Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

FEBSLetters582(2008)3335–3342

SimulationsofaGprotein-coupledreceptorhomologymodel

predictdynamicfeaturesandaligandbindingsite

Ste enWolfa,MarcusBo¨ckmanna,b,UdoHo¨welerc,Ju¨rgenSchlittera,KlausGerwerta,*

ab

DepartmentofBiophysics,UniversityofBochum,ND04North,44780Bochum,Germany

DepartmentofTheoreticalChemistry,UniversityofBochum,44780Bochum,Germany

c

CHEOPSMolecularModelling,48341Altenberge,Germany

Received14March2008;revised7July2008;accepted24August2008

Availableonline5September2008EditedbyRobertB.Russell

AbstractAcomputationalapproachtopredictstructuresofrhodopsin-likeGprotein-coupledreceptors(GPCRs)ispre-sentedandevaluatedbycomparisontotheX-raystructuralmod-els.Bycombiningsequencealignment,therhodopsincrystalstructure,andpointmutationdataontheb2adrenoreceptor(b2ar),wepredicta(À)-epinephrine-boundcomputationalmodeloftheb2adrenoreceptor.ThemodelisevaluatedbymoleculardynamicssimulationsandbycomparisonwiththerecentX-raystructuresofb2ar.Theoverallcorrespondencebetweenthepre-dictedandtheX-raystructuralmodelishigh.Especiallythepre-dictionoftheligandbindingsiteisaccurate.Thisshowsthattheproposeddynamichomologymodellingapproachcanbeusedtocreatereasonablemodelsfortheunderstandingofstructureanddynamicsofotherrhodopsin-likeGPCRs.

Ó2008PublishedbyElsevierB.V.onbehalfoftheFederationofEuropeanBiochemicalSocieties.

Keywords:Moleculardynamicssimulation;GPCR;Homologymodel;Beta(2)adrenoreceptor;Epinephrine;Ligandbinding

Inthepresentedapproachwecombinebinaryandmultiplesequencealignment,structuralfeaturesofrhodopsinandpointmutationdataon(À)-epinephrine-b2arinteraction[20]tocre-ateahomologymodel,calledB2AR,whichissubjectedtofreeMDsimulationsinanexplicitmembrane/solventenvironment.Incontrasttodockingofligandsintostatichomologymodels[11–14]orstaticmodelsfromframesoutofMDsimulations[16–19],B2ARcontainsanepinephrinemoleculeinourap-proachduringthewholesimulationperiod.Insodoingwewanttoevaluatethequalityofpossiblebindingmodespro-posedbyexperimentaldata[20].Wethenevaluatestructural,functionalandepinephrinebindingfeaturesofthedynamicmodelbycomparisonwiththehumanb2arcrystalstructures.Asthetwostructuresavailablearerathersimilar,wefocusonthecomparisonwiththePDBstructure2RH1[6],whichistheonewiththehighestresolution.

2.Materialsandmethods

2.1.Sequencealignment

Ratb2ar(UniProtKBaccessionnumberP10608)wassubjectedtobinaryalignmentwithbovinerhodopsin(P02699)usingBLAST(BLO-SUM62matrix)[21].FortheadditionalmultiplesequencecomparisonwithClustalW[22],theclassAGPCRsequencesP34971,P30546,P08911,andQ9H205wereincludedinthealignment.Thebinaryalign-mentwasusedasthebasisformodelling.Themultiplealignmentwasusedtocross-checkifmotivesfoundtobeconservedinthebinaryalign-mentcouldberegardedasbeingmodular.Sequencepartswiththesameresultsinbothalignmentsandcontainingthehighestconservedhelicalresidues[23]werede nedas‘‘anchorgroups’’.Gapswithinhelicalre-gionsweremovedtoloopregionsbyshiftingthesequencetowardsthenextanchorgroup.Furthermoreintraproteinhydrogenbondsandsaltbridges,andthepositioningofpositivecharges(Arg,Lys,His)atthelevelofphosphategroupswithinthemembraneweretakenintoconsiderationasstructuralrestraints.Hydrophilicresidueswithintheheptahelicaltransmembranedomainwereplacedatpositionsinwhichtheywereorientedtowardstheproteincore.Thesequenceofextracellularloop2(el2)wasshiftedtoallowtheformationofadisul-phidebondbetweenCys106(helixIII)andCys184(el2),asseenintherhodopsincrystalstructures.DuetotheirstructuralimportancefortheC-terminalendofhelixVIandtheel2,substitutionofTrp175andArg177byanalogousresiduesTrp173andArg175wasensured.Assmallligandbindingandstructuralchangesduringactivationtakeplacewithinthe7TMdomain[24–26],N-/C-terminaldomainsandintracellularloop3(il3)wereleftoutformodelling.

