Structure of the TRPA1 ion channel suggests regulatory mechanisms

StructureoftheTRPA1ionchannelsuggestsregulatorymechanisms

CandiceE.Paulsen1*,Jean-PaulArmache2*,YuanGao1,2,YifanCheng2&DavidJulius1

TheTRPA1ionchannel(alsoknownasthewasabireceptor)isadetectorofnoxiouschemicalagentsencounteredinourenvironmentorproducedendogenouslyduringtissueinjuryordrugmetabolism.Theseincludeabroadclassofelectrophilesthatactivatethechannelthroughcovalentproteinmodification.TRPA1antagonistsholdpotentialfortreatingneurogenicinflammatoryconditionsprovokedorexacerbatedbyirritantexposure.DespitecompellingreasonstounderstandTRPA1function,structuralmechanismsunderlyingchannelregulationremainobscure.Hereweusesingle-particleelectroncryo- resolutioninthepresenceofpharmacophores,microscopytodeterminethestructureoffull-lengthhumanTRPA1to4A

includingapotentantagonist.Severalunexpectedfeaturesarerevealed,includinganextensivecoiled-coilassemblydomainstabilizedbypolyphosphateco-factorsandahighlyintegratednexusthatconvergesonanunpredictedtransientreceptorpotential(TRP)-likeallostericdomain.ThesefindingsprovidenewinsightsintothemechanismsofTRPA1regulation,andestablishablueprintforstructure-baseddesignofanalgesicandanti-inflammatoryagents.TRPionchannelshavecrucialrolesinsomatosensationbyservingassensorsforthermalandchemicalstimuli1,2.Inmammals,theTRPA1subtype(sonamedforitsextensiveamino-terminalankyrinrepeatdomain)isexpressedbyprimaryafferentnociceptors,whereitdetectsstructurallydiversenoxiouscompoundsthatelicitpainandneuro-genicinflammation.Suchactivatorsincludepungentirritantsfrommustard,onionandgarlic,aswellasvolatileenvironmentaltoxinsandendogenouspro-algesicagents3–9.TRPA1isalsoactivateddown-streamofphospholipase-C-coupledreceptorsandhasbeenproposedtofunctionasasensorofnoxiouscold3,6,10,11.TRPA1isassociatedwithpersistentpain,respiratoryandchronicitchsyndromes12,13andisthereforeapromisingtargetfortreatingtheseandotherneurogenicinflammatoryconditions.WhileselectiveTRPA1antagonistshavebeendeveloped,theirsitesandmechanismsofactionremainunclear.ManyTRPA1agonistsarepotentelectrophilesthatactivatethechannelthroughcovalentmodificationofconservedcysteineorlysineresidueswithinthecytoplasmicNterminus14,15.WhiletheseandotherfunctionalpropertieshavebeendeducedfromelectrophysiologicalstudiesofTRPA1inwholecells,channelactivityisnotreadilyretainedinexcisedmembranepatches.This‘rundown’canbemiti-gatedifmembranesareexcisedintosolutionscontainingpolypho-sphates,suggestingthatobligatecytoplasmicco-factorssupportTRPA1functioninintactcells16–18.Therefore,determiningthebio-physicalandstructuralbasisofpolyphosphateregulationiskeytounderstandinghowTRPA1isregulatedinvivo,andhowitcanbeefficientlymanipulatedinartificialsystemsformoredetailedfunc-tionalcharacterization.

Atransformativestepinaddressingthesequestionswouldbetodeterminethethree-dimensional(3D)atomicstructureofthechan-nel.TRPchannelshaveposedparticularchallengesinthisregard,probablyreflectingtheirconformationallydynamicnatureanddiverseintracellularelements.Electronmicroscopyprovidesapoten-tialmeanstoachievethisgoal,althoughforTRPA1thisapproachhas

)19.untilnowyieldeddensitiesofrelativelylowresolution(,16A

However,advancesinsingle-particleelectroncryo-microscopy

1

(cryo-EM)haverecentlyenableddenovostructuralanalysisof

)resolution20,21.Here,weexploitthisTRPV1tonear-atomic(#4.0A

approachtodeterminethestructureofthefull-lengthhumanTRPA1

resolution,revealingthestructuralbasisofsubunitchannelto,4A

assembly,polyphosphateactionandantagonistbinding.

