Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

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Crystallographic

portrayal

of

different

conformational

states

of

a

Lys49

phospholipase

A

2

homologue:

Insights

into

structural

determinants

for

myotoxicity

and

dimeric

configuration

A. Ullah

a

_,

_T.A.C.B.

_Souza

b

_,

_C.

_Betzel

c

_,

_M.T.

_Murakami

b

_,

_R.K.

_Arni

a,∗

a_Centro_{Multiusuário}_de_Inovac¸_ão_{Biomolecular,}_Departamento_de_Física,_Universidade_Estadual_Paulista_(UNESP),_São_José_do_Rio_Preto-SP,_15054-000_SP,_Brazil b_Laboratório_Nacional_de_{Biociências,}_Centro_Nacional_de_Pesquisa_em_Energia_e_Materiais,_Campinas,_13083-970_SP,_Brazil

c_Institute_of_Biochemistry_and_Molecular_Biology,_University_of_Hamburg,_Laboratory_of_Structural_Biology_of_Infection_and_{Inflammation,}_c/o_DESY,_Notkestrasse_85,_Build._22a,

D-22603Hamburg,Germany

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13March2012

Receivedinrevisedform28April2012 Accepted5May2012

Available online 11 May 2012

Keywords:

Lys49phospholipaseA2homologue

Bothropsbrazili

Tetraethyleneglycol Conformationalstates Oligomerization

a

b

s

t

r

a

c

t

CatalyticallyinactivephospholipaseA2(PLA2)homologuesplaykeyrolesinthepathogenesisinduced

bysnakeenvenomation,causingextensivetissuedamageviaamechanismstillunknown.Although,the aminoacidresiduesdirectlyinvolvedincatalysisareconserved,thesubstitutionofAsp49byArg/Lys/Gln orSerpreventsthebindingoftheessentialcalciumionandhencetheseproteinsareincapableof hydrolyz-ingphospholipids.Inthiswork,thecrystalstructureofaLys49-PLA2homologuefromBothropsbrazili

(MTX-II)wassolvedintwoconformationalstates:(a)native,withLys49singlycoordinatedbythe back-boneoxygenatomofVal31and(b)complexedwithtetraethyleneglycol(TTEG).Interestingly,theTTEG moleculewasobservedintwodifferentcoordinationcagesdependingontheorientationofthenominal calcium-bindingloopandoftheresidueLys49.Thesestructuralobservationsindicateadirectrolefor theresidueLys49inthefunctioningofacatalyticallyinactivePLA2homologuesuggestingacontribution

oftheactivesite-likeregionintheexpressionofpharmacologicaleffectssuchasmyotoxicityandedema formation.DespitetheseveralcrystalstructuresofLys49-PLA2homologuesalreadydetermined,their

biologicalassemblyremainscontroversialwithtwopossibleconformations.Theextendeddimerwith thehydrophobicchannelexposedtothesolventandthecompactdimerinwhichtheactivesite-like regionisoccludedbythedimericinterface.IntheMTX-IIcrystalpackinganalysiswasfoundonlythe extendeddimerasapossiblestablequaternaryarrangement.

1. Introduction

PhospholipasesA2(PLA2s)representthemajorvenom compo-nentofsnakesbelongingtogenusBothropsandexhibit abroad range of biological effects includingneurotoxicity, myotoxicity, cardiotoxicity,hemolysis,anticoagulantandantiplateletactivities [1–4].SnakevenomPLA₂sareclassifiedintotwomainsubgroups basedonthepresenceofanasparticacidresidueatposition49in thecatalyticallyactiveAsp49-PLA2enzymes,oralysinein homo-logues(Lys49-PLA2s;reviewed in [5–7]) thatare consideredto becatalyticallyinactiveduetotheirinabilitytohydrolyzenatural phospholipids[8].Othersubstitutionssuchasarginineatposition 49havebeenobservedinthecatalyticallyinactivePLA2homologue fromZhaoermiamangshanensisvenom[9–11].

∗_{Corresponding} _author_at: _Departamento _de _Física, _Universidade _Estadual

Paulista(UNESP),SãoJosédoRioPreto,15054-000SP,Brazil. Tel.:+551732212460;fax:+551732212247.

