber that are life-threatening, and a leading causeof nosocomial (hospital-acquired) infections ofimmunocompromized individuals.1–3 P. aeruginosa,a ubiquitous environmental organism, is an adeptcolonist of damaged tissues and is of particular

d.

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http://dx.doi.org/10.1016/j.jmb.2009.12.021

909Pseudomonas aeruginosa LasA structure

significance in burns cases,4 in ocular keratitis arisingfrom long-term contact lens use5 and as a colonist ofcystic fibrosis lungs.6 Treatment of P. aeruginosainfection is complicated by its inherent resistance tomany classes of commonly used antibiotics and bythe readiness with which it can acquire additionalresistance factors carried onmobile genetic elements.7,8

P. aeruginosa infection is a growing clinical problemandmany of the fundamental processes related to theestablishment and maintenance of infection remainincompletely understood.P. aeruginosa secretes a wide array of toxins and

virulence factors implicated in the infectionprocess.9,10 These include a variety of hydrolyticenzymes with multiple functions involving bothdegradation of host surface and connective tissueand more subtle effects upon defence mechanisms.There is an increasing appreciation of the importanceof such enzymes to the establishment of P. aeruginosainfections but, in general, they have been littlestudied.11 One of the best characterised is LasA(also known as staphylolysin), an abundant 20 kDaprotease originally identified from a mutant labo-ratory strain with impaired elastinolytic activity12

that is secreted as a proenzyme and activatedextracellularly.13 However, in contrast to the prima-ry elastinolytic enzyme lasB elastase,14 LasA is a zincmetallopeptidase15 of far more restricted specificity

Fig. 1. Alignment of active M23 and related peptidase seqM23A (P. aeruginosa LasA; L. enzymogenes lytic protease) abacteriophage ϕ29 gp13 protein (C-terminal domain). NumbLasA (top; black) and full-length LytM (bottom; cyan) asassignments (obtained using DSSP91 to analyse the relevantalignment) and in cyan for LytM (pdb ID 2B1345, below alignand LytM (below alignment) are coloured as in Figs 2 and 3. Cother residues with potential mechanistic roles and yellow shadbonds. Blue shading denotes sequence conservation in M23shading denotes sequence conservation in M23B sequences. Findividual M23 subfamilies are coloured as above; residues comagenta. The alignment was constructed with ClustalW92 an

acting predominantly at glycine-glycine peptidebonds and at a relatively small number of sites inthe elastin polypeptide.16 LasA, however, signifi-cantly potentiates the elastinolytic activity of otherproteases, including LasB elastase,15,17 thereby con-tributing to the degradation of physical barriers tothe establishment of P. aeruginosa infection. It is clearthat LasA possesses other functions important to P.aeruginosa virulence. LasA, in vitro18 and in vivo in amouse lung model,19 stimulates shedding of theectodomain of the heparan sulphate glycoproteinsyndecan-1 from epithelial cells by an indirect,tyrosine-kinase mediated mechanism. Shed synde-can-1 ectodomains may enhance virulence by se-questration of cationic antimicrobial peptides or bysubverting the inflammatory response throughcomplexation of specific chemokineswith neutrophilchemotactic activity.20 LasA, together with LasBelastase, appears to be involved in modulating theinvasive phenotype of some P. aeruginosa strains.21

The amino acid sequence22 confirms LasA to be amember of the M23 metallopeptidase family23 ofstaphylolytic or β-lytic endopeptidases24 that isdistinguished by a His-x-His motif (Fig. 1).25 Theseenzymes are recognised primarily for their glycyl-glycine endopeptidase activity, leading to lysis ofGram-positive bacterial cell walls through cleavageof the pentaglycine interpeptides that cross-link

uences. The figure shows representative sequences fromnd M23B subfamilies, together with that of B. subtilisering is according to the sequences of mature, processedused in the respective PDB files. Secondary structurecrystal structures) are shown in black for LasA (above

ment). Loops 1–4 (see the text) of LasA (above alignment)yan shading denotes metal ligands, green shading denotesing denotes cysteine residues involved in LasA disulphideA sequences (LasA, L. enzymogenes lytic protease). Redor the gp13 sequence, residues conserved with respect tonserved with respect to both subfamilies are highlighted ind annotated with ALINE.93

910 Pseudomonas aeruginosa LasA structure

adjacent peptidoglycan chains.26 This so-called sta-phylolytic activity, which encompasses pathogenicand methicillin-resistant S. aureus (MRSA) strains,has resulted in family members, primarily Staphylo-coccus simulans bv. staphylolyticus lysostaphin but alsoincluding LasA,27,28 attracting considerable attentionas potential therapeutics for staphyloccal infection inboth human and animal patients.29–31 Physiologicalfunctions associated with staphylolytic activity mayinclude defence against competing organisms andturnover of peptidoglycan during normal growthand division.32,33 However it is clear that certainfamily members, such as LasA and the β-lyticendopeptidase from Lysobacter enzymogenes, aredistinguished by an ability to hydrolyse additionalpeptide substrates,34 against which enzymes such aslysostaphin are inactive.35 This apparent physiolog-ical distinction between M23 family members isconsistent with sequence alignment (Fig. 1),23 whichdivides M23 enzymes into two subclasses: M23A,containing LasA, and M23B to which the majority offamilymembers belong. It is clear that theM23 familyshares common structural features with bacterio-phage DD-endopeptidases that hydrolyse peptido-glycan cross-links as part of viral entry to the bacterialcell.36 A comparison of the active site of the S. aureusautolysin LytMwithmore distantly related D-Ala-D-Alametallopeptidases, such as VanX37 and S. albusGcarboxypeptidase,38 has led to the suggestion thatM23 metallopeptidases are part of a larger metal-loenzyme superfamily termed LAS enzymes, that actprimarily as peptidoglycan hydrolases.39

An increasing number of M23 metallopeptidasesare undergoing experimental characterisation,40–43

and crystal structures of some family members arenow available,44–46 but many aspects of these andrelated enzymes are poorly understood. In particu-lar, the relationship between structure and substratespecificity for the different classes of M23 andrelated enzymes remains to be established and,despite a number of proposals, there is no consensusregarding a catalytic mechanism for these enzymes.Furthermore, no M23A enzyme has been structur-ally characterized. In an attempt to address thesequestions, and as a step towards better understand-ing the multiple roles of LasA in P aeruginosavirulence, we have determined crystal structures ofLasA in the uncomplexed form and in complex withtartrate. Using this experimental information as astarting point, we present a model for substraterecognition by LasA that explains the differingspecificities of M23 metallopeptidases and, on thisbasis, we propose a catalytic mechanism.