UniquearchitectureofhumanTRPA1

AscreenofTRPA1orthologuesidentifiedthehumanchannelasthetopcandidateforstructuralanalysisbasedonparticlesizeandconforma-tionalhomogeneity(ExtendedDataFig.1a,b).Purified,detergent-solubilizedproteinappearedashomogeneous,mono-dispersedparticlesfromwhichgrossarchitecturalfeatures,suchasaputativetransmem-branecoreandextensiveintracellulardomain,couldalreadybedis-cerned(ExtendedDataFig.1b).Afterexchangeintoanamphipathicpolymer,TRPA1sampleswereimagedusingnegative-stainandsingle-particlecryo-EM,aspreviouslydescribed21(Fig.1a,bandExtendedDataFigs1gand2–5).Weimagedsamplesunderavarietyofconditions,ultimatelyobtaininghigh-resolution3Dreconstructionsonlyinthepresenceofanagonist(allylisothiocyanate,AITC)orantagonists

,3.9A and4.7A ,(HC-030031withandwithoutA-967079)to4.24A

respectively,usinggold-standardrefinementandFouriershellcorrela-tion(FSC)50.143criterionforresolutionestimation(Fig.1candExtendedDataFigs3–5).Indeed,eventwo-dimensional(2D)classaveragesofparticleswiththeseadditivesrevealedelementsofsecondaryandtertiarystructure,includingthetetramericorganizationofthechan-nel,well-resolveddensitiesfortheputativetransmembranecore,andacentralstalkflankedbyconvexstemsthattransitionintoahighlyflexiblecrescent-shapedelement(Fig.1aandExtendedDataFigs3b,4band5b).Thislatterelementwasroutinelyobservedbynegative-stainimagingforallorthologuesexamined(Fig.1bandExtendedDataFig.1b,g),andisthusabonafideandconservedstructuralfeature.In3Dreconstructions,mostsidechainswereseeninsuf-ficientdetailtoallowdenovoatomicmodelbuilding,whichwasinitiallyachievedwithsamplestreatedwithAITC(Fig.1eandExtendedDataFigs3and6).High-resolutioninformationsuitable

DepartmentofPhysiology,UniversityofCalifornia,SanFrancisco,California94158-2517,USA.2KeckAdvancedMicroscopyLaboratory,DepartmentofBiochemistryandBiophysics,UniversityofCalifornia,SanFrancisco,California94158-2517,USA.*Theseauthorscontributedequallytothiswork.

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RESEARCHARTICLE

a

c

Membrane

b

Intracellular

Sideview

Topview

Bottomview

d

TRPV1

e

Membrane

TRP

A1

β-sheet

βFigure1|3DreconstructionofhumanTRPA1.a,Representativecryo-EM2DclassaveragesofTRPA1(sideviews,leftandmiddle;end-onview,right).b,Representativenegative-stainparticlesinamphipol.c,3Ddensitymapof

resolutionwitheachTRPA1fromAITC-treatedsamplefilteredto3.5A

subunitcolour-coded.Threeviewsshowside,topandbottom.d,Ribbon

diagramofratTRPV1apo-stateatomicmodelforcomparison.e,RibbondiagramofTRPA1atomicmodelforresiduesLys446–Thr1078,includingthelastfiveankyrinrepeats.Channeldimensionsareindicated;side,topandbottomviewsare

shown.

formodelbuildingspannedresiduesLys446–Thr1078,excludingonlythemostdistalcytoplasmicregions,ashortS1–S2linkerthatextendsintotheextracellularspace,andashorthelixthatlinksaputativecarboxy-terminalb-strandtothecoiled-coildomain(ExtendedDataFig.7a,b).Thus,wehaveresolvedthestructureofthevastmajorityofthetransmembranecoreandproximalcyto-plasmicregionsinvolvedinsubunitassemblyandelectrophile

detection.Together,theseregionsrepresent,50%oftheprotein.Exceptwherenoted,discussionofthestructurepertainstotheAITC-treatedsample.