E-mailaddress:[email protected](R.K.Arni).

InspiteofthefactthatLys49-PLA2homologuesarecatalytically inactive, they induce a strong myonecrotic effect that is inde-pendentofphospholipidhydrolysisorarachidonicacidpathway [12–14].Anumberofmodelshavebeenproposedtoaccountforthe molecularbasisoftheirpharmacologicalactivities;howeverthe structuraldeterminantsremainelusive[10,11,14–21].Lys49-PLA2 homologueshavealsobeenimplicatedintheinhibitionofthe vas-cularendothelialgrowthfactor[22]andexhibitbroadantibacterial activities[23,24],indicatingtheirclinicalandbiomedicalrelevance. Bothrops brazili snake is widelydistributed in Braziland its venomcontainstwoPLA2s[25];myotoxin-I(MTX-I,Asp49)and myotoxin-II(MTX-II,Lys49).MTX-II,whichisenzymatically inac-tive,inducesstrongmyonecrosisanddose-timedependentedema [26]. This toxin also display cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichiacoli,CandidaalbicansandLeishmaniasp.[25].Inorder toshedlightonthemolecularbasis forthebiologicaleffectsof MTX-II,itscrystalstructurewasdeterminedat2.7 ˚Aresolution pro-vidingdetailsofthedimericconfigurationandtheroleoftheLys49 residueinthecoordinationofligands,whichsuggestsanimportant

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210 51 (2012) 209–214

Fig.1.(A)Molecularsize-exclusionchromatographicprofileofcrudevenomofB.brazili.(B)SDS-PAGEanalysisofpeaksfrommolecularsize-exclusionchromatography.M: molecularweightmarkers(kDa),Lane1;peak1,Lane2;peak2,Lane3;peak3,Lane4;peak4,Lane5;peak5,Lane6;peak6,Lane7;peak7.Lane6representsthepurified MTX-II.

contributionoftheactivesite–likeregionintheexpressionof bio-logicalactivities.

2. Materialsandmethods

2.1. Purification

Crude desiccated B. brazili venom waspurchased from Ser-pentarium SANMARU Ltda, Taquaral, São Paulo, Brazil. 100mg ofthis samplewassuspended ina 1.5mlsolution(0.02MTris; 0.15M NaCl, pH 8.0) and centrifuged at 10,000×g for 10min. The clear supernatant (1ml) was applied into a 16cm×60cm SephacrylS-100 column previously equilibratedwith thesame bufferandelutedataflowrateof0.2mlmin−1_(Fig._1A)._All frac-tionswereanalyzedbysodiumdodecylsulfate-polyacrylamidegel electrophoresis[27](Fig.1B).

2.2. Dynamiclightscattering

DLSmeasurementswereperformedonaDynaproMolecular Sizing instrument at 291K. The samples were previously cen-trifugedfor20minat20,000×gandthen,illuminatedwithlaserof 1␮m(diameter)andtheintensityfluctuationsfromthescattered lightweremeasuredina1cmpathlengthquartzcell.Thedatawere collectedwithintervalsof10swithatleast100acquisitions.The diffusioncoefficient(DT)wasdeterminedfromthedecayrate dis-tributionofintensitycorrelationprofilesandusedtocalculatethe hydrodynamicradius(Rh)oftheproteinviaStokes–Einstein equa-tion(DT=kbT(6Rh)−1,whereTisthetemperatureinKelvin,kbis theBoltzmann’sconstantandisthesolventviscosity).Analysis wasperformedusingthesoftwareDynamicsV6.3.40.

