Molecular insights into ago-allosteric modulation at cysteinyl leukotriene receptor 2

Domain: biology
Published: 2025-12-16
Editorial outcome: minor_revision

Abstract
Cysteinyl leukotriene receptor CysLT2R, which is activated by the endogenous cysteinyl leukotrienes (CysLTs) LTC4, LTD4, and LTE4, has emerged as a potential therapeutic target due to the involvement in various inflammatory diseases. Accumulating evidence indicates that CysLT2R is also involved in the pathogenesis of cardiovascular diseases and contribute to tumor progression in cancer. However, the structural basis underlying the ligand recognition and the receptor activation remains to be elucidated. Here, we present two cryo-electron microscopy (cryo-EM) structures of the human CysLT2R-Gq complexes bound to LTC4 and LTD4. CysLTs are characterized as ago-allosteric modulators (ago-PAMs) of CysLT2R. Our structures reveal that CysLTs are recognized by a lipid-facing pocket above intracellular loop 2 (ICL2) near the cytoplasmic side of the receptor. Furthermore, a noncanonical activation mechanism exists between the allosteric binding pocket and the Gq-binding site. Our findings provide comprehensive insights into the recognition of CysLTs and G protein signaling transduction by CysLT2R, which may facilitate rational design of drugs.

Introduction
The proinflammatory lipid mediators CysLTs, including LTC4, LTD4, and LTE4, exert their effects through interactions with two G protein-coupled receptors (GPCRs), CysLT1R and CysLT2R1–4. CysLT1R plays an important role in the allergic and inflammatory disorders, such as asthma, rhinitis, urticaria and atopic dermatitis, and also functions in cardiovascular diseases and cancers5–12. Meanwhile, CysLT2R has been implicated in treatments of severe asthma, cardiovascular and neurodegenerative disorders, as well as several types of cancers11–19. Notably, CysLT2R has recently been identified as the endogenous receptor for ceramides, with report of ceramide-bound activated structure emphasizing its critical role in atherosclerosis18. Moreover, the distinct tissue distribution and expression profiles of CysLTRs suggest the different spectra of receptor-associated disorder and corresponding therapy strategies2–4,13,20–22. Extensive efforts have been made to discover drugs targeting CysLTRs. Among them, three CysLT1R-selective antagonists (montelukast, zafirlukast and pranlukast) have been approved to treat asthma with high potency23. However, side effects and nonrespondency have been reported24. Although CysLT2R-selective or dual antagonists have been introduced to treat severe asthma and brain injuries25,26, the development of CysLT2R-targeted drugs still faces challenges due to the lack of understanding of ligand recognition and receptor activation mechanism triggered by CysLTs.

To facilitate the development of efficient therapies for diseases related to CysLT2R, a range of crystal structures bound to antagonists have been determined, providing atomic-level structural insights into the antagonist recognition and subtype selectivity27. However, the absence of CysLTs-bound CysLT2R structures has limited a complete mechanistic understanding of the endogenous agonist recognition and receptor activation.

In this study, we report two active structures of the human CysLT2R-Gq complexes bound to LTC4 and LTD4. Intriguingly, our structures demonstrate that CysLTs interact with CysLT2R at allosteric binding pocket formed by TM3, TM4, TM5 and ICL2, which is distinct from the extracellular pocket occupied by ceramides and antagonists. While this manuscript was in preparation, a cryo-EM structure of LTD4-bound CysLT2R-Gq complex was reported28. In that structure, LTD4 was modeled in the orthosteric binding pocket, similar to CysLT2R antagonists27. Therefore, to elucidate the orthosteric and allosteric action of chemically diverse agonists, here we also provide a comprehensive functional assay triggered by LTC4 and ceramide coordinately. CysLTs are characterized as ago-PAMs for CysLT2R. Together with mutational studies, the structural information provides a framework for understanding the ago-allosterism, noncanonical activation mechanism and signaling transduction of CysLT2R and a structural basis for designing new anti-CysLTs drugs.