Results and Discussion

Purification, crystallization and structuredetermination

LasA was readily purified from culture super-natants of P. aeruginosa strain PAO1 by ion-exchange

and size-exclusion chromatography, yielding be-tween 1 mg and 2 mg of purified protein per litre ofbacterial culture. N-terminal sequencing revealedthe expected sequence (APPSNLMQLP) corres-ponding to cleavage of the proLasA precursor atposition 237 of the full-length gene product,47 andelectrospray mass spectrometry confirmed theexpected molecular mass of 19,963 Da. Crystalsobtained after overnight incubation of concentratedprotein at 4 °C belonged to space group P21 with celldimensions a=105.80 Å, b=34.08 Å, c=105.83 Å,β=102.04° and there are four molecules in theasymmetric unit. Whilst spontaneous crystallizationat low concentrations of protein and precipitant hasbeen reported for disease-state mutants of structuralproteins such as the human eye lens γD-crystallin,48

such behaviour would appear to be unusual for anon-mutated, secreted, soluble protein such asLasA. Crystals obtained from sodium potassiumtartrate by the hanging-drop, vapour-diffusionmethod belonged to space group P212121 witha=34.96 Å, b=58.84 Å c=146.44 Å and containedtwo molecules in the asymmetric unit.The structure of the tartrate complex was solved in

a three-wavelength multiwavelength anomalousdiffraction experiment using the Zn2+ K-edge. Thestructure of the uncomplexed native protein wasthen solved by molecular replacement. After densitymodification49 and automated model building50,51

in RESOLVE, manual rebuilding, addition of watermolecules and refinement of the final structurescontain 2872 (5627) protein atoms, 391 (734) watermolecules, 2 (4) zinc ions, 2 (0) tartrate moleculesand 3 (0) molecules of glycerol in each asymmetricunit. Values in parentheses refer to the uncomplexedstructure (P21 crystal form). Refinement statistics arepresented in Table 1B.

Overall structure

The excellent quality of the final electron densitymaps obtained for both the multiwavelength anom-alous diffraction (tartrate complex) and molecularreplacement (native protein) structures permitteduninterrupted tracing of the main chain. In thetartrate complex, residues 1–182 (numbering isgiven for the mature protein taking Ala237 of thepreproprotein precursor as 1) of each chain in theasymmetric unit were modelled, with alternativeconformations present for the first six residues ofchain A. For the native structure, N-terminal Ala1was omitted from chains C andD, and theC-terminalLeu182 was omitted from chains B and D. For thetartrate complex, 88.7% of residues have geometrylying within the core regions of the Ramachandranplot with the remainder within the additionallyallowed areas. For the native structure, the respectivevalues are 87.8% and 11.7%, with 0.5% of residuesplaced within the generously allowed region.The LasA structure (Fig. 2a) is a three-layered

sandwich of antiparallel β-sheets in which a centralsheet of eight strands (β16, β1, β2, β10, β6, β5, β4

†http://blast.ncbi.nlm.nih.gov/Blast.cgi

Table 1. Data collection and Refinement statistics

Inflection Peak Remote Native

A. Data collection statisticsBeamline A.P.S. 19ID A.P.S. 19ID A.P.S. 19ID S.R.S. 14.1Wavelength (Å) 1.28402 1.28348 1.2574 1.488Resolution (Å) 50 – 2.14 50 – 2.14 50 – 2.09 20.0 – 2.01Total reflections 109,259 119,016 74,203 140,482Unique reflections 31,654 32,280 32,180 28,824Completeness (%) 99.7 (98.0) 100 (100) 92.4 (89.3) 96.4 (90.7)Redundancy 3.5 (2.6) 3.7 (3.6) 2.3 (2.2) 3.2 (3.0)I / (σ I) 13.8 (3.9) 23.4 (14.7) 17.5 (8.2) 16.6 (10.5)Rmerge (%) 9.5 (29.9) 5.4 (10.0) 5.6 (13.5) 6.2 (11.9)

Tartrate complex NativeB. Refinement statisticsResolution (Å) 50.00 – 2.14 10.00 – 2.00No. reflections 16,554 22,972No. protein atoms 2872 5627No. water molecules 391 734RMSD from idealityBond lengths (Å) 0.008 0.007Bond angles (°) 1.138 1.090Rcryst (%) 14.9 18.6Rfree (%) 19.5 26.2Average B-factorsProtein atomsa; (Å2) 7.098 7.547Water molecules (Å2) 20.370 10.193

The values in parentheses relate to data collected in the highest resolution shell. Note that Bijvoet pairs were not merged for the threeMAD datasets.

a Values include Zn2+, tartrate and glycerol molecules.


and β8) is flanked by two shorter sheets of three (β3,β7 and β9) and four (β12 – β15, forming a distinct C-terminal subdomain) strands each. A single zinc ionlies at the bottom of a prominent deep grooverunning across one face of the protein. The floor ofthis groove is formed by the β-core of the protein44

and itswalls by four connecting loops: loop 1 (Pro19–Ser34, linking β1 and β2)); loop 2 (Ser63–Cys65,β4–β5); loop 3 (Ala103–Pro119, β9–β10); and loop 4(Gly129–Phe131, β10–β11) Two disulphide bridges,Cys65 – Cys111 and Cys155 – Cys170, anchor thebase of loop 3 to the central β core and link strandsβ14 and β15 in the C-terminal subdomain. Loop 3and the β14–β15 loop (Asn162–Thr167) occupyslightly different positions in the two, independentlyrefined, molecules of the tartrate complex structure.The conformation of loop 3 varies between the nativeand tartrate complex structures. In the native(uncomplexed) structure, loop 3 is held in positionby a hydrogen bond between the Tyr80 side chainhydroxyl and the backbone carbonyl oxygen of Gly-113. In the tartrate complex this contact is lost and theactive site groove widens (Fig. 2a), suggesting thatchanges in the conformation of loop 3 might beassociated with substrate binding.DALI52 and manual searching of the protein

databank53 identified five structures related toLasA: domain IIA of Bacillus subtilis glucose perme-ase (whose structural relation to M23 peptidases hasalready been noted44);54 the LytM glycylglycineendopeptidase from Staphylococcus aureus;44,45 twoother putative M23B peptidases of unknown func-tion from Pseudomonas aeruginosa (PDB accessionnumber 2HSI) and Vibrio cholerae;46 and the C-