2.2.Homologymodelling

ChainAofrhodopsincrystalstructure1U19[4]wasusedasabasisstructure.Internalwatermoleculeswereignoredduringmodelbuild-ing.AspalmitylresiduesattheendofhelixVIIIarelocatedontheproteinsurface,formingtheproteindimerisationinterface[6],and

1.Introduction

GPCRsformthelargestgroupofmembranereceptorsandshareacommonstructuralmotifofseventransmembranea-helices(7TMdomain)[1].Membersofthisproteinfamilyarethetargetofmorethan50%ofdrugssoldworldwide[2].Milestonesinunderstandingoftheirmolecularreactionmech-anismsarethedeterminationsoftheX-raystructuresfortwoGPCRs:thevisualreceptorrhodopsin[3,4],andveryrecentlytheb2adrenoreceptor(b2ar)[5,6].TherhodopsinstructuresstimulatedtheoreticalstudiesinwhichitwasusedasthemajortemplatetoexplorestructuralfeaturesandmechanismsofclassAreceptors[1,7–10],includingb2ar[11–14]andotherGPCRs.OurgoalhereistosetupcomputationalmodelsofclassAGPCRsviahomologymodelling.Theyshouldprovideinsightintotheirstructureanddynamicpropertiesinbiologi-calmembranesviaunrestrained(free)moleculardynamics(MD)simulations.Sofar,thisgoalhasbeenapproachedinvariousways[15–19],butduetothelackofinformationonasecondGPCRstructure,theaccuracyofthestructurepredic-tionwasunabletobeassessed.Therecentpublicationofthe rstb2arcrystalstructurenowallowsustoevaluatesuchGPCRmodellingprocedures.

Correspondingauthor.Fax:+492343214626.E-mailaddress:gerwert@bph.rub.de(K.Gerwert).

*

0014-5793/$34.00Ó2008PublishedbyElsevierB.V.onbehalfoftheFederationofEuropeanBiochemicalSocieties.doi:10.1016/j.febslet.2008.08.022

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

3336

glycosylationsdonotin uencetheligandbindingofb2ar[27],post-translationalmodi cationswerenottakenintoaccountformodelling.SequencereplacementwasperformedwithSCWRL[28].RemainingstericalclasheswereremovedwithMOBY[29].Sequencegaps/insertsintheloopregionswereresolvedbyaddition/removalofaminoacids,followedbyshortperiodsofsimulatedannealing(5ps),energymini-misationoftherespectiveloopsandfullmodelminimisation.2.3.Additionofepinephrine

TheminimalenergystructureandatomicchargesofepinephrineweredeterminedbyGAUSSIAN03[30]vacuumDFTcalculationswithB3LYP/6-31++G(d,p)andRESPatomicchargescalculation[31].TheproteinmodelwascheckedforinternalcavitieswithMOBY.Epinephrineinitsvacuumminimalenergystructurewasplacedintotheonlycavitylargeenoughtocontaintheligandaccordingtohydro-genbondcontactsdeterminedinpointmutationanalysis[20].Thesecontactswere:Asp113/ammoniummoiety;Asn293/bhydroxylgroup;Ser203,Ser204/metahydroxylgroup;andSer207/parahydroxylgroup.Epinephrinewassubjectedtoamolecularmechanicssteepestdescentminimisationwithdistancerestraintsonpolarresiduesinproteinsidechainsandligandmentionedabove,followedbyminimisationofthe

.Theresultingprotein/li-ligandplussurroundingresidueswithin4A

gandmodelwasusedinthefollowingMDsimulations.

