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首页 Autoinhibition and activation mechanisms.pdf

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,主题内容包含NATURE|VOL|MARCH|wwwnaturecomarticlesAutoinhibitionandactivationmechanisms符等。

NATURE|VOL|MARCH|wwwnaturecomarticlesAutoinhibitionandactivationmechanismsoftheWiskottAldrichsyndromeproteinAnnetteSKim,LazarosTKakalis,NorzehanAbdulManan,GraceALiuMichaelKRosenCellularBiochemistryandBiophysicsProgram,MemorialSloanKetteringCancerCenter,YorkAvenue,NewYork,NewYork,USATheRhofamilyGTPase,Cdc,canregulatetheactincytoskeletonthroughactivationofWiskottAldrichsyndromeprotein(WASP)familymembersActivationrelievesanautoinhibitorycontactbetweentheGTPasebindingdomainandthecarboxyterminalregionofWASPproteinsHerewereporttheautoinhibitedstructureoftheGTPasebindingdomainofWASP,whichcanbeinducedbytheCterminalregionorbyorganiccosolventsIntheautoinhibitedcomplex,intramolecularinteractionswiththeGTPasebindingdomainoccluderesiduesoftheCterminusthatregulatetheArpactinnucleatingcomplexBindingofCdctotheGTPasebindingdomaincausesadramaticconformationalchange,resultingindisruptionofthehydrophobiccoreandreleaseoftheCterminus,enablingitsinteractionwiththeactinregulatorymachineryThesedatashowthat`intrinsicallyunstructured'peptidessuchastheGTPasebindingdomainofWASPcanbeinducedintodistinctstructuralandfunctionalstatesdependingoncontextProteinsintheRassuperfamilyofsmallGTPasesregulateavastarrayofcellularprocesses,rangingfromgrowthanddifferentiationtovesicletrafckingMembersofthefamilycyclebetweenactiveGTPboundandinactiveGDPboundstates,andtherebyactasmolecularswitchesinsignaltransductionpathwayslinkingcellsurfacereceptorstointracellularmachineryTheRhosubfamilyofRaslikeGTPasesfunctionsinsignallingpathwaysthatcontrolcytoskeletalstructure,geneexpression,cellcycleprogressionandtumourmetastasisThethreeprincipalmembersofthisgroup,Cdc,RacandRho,areeachcapableofinducingdistinctactinbasedstructuresincells,lopodia,lamellipodiaandstressbres,respectivelyRhoactspredominantlythroughthebundlingofpreexistingactinstructures,whereasCdcandRacstimulatedenovoactinpolymerization,aprocessthatcanbemediatedbymembersoftheWiskottAldrichsyndromeprotein(WASP)familyMutationsinthearchetypalmemberofthisgroup,WASP,leadtotheWiskottAldrichsyndrome,apaediatricdisordercharacterizedbyactincytoskeletaldefectsinhaematopoieticcells,leadingclinicallytothrombocytopenia,eczemaandimmunodeciencyTheWASPproteinssignaltothecytoskeletonthroughtheArpcomplex,anactinnucleatingassemblythatregulatesthestructureanddynamicsofactinlamentnetworksattheleadingedgeofthecellWASPfamilymembers,includingWASP,thewidelyexpressedneuronalWASP(NWASP),severalScarWAVEproteinsandBeepLasp,arecharacterizedbyacentralsegmentthatisrichinprolinefollowedbyaCterminalVCAregion(forverprolinFigureAminoacidsequencesofhumanWASPandNWASPwithconsensussecondarystructureoftheGBDconstructsofWASPshownaboveInthesequence,theCRIBmotifisboxedinyellowandtheconservedArpbindingsequencesareboxedinredSecondarystructuresarecolouredaccordingtothefunctionallayersdescribedinthetext:yellow,layer(residues)blue,layer()andred,layer()AlthoughinourinitialreportoftheCdcWASPcomplexwedescribedGBDasformingabhairpinwithshortbstrandsand,recentanalyseshaverevealedtwoadditionalbackbonehydrogenbondsbetweenGlyandAspandsheetlikeNOEsbetweenValandVal,consistentwiththelongerdemarcationResiduesinvolvedinthelayerlayerinterface()andinthelayersandlayer(lledcircles)arehighlightedMacmillanMagazinesLtdhomologyregion(VHR),colinhomologyregion(CHR),andacidicregion(AR))TheVCAregionbindstheArpcomplexandactin,andtogethertheseinteractionsleadtoenhancementofactinnucleationbytheArpcomplexandrapidformationofnewactinlamentsIncontrasttotheircommoncarboxytermini,theaminoterminiofWASPproteinsaredistinct,enablingfamilymemberstoactivateArpinresponsetodifferentupstreamsignalsTheNterminiofWASPandNWASPcontainaresiduesequence,termedaCRIB(forCdcRacinteractivebinding)motif,whichisfoundinanumberofotherwiseunrelatedeffectorsofCdcandRacBothWASPandNWASPbindactivatedCdcthroughaGTPasebindingdomain(GBD)thatconsistsoftheCRIBmotifandsurroundingsequencesWehavereportedthestructureofaCdcWASPGBDcomplexwhichrevealsthattheconservedCRIBresiduesformalinearepitopeattheNterminusthatannealstothebstrandoftheGTPase,whereasCterminalresiduesfoldintoabhairpinandahelixthatpackagainstSwitchIandII(ref)InNWASP,theGBDisalsocapableofbindingtheCterminalVCAregion,resultinginautoinhibitionoftheArpstimulatoryactivityoftheprotein,BindingofCdcisbelievedtocauseastructuraltransitioninNWASPthatresultsindissociationofitsintramolecularcontactsandenhancedArpmediatedactinpolymerization,Thus,byactivatingNWASP,CdccontrolstheactivityoftheArpcomplexandsubsequentactindynamicsnecessaryforformationoflopodiaHerewereportthat,likeNWASP,theWASPGBDcanbinditsownVCAdomaininamannerthatiscompetitivewithCdcAlthoughtheGBDislargelyunstructuredinthefreestate,bindingoftheVCAregionoradditionofsmallamountsoforganiccosolventinducesformationofacompact,foldeddomainstructureinresiduesimmediatelyCterminaltotheCRIBmotifInthisautoinhibitedstate,theCHRformsanamphipathichelixthatpacksagainstahydrophobicsurfaceoftheGBD,explainingitsdecreasedafnityforactinandtheArpcomplexNotably,thisfoldoftheGBDisstericallyincompatiblewiththeactivatedconformationseenintheCdccomplexComparisonoftheconformationsandbiochemicalpropertiesofthesetwoGBDstatesrevealsthestructuralandthermodynamicbasisofactivationofWASPfamilyproteinsbyCdcWASPGBDbindingtotheVCAregionTodeterminewhetherWASPandNWASPcanberegulatedsimilarly,weexaminedthebindingofaseriesofWASPVCAderivedfragmentstoseveralGBDconstructsTheVCAconstructswerepreparedasglutathioneStransferase(GST)fusionproteins,spanningresidues(VCA),(VCDA),(DVCDA),(CDA),(C),(VC)and(VDC)BindingpartnersforthesesequencesincludedGBD(residues,theminimalhighafnityCdcbindingdomain,),GBD(residues),GBD(residues)andGBD(residues,lackingaportionoftheCRIBmotif)(Fig)ToidentifytheminimalGBDbindingregionoftheVCAdomain,wecarriedoutanafnitytitrationassaythatwasbasedonthesaturablechangeintryptophanuorescenceofGBDupontheadditionofthrombincleavedVCAconstructs(Figa)AllCHRcontainingconstructsboundGBDwithsimilarafnities,whereastheVDCpeptidethatlacksthelatterportionoftheCHRprovedunabletobindGBDTheseresultsindicatethattheCHRoftheVCAdomainisbothsufcientandnecessaryforinteractionwiththeWASPGBDWethensoughttoidentifytheminimalVCAbindingregionoftheGBDGBD,GBDandGBDallboundimmobilizedCHRcontainingconstructsequallywell,indicatingthatthebasicregionattheNterminusofGBDisnotrequiredforformationofthecomplex,incontrasttoprevioushypotheses(FigaFigb,lanesand)However,CterminalGBDresiduesareessentialforbinding,asGBDfailedtointeractsignicantlyintheGSTafnityassays(Figb,lanesand)TheseresultsindicatethatformationoftheintramolecularcomplexrequiressequencesoftheGBDthatareCterminaltotheCRIBmotif,butnottheentireCRIBmotifBindingoftheNWASPGBDtoeitherCdcortoVCApeptidesismutuallyexclusiveWASPbehavesinasimilarmanner:incubationoftheimmobilizedGSTCGBDcomplexwithCdcGMPPNP(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)CA(GBD)VCA(GBD)VCA(GBD)VC(GBD)VC(GBD)ndCA(GBD)GBD(FF)abcdVCA–VCA–VCA–CA–VC–VC–––C–––––TFE(vv)Tm(C)()–Θ–(degcmdmol–)()FigureBiochemicalcharacterizationoftheWASPGBDa,NormalizedchangeintryptophanuorescenceofGBDandGBDonadditionofVCApeptidesDissociationconstant(Kd)wascalculatedasdescribedTryptophanuorescenceoftheGBDwasquenchedinallcasesby,(exceptinthecaseofVDC)uponbindingtothrombincleavedVCAconstructsthatlacktryptophannd,notdeterminedb,BindingofGBDconstructstoVCApeptides,andcompetitionbyCdcImmobilizedGSTVCA(lane)orGSTC(lanes)wereincubatedwithGBDconstructs,followedbyCdcGDPorCdcGMPPNPasindicatedProteinsretainedonthebeads(`B'lanes)orreleasedinthesupernatant(`S'lanes)wereseparatedbySDSPAGEandvisualizedbyCoomassiebluestainingBecauseGBDconstructsretainCoomassiebluepoorly(ASKandZAM,unpublisheddata),therelativegelbandintensitiescomparedwiththeGSTfusionconstructsdonotaccuratelyreecttheirbindingstoichiometryc,Summaryofbindingdatad,EffectsofTFEadditiononGBDFarultravioletCDmolarellipticityrecordedatCatnm(lledsquares)andmeltingtemperature(opensquares)ofmMGBDatdifferentconcentrations(vv)ofTFEMacmillanMagazinesLtdconditionsinvitro,Asexpected,smallamountsofalcoholicsolvents(forexample,,,triuoroethanol(TFE),methanol,isopropanol)causeamarkedincreaseinthehelicalcontentofthepeptideasindicatedbycirculardichroism(CD)spectra,whichreachesaplateauat,cosolvent(Figd)However,thisincreasedhelicitycorrelateswithasignicantincreaseindenaturationtemperatureofthepeptide,whichrisesfromCintheabsenceoforganicsolventstoamaximumvalueofCatTFE(Figd),andingmoreconsistentwithinductionofafoldedcorethanwithinductionofrandomhelicalcoilsWereasonedthatthisfoldmightmimicthatoftheGBDVCAcomplexTheNMRspectraofthesestatessupportedthishypothesisMultidimensionalspectraofaqueousGBDshowpoordispersionofamideandaliphaticprotonchemicalshifts,withlargevariationsinpeakshapeandintensity(seeSupplementaryInformation)Only,oftheexpectedbackboneamidepeaksarevisibleinHNHSQC(heteronuclearsinglequantumcoherence)spectra,andallamideprotonsexchangerapidlywithsolventHowever,thepeptidechainismonomericandrelativelycompactbasedonanalyticalultracentrifugationandgelltrationanalyses,respectively(notshown)Together,thesedatasuggestthatGBDsamplesmultiple,looselypackedconformationsinaqueoussolutionIncontrast,HNHSQCspectraofNGBDinthepresenceofTFEandofNGBDinthepresenceofsaturatingunlabelledVCA(seeSupplementaryInformation)revealamarkedincreaseinamidechemicalshiftdispersion,adecreaseinmostlinewidths,developmentofrelativeuniformityinpeakshapes,andappearanceofvirtuallyallexpectedbackboneamideresonancesUnderbothconditions,anumberofbackboneamideprotonsexchangeslowlywithsolvent(withTFE,withVCApeptide),basedontheirobservationinHNHSQCspectraoflyophilizedsamplesfreshlydissolvedinDOHTFEorinDOSignicantly,amideandaliphaticresonancesarehighlysimilarinthetwosystems,includingHNHSQCcrosspeakpatternsandadistinctiveupeldmethylresonanceatppm,whichsuggestspackingofthisgroupagainstthefaceofanaromaticringThisglobalsimilaritywaspreservedinspectraoftheminimalGBDCcomplex,withtheabsenceofonlyseveralintense,poorlydispersedsignalsrepresentingdisorderedresiduesThesefeaturesoftheGBDTFEandGBDVCANMRspectrastronglysuggestthatTFEandtheVCApeptideinduceacommondiscreteGBDfoldwithawellpackedhydrophobiccoreNotably,GBDspectrarecordedinthepresenceofCdcrevealaverydifferentcrosspeakpattern,andtheabsenceofthehigheldmethylresonance,indicatingthatthefoldofthepolypeptideisdistinctinthetwostates(seeSupplementaryInformation)AutoinhibitedstructureoftheWASPGBDTounderstandthenatureoftheautoinhibitedWASPconformationanditsrelationshiptothecomplexwithCdc,wedeterminedtheNMRsolutionstructuresofaGBDVCAcomplexandtheGBDaloneinthepresenceofTFEIntheformercase,wecovalentlyjoinedtheGBDandCconstructsintoasinglechainthroughaexible(GlyGlySer)sequence(GBDC)Thislinkedconstructwasmonomericbyanalyticalultracentrifugationandgelltration(notshown)CrosspeaksinHNHSQCspectraofGBDCnearlysuperimposewithasubsetofpeaksinspectraofthelargerGBDVCAcomplexandofawildtypeconstructspanningresidues(notshown),indicatingthattheminimalconstructisrepresentativeoftheintramolecularcomplexinfulllengthWASPTablegivesstatisticsdescribingthenalsetofconvergedconformersforGBDinTFEandforGBDCFigureshowsthebesttsuperpositionsoftheconformersResiduesinthewelldenedcoreoftheTFEstructure,,showAÊrootmeansquaredeviation(rmsd)fromtheaveragecoordinatesforbackboneatoms,andAÊrmsdforallheavyatoms,whereasthecorrespondingandregionsinthecomplexdisplayAÊrmsdforbackboneatomsandAÊrmsdforallheavyatomsInbothstructures,theNterminalresidues,whichencompasstheconservedCRIBmotif,shownolongrangenuclearOverhausereffects(NOEs)untilresidue,poorchemicalshiftdispersion,andnegativeorweaklypositivebackboneamideheteronuclearN{H}NOEs(notshown),indicatinghighlevelsofmobilityinsolutionIncontrast,Cterminalresiduesformaglobulardomainconsistingofashortibhairpin(and)andfourahelices(roughly,a,a,aand,aseeFigs,a,c)Inthesefourhelices,thefwanglesforthetwostructuresarenearlyidentical,withminorexceptionsattheinitialandnalresiduesofaandaTheintramolecularcomplexalsocontainsafthhelix,a,whichencompassesresiduesinthersthalfofthedesignatedCHR()ResiduesafteralsoappeartobemobileinsolutiononthebasisofthecriterialistedaboveThus,althoughresiduesareclearlyneededforhighafnitybindingtotheGBD(Figac),theircontributiontobindingenergymustoccurthroughmechanismsthatdonotrelyonorderingofthepeptidebackboneThebhairpin,demarcatedbytwobackbonehydrogenbondsbetweenAspandGly,formsa,anglewiththeahelix,creatinganLshapedamphipathicsurface(Figc)AshortturnatresiduesseparatestheendsofthehairpinanddirectstheValsidechaintowardthecoreofthedomainTheturnisstabilizedbyabackbonehydrogenbondbetweentheAspcarbonylandtheAsnamide,andbyhydrophobicpackingofValagainstLeuandThr(Figc)Thisrstlayerofelementspacksagainstasecondlayerofthreeroughlyplanarhelicesthatformthreesidesofarectangle,withaalignedwiththestrandsofthebhairpinandaandalyingperpendiculartothemAhydrogenbondbetweentheamidenitrogenofPheandthecarbonyloxygenofLeuordersthebhairpinagainsttheaandahelicesandprovidesaCterminalcapforaOtherwise,interactionsbetweenthesetwolayersaremostlystabilizedbyhydrophobiccontacts,dominatedbyinteractionsbetweenTrparticlesNATURE|VOL|MARCH|wwwnaturecomFigureStereoviewsofthebesttsuperpositionsofthebackbone(N,Ca,C)atomsofthenalNMRstructuresofGBDinTFE(a)andGBDC(b)Residuesina,and,andthe(GlyGlySer)linkerinbarenotdenedbytheNMRdata,appearmobileinsolutionandhavebeenomittedforclarityStructurallayersarecolouredasinFigMacmillanMagazinesLtdandPheofthebhairpinValandLeuofthebaturnLeu,Leu,PheandIleofaLeuofaIle,TyrandIleofaandLeu,ValandMetofaTheclosecorrespondenceofthexrotamersoftheseresiduesinthesolventinducedconformationandtheintramolecularcomplex,withtheexceptiononlyofTyr,showsthatthehydrophobiccoresofthetwostructurespackinanearlyidenticalfashionThisjuxtapositionoftherstandsecondlayersburiesasurfaceareaof,AÊandplacesoneofthemethylgroupsofLeuabovethearomaticringofPhe,accountingforthelargeupeldchemicalshiftofitsmethylprotons(seeSupplementaryInformation)Theoppositefaceofthesecondlayerconsistsofabedofhydrophobicsidechainsemanatingfromhelicesa,whichareexposedtosolventintheTFEstructureIntheintramolecularcomplex,theahelixoftheCHRliesnestledonthisbed,formingathirdstructurallayerandcompletingthecoreofthedomainResiduesLeu,LeuandMetofthehydrophobicfaceoftheCHRhelixllthreedeeppocketsonthelayersurface,withadditionalextensivecontactsmadebyVal,Met,ValandArg(Figd)Thesecontactsbury,AÊofaccessiblesurfacebetweenlayerandlayersandTheorientationoftheahelixrelativetothehydrophobicplaneisstabilizedbybothelectrostaticinteractionsbetweenthesidechainsofArgofthehelixandGlulocatedatthetopoftheaaturn,andhydrophobicinteractionsbetweenL

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