terminal domain of the gp13 protein of Bacillussubtilis bacteriophage ϕ29.36 As the P. aeruginosa andV. cholerae structures closely resemble that of LytM(maximal RMSD of 1.38 Å), and little biochemicalinformation is available for these proteins, ourdiscussion is concentrated upon comparisons ofLasA with the LytM and gp13 structures (Fig. 2band c).Superposition of the LasA, LytM (PDB accession

number 2B13) and gp13 (PDB accession number3CSQ) structures reveals distinct differences. Firstly,both the two disulphide bridges and the C-terminalsubdomain are unique to the LasA structure.Database searches using BLAST† indicate that theLasA-like C-terminal subdomain, including the 155–170 disulphide bridge, occurs in M23A enzymesfrom Lysobacter enzymogenes,55 Achromobacterlyticus56,57 and Aeromonas hydrophila58, as well as inuncharacterised M23A family members from arestricted range of environmental bacteria (e.g.Shewanella spp.), including some pathogenic species(V. cholerae). The 65 – 111 disulphide is also found ina series of uncharacterized M23A sequences fromBurkholderia spp. that lack the C-terminal domain.Secondly, the length of the spacer sequence betweenthe first histidine (LasA His23) and aspartate (LasAAsp36) Zn2+ ligands varies from three (LytM) to six(gp13) to 12 (LasA) residues. Sequence alignment ofbiochemically characterized M23 peptidases (Fig. 1)suggests that together these features might differen-tiate the two subclasses of M23 peptidases from one

Fig. 2. Structure of LasA and comparison with LytM and gp13. a, A stereoview of the LasA tartrate complex structure.The β-sheet core is coloured blue and the zinc ion is rendered as a grey sphere. Subdomains are coloured pink (strands β3,β7 and β9) and cyan (C-terminal subdomain, strands β12 – β15). Alternative conformations of loop 3 are shown in dark(tartrate complex) and pale (native) red. Active site residues and LasA disulphide bonds are rendered as sticks (side chainatom colours as standard). b, Structures of LasA (left), LytM (centre; PDB accession 2B13) and gp13 (right). The colourscheme is described above. Loops defining the active site groove (see the text) are coloured equivalently in the threestructures. c, Surface representations of LasA (left), LytM (centre) and gp13 (right) coloured by electrostatic potential. Zincions are rendered as grey spheres. Figs 2a and b, 3 and 4 were generated using PyMol [http://www.pymol.org]. Figure 2cwas generated using CCP4MG.94


another and from related bacteriophage enzymessuch as gp13. The relationship between these struc-tural motifs and the apparent broader specificity and

more diverse biological function(s) of the M23Aenzymes remains to be fully elucidated, althoughboth LasA15 and the A. hydrophila enzyme58 are inhi-


bited by disulphide reducing agents, suggesting thatdisulphide bonds might be essential to their activity.Thirdly, the lengths and conformations of the

connecting loops (loop 1 – loop 4; above) that definethe active site groove vary between the threestructures. The two walls of this groove are formedby loops 1 and 4 (right-hand side as viewed in Fig.2b) and loops 2 and 3 (left-hand side, Fig. 2b). InLasA, loops 1 and 3 are long and loops 2 and 4 areshort, resulting in an active site groove that iscentrally constricted in the vicinity of the boundZn2+ but widens between loops 2 and 4 (Fig. 3b). InLytM, loops 2 and 4 are both longer, creating anactive site groove that is more constricted along agreater proportion of its length (Fig. 3c). LytM loop 1

Fig. 3. Model of LasA–substrate interactions. a, Interactionsite residues and peptide are rendered as sticks with atom coloions are rendered as grey spheres and water molecules are reninteractions are shown as broken lines. The LasA main chain ispeptide on surface representations of LasA (b), LytM (c; PDcolours are as described above; surfaces of loops 1–4 are colsurface of the C-terminal domain of LasA is coloured cyan. Npeptide by LytM loop3 (c) and extension of loops 1 and 4 acr

projects away from the core of the protein andadopts different conformations in the variousavailable structures. The side chain of LytM Tyr-204 points back into the active site and, in the LytMphosphate complex structure (2B44), makes ahydrogen bond with Asn286 of loop 3 that dividesthe active site groove in two.45 In the gp13 structure,the conformation of the central portion of loop 3 isnot defined and loop 2 is relatively short. However,loop 1 is elongated and projects over the active siteand loop 4 is much longer than its equivalents in theother two structures. In consequence, the entrance tothe gp13 active site groove lies at an angle to theplane of the central β-core of the protein, rather thanperpendicular to it as is the case in LasA or LytM

s of Gly-Gly-Phe-Gly-Gly with the LasA active site. Activeurs as standard, except peptide carbon atoms (cyan). Zincdered as red spheres. Zinc–ligand and hydrogen bondingcoloured as in Fig. 2a. b and c, Superposition of modelled

B accession 2B1345) and gp13 (d). Peptide and zinc atomoured according to the scheme used in Figs 1 and 2. Theote occlusion of the P2’ segment of modelled LasA-boundoss the more open gp13 active site groove (d).


(Figs 2c and 3d). It is likely that the differentarchitectures of the active site groove in the threeenzymes reflect their differing substrate specificitiesand physiological roles.

Active site

In common with other M23 peptidases of knownstructure, the LasA active site contains a single Zn2+

Fig. 4. LasA active site and comparison with uncompleuncomplexed (a) and tartrate complex (b) structures. Electronbonding and Zn2+–ligand interactions are shown as brokenmolecules are rendered as red spheres. Atom colours are as sta(blue) terminus). c, Active sites of (left) gp1336, (centre) V. choleand (right) P. aeruginosa putative M23B peptidase (PDB acccoloured as described above.

coordinated by conserved His and Asp proteinligands. Details of this coordination vary betweenthe native and tartrate complex structures. Inuncomplexed LasA (Fig. 4a), Zn2+ is coordinatedin a slightly distorted trigonal bipyramidal geome-try by His23 (Zn2+–Nɛ2 2.23 Å), His122 (of the M23His120 - Xaa - His122 sequence motif; Zn2+–Nδ1