Results
StructuredeterminationandoverallstructuresofCysLTs-CysLT2R-Gqcomplexes
TheCysLTsLTC4,LTD4,andLTE4arealipidfamilyconjugatedwithapeptidemoiety(Fig.1a),whicharederivedfromarachidonicacid1,29.BothCysLTRsrecognizethreeCysLTswithdifferentpotencies,predominatelyactivatingGq(Fig.1b),andalsosignalingthroughGi/o2.ToelucidatethebindingmodeandsignalingprofileofCysLTstoCysLT2R,weemployedsingle-particlecryo-EMtodeterminetwostructuresofCysLT2R-GqproteincomplexesboundtoLTC4andLTD4,respectively.Inourstructuralstudies,weusedthewild-typehumanCysLT2R.WealsousedanengineeredminiGαqsubunit30withtheN-terminal35aminoacidsreplacedbytheircorrespondingresiduesinGαi,whichhasbeensuccessfullyusedtoobtaincryo-EMstructuresofseveralotherGq-coupledGPCRs31–33.Theengineeredmini-Gαqconstructexhibitsfunctionalactivitycomparabletothatofwild-typeGαq31.OwingtoitsenhancedtendencyforformingstablecomplexeswithGPCRs,themini-Gαqvariant,whoseC-terminalα5helixremainsunchanged,effectivelymimicswild-typeGαqinreceptorcoupling.Consequently,mini-Gproteinshavebeenservingasidealtoolsforbiophysicalinvestigations ofGPCRsintheiractivestate34.Unlessotherwisestated,Gqreferstotheengineeredmini-Gαq-βγheterotrimerusedforstructuraldetermination.ToimprovethestabilityofCysLT2R-Gqcomplexesforcryo-EMstudies,weassembledthecomplexesusingtheNanoBiTtetheringstrategy35togetherwithasingle-chainvariablefragment(scFv16),whichhasbeendevelopedpreviouslytostabilizetheGiheterotrimer36.WepreparedtheCysLT2R-Gqcomplexesinlaurylmaltoseneopentylglycol(LMNG)/cholesterolhemisuccinate(CHS)detergentconditionforcryo-EMstudies(SupplementaryFig.1a,b).Ultimately,LTC4-andLTD4-boundCysLT2R-Gqcomplexesweredeterminedtooverallresolutionsof3.3and3.5Å,respectively(Fig.1c,d,SupplementaryFig.2,Table1).Thehigh-qualitycryo-EMmapsenabledustomodelthemajorityofCysLT2R,GqandscFV16antibodyinthestructures.ApartfromtheNterminusandCterminus,alargepart(232-237)ofICL3wasalsonotmodeledduetoweakdensity,indicatingthatICL3wasnotinvolvedintheinteractionwithGqprotein.However,ICL2,whichishighlyflexibleininactivestructures,becamevisibleuponligandbindingduetoitsdirectinteractionwithGq.