2.3. Crystallization

TheMTX-IIsamplewasconcentratedto15mgml−1_in

micro-concentrators (AMICON) and crystallization was performed by thehanging-dropvapor-diffusionmethodat291Kusing24-well tissue-cultureplates[28].Thesamplewasscreenedagainst com-merciallyavailablecrystallizationkitsincludingcrystalscreen1 and2,gridscreenammoniumsulfateandgridscreenpolyethylene glycol(PEG)6000(HamptonResearch).Typically,1␮lofprotein solutionwasmixedwithanequalvolumeofthescreeningsolution andequilibratedoverareservoircontaining0.5mlofthe crystal-lizingsolution.Initialtestsyieldedmicro-crystalsinthecondition consistingof0.1MTris–HClpH8.5,0.2MMgCl2and18%(w/v)PEG 4000.Thisconditionwasoptimizedandsinglecrystalsadequate forX-raydiffractionwereobtainedwhena 2␮lproteindroplet wasmixedwithanequalvolumeofreservoirsolutioncontaining 0.1MTrispH8.2,25%(w/v)PEG4000(Fig.2).

Fig.2.PhotomicrographofMTX-II(maximumdimension:200␮m).

2.4. Datacollection,processingandstructuredetermination

AMTX-IIcrystalwasflash-cooledina100Knitrogen-gasstream andX-raydiffractiondatawerecollectedontheW01B-MX2 beam-lineattheBrazilianSynchrotronLightLaboratory(LNLS-Campinas, Brazil).Thewavelengthoftheradiationsourcewassetto1.458 ˚A anda MarMosaic225mmCCDdetectorwasusedtorecordthe intensities.Thecrystalwasexposedfor90switharotationof2◦

perframeandatotalof90imageswerecollectedusinga sample-to-detectordistanceof100mm.Thedatawereindexed,integrated andscaledusingtheprogramsDENZOandSCALEPACKfromthe HKL-2000package[29].Data-processingstatisticsarepresented inTable1.Molecularreplacementwascarriedoutusingthe pro-gramMOLREP[30]andamodelbasedontheatomiccoordinates ofbothropstoxin-IfromBothropsjararacussu(PDBID:2H8I[10]). Thestructurefactorsandatomiccoordinatesweredepositedinthe ProteinDataBankundertheaccessioncode4DCF.

3. Resultsanddiscussion

3.1. Purification

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I/(I) 8.1(2.6) Datacompleteness(%) 94.2(86.1)

Redundancy 3.0(2.7)

Numberofmeasuredunique reflections

12,516

Dataanalysis

VM(Å3Da−1) 2.21

Solventcontent(%) 44.26

Moleculesperasymmetricunit 4

Modelrefinement

Resolutionrange(Å) 19.72–2.70

Rfree(%) 31.98

Rfactor(%) 26.27

Numberofreflectionsusedin refinement

11,881

Numberofproteinatoms 3843

Watermolecules 108

Ligands 2

R.m.sbond-lengthdeviation(Å) 0.012 R.m.sbond-angledeviation(%) 1.472 MeanB-factor(Å2₎ _36.95

Ramachandranplotanalysis

Mostfavoredregions(%) 85

Allowedregion(%) 15

Disallowedregion(%) 0

a_R merge=

hkl

i|Ii(hkl)−I(hkl)|/

hkl

iIi(hkl), where Ii(hkl)is the ith

observationofreflectionhklandI(hkl)istheweightedaverageintensityforall

observationsIofreflectionhkl.

massof14kDa,whichisinagoodagreementwiththecalculated value(13.7kDa).

3.2. Structuraldescription

MTX-II was sequenced and characterized by 25 and 26, respectively. The sequence comprises all the features of inac-tiveLys49-PLA2homologuesincludingtheresiduesLeu5,Gln11, Asn28, Leu32 and Lys49. Sequence alignment of MTX-II with otherBothropsLys49-PLA2 homologues indicatedthatthe puta-tivecatalyticapparatus(catalytictriad+Tyr73)isfullyconserved as well as the nominal calcium-binding loop. The most vari-able region is the C-terminus that may have a key role in myotoxicity[31–34].

ThestructureofMTX-IIwassolvedat2.7 ˚Aresolution(Table1) by molecular replacement using the atomic coordinates of bothropstoxin-IfromB.jararacussu(PDBID:2H8I[10])asa tem-plate.Theatomicmodeldisplayedgoodoverallstereochemistry withRMSD values of 0.012 ˚A and 1.472◦ _for _bond _lengths _and

angles,respectively.Theaveragetemperaturefactor(B-value)for allatomsis36.95 ˚A2 _(Table₁₎_and_85.0% _of_the_dihedral_angles are situated in the mostfavored regions, whereas the remain-ing15.0%ofresiduesarefoundineitherpermittedorgenerously allowedregionsof theRamachandranplot(Table1).The asym-metricunitconsistsoftwodimersrelatedbyatwo-foldaxisof rotation.