2.4.Molecularmechanicssimulationsanddataanalysis

SimulationswerecarriedoutwithGROMACS3.3[32]bymergingtheGROMOS96force eldandlipidparametersofBergeretal.[33,34]accordingtoSchlegeletal.[9].Allacidic/basicsidechainswerefullychargedexceptAsp79,whichisprotonatedinrhodopsin[35].Atopologyfor(À)-epinephrinewasobtainedfromthePRODRGserver[36]withtheatomicchargesmentionedabove.Theprotein/ligandmodelwasintroducedintoanequilibratedbilayerof256POPCmol-ecules,surroundedbya154mMNaClsolution,followingtheproce-dureofKandtetal.[37].Theresultingsystemcontains68770atoms.Aftersystemequilibration,trajectorieswithalengthof10nswererecorded.Ananalysisofthetrajectories(energies,rootmeansquaredisplacement(RMSD)and uctuations(RMSF)ofatomcoor-dinates)wasperformedusingGROMACS[29]andMOBY[32]anal-ysistools.Therootmeansquare(RMS)deviationofthecrystalstructure2RH1wascalculatedfromtheBfactorsgiveninthecrystalstructure leby

r Bi

ri¼

8p2

S.Wolfetal./FEBSLetters582(2008)3335–3342

withrbeingtherootmeansquaredeviationofthepositionofatomi,andBtherespectiveBfactor.

3.Results

3.1.Equilibrationofmodelstructure

AnalysisbyMOBYcon rmedthatthetotalsystemenergy(Etot)droppedtoits nalvalueduringunrestrainedMDsim-ulationafter6ns.Thetotalenergyoftheprotein(Eprot)reachedaminimumafter8ns.Theseventransmembraneheli- fromthecesremainedwithinanCaatomRMSDof2.3A

startingstructure.Incomparisonto2RH1,itrosetoanaver- within1ns,andclimbedonlyslightlytoanagevalueof2.5A

after7ns.BecauseoftheCa-RMSDaveragevalueof2.8A

comparisonwith2RH1,wetookthelast3nsofthetrajectoryintoaccountforfurtheranalysis.Duringthisperiod,theloopregionsshowedpronouncedmovementswitharesultingrootmeansquare uctuation(RMSF)ofCaatomcoordinatesof

,whilethetransmembranehelicesremainedstable1.2–2.0A

).inposition(RMSFof0.5–0.8A

http://www.77cn.com.cnparisonofX-rayandmodelstructure

InFig.1theX-raystructureandthemean3-DstructureofourB2ARmodelduringthelast3nsofMDsimulationarecomparedwitheachother.TheRMSDresolvedforeachCaatomofthemodelledstructureisshown.Ahighcorrespon-denceisfoundforthepositionsoftheseventransmembranehelices(I-VII).Onehundredandthirtyeightof198(70%)Ca

oftheatomsofthetransmembranehelicesarewithin2.0A

crystalstructure.Thereof75(38%)arewithintheRMSdevia-tioncalculatedfromtheBfactorsofatomcoordinatesinthecrystalstructureandthuswithinthestatisticalsigni canceofthe2RH1coordinates(errorbaroftheB2ARmodelinFig.1containstheRMSdeviationcurveoftheb2arcrystalstructuralmodel).Theyaremostlylocatedinthemiddleofthehelicalbundle.Furthermore,theadditionalhelix

VIII

Fig.1.Rootmeansquaredisplacement(RMSD)ofthemodelledB2ARstructurefrom2RH1foreachCaatom(red)duringlast3nsofMDsimulationcomparedtotherespectiverootmeansquare(RMS)deviationof2RH1calculatedfromBfactorsofthecrystalstructure(black).Therootmeansquare uctuation(RMSF)oftherespectiveB2ARmodelatomcoordinates,equallingtheRMSdeviationofthecoordinatesduringsimulation,isshownasblueerrorbars.Positionsoftheheliceshighlightedingrey.LettersA–FrefertositesshowninFig.2.The7TMmotifcloselyresemblesthecrystalstructure.