2.22 Å), Asp36 (Zn2+–Oδ1 2.09 Å) and two watermolecules (Wat1, 2.10 Å and Wat2, 2.70 Å; the

xed M23 structures. Stereoviews of LasA active site indensity is 2|Fo|−|Fc|.fφcalc, contoured at 1.2σ. Hydrogenlines. Zinc ions are rendered as grey spheres and waterndard, except Cα atoms (colour ramped fromN- (red) to C-raeM23B putative peptidase46 (PDB accession code 2GU1)ession code 2HSI) shown in equivalent orientations and


distances given are for subunit A). This five-coordinate geometry with two Zn2+-bound watermolecules, where Asp36 and Wat2 are apically andHis23, His122 and Wat1 are axially positioned in atrigonal bipyramidal coordination shell, is observedin three of the four molecules in the nativeasymmetric unit. Wat2 is absent from the fourth(molecule B). Both Wat1 and Wat2 make additionalinteractions with conserved amino acids close to themetal centre. Wat1 forms a hydrogen bond to theNɛ2 atoms of His120 (2.83 Å) and, more weakly,His81 (3.46 Å) and Wat2 to the side chain hydroxylof Tyr151 (2.87 Å). In the tartrate complex (Fig. 4b)Zn2+ co-ordination is better described as tetrahedral,and the positions of both active site water moleculesare approximately occupied by tartrate oxygenatoms. Wat1 is displaced by tartrate O11, whichoccupies an almost identical position relative toHis81 (O11 – His81 Nɛ2 3.41 Å) and His120 (O11 –His120 Nɛ2 2.77 Å) but is 0.44 Å further away fromthe Zn2+ (O11 – Zn2+ 2.54 Å; distances for subunitA). Tartrate O1 lies 1.19 Å from the positionoccupied by Wat2 and is closer (O1 – Zn2+ 2.09 Å)to both Zn2+ and Tyr151 (O1 – Tyr151 OH 2.71 Å).In addition to their resemblance to viral DD-

endopeptidases such as gp13, M23 peptidases suchas LasA and LytM have been proposed to share acommon active site structure with a wider group ofproteins (termed LAS enzymes) that includes sonichedgehog (shh), Streptomyces albus D-Ala-D-Alapeptidase, the D-Ala-D-Ala carboxypeptidaseVanX from Enterococcus faecium, the Listeria mono-cytogenes bacteriophage A500 L-alanoyl-D-gluta-mate peptidase Ply50059 and the peptidoglycanamidase MepA.39,60 Our observation of a five-,rather than four-coordinate zinc ion, in a site thatcontains two bound water molecules, varies fromother available crystal structures for LAS enzymesthat either show tetrahedral coordination or, in thecase of the LytM–tartrate complex, are described assemi- pentacoordinate.45 However, in the availableuncomplexed structures of other LAS enzymes, thelocation, and interactions made by, the single Zn2+-bound water molecule differ: in gp13 (Fig. 4c, left)this water is hydrogen bonded to Ser187, and mightbe considered equivalent to LasA Wat2, whereas inthe V. cholerae M23B enzyme (Fig. 4c, centre) it ishydrogen bonded to His311 (equivalent to LasAHis120) and thus occupies an environment similar toLasA Wat1. Taken together, these data suggest thatZn2+ coordination might be flexible, where boundwater molecules can occupy either or both of twoalternative sites. Importantly, forVanX, the only suchenzyme to be subject to detailed spectroscopiccharacterisation, extended X-ray absorption finestructure and electron paramagnetic resonance spec-troscopy of Zn2+ and/or Co2+-substituted materialindicate a five-coordinate metal centre in bothuncomplexed and phosphinate-inhibited enzymes.61

Alternatively, Zn2+ coordinationmight vary betweenfamily members. In this context, it is notable that thecrystal structure of the otherwise uncharacterizedM23 enzyme fromP. aeruginosa (PDB accession 2HSI)

has Zn2+ coordinated by four protein ligands (His180(equivalent to LasA His23), Asp184 (LasA Asp36),His259 (LasA His120) and His261 (LasA His122) aswell as one weakly interacting (3.08 Å) watermolecule that interacts indirectly withHis228 (equiv-alent to LasA His81) via a further intervening watermolecule (Fig. 4c, right). This further suggests thatboth Zn2+ coordination geometry, and the identity ofcoordinating residues, can vary in M23 enzymes.

Substrate binding and specificity

Lysostaphin-like peptidases, such as LytM, showstrong preference for peptide bonds that link glycineresidues, as evidenced by the substitution of Ser forGly in the pentapeptide interbridges of peptidogly-can from lysostaphin-resistant staphylococci.62,63 Incontrast, LasA, whilst possessing significant staphy-lolytic activity,64 also cleaves a wider range ofglycine-containing sequences, including tropoelas-tin-derived pentapeptides such as Pro-Gly-Gly↓Tyr-Gly (16; ↓denotes cleavage site) and derivatives ofthe tripeptides Gly-Gly↓Leu and Gly-Gly↓Phe35.Lysostaphin was found to be entirely inactiveagainst the latter two substrates. Analysis of activityagainst 19 tropoelastin-derived peptide sequencessuggests that whilst LasA is tolerant of aromatic orbranched amino acids at the P1’ position, suchsubstitutions are less favoured at P2’. Glycine waspreferred at P1.16

Our structure provides some insights into thesepreferences, and into the differing specificities ofM23 peptidases. Using the position of the boundtartrate molecule as a guide, we manually dockedthe pentapeptide Gly-Gly-Phe-Gly-Gly into theactive site so that the carbonyl oxygen of the scissilepeptide occupied the approximate position of(Tyr151-bound) tartrate oxygen O1 and the P2 andP1 glycines follow the path of the tartrate carbonskeleton, and subjected this complex to energyminimization. The results are shown in Fig. 3.Two features of this model for substrate binding

are immediately apparent (Fig. 3a). Firstly, the depthof the P1 site is largely defined by the side chain ofthe active site His120 and, to a lesser degree, by theside chain of Ser116 and the main chain of loop 3.These two factors combine to sterically hinderbinding of amino acids with side chains largerthan hydrogen and hence impose selection forglycine at P1. Second, at the P1’ position, thearomatic side chain of phenylalanine occupies ahydrophobic cavity defined by the side chains ofTyr80, His81 and the Zn2+ coordinating His122,where it can make favourable edge–face interactionswith each of these residues. Substrates bearingbranched-chain amino acids such as leucine at P1’could also make hydrophobic contacts with Tyr80and His122, but available experimental evidencesuggests that these are less tightly bound,35 consis-tent with the possibility that the shorter side chaincan make less extensive interactions. AlthoughTyr80 is conserved only within the M23A peptidasesubfamily,23 its substitution, for example by methi-


onine in LytM, would not be expected to occlude theP1’ pocket, although its loss might significantlyreduce affinity for substrates with aromatic (e.g.Phe) or other hydrophobic (e.g. Leu) amino acids atthe P1’ position.Rather, we suggest that the major factor dictating