The molecular topology of MTX-II conserves all the main features of its homologues (Fig. 3A): the N-terminal ␣-helix H1 (residues Ser1–Thr13); the short helical turn (residues

117) and the different conformational states observed for Lys122.

3.3. Lys49coordinationanditsroleinthestabilizationof tetraethyleneglycol

Theactivesite-likeregionofLys49-PLA2homologuesisformed byHis48,Lys49,Tyr52,Tyr73andAsp99,andthecatalyticwater molecule.TheresiduesGly28,Gly29,Arg32andGly33areinvolved inthecoordinationofCa2+_ion_in_Asp49-PLA

2s[35–38],whichplays aroleinthestabilizationofthetetrahedralintermediateduring catalysis.InLys49-PLA2homologues,theLys49-NZatomislocated atthepositionoftheCa2+_ion_and_is_coordinated_by_the_backbone atomsfromtheCGVG/LGRmotif.

IntheMTX-IIstructure,thetwodimersformingthe asymmet-ricunitdisplaydifferentconformationalstatesduetothepresence of tetraethylene glycol (TTEG) bound to the dimer formed by molecules Aand B. Inthe caseof thesecond dimerformedby moleculesCandDwheretheactivesite-likeregionisempty,the Lys49side-chainismono-coordinatedbythecarbonyloxygenatom of Val31 (Fig. 4A).In moleculeB, a TTEG moleculeis foundat theactivesite-likeregionanditisstabilizedbya single hydro-genbondformedbetweenitsterminalhydroxylgroupandGly30 (Fig.4B).InmoleculeA,TTEGisanchoredtothenominal calcium-bindingloopbythebackboneofGly30analogouslytomolecule Band additionallyby theNZ atomof Lys49,suggesting a con-tributionofLys49inthefunctioningofLys49-PLA2 homologues (Fig.4C).InBthTX-Istructure,aPEGfragmentwasalsoencountered boundtotheactivesite-likeregion,conservingthehydrogenbond withGly30andmakingwater-mediatedinteractionswithHis48 [10].Fattyacids[39,40]andstearicacid[41]werealsoobservedat theactivesite-likeregionofotherLys49-PLA2homologues,which supports theimportance of this interfacefor the expressionof theirpharmacologicalactivitiesincludingmyonecrosisandedema formation.

Acorrelationbetweenthepresenceoftheligandattheactive site-likeregionandthecoordinationofLys49wasobservedinthe MTX-IIstructure.WhenLys49participatesinthestabilizationof TTEG,thecarbonyloxygensofAsn28andVal31arecoordinatedby theLys49side-chain,whereasintheunboundstateLys49 coor-dination is done by the carbonyloxygens of Val31and Gly33. Theseconformationalchangesinthenominalcalcium-bindingloop reflectinarearrangementoftheC-terminalregion,whichmodifies thepolarizationandsurfacehydrophobicity.

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212 51 (2012) 209–214

Fig.3. (A)OverallstructureofMTX-II.N-terminal␣-helixH1(residuesSer1–Thr13);oneshorthelicalturn(residuesPro17–Tyr21);thetwolonganti-paralleldisulfide-linked ␣-helicesH2(residuesAsp39-Lys57)andH3(residuesSer90-E108)thatareseparatedby9 ˚A;the␤-wingregion(residuesSer74–Gly85)andtheflexibleC-terminalregion (Leu110-Cys133).Nominalcalciumbindingloopareinblue.(B)MTX-IIflexibility.RibbonrepresentationcoloredbyB-factor.(Forinterpretationofthereferencestocolorin thisfigurelegend,thereaderisreferredtothewebversionofthisarticle.)