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

S.Wolfetal./FEBSLetters582(2008)3335–33423337

oftheb2arcrystal[3,6]isidenti edandwithinmax.3.0A

structuralmodel.

AscanbeseeninFig.2A,theremaining30%oftheheliceswheretherewasnomatchbetweenthemodelandtheX-raystructuremostlycorrespondtoproteinpartswithinterproteincontacts,whicharti ciallystabilisetheproteininthecrystal.Suchpositions(denotedA–FinFigs.1and2)clusterattheextracellularendsofhelicesI(A)andVI(E),andtheintracel-lularendsofI(B),III(C),andVII(F).WhilecontactsBandFconnecttwoproteinsintheproposedb2ardimer[6],Aisformedbetweentwob2arproteinsnotengagedinadimer.CandEareformedbetweenb2arandneighbouringT4lysozymeportionsofthefusionprotein.Foramoredetailedcomparisonofthetertiarystructureofthetwoadrenergicreceptormodels,their3-DstructuresaresuperposedinFig.2B–D.Incompar-isonwiththecrystalstructure,helixIshowsatiltof33°attheextracellularand13°attheintracellularhalf,withIle43being

thecentreofrotation,leadingtoapositionaldeviationof10A

oftheintracellularend.Furtheroftheextracellularand5A

shiftsinpositionsawayfromtheircorresponding2RH1posi- out-tionscanbeobservedfortheextracellularendsofII(5A

inwardmovement),VI(4A inwardwardmovement),III(4A

outwardmovement)andtheintracel-movement)andVII(4A

inwardmovement)andVII(5A outwardlularendsofIII(4A

movement).ThetiltingmovementofhelixIisfollowedbyashiftoftheextracellularendofIIandtheintracellularendofhelixVII,leadingtotheirdeviationfromthecrystalstruc-ture.Althoughtheil3ismissing,theintracellularendsofheli- ofandthereforeclosetocesVandVIremainwithin4A

2RH1.

Asexpected,largerdeviationsareobservedfortheloopre-gions(extracellularloops(el)1–3andintracellularloops(il)1–3),butremainreasonablyclosetothecrystalstructure.One

(peakDinexceptionisel2,whichisdisplacedbyupto20A

Fig.1).Aswestartwithrhodopsinasthebasisstructuralinformation,theloopispresentasabhairpinwhichentersdeepintotheproteincore.Asaresult,thedisulphidebondob-servedinrhodopsinbetweenel2andhelixIIIisreproducedinthemodelbyconnectingCys106(helixIII)andCys184(el2).However,crystalstructure[6]andpointmutationanalysisofb2ar[38]pointtotwodisulphidebonds,onebetweenCys106/Cys191,andtheotherbetweenCys184/Cys190.Be-causethedisulphidebondsarenotcorrectlyrecognizedinthemodel,theel2seemsthusincorrectlypredicted,whichcausesthelargestdeviationbetweenmodelandX-raystruc-ture.In2RH1,theel2formsahelixontopofthebindingcre-viceinsteadofthebhairpindeepinsidetherhodopsinmolecule.However,theel2formsvariouscontactstotheT4lysozymeportionofneighbouringproteinswithin2RH1(con-tactDinFig.2),andthusmaynotrevealitsnativestructureinthecrystalstructure.Inaddition,theloopisnotobservedinthesecondcrystalstructureavailable[5]becauseoflocaldisor-der.Duetoitsdisulphidelinktotheel2,themodelledextracel-lulartopofhelixIIIshowsashiftinpositionrelativeto2RH1aswell.