the relative lack of activity of lysostaphin againstsubstrates with larger side chains at the P1’ positionis the narrower active site groove of LytM-likeenzymes.35 In comparison with LasA, the mostconstricted part of the LytM active site groove isconsiderably longer (above; Figs 2c and 3b and c).Notably, LytM loop 3 (Gly282–Pro290) forms aprominent central protrusion that makes stericclashes with both P1’ Phe and P2’ Gly of themodelled peptide in the conformation imposedwhen the phenylalanine side chain is positioned inthe P1’ pocket (Fig. 3c). In of contrast, such clashesare avoided in the shorter, more open active sitegroove of LasA (Fig. 3b), although the proximity ofbound peptide to the side chain of Ser115 (Fig. 3a)might explain, in part, why peptides with bulky orbranched amino acids at the P2’ position are notfavoured substrates.16 Our model suggests that,while the LasA active site is wide enough toaccommodate limited amino acid substitutions atsome substrate positions, the long, narrow LytMgroove sterically hinders binding of substitutedpeptides while also requiring that these substratesare bound in extended conformations.Similar considerations might apply to the specific-

ity of related enzymes. It has recently becomeapparent that bacteriophage DD-endopeptidasessuch as gp13 share many common features withM23 peptidases. gp13 from B. subtilis ϕ29 bacterio-phage cleaves the cross-linking amide bondbetween meso-diaminopimelate and D-alanine ofpeptidoglycan,65 and is thus active against asubstrate bearing a methyl group at the P1 position(D-Ala) and extended side chains at P2 and P1’ (meso-diaminopimelate) and P2’ (D-glutamate). Inspectionof the gp13 structure (Fig. 3d) supports our proposalsregarding specificity. Although superposition ofmodelled LasA-bound peptide indicates that thegp13 fold, particularly loops 1 and 4, will impose adifferent orientation on bound substrate (see above),it is clear that gp13 possesses a wider and more openactive site than either LasA or LytM. In particular,loop 3 is shorter in gp13 than in LasA (9, rather than17, residues) and (unlike both LasA structures,where loop 3 is involved in crystal contacts) thecentral portion (residues 272–275 inclusive) is notdefined in the gp13 crystal structure, suggesting thatit might be flexible. Further, loop 2 of gp13 (fourresidues) is shorter than loop 2 of LytM (eightresidues). Together, the truncated, mobile loop 3 andthe shorter loop 2 of gp13 create a more open activesite in gp13 that might enable binding of bulkierpeptidoglycan substrates bearing meso-diaminopi-melate at the P1’ and D-glutamate at the P2’positions. Similarly, in comparison with LasA,repositioning of the main chain of gp13 loop 3,together with a 0.4 Å displacement of His278

towards His247 (gp13 His247 Nɛ2–His278 Nɛ2

3.43 Å; LasA His81 Nɛ2–His120 Nɛ2 3.79 Å) mightexpand the P1 pocket of gp13 sufficiently toaccommodate the methyl group of D-Ala whilst stilldiscriminating against larger side chains. Verifica-tion of these proposals awaits determination ofstructures for the relevant complexes.

A mechanism for LasA

A striking feature of the M23 metallopeptidaseactive site is the presence of two histidine residues(LasA His81 and His120) that are highly conservedbut do not co-ordinate Zn2+. Earlier mutagenesisstudies on M23 family members (LasA47, LytM44

and Staphylococcus capitis ALE-141) and relatedenzymes (MepA66) showed that both histidines arerequired for catalysis. Accordingly, we have exam-ined the interactions made by these residues in thetartrate complex and uncomplexed structures. In theuncomplexed structure, the Nδ atoms of His81 andHis120 make tight hydrogen bonds with the mainchain carbonyl oxygen atoms of Ala109 (range 2.67 –2.77 Å for the four molecules in the asymmetric unit)and Ala118 (2.61 – 2.81 Å), respectively. Weconclude that both of these residues are tautomer-ized at physiological pH, such that Nδ is protonatedand Nɛ is not. By analogy with other Zn2+-dependent hydrolases,67,68 we expect a Zn2+-bound water molecule to act as the nucleophile inthe hydrolytic reaction. Our finding of a five co-ordinate Zn2+ in uncomplexed LasA provides twocandidates for this role, Wat1 and Wat2. We suggestthat Wat1, being more tightly bound (Wat1 – Zn2+

2.10 Å; Wat2 – Zn2+ 2.70 Å), is the more likelynucleophile, and thus that incoming substratedisplaces Wat2, as in our model for binding ofsubstrate pentapeptide. We further suggest thatinteractions made by bound tartrate approximate tothose of the hydrolysed product. (In steady-statekinetic experiments, we observed that hydrolysis ofthe fluorescent substrate Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans69 is not affected by sodium L-tartrate (theform observed in the crystal structure) at concentra-tions up to 2 mM (data not shown). These results areconsistent with the observations reported by Firczuket al.45 on LytM and the lack of observable productinhibition for LasA,69 which suggest that binding ofproduct is relatively weak.Taken together, these conclusions enable us to

propose a catalytic mechanism for LasA (Fig. 5).Substrate carbonyl oxygen displaces Zn2+-boundWat2, thereby enabling direct interaction with Zn2+

to polarize the peptide carbonyl bond (Fig. 5b),rendering it susceptible to nucleophilic attack bythe second Zn2+-bound water molecule, Wat1.Wat1 is oriented by interactions with the Nɛ

atoms of both His120 and His81, while that withZn2+ is weakened (Wat1 – Zn2+ distance increasesfrom 2.10 Å in the uncomplexed structure to 2.76 Åin the model for bound peptide). Importantly, inthe model Wat1 lies almost equidistant from bothHis81 (Wat1 O – Nɛ 2.76 Å) and His120 (Wat1 O –

Fig. 5. Proposed mechanism of peptide bond hydrolysis for M23 metallopeptidases. Broken lines denote Zn2+–ligandand hydrogen bonding interactions. See the text for a full description.


Nɛ 2.85 Å). We suggest, therefore, that either ofthese residues is capable of abstracting a protonfrom Wat1 (Fig. 5b), enabling its addition, ashydroxide, to the substrate carbonyl carbon togenerate an oxyanion (Fig. 5c) that is stabilized bybidentate co-ordination of Zn2+ and by interactionwith the unprotonated His residue. Proton transferfrom the histidine general base to the departingamide nitrogen (Fig. 5c) facilitates cleavage of thepeptide bond to generate a product complex (Fig.5d) in which the product carboxylate is boundanalogously to the crystallographically observedtartrate. A key feature of our model is that, whileeither His81 or His120 can act as general base/acidin the hydrolysis reaction, the same residue performsboth functions in a given catalytic cycle, whereas

the other remains unprotonated and stabilizes theoxyanion.Such a mechanism is attractive for several reasons.