Fromabird’s-eyeview,TRPA1resemblesTRPV1anddistantlyrelatedvoltage-gatedpotassium(Kv)channels:eachsubunitconsistsofsixtransmembranea-helices(S1–S6)plusare-entrantporeloopbetweenS5andS6;homotetramersareformedthrough

‘domain

β-sheet

Ankyrin repeats

b

S4S4–S5S3

S1

Linker domain

C-terminalcoiled-coil

AR16AR15AR14AR13AR12

S1

S2

S2

LinkerPre-S1domainhelixS4–S5linker

TRP-like domain

Coiled-coil

S2

S4

S5

S3

S6

Pre-S1β180Linker domainAR16AR15AR14AR13AR12

C-terminalcoiled-coil

45AR16AR15AR14AR13

AR12

Linker domain

S1

S3

S5

S6

C-terminalcoiled-coil

Figure2|StructuraldetailsofasingleTRPA1subunit.a,Lineardiagramdepictingmajorstructuraldomainscolour-codedtomatchribbondiagramsbelow.Dashedlinesandboxesdenoteregionsforwhichdensitywasinsufficienttoresolvedetailedstructure(sequencebeforeAR12,loopcontainingCys665,

512|NATURE|VOL520|23APRIL2015

S1–S2,S2–S3andS3–S4linkers,connectionbetweenthirdb-strandandcoiled-coil,Cterminussubsequenttocoiled-coil),orwherespecificresiduescouldnotbedefinitivelyassigned(portionofthelinkerbeforeandafterthecoiled-coil).b,RibbondiagramsdepictingthreeviewsoftheTRPA1subunit.

ARTICLERESEARCH

a

Destabilizing

b

c

Stabilizing

d

e

InsP6

Figure3|C-terminalcoiled-coilmediatescytosolicinteractionsand

polyphosphateassociation.a,SideviewofTRPA1coiled-coilwithtwocoreglutamineresiduesboxedinred(destabilizing)orblue(stabilizing).b,c,Crosssectionsofcoiled-coilatindicatedregionswithcoreresiduesdepictedinstickformat.Dashedredlinesshowresidueinteractions.d,HelicalwheelpresentationofresiduesLys1046–Lys1052.Gln1047fromeachsubunitisindicatedwithanarrow.Basicresiduesin‘b,e’and‘c,g’positionsofneighbouringhelicesformthebindingsiteforInsP6.Coloursdifferentiateclassofresidues:lightgrey,aliphatic;darkgrey,aromatic;lightpink,polar;purple,basic.e,DensityforInsP6adjacenttopositively-chargedpocketformedbyLys1046andArg1050fromonecoil,andLys1048andLys1052fromanadjacentcoil.Althoughnotmodelled,InsP6

probablydocksparalleltothecoiled-coilsuchthateachpositivelychargedresiduecoordinatesanindividualphosphatemoiety.