3.3.Dynamicfeatures

InFig.3featuresofthemodelledstructure,whichareeluci-datedbydynamics,areshownindetail.Themodelexhibitsasta-bleinterhelicalhydrogenbondbetweenthehighly

conserved

Fig.2.(A)Interproteincontactsinthecrystalstructuralmodel.PeakpositionsA–FinFig.1coincidewithcrystalcontactsin2RH1closetothe7TMmotif.(B–D)Comparisonofb2arcrystalstructureandmeanmodelstructureduringlast3nsofMDsimulation:side(B),extracellular(C)andintracellular(D)view.2RH1inblue,meanstructureofthedynamicmodelingreen.

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

3338S.Wolfetal./FEBSLetters582(2008)3335–3342

Fig.3.(A)InterhelicalcontactbetweenAsn51andSer319duringfreeMDsimulation.Right:Distanceofhydrogenbonddonor/acceptoratoms.Hydrogenbonddistanceshighlightedinyellow.Thebondremainsstableduringsimulation.(B)Comparisonofioniclockmotifs.Topleft:rhodopsincrystalstructure1U19.Topright:b2arcrystalstructure2RH1.Bottomleft:Modelafter1.5nsMDsimulation.Bottomright:DistanceplotofArg131andGlu268duringfreeMDsimulation.After0.5ns,theGlu/Argionpairlosesitsconnection.

Asn51inhelixIandSer319inhelixVII,ascanbeseeninFig.3A.Thisbondcreatesastablehydrophilicconnectionbetweenheli-cesIandVII.Theioniclock,involvingthehighlyconservedE/DRYmotifinhelixIII[5],opensduringfreeMDsimulationinniceagreementwiththeX-raystructure(Fig.3B):after0.5ns,Arg131(helixIII)losesitsconnectiontoGlu268(helixVI)be-causeofelectrostaticinteractionwithAsp130.

3.4.Ligandbindingsite

TheligandbindingsiteintheX-rayandthesimulatedmodelisshowninFig.4.TheB2ARmodelshowsonlyonecavity

withintheproteinlargeenoughtoaccommodatethenativeli-gandepinephrine.Residuesknowntobeinvolvedinligandbinding[20]arefoundatthesurfaceofthecavity.Itslocationandform twellthedemandsofepinephrine,andiscompara-bletotheonein2RH1.Deviationsinitsformareduetoadif-ferentboundaryformedbyel2inourmodelandthecrystalstructure,respectively:whileinthecrystalstructuralmodel2RH1,thenicheisopentotheextracellularmedium,itisclosedcompletelybytheloopinthesimulatedmodel.How-everthereisahighcorrespondenceinthepositionsoftheli-gands.Thesmalldi erencesobservedmayalsoberelated

to

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

S.Wolfetal./FEBSLetters582(2008)3335–33423339

Fig.4.ProteinsurfaceofthebindingpocketofB2ARinperpendicularview.Top:crystalstructure2RH1.Carazololshowninsticks.Bottom:dynamicmodelafter10ns.Epinephrineshowninsticks.Thebindingpocketinthemodeliscomparablebylocationandformtotheonein2RH1.

thee ectoftherespectiveligandonthereceptor:whileepi-nephrineusedinthemodelisanagonist,carazololinthecrys-talstructuralmodelisapartialinverseagonist,whichmightbindinaslightlydi erentmode.Wethereforefocusonade-tailedevaluationofthebindingmodeofepinephrineobservedinourB2ARmodel.Notethatdockingoftheagonistisopro-teronolinto2RH1[26]didnotresultinareasonablebindingmode,so2RH1mightbealesssuitedtargetforanalysisofagonistbindinginb2ar.Fig.5showsthecontactpatternbetweenepinephrineandB2ARduringfreeMDsimulationandarepresentativebindingstructureafter10nsofsimulation.Epinephrineformshydro-phobiccontactstoVal114,Phe289,andPhe290,andhydrogenbondstoAsp113,Asn293,Ser203,Ser204,andSer207.Allres-iduesareknownfrompointmutationanalysisandarepro-posedtointeractwithepinephrine[20,39,40].Concerningthepredicteddynamicsofthosecontacts:epinephrinelosesitsini-tialcontactstoSer207andAsn293duringthe rst0.1ns.

