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Autoinhibition and activation mechanisms.pdf

Autoinhibition and activation m…

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简介:本文档为《Autoinhibition and activation mechanismspdf》,可适用于自然科学领域

NATURE|VOL|MARCH|wwwnaturecomarticlesAutoinhibitionandactivationmechanismsoftheWiskott±AldrichsyndromeproteinAnnetteSKim,LazarosTKakalis,NorzehanAbdulManan,GraceALiuMichaelKRosenCellularBiochemistryandBiophysicsProgram,MemorialSloanKetteringCancerCenter,YorkAvenue,NewYork,NewYork,USATheRhofamilyGTPase,Cdc,canregulatetheactincytoskeletonthroughactivationofWiskott±Aldrichsyndromeprotein(WASP)familymembersActivationrelievesanautoinhibitorycontactbetweentheGTPasebindingdomainandthecarboxyterminalregionofWASPproteinsHerewereporttheautoinhibitedstructureoftheGTPasebindingdomainofWASP,whichcanbeinducedbytheCterminalregionorbyorganiccosolventsIntheautoinhibitedcomplex,intramolecularinteractionswiththeGTPasebindingdomainoccluderesiduesoftheCterminusthatregulatetheArpactinnucleatingcomplexBindingofCdctotheGTPasebindingdomaincausesadramaticconformationalchange,resultingindisruptionofthehydrophobiccoreandreleaseoftheCterminus,enablingitsinteractionwiththeactinregulatorymachineryThesedatashowthat`intrinsicallyunstructured'peptidessuchastheGTPasebindingdomainofWASPcanbeinducedintodistinctstructuralandfunctionalstatesdependingoncontextProteinsintheRassuperfamilyofsmallGTPasesregulateavastarrayofcellularprocesses,rangingfromgrowthanddifferentiationtovesicletraf®ckingMembersofthefamilycyclebetweenactiveGTPboundandinactiveGDPboundstates,andtherebyactasmolecularswitchesinsignaltransductionpathwayslinkingcellsurfacereceptorstointracellularmachineryTheRhosubfamilyofRaslikeGTPasesfunctionsinsignallingpathwaysthatcontrolcytoskeletalstructure,geneexpression,cellcycleprogressionandtumourmetastasisThethreeprincipalmembersofthisgroup,Cdc,RacandRho,areeachcapableofinducingdistinctactinbasedstructuresincells,®lopodia,lamellipodiaandstress®bres,respectivelyRhoactspredominantlythroughthebundlingofpreexistingactinstructures,whereasCdcandRacstimulatedenovoactinpolymerization,aprocessthatcanbemediatedbymembersoftheWiskott±Aldrichsyndromeprotein(WASP)familyMutationsinthearchetypalmemberofthisgroup,WASP,leadtotheWiskott±Aldrichsyndrome,apaediatricdisordercharacterizedbyactincytoskeletaldefectsinhaematopoieticcells,leadingclinicallytothrombocytopenia,eczemaandimmunode®ciencyTheWASPproteinssignaltothecytoskeletonthroughtheArpcomplex,anactinnucleatingassemblythatregulatesthestructureanddynamicsofactin®lamentnetworksattheleadingedgeofthecellWASPfamilymembers,includingWASP,thewidelyexpressedneuronalWASP(NWASP),severalScarWAVEproteinsandBeepLasp,arecharacterizedbyacentralsegmentthatisrichinprolinefollowedbyaCterminalVCAregion(forverprolinFigureAminoacidsequencesofhumanWASPandNWASPwithconsensussecondarystructureoftheGBDconstructsofWASPshownaboveInthesequence,theCRIBmotifisboxedinyellowandtheconservedArpbindingsequencesareboxedinredSecondarystructuresarecolouredaccordingtothefunctionallayersdescribedinthetext:yellow,layer(residues±)blue,layer(±)andred,layer(±)AlthoughinourinitialreportoftheCdc±WASPcomplexwedescribedGBDasformingabhairpinwithshortbstrands±and±,recentanalyseshaverevealedtwoadditionalbackbonehydrogenbondsbetweenGlyandAspandsheetlikeNOEsbetweenValandVal,consistentwiththelongerdemarcationResiduesinvolvedinthelayerlayerinterface()andinthelayersandlayer(®lledcircles)arehighlighted©MacmillanMagazinesLtdhomologyregion(VHR),co®linhomologyregion(CHR),andacidicregion(AR))TheVCAregionbindstheArpcomplexandactin,andtogethertheseinteractionsleadtoenhancementofactinnucleationbytheArpcomplexandrapidformationofnewactin®lamentsIncontrasttotheircommoncarboxytermini,theaminoterminiofWASPproteinsaredistinct,enablingfamilymemberstoactivateArpinresponsetodifferentupstreamsignalsTheNterminiofWASPandNWASPcontainaresiduesequence,termedaCRIB(forCdcRacinteractivebinding)motif,whichisfoundinanumberofotherwiseunrelatedeffectorsofCdcandRacBothWASPandNWASPbindactivatedCdcthroughaGTPasebindingdomain(GBD)thatconsistsoftheCRIBmotifandsurroundingsequencesWehavereportedthestructureofaCdc±WASPGBDcomplexwhichrevealsthattheconservedCRIBresiduesformalinearepitopeattheNterminusthatannealstothebstrandoftheGTPase,whereasCterminalresiduesfoldintoabhairpinandahelixthatpackagainstSwitchIandII(ref)InNWASP,theGBDisalsocapableofbindingtheCterminalVCAregion,resultinginautoinhibitionoftheArpstimulatoryactivityoftheprotein,BindingofCdcisbelievedtocauseastructuraltransitioninNWASPthatresultsindissociationofitsintramolecularcontactsandenhancedArpmediatedactinpolymerization,Thus,byactivatingNWASP,CdccontrolstheactivityoftheArpcomplexandsubsequentactindynamicsnecessaryforformationof®lopodiaHerewereportthat,likeNWASP,theWASPGBDcanbinditsownVCAdomaininamannerthatiscompetitivewithCdcAlthoughtheGBDislargelyunstructuredinthefreestate,bindingoftheVCAregionoradditionofsmallamountsoforganiccosolventinducesformationofacompact,foldeddomainstructureinresiduesimmediatelyCterminaltotheCRIBmotifInthisautoinhibitedstate,theCHRformsanamphipathichelixthatpacksagainstahydrophobicsurfaceoftheGBD,explainingitsdecreasedaf®nityforactinandtheArpcomplexNotably,thisfoldoftheGBDisstericallyincompatiblewiththeactivatedconformationseenintheCdccomplexComparisonoftheconformationsandbiochemicalpropertiesofthesetwoGBDstatesrevealsthestructuralandthermodynamicbasisofactivationofWASPfamilyproteinsbyCdcWASPGBDbindingtotheVCAregionTodeterminewhetherWASPandNWASPcanberegulatedsimilarly,weexaminedthebindingofaseriesofWASPVCAderivedfragmentstoseveralGBDconstructsTheVCAconstructswerepreparedasglutathioneStransferase(GST)fusionproteins,spanningresidues±(VCA),±(VCDA),±(DVCDA),±(CDA),±(C),±(VC)and±(VDC)BindingpartnersforthesesequencesincludedGBD(residues±,theminimalhighaf®nityCdcbindingdomain,),GBD(residues±),GBD(residues±)andGBD(residues±,lackingaportionoftheCRIBmotif)(Fig)ToidentifytheminimalGBDbindingregionoftheVCAdomain,wecarriedoutanaf®nitytitrationassaythatwasbasedonthesaturablechangeintryptophan¯uorescenceofGBDupontheadditionofthrombincleavedVCAconstructs(Figa)AllCHRcontainingconstructsboundGBDwithsimilaraf®nities,whereastheVDCpeptidethatlacksthelatterportionoftheCHRprovedunabletobindGBDTheseresultsindicatethattheCHRoftheVCAdomainisbothsuf®cientandnecessaryforinteractionwiththeWASPGBDWethensoughttoidentifytheminimalVCAbindingregionoftheGBDGBD,GBDandGBDallboundimmobilizedCHRcontainingconstructsequallywell,indicatingthatthebasicregionattheNterminusofGBDisnotrequiredforformationofthecomplex,incontrasttoprevioushypotheses(FigaFigb,lanesand)However,CterminalGBDresiduesareessentialforbinding,asGBDfailedtointeractsigni®cantlyintheGSTaf®nityassays(Figb,lanesand)TheseresultsindicatethatformationoftheintramolecularcomplexrequiressequencesoftheGBDthatareCterminaltotheCRIBmotif,butnottheentireCRIBmotifBindingoftheNWASPGBDtoeitherCdcortoVCApeptidesismutuallyexclusiveWASPbehavesinasimilarmanner:incubationoftheimmobilizedGSTC±GBDcomplexwithCdcGMPPNP(anonhydrolysableGTPanalogue)effectsreleaseoftheGBDintosolution(Figb,lanesand)CdcGDP,whichbindsWASPonlyweakly,doesnotdissociatetheGBDfromtheVCApeptides(Figb,lane)Thus,forbothWASPandNWASP,interactionsbetweentheGBDandVCAregionsareincompatiblewithCdcbinding,andGTPasemediateddisruptionofanautoinhibitorycomplexprobablyhasanimportantroleinsignallingtothecytoskeletonToelucidatethemechanismofWASPfamilyactivationbyCdc,weexaminedtheconformationalstatesofboththeunboundandVCAboundGBDInductionofafoldeddomainintheWASPGBDWeandothershavereportedthatisolatedGBDpeptidesofWASPdonotassumeasingle,discretestructureunderphysiologicarticlesNATURE|VOL|MARCH|wwwnaturecomGBD#CdcGBDKKKKMrCdcGMPPNPGST–VCAGST–CGBDGBDGBDCdc–––––––––––––BBBBSBBSVCAconstructs(µM)VCAconstructGBDconstructsGBDGBDGBDKd((µM)C∆A(GBD)±VC∆A(GBD)±∆VC∆A(GBD)±VC(GBD)±V∆C(GBD)ndC∆A(GBD)±GBD(∆FF)abcdVCA–VC∆A–∆VC∆A–C∆A–VC–V∆C–––C–––––TFE(vv)Tm(°C)()–Θ–(degcmdmol–)()FigureBiochemicalcharacterizationoftheWASPGBDa,Normalizedchangeintryptophan¯uorescenceofGBDandGBDonadditionofVCApeptidesDissociationconstant(Kd)wascalculatedasdescribedTryptophan¯uorescenceoftheGBDwasquenchedinallcasesby,(exceptinthecaseofVDC)uponbindingtothrombincleavedVCAconstructsthatlacktryptophannd,notdeterminedb,BindingofGBDconstructstoVCApeptides,andcompetitionbyCdcImmobilizedGST±VCA(lane)orGST±C(lanes±)wereincubatedwithGBDconstructs,followedbyCdcGDPorCdcGMPPNPasindicatedProteinsretainedonthebeads(`B'lanes)orreleasedinthesupernatant(`S'lanes)wereseparatedbySDS±PAGEandvisualizedbyCoomassiebluestainingBecauseGBDconstructsretainCoomassiebluepoorly(ASKandZAM,unpublisheddata),therelativegelbandintensitiescomparedwiththeGSTfusionconstructsdonotaccuratelyre¯ecttheirbindingstoichiometryc,Summaryofbindingdatad,EffectsofTFEadditiononGBDFarultravioletCDmolarellipticityrecordedatCatnm(®lledsquares)andmeltingtemperature(opensquares)ofmMGBDatdifferentconcentrations(vv)ofTFE©MacmillanMagazinesLtdconditionsinvitro,Asexpected,smallamountsofalcoholicsolvents(forexample,,,tri¯uoroethanol(TFE),methanol,isopropanol)causeamarkedincreaseinthehelicalcontentofthepeptideasindicatedbycirculardichroism(CD)spectra,whichreachesaplateauat,cosolvent(Figd)However,thisincreasedhelicitycorrelateswithasigni®cantincreaseindenaturationtemperatureofthepeptide,whichrisesfromCintheabsenceoforganicsolventstoamaximumvalueofCat±