interactionsat‘a’and‘d’positionsthroughoutthecore,consistentwithcoiled-coilgeometry,butdistinctfromthecanonicalcoiled-coilheptadrepeat,inwhich‘a’and‘d’positionsaretypicallyoccupiedbyhydrophobicresidues.Instead,TRPA1containstwoglutaminesinstaggered‘d’positionsthatmaydestabilizethestructure(Gln1047)and/orinteractthroughintra-planarhydrogenbonds23(Gln1061)(Fig.3a–c).Furthermore,residuesontheexteriorsurfaceofcoiled-coils(‘b,c,e–g’positions)arecommonlypolarorchargedtofacilitateinter-helixandsolventinteractions,buthereweseeseveralhydro-phobicoraromaticresiduesintheselocations,ofwhichonlyhalfinteractwithanotherchanneldomain(seebelow).Thiscoiled-coil-basedmechanismofsubunitinteractiondiffersfromthatseeninTRPV1,inwhichankyrinrepeatsononesubunitformcontactswithathree-strandedb-sheetontheadjacentsubunit(Fig.1d).Thus,TRPchannelsassociatethroughatleasttwobroadstructuralmechanisms,irrespectiveofwhethertheycontainanankyrinrepeatdomain(ARD).Physiologicalstudieshaveshownthatsolublepolyphosphatessus-tainTRPA1channelactivityinexcisedmembranepatches16–18.Indeed,wefoundthatinclusionofinositolhexakisphosphate(InsP6)throughoutchannelpurificationwasaprerequisitetoobtain-ingmono-dispersedprotein.Nevertheless,amechanisticexplanationforthisphenomenonhasbeenlacking.Remarkably,wealwaysobservedstrongdensitiesnearthecoiled-coilthatprobablycorre-spondtoInsP6(ExtendedDataFig.7d,e).Positivelychargedresidues,namelyLys1046andArg1050fromonecoilandLys1048andLys1052fromanadjacentcoil,associatewiththisdensityviafour-coordinatecharge–chargeinteractions(Fig.3d,e).Thisisconsistentwiththeobservationthatpolyphosphateshavingatleastfourphos-phatemoietiesaremosteffectiveatsupportingTRPA1function16.Interestingly,thepresumptivedestabilizingcoreresidue(Gln1047)islocatedbetweenInsP6-coordinatingsidechains(Lys1046andLys1048),andthusinter-helicalcrossbridgingbyInsP6couldcounteractthisglutamine-mediatedcoiled-coildestabilization(Fig.3a,d).ThisinteractionisreminiscentofapreviouslyestablishedroleforInsP6asanessentialco-factorforadenosinedeaminase24,andfurtherillustrateshowcellularpolyphosphatescanfunctionasprim-itiveproteinstabilizationfactors25.

swap’interactions(Fig.1d,e).Apartfromthisconservedtransmem-branecore,TRPA1exhibitsnumerousdistinctivefeatures,particu-larlywithinthelargeintracellularN-andC-terminaldomains,whichtogetheraccountfor,80%ofthechannel’smass(Fig.2).Forexample,aC-terminaltetramericparallelcoiled-coilmediatesextens-ivesubunitinteractions(Figs1eand2b).Also,adomainthatcontainsfiveankyrinrepeatssurroundsthecoiled-coilandislinkedwithanotherextendedfeaturethatformsthecrescent-shapedelement(Figs1e,2bandExtendedDataFig.8).Keycysteineresiduesthatcontributetoactivationbyelectrophilesarelocatedwithinthepre-S1regionatsolvent-accessiblesites,probablyaccountingfortheirrelativechemicalreactivity.Moreover,theseresiduesareincloseappositiontoa‘TRP-like’allostericmodulatorydomaincharacteristicofotherTRPchannels(butnotpredictedtoexistinTRPA1),provid-ingmechanisticinsightintoelectrophile-evokedchannelgating.

Coiled-coilandpolyphosphatebinding

)AnotablefeatureoftheTRPA1structureisawellresolved(,4A

tetramericcoiled-coillocatedatthecentreofthechannel,belowtheionpermeationporeneartheCterminus,whereitformsastalk-likeinteractionlocusforallfoursubunits(Fig.1eandExtendedDataFig.7c,d).Althoughcoiled-coilshavebeenshowntomediatesub-unitassemblyforsomeTRPsubtypes,theprimarysequencewithinthisregionofTRPA1ispredictedtocontaina-helices,butnotacoiled-coil,perse22.Nonetheless,ourstructurerevealsside-chain