After

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

3340S.Wolfetal./FEBSLetters582(2008)3335–3342

Fig.5.Ligandbindingindynamichomologymodel.Left:Contactanalysisofreceptormodelandepinephrine.A:alkylgrouporacceptorcontact;B:proteinbackbonecontact;D:donorcontact;E:contactonedgeofring;H:heterogroup(epinephrine);R:contactonringplane;S:aminoacidsidechain.Epinephrineammoniummoietyandbhydroxylgroupcontactsremainstableduringsimulation.TheterminalmethylmoietyisintercalatedbetweenPhe289andPhe290.After5ns,thecatecholringformscontactstoVal114andPhe290.Thecatecholhydroxylgroupbondpatternchangesafter5ns.Right:representativebindingmodeofepinephrineat10nsoffreeMDsimulation.Epinephrineinorange.EpinephrineformshydrophobiccontactstoVal114,Phe290,andPhe290,andhydrophiliccontactstoAsp113,Asn293,Ser203,Ser204,andSer207.

5ns,thecatecholringofepinephrine ipstoapositionwhereitissandwichedbetweenVal114andPhe290.TheterminalmethylmoietyformsastablevanderWaalscontactwiththephenylringsofPhe289andPhe290.Unlikeinthestartingstructure,Asn293formsahydrogenbondtotheammoniummoietyinsteadofthebhydroxylgroupofepinephrine,whichengagesinahydrogenbondwithAsp113,attachingtheligandinaclamp-likemannertothecarboxylicresidue.Ser203andSer207formhydrogenbondcontactstothemetaandparahy-droxylgroupsofepinephrine,respectively,whileSer204bindsoccasionallytothemetahydroxylgroup.

4.Discussion

Whiledevelopingourmodellingtechniquebeforethepubli-cationoftheb2arcrystalstructures,weappreciatetheniceagreementbetweenourmodelandthecrystalstructureduringsimulation(seeFigs.1and2).Thedynamicmodelisparticu-larlyaccurateatreproducingthe7TMdomains,andtheligandbindingsite.The exibleloopsshowlargerdeviations,andel2,whichshowsthelargestdeviationbetweenrhodopsinandb2ar,togetherwiththeintramoleculardisulphidebondpatternisnotwellpredicted.However,thedeviationsbetweenthemodelledandtheX-raystructurearemostlyrelatedtointer-proteincontactsobservedinthecrystal.

Withrespecttodynamicproperties,amoredetailedcompar-isonshowsthatthecontactofAsn51andSer319connectinghelicesIandVIIiscontinuouslymaintained.Thiscontactcanalsobeobservedinthecrystalstructuralmodel[6].ItwasalreadyreportedtobestableinMDsimulationsonrho-dopsinandisbelievedtobeanimportantandconservedcon-nectionmotifofGPCRs[9].Asn51isthemostconservedresidueinHelixI[23].Pointmutationstudiesontheanalogousa1B-adrenergicreceptor[41]demonstratedthatamutationto

Alaresultsinaconstitutivelyactivereceptor,whilemutationtoAsp,whichcanexhibittheobservedhydrogenbondaswell,didnothaveanimpactonthereceptorfunction.Thehydrogenbondthereforeseemstobeimportantfordi erentiatingbe-tweenactiveandinactiveconformationofthereceptor.