3.4. UnpolarizedstateoftheC-terminalregioninthepresenceof ligands

Differentmechanismshavebeenproposedtothemyonecrotic activityofLys49-PLA2homologuesrelatedtotheirdimeric confor-mation[6,10,42]andformationofthehydrophobicknuckle[40]. In the lattermodel, the hyperpolarization of thepeptide bond betweenresiduesCys29andGly30duetothepresenceofLys122 causesalocalrearrangementoftheC-terminalresidues(Fig.4D).

This is then lockedinto a conformation with thehydrophobic residues,Phe121andPhe124formingahydrophobicknucklethat isexposedtothesolventandisconsideredessentialforinteraction withthemembrane (Fig.4D).However,intheMTX-IIstructure, thepresenceoftetraethyleneglycolinteractingwiththenominal calcium-bindingloopandLys49doesnotresultintheformation ofthehydrophobicknuckle.AnalogouslytoMTX-II,the Zhaoer-miatoxin(Arg49-PLA2)incomplexwithaligandalsoexhibitsa C-terminalconformationwherethehydrophobicresiduesarenot

Fig.4. (A)RelaxedconformationalstateofmoleculesCandDofMTX-II,wheretheLys49side-chainismono-coordinatedbycarbonyloxygenofVal31.(B)Inthemolecule B,thetetraethyleneglycol(green)isonlystabilizedbyGly30amidebond.(C)ThemoleculeAofMTX-IIwithtetraethyleneglycol(green)anchoredtothenominal calcium-bindingloopbythebackboneofGly30andbytheLys49NZatom.(D)ComparisonoftheunpolarizedstateoftheC-terminalregioninthepresenceofligands(MTX-II,blue) withthehyperpolarizedstateoftheLys49-PLA2homologuefromAgkistrodoncontortrix(green).ThepresenceofLys122intheactivesitehyperpolarizestheamidebond

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Fig.5.DimericconfigurationofLys49-PLA2homologues.(A)ExtendeddimerofMTX-II.(B)CompactdimerofBthTX-I(PDBID:2H8I).

exposedtothesolvent.TheseresultsshowthatLys49-PLA2 homo-loguesundergostructuralrearrangementsintheC-terminuswhen theactivesite-likeregionisoccupied,providingnewinsightsinto theirmolecularmechanisms.

3.5. Oligomerization

All Lys49-PLA2 homologues are characterized as dimers in solutionandtwodimericconfigurationshavebeenproposedfor Lys49-PLA₂ homologues;anextendedanda compact conforma-tion[10,18,43](Fig.5).Intheextendeddimer,formedbyhydrogen bondsbetweenthe␤-wings,thehydrophobicchanneland active-sitecleftareexposedtothesolvent[18,43],formingacommon, largehydrophobic surface enabling the interaction with mem-branes. In the compact dimer, non-polar contactspredominate andthehydrophobicsurfacesurroundingthePLA2-activesite-like regionsareshieldedfromthesolvent[10,20].Todate,all Lys49-PLA2homologuestructuresavailableintheProteinDataBankcan beinterchangeablyassembledinthetwopossibleconfigurations. SAXSstudiesandcrystalstructureofseveralligandcomplexeshave beenindicatingthecompactdimerasthebiologicalassemblyfor Lys49-PLA2homologues.

DLS experiments also confirmed the dimeric state of MTX-II(resultsnotshown)and crystalpackinganalysisshowedthat onlytheextendeddimercanbeformedusingallpossible sym-metryoperations.Thisistheuniquecrystalstructureinwhichthe compactdimercannotbeassembledbysymmetryoperations, sug-gestingthatstructuralrearrangementsbythepresenceofligand couldinduceaswitchbetweenthesetwopossibleconformations orsimplyindicatesthattheextendeddimeristhemorelikely bio-logicalunitofLys49-PLA2homologues.

Authors’contributions

AUpurifiedandcrystallizedtheprotein.TACBSandMTM col-lected X-raydiffraction data and refined the structure. CB and RKAanalyzedthedata,wrotethemanuscriptandsupervisedthe project.

Acknowledgments

This research was supported by grants from CNPq, TWAS, FAPESP,DAADandCAPES.

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