Pre-S1,TRPdomainandreactivesites

Thepre-S1regionconnectstheARDtoS1andisofparticularinterestbecauseitcontainsresiduestargetedbyelectrophilicagonists14,15(Fig.4a).Thisregionconsistsoftwoelements,includingthepre-S1helixandaprecedinglinkerregion,theprimarysequenceofwhichyieldslittleinsightintoitsstructureormechanisticconnectiontochannelgating.Our3Dstructuresrevealanoveralltopologyforthelinkerconsistingoftwohelix-turn-helixmotifsseparatedbytwoputativeanti-parallelb-strands(Fig.4a).AlthoughTRPA1wasnotpredictedtocontainaTRPdomainbecauseitlacksacanonical‘TRPbox’motif,ana-helixdirectlyafterS6isstructurallyandtopologicallyanalogoustotheTRPdomaininTRPV1,althoughlocatedfurtherbelowtheinnermembraneleafletcomparedtoTRPV1.

ThelinkerisintegratedwiththeoverlyingTRP-likedomainthroughtwomainstructuralfeatures:severalhydrophobicinterac-tionsbetweenstackeda-helices,andformationofaputativethree-strandedb-sheetinwhichtwostrandsarecontributedbythelinkerandonebythesequenceaftertheTRP-likedomain(Fig.4a,b).Whileananalogousthree-strandedb-sheetinTRPV1mediatesinter-subunitinteractions,thisputativemotifinTRPA1seemstobeafreestandingstructure(Fig.1e).Interestingly,thethirdb-strandcon-nectstoashort,poorlyresolveda-helixthatisalmostburiedintheinnerleafletofthemembraneandformspartofapoorlyresolvedloopconnectingtheTRP-likedomainwiththeC-terminalcoiled-coil(ExtendedDataFig.7b).ManyTRPchannelsaremodulatedbymembranephospholipids(suchasphosphatidylinositol-4,5-bisphosphate)26andthisconnectinghelixinTRPA1mayprovideastructuralbasisforsuchregulation.

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a

Pre-S1 helix

S4–S5 linker

c

TRP-like domainHelix-turn-helix 2C621

W711

K710

E987

Helix-turn-helix 1

AR16

b

L686

L973d

chemicalmodification.Furthermore,thiskeycysteineislocatedatthebeginningofana-helixandadjacenttoalysine,probablyreducingthepKaofthethiolmoietytoenhanceitsnucleophiliccharacter27.WewereunabletoresolveAITC-mediatedadductformationowingto

)and/orinsufficientresolutionatthesesites(estimatedat4–4.5A

instabilityoftheresultingmodification,andthusthismechanismremainsspeculativeuntilabonafideligand–channelcomplexcanbeclearlyvisualized.Moreover,wehavenotobservedmarkedcon-formationalchangeswithinthisregionwhencomparingstructuresinthepresenceofAITCversusantagonists,butthismaysimplyreflectlackofchannelactivityunderconditionsamenabletocryo-EMana-lysis.Additionally,residuesinthedistalNterminus(forexample,Cys421inmouseTRPA1)ortransmembranecore(forexample,Ser943andIle946inhumanTRPA1)havebeensuggestedtocon-tributetoelectrophilesensitivity15,28,29.Theformerregionisunre-solvedinourstructure,andthelatterresiduesareunlikelytobedirectlymodifiedbyelectrophiles.Thetransmembranecorealsocon-tainsfivecysteineandlysineresidues,threeofwhich(Cys727,Lys771andCys834)probablyfacethelipidenvironmentandmaythereforebemodifiablebylipophilicelectrophiles.

T984

C621

Ankyrinrepeatdomain

TheextensiveN-terminalankyrinarrayisthenamesakeforTRPA1,yetwehavelittleunderstandingofitsfunctionality.Indeed,amongvertebrateTRPproteins,TRPA1boaststhelongestARD,variablyestimatedtoconsistof14–18ankyrinrepeats1,17,30,31.Inourrawmicrographs(negativestainandcryo-EM)and2Dclassaverages,theTRPA1Nterminusisdistributedintotwodistinctdensitiescon-sistingofwell-resolvedconvex‘stems’followedbyaflexible‘crescent’(Fig.1a,b).Inallofour3Dreconstructions,weseedensityforfive

)thatcontributetowell-definedankyrinrepeats(resolvedto,4–5A

thestems(Fig.1candExtendedDataFigs3–5).Ahomologymodelpredictsthatthecrescentconsistsof11ankyrinrepeatsspanning

,whichmayadoptapropellerarrangementthatfacilitates,100A

intersubunitinteractions(ExtendedDataFig.8).