TFE(Figd),a®ndingmoreconsistentwithinductionofafoldedcorethanwithinductionofrandomhelicalcoilsWereasonedthatthisfoldmightmimicthatoftheGBD±VCAcomplexTheNMRspectraofthesestatessupportedthishypothesisMultidimensionalspectraofaqueousGBDshowpoordispersionofamideandaliphaticprotonchemicalshifts,withlargevariationsinpeakshapeandintensity(seeSupplementaryInformation)Only,oftheexpectedbackboneamidepeaksarevisibleinHNHSQC(heteronuclearsinglequantumcoherence)spectra,andallamideprotonsexchangerapidlywithsolventHowever,thepeptidechainismonomericandrelativelycompactbasedonanalyticalultracentrifugationandgel®ltrationanalyses,respectively(notshown)Together,thesedatasuggestthatGBDsamplesmultiple,looselypackedconformationsinaqueoussolutionIncontrast,HNHSQCspectraofNGBDinthepresenceofTFEandofNGBDinthepresenceofsaturatingunlabelledVCA(seeSupplementaryInformation)revealamarkedincreaseinamidechemicalshiftdispersion,adecreaseinmostlinewidths,developmentofrelativeuniformityinpeakshapes,andappearanceofvirtuallyallexpectedbackboneamideresonancesUnderbothconditions,anumberofbackboneamideprotonsexchangeslowlywithsolvent(withTFE,withVCApeptide),basedontheirobservationinHNHSQCspectraoflyophilizedsamplesfreshlydissolvedinDOHTFEorinDOSigni®cantly,amideandaliphaticresonancesarehighlysimilarinthetwosystems,includingHNHSQCcrosspeakpatternsandadistinctiveup®eldmethylresonanceatppm,whichsuggestspackingofthisgroupagainstthefaceofanaromaticringThisglobalsimilaritywaspreservedinspectraoftheminimalGBD±Ccomplex,withtheabsenceofonlyseveralintense,poorlydispersedsignalsrepresentingdisorderedresiduesThesefeaturesoftheGBDTFEandGBDVCANMRspectrastronglysuggestthatTFEandtheVCApeptideinduceacommondiscreteGBDfoldwithawellpackedhydrophobiccoreNotably,GBDspectrarecordedinthepresenceofCdcrevealaverydifferentcrosspeakpattern,andtheabsenceofthehigh®eldmethylresonance,indicatingthatthefoldofthepolypeptideisdistinctinthetwostates(seeSupplementaryInformation)AutoinhibitedstructureoftheWASPGBDTounderstandthenatureoftheautoinhibitedWASPconformationanditsrelationshiptothecomplexwithCdc,wedeterminedtheNMRsolutionstructuresofaGBD±VCAcomplexandtheGBDaloneinthepresenceofTFEIntheformercase,wecovalentlyjoinedtheGBDandCconstructsintoasinglechainthrougha¯exible(GlyGlySer)sequence(GBD±C)Thislinkedconstructwasmonomericbyanalyticalultracentrifugationandgel®ltration(notshown)CrosspeaksinHNHSQCspectraofGBD±CnearlysuperimposewithasubsetofpeaksinspectraofthelargerGBD±VCAcomplexandofawildtypeconstructspanningresidues±(notshown),indicatingthattheminimalconstructisrepresentativeoftheintramolecularcomplexinfulllengthWASPTablegivesstatisticsdescribingthe®nalsetofconvergedconformersforGBDinTFEandforGBD±CFigureshowsthebest®tsuperpositionsoftheconformersResiduesinthewellde®nedcoreoftheTFEstructure,±,showAÊrootmeansquaredeviation(rmsd)fromtheaveragecoordinatesforbackboneatoms,andAÊrmsdforallheavyatoms,whereasthecorresponding±and±regionsinthecomplexdisplayAÊrmsdforbackboneatomsandAÊrmsdforallheavyatomsInbothstructures,theNterminalresidues,whichencompasstheconservedCRIBmotif,shownolongrangenuclearOverhausereffects(NOEs)untilresidue,poorchemicalshiftdispersion,andnegativeorweaklypositivebackboneamideheteronuclearN{H}NOEs(notshown),indicatinghighlevelsofmobilityinsolutionIncontrast,Cterminalresidues±formaglobulardomainconsistingofashortibhairpin(±and±)andfourahelices(roughly±,a±,a±,aand±,aseeFigs,a,c)Inthesefourhelices,thefwanglesforthetwostructuresarenearlyidentical,withminorexceptionsattheinitialand®nalresiduesofaandaTheintramolecularcomplexalsocontainsa®fthhelix,a,whichencompassesresiduesinthe®rsthalfofthedesignatedCHR(±)ResiduesafteralsoappeartobemobileinsolutiononthebasisofthecriterialistedaboveThus,althoughresidues±areclearlyneededforhighaf®nitybindingtotheGBD(Figa±c),theircontributiontobindingenergymustoccurthroughmechanismsthatdonotrelyonorderingofthepeptidebackboneThebhairpin,demarcatedbytwobackbonehydrogenbondsbetweenAspandGly,formsa,anglewiththeahelix,creatinganLshapedamphipathicsurface(Figc)Ashortturnatresidues±separatestheendsofthehairpinanddirectstheValsidechaintowardthecoreofthedomainTheturnisstabilizedbyabackbonehydrogenbondbetweentheAspcarbonylandtheAsnamide,andbyhydrophobicpackingofValagainstLeuandThr(Figc)This®rstlayerofelementspacksagainstasecondlayerofthreeroughlyplanarhelicesthatformthreesidesofarectangle,withaalignedwiththestrandsofthebhairpinandaandalyingperpendiculartothemAhydrogenbondbetweentheamidenitrogenofPheandthecarbonyloxygenofLeuordersthebhairpinagainsttheaandahelicesandprovidesaCterminalcapforaOtherwise,interactionsbetweenthesetwolayersaremostlystabilizedbyhydrophobiccontacts,dominatedbyinteractionsbetweenTrparticlesNATURE|VOL|MARCH|wwwnaturecomFigureStereoviewsofthebest®tsuperpositionsofthebackbone(N,Ca,C)atomsofthe®nalNMRstructuresofGBDinTFE(a)andGBD±C(b)Residues±ina,and±,±andthe(GlyGlySer)linkerinbarenotde®nedbytheNMRdata,appearmobileinsolutionandhavebeenomittedforclarityStructurallayersarecolouredasinFig©MacmillanMagazinesLtdandPheofthebhairpinValandLeuofthebaturnLeu,Leu,PheandIleofaLeuofaIle,TyrandIleofaandLeu,ValandMetofaTheclosecorrespondenceofthexrotamersoftheseresiduesinthesolventinducedconformationandtheintramolecularcomplex,withtheexceptiononlyofTyr,showsthatthehydrophobiccoresofthetwostructurespackinanearlyidenticalfashionThisjuxtapositionofthe®rstandsecondlayersburiesasurfaceareaof,AÊandplacesoneofthemethylgroupsofLeuabovethearomaticringofPhe,accountingforthelargeup®eldchemicalshiftofitsmethylprotons(seeSupplementaryInformation)Theoppositefaceofthesecondlayerconsistsofabedofhydrophobicsidechainsemanatingfromhelicesa±,whichareexposedtosolventintheTFEstructureIntheintramolecularcomplex,theahelixoftheCHRliesnestledonthisbed,formingathirdstructurallayerandcompletingthecoreofthedomainResiduesLeu,LeuandMetofthehydrophobicfaceoftheCHRhelix®llthreedeeppocketsonthelayersurface,withadditionalextensivecontactsmadebyVal,Met,ValandArg(Figd)Thesecontactsbury,AÊofaccessiblesurfacebetweenlayerandlayersandTheorientationoftheahelixrelativetothehydrophobicplaneisstabilizedbybothelectrostaticinteractionsbetweenthesidechainsofArgofthehelixandGlulocatedatthetopofthea±aturn,andhydrophobicinteractionsbetweenL

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