Firstly, the proposal requires that both conservedhistidines (LasA His81 and His120) present unpro-tonated Nɛ atoms towards Wat1, suggesting thatLasA and related enzymes should readily beinactivated by acid. To test this inference, we haveinvestigated hydrolysis of the fluorigenic peptidesubstrate Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans69 bypurified LasA (Fig. 6). Our experiments revealedthat LasA activity is high at pH 9 (kcat 5.1s

-1; KM58μM) and pH 8 (kcat 5.3s

-1; KM 61μM), reduces atpH 7 (kcat 1.7s

-1; KM 105μM) and is abolished at pH 6and below. This behaviour is consistent with thehypothesis above, i.e. that ionizable group(s), such

Fig. 6. pH dependence of LasA activity. Hydrolysis of200 μM Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans by 39 nMLasA in 1 mM sodium citrate, 10 mM sodium phosphate,20 mM boric acid at 25 °C.


as histidine side chains, with pKa values close toneutral play a key role in the LasA hydrolysis reac-tion, and with previously reported data for LytM45

and ALE-1.41

Secondly, our observation of a five-coordinateZn2+ site, in which Wat1, one of two metal-boundwater molecules, hydrogen bonds to both His81 andHis120, clarifies several aspects of LAS/M23 pepti-dase mechanism. A number of previous proposalshave suggested that one of the two conservedhistidine residues (LasA His81; His120) could actas a general base to activate the hydrolytic watermolecule.39,46,65,66 However, as interaction of Zn2+

with the substrate carbonyl carbon is also expectedto be important to substrate binding and activa-tion,66 it is difficult to reconcile the presence of bothsubstrate and Zn2+-bound water with the availablecrystallographic evidence for a tetrahedral Zn2+

centre. Our structure of uncomplexed LasA nowshows how incoming substrate could bind Zn2+ (bydisplacing Wat2) whilst retaining a metal-boundwater molecule (Wat1) that is appropriately posi-tioned and oriented to act as the nucleophile in thehydrolytic reaction by virtue of its interaction withboth conserved histidine residues. Zn2+ coordina-tion, particularly with respect to the positions ofactive site water molecules, differs between thevarious LAS structures36,37,45,46,59,60,70 (Fig. 4) andbetween crystallographic (four-coordinate37) andspectroscopic (five-coordinate61) studies of theVanX enzyme. These data suggest that five-coordi-nate Zn2+, as found in our proposed substratecomplex, can be accommodated in the LAS/M23active site and thus that the essential features of ourmechanism might hold true for other members ofthis enzyme family, even where Zn2+ coordination istetrahedral in the resting state. Our suggestion thateither His81 or His120 can act as the general base/acid is in accord with previous proposals, where thisfunction has been ascribed to His311 of the V.cholerae putative M23B peptidase (equivalent toLasA His120)46; His-247 of gp13 (LasA His81) orGlu-181 (LasA His81) of the more distantly relatedVanX enzyme.37,71,72 Our scheme also satisfies

available mutagenesis data by providing roles forboth conserved histidine residues.41,66

Other catalytic schemes might also be proposedon the basis of the data presented here. A mecha-nism in which Wat1 is displaced by the substratecarbonyl and Wat2 acts as the nucleophile isattractive on the grounds that the more labile water(in the uncomplexed structure) might be the betternucleophile. However, this mode of substratebinding would place the incoming carbonyl oxygenclose to the Nɛ atoms of both His81 and His120,requiring these to be protonated in order to makehydrogen bonds with substrate and oxyanionspecies and suggesting, contrary to our experimentalobservations, that activity should be maintained, oreven increase, with a decrease in pH. A secondpossibility is that one conserved histidine orients andactivates the nucleophilic Wat1 and the other posi-tions substrate; for example, by making a hydrogenbond with the amide of the scissile peptide. This iscompatible with the available experimental evi-dence, but would require a mode of substrate bind-ing different from what is observed in our model ofthe substrate complex.Metallopeptidases such as thermolysin and car-

boxypeptidase A (CPA) are extensively studiedparadigms of enzymes that utilise a single Zn2+ incatalysis of (peptide) hydrolysis reactions.67,73,74 Thegenerally accepted mechanisms for these twoenzymes use an active site carboxylate group(Glu143 and Glu270, respectively) that does notcoordinate Zn2+ to abstract a proton from Zn2+-bound water and back-donate it to the departingamine leaving group. Zn2+ is proposed to functionprimarily to promote nucleophilic attack of boundwater upon the substrate carbonyl and could assist inpolarizing this bond, although electrophilic aminoacid side chains such as Arg127 of CPA or His231 inthermolysin might also make important contribu-tions to this. Our mechanism for LasA shows somesimilarities to these proposals. The histidine (His81or His120) general acid/base is equivalent toGlu143/Glu270, and it is possible that LasA Tyr151(Fig. 3a) could orient and/or polarize the substratecarbonyl in a fashion similar to that of CPA Arg127.Interestingly, however, the LytM–tartrate complexstructure (PDB accession 2B1345) shows the equiva-lent residue, Tyr204, to be located significantlyfarther from the metal centre or bound tartratethan LasA Tyr151. In addition, this residue is notcompletely conserved in the known M23 metallo-peptidase sequences (Fig. 1), suggesting that itsinteraction with substrate is not essential to cataly-sis. Furthermore, the structures of uncomplexedthermolysin75 or CPA76 contain tetrahedral zinccentres rather than the five-co-ordinate arrangementobserved here. In this respect, LasA and related M23peptidases might better resemble peptidoglycandeacetylases such as LpxC68 or Mycobacteriumtuberculosis MshB,77 although in the proposedcatalytic mechanisms for these enzymes the rolesof general base and acid are carried out by twodifferent amino acids rather than a single residue.