Insomenon-mammalianspecies,suchasinsectsandsnakes,TRPA1exhibitsrelativelylowsensitivitytoelectrophilesandis,instead,activatedbyheat32–35.ChimaericandmutagenesisstudieshaveidentifiedregionswithintheARDthatspecifythermalorchemicalresponsiveness36,37,suggestingthattheARDcancommun-icatewiththepore.Inthisregard,packinginteractionsstericallylinktheankyrinrepeatsinthestemwiththeoverlyinghelix-turn-helixmotifsofthelinkerregionthroughhydrophobicandsomepolarinteractions(Fig.5a).ThisispropagatedupwardsandterminateswithintheTRP-likedomain,therebyforminganetworkofpackedinteractionscapableoftransducinginformationfromtheARDtothepore(Fig.5a).Alsoofnotearecloseside-chaininteractionsbetweenthecoiled-coilregionandAR12,aswellasAR16andthefirsthelix-turn-helix,whichprobablystabilizethewell-resolvedintracellularstems,contributingtochannelassembly(Fig.5b,c).Inadditiontoelectrophilicirritants,mammalianTRPA1canbeactivatedormodulateddownstreamofphospholipase-C-coupledreceptorsthroughincreasedcytosoliccalciumorpossiblydirectinter-actionwithGbc.TheseandothermodulatoryactivitiesmayalsobespecifiedbytheARD4,6,11,18,inwhichcasefurthermechanisticinsightswillrequirehigh-resolutionstructuresofseveralTRPA1speciesortho-loguesthatincludethisentiredomain.

Figure4|Cytoplasmicdomainsformanintegratednexus.a,DomainarchitectureandwebofinteractionsbetweentheTRP-likedomain(blue)andpre-S1helix(orange),theoverlyingS4–S5linker(purple)andunderlyinglinkerregion,consistingoftwohelix-turn-helixmotifs(greenandyellow)separatedbytwoputativeanti-parallelb-strands(pink).Athirdb-strand(pink)iscontributedbyresiduesfollowingtheTRP-likedomain.Structurallyresolvedreactivecysteinesandlysine(Cys621,Cys641andLys710)areshowninball-and-stickformat.Thehelix-turn-helicesarestackedabovetheARD(rose).b,TheTRP-likedomainformshydrophobicinteractionswiththesecondhelix-turn-helixmotifandS4–S5linker.Thefirsthelix-turn-helix(containing

Cys621)isintegratedwiththeTRP-likedomainthroughinteractionswiththeinterveningsecondhelix-turn-helix.c,TheTRP-likedomainalsointeractswiththepre-S1helix.d,Cys621islocatedinacloselypackedpocketlinedbyAR16belowandthesecondhelix-turn-helixabove.Cys621isshownasahydrophobicitysurface.

TheTRP-likedomaininTRPA1makesadditionalcontactswithothernon-contiguousstructures,includingthepre-S1helixandS4–S5linker,consistentwithproposedrolesforTRPdomainsassitesofallostericmodulation20(Fig.4b,c).InTRPV1,theTRPdomaininter-actswiththepre-S1helixandS4–S5linkerviapolarinteractions20,whereasinTRPA1theseinteractionsareexclusivelyhydrophobic,andmoreextensive(Fig.4b,c).Nonetheless,ourstructuresuggeststhattheTRP-likedomaininTRPA1alsosubservesallostericregu-lation,whichwasnotreadilyapparentwithouta3Dmodel.