ThesaltbridgeformedbetweenAsp130andArg131isimportantforkeepingthereceptorinaninactivestate.ItisrupturedviaaprotonationofAsp130uponreceptoractiva-tion.MutationofAsp130toAsnshowsanincreaseinbasalactivityofthereceptor[42],andArg131isakeyresidueforGproteinbinding[24].TheopeningofthesaltbridgebetweenArg131andGlu268,afeaturefoundintheX-raystructuresoftheb2adrenergicreceptor[5,6]butnotinrhodopsin,isaccu-ratelypredictedinthesimulatedmodel.Interestingly,pointmutationanalysisshowsthatmutationofGlu268toGlnorAlaresultsinaconstitutivelyactivereceptor,whichwasinter-pretedtoberelatedtoaninteractionbetweenAsp130/Arg131andGlu268[42].Crystalstructuresandsimulationalikepointtoaweakeningofthisinteractioninb2arcomparedtorhodop-sin.Itseemstoberelatedtothebasalactivityofb2ar,whilerhodopsindoesnotexhibitsuchactivityintheinactivestate[5].

AscanbeseeninFig.4,theligandbindingnicheisalsowellpredicted.Thebehaviourof(À)-epinephrineduringsimulation(showninFig.5)isremarkable:although(À)-epinephrinewasdockedintoamodelderivedfromrhodopsininaninactiveconformation,epinephrine tswellintothecavityappearingduringmodelbuildinginitslowest-energyconformation.Thereorientationafter5nspositionstheligandintoabindingmodewhichagreeswellwithpointmutationanalysisandli-gandbindingassays.EpinephrineestablishestheproposedhydrogenbondpatternwithSerines203,204and207men-tionedinRef.[43].Thesigni canceofhydrophobiccontactstoPhe289andPhe290foragonistbindingiscon rmedbypointmutationanalysis[40].Val114ishighlyconserved

in

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand bin

S.Wolfetal./FEBSLetters582(2008)3335–3342theadrenergicreceptorfamily,andmutationtoAlaleadstoa300-foldlossofagonista nitycomparedtoa3-foldlossofantagonista nity[39].Theclamp-likeconnectionofammo-niumgroup,bhydroxylgroupandAsp113sidechainwithAsn293bondedtotheammoniumgroupexplainspointmuta-tiondataonAsn293[44]inawaynotyetreported.AswitchofenantiomerwouldinterferewiththeconnectiontobothAsp113andAsn293.MutationofAsn293toleucineandsub-sequentlossofaconnectinghydrogenbondwoulda ectbind-ingnegativelyaswell.Furthermore,mutationofAsn293toAspwouldputanegativelychargedresidueclosetoAsp113andperturbthebindingpatternnecessaryforreceptoractiva-tion,whicho ersanexplanationtothedatainRef.[45].Asimilarring-likemodecanbeseenin2RH1betweencarazololandAsp113(seeFig.4DinRef.[26]).Dockingofepinephrineintotheproteinmodelthereforeleadstoadynamicbindingmodewhichisinagreementwithdataonligandbindingdur-ingfreeMDsimulation.

5.Conclusions

DuringfreeMDsimulation,ourdynamichomologymodelofB2ARreproducesstructuralanddynamicpropertieswhicharereportedfortheX-raystructuralmodel.Themodelexhib-itsacavitywhichmeetsthestericalandelectrostaticdemandsofthenativeagonistepinephrine,whichbindstotheproteininastablebindingmodeduringsimulation.Therefore,GPCRmodelscreatedthiswaycanbeusedtogaininsightintopro-teinstructure,andreceptor/ligandbindingdynamicswhicharenotaccessiblebystatichomologymodels.

Acknowledgements:TheauthorswouldliketothanktheNICJu¨lich(ProjectNo.hbo26)andtheRRZKKo¨lnforprovidingcomputingtime,andtheRuhr-UniversityResearchSchoolforadditionalfunding.TheyarealsogratefultoUlrikeKru¨gerandKeithHeysmondforimprovingtheEnglishofthemanuscript.AllmolecularimageswereproducedwithPyMOL[46].

AppendixA.Supplementarydata

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