CysLTsareago-PAMsforCysLT2R
CysLTshavelongbeenidentifiedasendogenousligandsofCysLTRs3,4,andithasbeenwidelyacceptedthattheyarerecognizedthroughtheextracellularregionsofCysLTRs.IntherecentlyreportedLTD4-boundCysLT2R-Gqstructure(PDBID:9IXX),aclusterofnoncontinuouselectrondensityobservedatorthostericbindingsitewasassignedtoLTD428.However,atthecorrespondingpositionofourstructures,CHSmoleculesweremodeled(Figs.1c,dand2a).EspeciallyinourLTC4-boundCysLT2R-Gqstructure,theunambiguousstrongdensityofCHSinorthostericbindingsiteisprecludedfromCysLTsbinding.WenoticedthatLTD4wasonlyaddedintotheproteincomplexaftersizeexclusionchromatography(SEC)inthatstudy28,whichmaybeaccountforthedifferentstructuralcharacterization.Usually,theGPCR-Gproteinsignalingcomplexispreparedinthepresenceofagonistduringthewholepurificationprocedure.Furthermore,ourCysLTs-boundCysLT2R-GqstructuressuggestthattheCysLT2R-GqsignalingpathwayissynergisticallyregulatedbyceramidesandCysLTsthroughboththeorthostericandallostericmechanisms,whichisalsoimpliedbytheirdistinctchemicalstructures(Fig.1a,SupplementaryFig.3c).TounderstandtheallostericregulationmechanismofceramidesbyCysLTs,weconductedfunctionalassaystostudythecooperativityofthesetwokindsofligands.Firstly,weemployedNanoBiT-basedassaystodemonstratethefunctionaldifferencesbetweenCysLTsandC16:0ceramideonCysLT2R(Fig.2b).Consistentwithpreviousfindings18,thepotency(EC50,half-maximumEffectiveConcentration)ofceramide(2.749μM)issignificantlylowerthanthatofLTC4toCysLT2R(3.9nM).Ofinterest,LTC4wereobservedtogivehighermaximalG-proteincoupling(Emax,maximumEffect)comparedtoceramide,indicatingthatceramidefunctionsasapartialagonistforCysLT2R.Next,weassessedLTC4effectsonthesignalingbehaviorofceramide.Thepotency(EC50)andefficacy(Emax)ofceramideweresignificantlyenhancedinthepresenceofLTC4(Fig.2c–e,SupplementaryTable3),inlinewithourprediction,supportingthepositiveallostericeffectsofCysLTs.Meanwhile,wefoundthatahighconcentrationofceramidecouldaugmenttheEmaxbyCysLTs,indicatingthattheachievementoffullactivationofCysLT2RneedsthepresenceofbothCysLTsandceramide.Takentogether,inadditiontoactingasagonistsontheirown,CysLTsalsoenhancethebindingpropertiesofceramide,therebybeingclassifiedasago-PAMsofCysLT2R.

RecognitionofCysLTsbyCysLT2R
Themembrane-embeddedallostericbindingsitesofLTC4andLTD4inCysLT2Rarelargelyoverlapped(Fig.3a),whichareprimarilyconstitutedbyaclusterofhydrophobicresiduesfromTM3,TM4,TM5andICL2,includingY1273.41,T1303.44,V1313.45,V1343.48,F1373.51,L1383.52,L147ICL2,L1584.45,L2125.49,T2165.53,C2205.57,andL2235.60(Fig.3b,c).Mostimportantly,thenegativechargedcarboxylheadgroupofglycineofLTC4andcysteinylcarboxylgroupofLTD4formsapolarinteractionnetworkwithH1423.56stabilizedbyR145ICL2,whichiscrucialforCysLTs-inducedCysLT2Ractivation(Fig.3b,c,SupplementaryFig.3a).WeusedaNanoBiT-basedassaytoexaminethecontributionofthoseresidueswithinthepocket.Themutagenesisdatashowedthatmutationsoftheseresidueshadvariouseffectsonreceptoractivity(Fig.3d,e).Mostmutantsexhibitednearwild-typeexpressionlevel(SupplementaryFig.4g).Interestingly,mutationatresiduesY1273.41,L1584.45andL2125.49,whicharedistantformLTD4,stillaffectedreceptoractivation,indicatingtheimportanceofintactstructuralshapeofthisbindingpocketforreceptoractivity.
ComparedtoLTC4,LTD4occupiedasmallerbindingpocketwithadifferentorientationofcysteinylcarboxylgroup(Fig.3c).Nevertheless,thepolarinteractionnetworkwithH1423.56andR145ICL2maintainssimilarpotencyforLTD4asLTC4,highlightingtheimportantroleofICL2inligandrecognitionandGPCRsignaling.ThestructuresmayexplainthelowerpotencyofLTE4,whichisattributedtoitssmallersizeandthelackofshapecomplementaritywiththebindingpocket.ThestructureofLTE4-CysLT2Rcomplexisneededtoelucidatethepreciseselectivitymechanism.
Interestingly,CHSmoleculeintroducedinpurificationbufferwasobservedinthecleftbetweenTM4andTM5intwoCysLT2Rstructures,whichwasoccupiedbyceramideandantagonists(Figs.1c,dand2a,SupplementaryFig.3b).Withthelargelystudiedligandbindingpropertiesatthissite,weconductedastudytocharacterizetheeffectsofCHSonCysLT2Rsignaling.However,CHSdidnotplayaroleinactivitymodulationinourfunctionalassays.CHScannotactasanagonisttoactivateCysLT2R,orinhibitreceptoractivityasanantagonistlikepranlukastpranlukast(SupplementaryFig.3d,e).WeproposethatCHSactedasaplaceholderthathelpedmaintainthearchitectureofextracellularandtransmembranedomainstoenhancethestabilityandconformationalhomogeneityofCysLT2R–Gqcomplexes,enablinghigh-resolutionstructuredetermination.