Concluding remarks

The structures we present here provide newinsights into structure, mechanism and specificity inthe M23 metallopeptidases and related enzymes,such as DD-endopeptidases involved in bacterio-phage entry and the various LAS enzymes. Ourresults show that M23A metallopeptidases such asLasA are distinguished from the more numerousM23B enzymes like LytM, and from bacteriophageenzymes such as gp13, by structural features thatinclude disulphide bridges and possession of anadditional C-terminal subdomain as well as altera-tions to the active site region that are manifest indifferences in the sequence spacing of residues thatform the metal-binding site and in the overall sizeand shape of the likely substrate-binding cleft. Asbiochemical evidence shows LasA to possess signif-icant hydrolytic activity against a wider range ofsubstrates than the related enzymes that have beencharacterised so far, these structural differences areconsistent with the expanded range of substratesagainst which LasA exhibits hydrolytic activity and,by implication, with the multiple roles suggested forLasA in P. aeruginosa virulence. Our structureprovides a starting point for further investigationsinto the role of LasA in P. aeruginosa pathogenesis,and a basis for the design of smallmolecule inhibitorsas a possible new means of therapeutic interventionin the early stages of P. aeruginosa infection.

Materials and Methods

Unless stated otherwise, all reagents were of analyticalgrade and were purchased from Sigma (Poole, U.K.). P.aeruginosa strain PAO1 was a generous gift from thePseudomonas Genetic Stock Center‡.

Protein expression and purification

LasA was purified from P. aeruginosa strain PAO1culture supernatants by a modified version of theprocedure described by Kessler.34 A single bacterialcolony was used to inoculate 100 ml of nutrient broth,which was grown overnight at 37 °C with shaking at200 rpm. Nutrient broth (1 l) was inoculated with 10 ml ofthe resulting culture and grown for 16 h as describedabove. Cellular debris was removed by centrifugation for20 min at 4700g, ammonium sulfate added to thesupernatant to 80% saturation and the whole left tostand for 1 h at 4 °C with gentle stirring. Centrifugationfor 1 h at 13,000g yielded a greenish pellet that wasresuspended in 100 ml of 50 mM Tris–HCl, pH 7.0 anddialysed overnight against frequent changes of the samebuffer. After insoluble material was removed by centrifu-gation (30 min at 20,000g) the dialysate was loaded onto afast-flow SP-Sepharose column (Amersham GE Health-care) and eluted with a 0 – 500 mM NaCl gradient.Fractions containing LasA were identified by SDS-PAGE,78 pooled and dialysed overnight against 50 mMMes, pH 6.0 then loaded onto a 5 ml ReSOURCE S column

‡http://www.pseudomonas.med.ecu.edu/

(Amersham GE Healthcare) and eluted with a 0 – 300 mMNaCl gradient. LasA-containing fractions were identifiedby SDS-PAGE, pooled and the total volume reduced to∼1.5 ml in a Vivaspin centrifugal filtration device(Sartorius). This was loaded onto a 125 ml Superdex 75size-exclusion column (Amersham GE Healthcare) andeluted with 50mMTris–HCl, pH 7.0, 100 mMNaCl. LasA-containing fractions were pooled and concentrated bycentrifugation to ∼8 mg/ml as described above. Crystal-lization experiments were set up immediately to avoidprecipitation of concentrated material. The presence andidentity of LasA was confirmed by an assay of staphylo-lytic activity as described,34 and by electrospray massspectrometry. LasA-containing bands were transferred byelectroblotting from an SDS-PAGE gel onto a PVDFmembrane (ProBlott, Applied Biosystems) according tothe manufacturers' instructions and the ten amino-terminal residues were sequenced by Edman degradation.

Crystallization and data collection

Needle-like crystals (approximate dimensions 25 μm×25 μm×300 μm) appeared spontaneously overnight at 4 °Cin protein samples pooled before gel-filtration chromato-graphy and concentrated to ∼8 mg/ml in 50 mM Mes.pH 6.0. 150 mM NaCl. The largest of these was mountedin a rayon loop (Hampton Research) and cryoprotectedby transient exposure to buffer supplemented with 30%(v/v) glycerol. A data set, 96.4% complete overall to 2.01 Åresolution, was collected on a single crystal cooled to 100 Kusing the Quantum 4 CCD detector (Area DetectorSystems Corporation) mounted on station 14.1 of theSynchrotron Radiation Source, Daresbury, U.K.Problems with control of the (often excessive) degree of

nucleation made these crystals difficult to reproduce.Further, preliminary experiments on the BM14 beamlineof the European Synchrotron Research Facility (ESRF; datanot shown) indicated that these were of insufficient qualityto permit experimental phase determination using theanomalous signal from the intrinsic zinc ion. We thereforeinitiated a search for additional crystal forms by sparse-matrix screening using protein at ∼8 mg/ml immediatelyafter concentration in hanging-drop, vapour-diffusioncrystallization experiments. Sparse-matrix screens (Hamp-ton Research Crystal Screens 1 and 279,80) were used in 24-well plates (Hampton Research) with 1 μl of proteinsolution and 1 μl of well solution in the drop and 500 μlin the reservoir. These were maintained at a constant 20 °C.A very high number of qhitsq from the screening procedure(51 of 96 conditions yielded spherulites, needles or crystals)confirmed low protein solubility and ready nucleationacross a wide range of conditions. Inspection suggestedthat the most promising crystals appeared within two daysfrom 400 mM sodium potassium tartrate and were ofrectangular prismatic habit with a maximal dimension of50 μm. These were removed from the drop, cryoprotectedby transient exposure to mother liquor containing 30%glycerol, mounted in rayon loops (Hampton Research) andfrozen directly on the X-ray set. Three data sets at wave-lengths corresponding to the maximum values for the f''(1.28348 Å) and f’ (1.28402 Å) components of the Zn2+anomalous scattering edge, and at a high-energy remotewavelength (1.2574 Å)were collected on the SBC-2 custom-built 3×3 mosaic CCD detector mounted on beamline 19IDof the Structural Biology Center, Advanced Photon Source,Argonne National Laboratory, IL, USA. Wavelengths atwhich data were collected were determined from an X-rayfluorescence scan of a somewhat larger crystal ofP. aeruginosa SPM-1 Zn2+ β-lactamase.81


All data were integrated, scaled and merged usingHKL2000.82 Data collection statistics are given in Table 1A.