Ourstructurealsorevealsthespatialdistributionwithinthepre-S1regionofkeycysteineandlysineresiduesthatcontributetoelectro-philesensitivity:Cys621residesinthefirsthelix-turn-helix;Cys641islocatedinthefirststrandoftheputativeb-sheet;Cys665isinaflexibleloopconnectingtheb-strandstothesecondhelix-turn-helix;andLys710islocatedwithinthepre-S1helix(Fig.4a).Eachofthesesitesissolventaccessible,andtheirlocationswithinthisallostericnexusmakesthemwellsuitedtodetectreactivechemicalagonistsandtransmittheseeventstotheTRP-likedomain(Fig.4a–c).Forexample,Cys621ispackedinapolarpocketbetweenankyrinrepeat16(AR16)andtheoverlyinghelix-turn-helix(Fig.4a,d).Assuch,electrophilesmayprovideadrivingforceforconformationalchangethatrelievessterichindranceand/orelectrostaticrepulsionafter

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Poreandantagonistbindingsite

ThecentralcavityintheionpermeationpathwayofAITC-treated

)grosslyresemblesthatofTRPV1inTRPA1(resolvedto3.5–4.5A

exhibitingtwomajorconstrictions(Fig.6a,b).Thereare,however,somenotabledifferences.Forexample,theouterporedomainofTRPA1containstwoporehelices,reminiscentofbacterialvoltage-gatedsodiumchannels,wherethesecondhelixprobablyactsasanegativelychargedconduitforattractingcationsandrepellinganions

ARTICLERESEARCH

TRP-like domain

Coiled-coilhelices

AR13

ab

Y456

I1069

Helix-turn-helix 2

C621

Helix-turn-helix 1

I623L584Y629

C633

I599L568

AR16

AR12

L630

c

Helix-turn-helix 1

Coiled-coil

Figure5|StructuralintegrationoftheARD.a,Theinterdigitatedconvex‘stem’regionoftheARDconsistingofAR12–AR16(onlyAR15andAR16areshown;rose)couplestotheallostericTRP-likedomain(blue)throughinteractionswithtwointerveninghelix-turn-helixmotifs(greenandyellow)ofthelinkerregion.AR15–AR16stackingisstabilizedthroughhydrophobicinteractions.AR16isalsoconnectedtotheoverlyingfirsthelix-turn-helixmotifthroughhydrophobicandpolarinteractions.b,c,TheARDandlinkerregionmakeconnectionswiththecoiled-coilthroughaseriesofhydrophobic,polarandpotentiallyp-cation

interactionsinvolvingresiduesinAR12andAR13(b)aswellasAR16andthefirsthelix-turn-helixofthelinkerregion(c).Coiled-coila-helicesfromthesameandneighbouringsubunitarecolouredorangeandpurple,respectively.

F583L552

AR15

fromthechannelmouth38(Fig.6a).Bycontrast,TRPV1andKvchannelscontainonlyasingleporehelix21,39.Moreover,thesteepershapeoftheouterporeregioninTRPA1isdistinctfromthewideroutervestibuleseeninTRPV1(closedstate)andbacterialNavchan-nels,butmorereminiscentofKvchannels38,39.

Differencesarealsoseeninthepresumptivegates.First,theuppergateinTRPV1isformedbytworesidues(Gly643andMet644),whereasthatofTRPA1isformedbyone(Asp915).Here,weseea

betweendiagonallyopposedrestrictionpointmeasuring7.0A

Asp915residues,whichisnarrowerthanthatseeninTRPV1

)intheactivatedstate,butpotentiallywideenough(.6A )(7.6A

20,40,41

(Fig.6a,btoaccommodatepartiallydehydratedcalciumions

andExtendedDataFig.7f).Interestingly,Asp915haspreviouslybeenimplicatedincontrollingcalciumpermeabilityinmouseTRPA1(ref.18).Second,thelowergateinratTRPV1isformedbyasingleconstrictioninS6atresidueIle679,whereasthatofhumanTRPA1consistsoftwohydrophobicsealsformedbyIle957andVal961,creatinganincreasinglyconstrictedfunnel

)issufficienttoblockconductionofwhosenarrowestpoint(6.0A

rehydratedcations(Fig.6a,bandExtendedDataFig.7g).Thus,wemayhavecaughtTRPA1inanintermediateconfigurationinwhichtheuppergateispartiallyopenandthelowergateclosed.Alternatively,theupperconstrictionmaynotconstitutearegulatedgateowingtoamorehighlyscaffoldedstructureaffordedbythesecondporehelix.DistinguishingbetweenthesepossibilitieswillrequirestabilizingTRPA1indistinctfunctionalstatesundercondi-tionsamenabletostructuralanalysis.