Structure solution

Automated Patterson searches as implemented in theprogram SOLVE83 using all three data sets collected on acrystal obtained from sodium potassium tartrate, readilylocated both Zn2+ sites of the asymmetric unit, enablingmultiwavelength anomalous diffraction phases to becalculated to 2.14 Å resolution. Density modification49

and automated model-building50,51 were done withRESOLVE; 78% of main chain residues (54% with sidechains) were successfully built by this method. Remainingresidues (predominantly loops) were built manually usingO84 or COOT.85 Crystallographic water molecules wereadded automatically using the ARP/wARP version 5.0package86 as implemented in the CCP4 suite for proteincrystallography87 with subsequent manual modifications.Models were refined using REFMAC 588 against the“peak” data set.The uncomplexed structure (S.R.S. 14.1 data set) was

solved by molecular replacement in AMoRe89 using onemonomer of the final lasA–tartrate structure as a searchmodel. The positions of four monomers were identifiedunambiguously by molecular replacement and initialelectron density maps were of good quality. However,the refinement statistics of the starting model did notimprove following cycles of Refmac5. Inspection of thenative Patterson for this dataset revealed a peak of 2/3origin height at approximately (0.5, 0, 0.5) and, corre-spondingly, that two of the monomers in the molecularreplacement solution were related to the other two by thesame translation vector. With these observations in mind,we examined the intensity distribution of measuredreflections in different projections using HKLVIEW. Inkeepingwith the Patterson peak and the vector relating themolecules within the asymmetric unit, it was clear thatreflections belonging to (h + l=odd) were systematicallymuch weaker than for reflections (h+ l=even). Reasoningthat the inclusion of these systematically weak reflectionswas interfering with the refinement process, we filteredthem from the final dataset. Resulting electron densitymaps were again of good quality and subsequentrefinement to produce improved maps and statisticsproceeded smoothly. Although the completeness of thefiltered data is only half of the original, the filtered termsbeing systematically weak would make little impact onelectron density maps and effectively add only noise to X-ray gradients for refinement. Moreover, the relatively highobservation to parameter ratio at 2 Å Bragg spacing, andthe fact that the omitted reflections are not clustered in anyparticular area of reciprocal space, mean that the mapquality, refinement progress, and reliability of the finalmodel are not unduly affected by the proceduresdescribed. Model building and refinement was subse-quently done as above, except that during refinement non-crystallographic symmetry restraints were imposed andeach monomer was treated as a TLS unit.90 Data collectionstatistics are given in Table 1A for the complete dataset andcrystallographic refinement statistics are given in Table 1B.

Modelling of the LasA substrate complex

The coordinates of the A subunit of the lasA tartarecomplex were chosen as the basis for modelling a complexwith the pentapeptide substrate Gly-Gly-Phe-Gly-Gly.

Hydrogen atoms were added consistent with pH 7. Amodel of the peptide was built and partial chargesassigned on te basis of AM1 calculations. This was thendocked manually into the LasA active site of the proteinsuch that carbonyl oxygen of the scissile peptide (Gly-Phe)bond occupied the approximate position of (Tyr151-bound) tartrate oxygen O1 and the P2 and P1 glycinedipeptide followed the path of the tartrate carbonskeleton. The tartrate molecule and any crystallographicwater molecules overlapping the peptide substrate wereremoved and the resulting complex was soaked with a 5 Ålayer of water molecules. This complex was subjected toenergy minimization. The distances between the proteinligand atoms and the zinc ions and between the twozincs were constrained to their values in the crystalstructure by a harmonic potential with a force constant of1000 kcal/Å2. (This treatment is necessary to deal with theinadequate treatment of a Zn2+ in this forcefield as a hardsphere with a point charge.) The protein backbone atomswere tethered to their crystal structure positions with aharmonic potential. Successive rounds of template-forcingenergyminimization, in which the tethering force constantfrom was reduced from 1000 to 5 kcal/Å2, were thenperformed for a total of 5000 iterations. Structures wereviewed and manipulated with InsightII (Accelrys Soft-ware Inc., San Diego, 2005) and energy minimizationcalculations were performed with Discover 2.98. Calcula-tions were performed on a dual-core i686 PC running theCentos 3 Linux operating system.

Enzyme kinetics

LasA activity was assayed by monitoring the increase influorescence obtained on hydrolysis of the internallyquenched fluorigenic peptide Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans as described.69 Kinetic experiments were doneat 25 °C in a Spectramax M2 fluorescence plate reader(Molecular Devices) using excitation and emission wave-lengths of 340 nm and 460 nm, respectively. Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans substrate was dissolved in DMSOto a concentration of 20 mM and diluted to achieveconcentrations of between 6.25 and 200 μM in a total assayvolume of 100 μl in standard opaque 96-well plates(Greiner). The concentration of LasA was maintained at 39nM. All experiments were done in triplicate using 1 mMsodium citrate, 10 mM sodium phosphate, 20 mM boricacid buffer with the concentration of DMSOmaintained at1% (v/v). Control experiments carried out at pH 8.5established that these conditions do not affect LasAactivity when compared to 5 mM Tris as used by Elstonet al. Initial hydrolysis rates were determined by linearregression fitting of the linear portion of the reaction timecourse, plotted against substrate concentration and thekinetic parameters Vmax and KM obtained by fitting to theMichaelis–Menten equation using non-linear regression.Data fitting and manipulation were carried out usingGraphpad Prism. Values for kcat were derived by convert-ing changes in fluorescence signal to concentrations ofhydrolysed product using standard curves established bymeasuring the fluorescence of known concentrations ofunhydrolysed and hydrolysed peptides.

Protein Databank accession codes

Coordinates and structure factors have been depositedwith the Protein Databank (www.rcsb.org) with accessionnumbers 3IT7 for the tartrate complex and 3IT5 for theuncomplexed structures, respectively.

www.rcsb.org


Acknowledgements

We acknowledge funding from the Beit MemorialFellowships for Medical Research (to J.S.) and theUK Central Laboratories of the Research Councils(to J.S. and L.R.M.). We thank Will Mawby, GinnyShaw and Steven Howell for mass spectrometry andprotein sequencing, Norma Duke for assistance withX-ray data collection, Wladek Minor for assistancewith X-ray data processing, Graham Bloombergand Will Mawby for synthesis of the Dabsyl-Leu-Gly-Gly-Gly-Ala-Edans substrate, Ian Collin-son for the use of the fluorescence plate reader, Dr.Darryl Hill for his careful reading of the manuscriptand Professor Guy Dodson, Patrick Murphy andAnna Croft for helpful discussions. Pseudomonasaeruginosa strain PAO1 was a generous donationfrom the Pseudomonas genetic stock center (www.pseudomonas.med.ecu.edu). Use of the AdvancedPhoton Source was supported by the US Depart-ment of Energy, Basic Energy Sciences, Office ofScience, under contract no. W-31-109-Eng-38.

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Crystal Structure of the LasA Virulence Factor from Pseudomonas aeruginosa: Substrate Specificity and Mechanism of M23 Metallopeptidases