HC-030031andA-967079representthetwomainclassesofTRPA1antagonists7,42,43.WedeterminedstructuresofTRPA1inthepresenceofHC-030031,alone,ortogetherwithA-967079.Remarkably,thedouble-antagoniststructurerevealedauniquedensitywithinapocketformedbyS5,S6andthefirstporehelix(Fig.6candExtendedDataFig.7h–j).ThisdensitywasnotobservedinthestructurewithHC-030031alone,andthusprobablycorrespondstoA-967079.PhylogeneticcomparisonstogetherwithmolecularmodellinghaveidentifiedsixresiduesrequiredforA-967079sensitivity44–47,allofwhichsurroundtheobservedden-sity,asdoesPhe909,whichishighlyconservedandthereforenotpreviouslyimplicatedinA-967079binding(Fig.6c).Indeed,muta-tionofthisresidue(Phe909Thr)abrogatedA-967079inhibitionof

AITC-evokedresponses(Fig.6dandExtendedDataFig.9),furthercorroboratingassignmentofthisantagonistsite.Ithasbeenpro-posedthatA-967079formsH-bondinteractionswithSer873and/orThr874,whicharealsolocatedatthebottomoftheputativebindingpocket47(Fig.6c).Consequently,orientationofthephenylringoftheantagonistinproximitytoPhe909maystabilizeligandbindingthroughp–pinteractions.Infact,inthedouble-antagoniststructure,Phe909andadditionalkeyresiduesmoveclosertotheA-967079density,suggestingthatdrugbindingoccursviaan‘inducedfit’mechanisminvolvingmovementsinallthreeafore-mentionedregionsthatcomprisethepocket(Fig.6c,comparetoAITCmodelinwhite).

A-967079mayhinderchannelactivitythroughcoordinatedbindingtoS5,S6andfirstporehelixdomains,whichinTRPV1aremobileelementsinvolvedingating.Assuch,A-967079mayactasamolecularwedgetoinhibitopeningofthelowergatebyimped-ingmovementoftheseelements.InthecaseofTRPV1,classicvanilloidligandsoccupyasitewithinthelowerS2–S4bundle.Moreover,localanaestheticsinhibitNavchannelsbybindingtodiscretesitesalongtheS6pore-liningsurfacetoblocktheselectivityfilteroractivationgate48,49.Therefore,http://wendang.chazidian.comstly,themutationsdescribedabovedonotimpairHC-030031antagonism,suggestingthatthesetwocompoundsbindtodiscretesites(Fig.6dandExtendedDataFig.9g).WewereunabletoidentifyaseconddensitycorrespondingtoHC-030031,perhapsowingtoitsloweraffinity,leavingitsbindingsiteandmechanismofactionunre-solved.

Concludingremarks

TRPA1isasensorforchemicalirritantsandamajorcontributortochemonociception.Wenowshowthatkeyresiduesinvolvedinirrit-antdetectionaresolventaccessibleandliewithinaputativeallostericnexusconvergingonanunpredictedTRP-likedomain,suggestingastructuralbasisinwhichTRPA1functionsasasensitive,low-thresholdelectrophilereceptor.Animportantnextstepistovisualizeelectro-phile-evokedconformationalchangesthatareassociatedwithgating,agoalthatwillrequirerobuststabilizationofTRPA1underconditionsamenabletostructuralstudies.OuranalysisofhowInsP6stabilizesthechannelrepresentsastepinthisdirection.Thephysiologicalpurposeof

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