ConformationalchangesofCysLT2Rfrominactivetoactivestate
Thecomparisonofourstructureswithpreviousreportedinactive(11a-bound),intermediateactive(11c-bound)andceramide-activatedstructureshasenabledustoinvestigatetheago-allostericactivationprocessofCysLT2RbyCysLTs18,27.TheLTC4-boundandLTD4-boundCysLT2R-Gqcomplexstructuresshowhighoverallsimilarity(Fig.4d).Withahigherresolution,weusetheLTC4-boundCysLT2R-Gqstructureforfurtherstructuralcomparison.
IntheLTC4-boundactivestructure,theabsenceofligandbindingintheorthostericbindingpocketreleasedtheresiduesinvolvedinligandbinding,allowingthemtoundergoconformationalchangesandenablingfreeconformationtransmission.StructurecomparisonshowedthatthesignificantconformationaldifferencebetweenLTC4-boundandceramide-boundstructuresoccurredatextracellularpart(Fig.4a,b).Especially,theECLsundergolargeconformationalreplacementbetweenthesetwostructures(Fig.4a,b,d).Additionally,theextracellularpartofTMsexhibitsmarkedlydifferentconformations.Fordetailedinstance,thepolarinteractionsbetweenY1193.33-Y2636.51andY1233.37-H2646.52,whicharebrokenuponactivationinceramide-boundstructuretoinduceF2606.48intoactivestate,areretainedinLTC4-boundstructure(Fig.4c).Surprisingly,structuralsuperpositionshowsthattheseresiduescriticalforceramide-inducedactivationadoptaninactiveconformationintheLTC4-boundstate(Fig.4c).Importantly,thetoggleswitchF2606.48isalmostidenticalinbothLTC4-boundand11a-boundstructures(Fig.4c),indicatingthatLTC4employsanoncanonicalactivationmechanismdifferentfromceramide.Therefore,comparisonanalysisrevealedthattheextracellularpartofLTC4-boundCysLT2Rresemblesthe11a-boundinactivestatewithahigherstructuralsimilaritythanactivestateintheceramide-boundstructureexhibitedbyresiduemeansquaredistance(RMSD)value(0.899Åand1.099Å,respectively)(Fig.4d).TheprominentconformationalchangesuponLTC4activationoccurredatcytoplasmicside,suggestingthattheactivationmechanismCysLT2RbyLTC4beginsattheallostericbindingandextendstoGqinterfaceregiontofacilitatetheGqproteincoupling(Fig.4d,SupplementaryFig.4a,b).Notably,theintermediatestate11c-boundCysLT2Rstructureperfectlycompletestheconformationchangetrajectoryforminactivetoactivestate(SupplementaryFig.5).
Ofnote,althoughtheresiduesY1193.33,Y1233.37,Y2636.51andH2646.52inorthostericpocketarenotinvolvedinactivationpropagationpath,mutationsintheseresiduessignificantlyaffectedthereceptoractivitysimulatedbyLTC4andLTD4(Fig.4c,SupplementaryFig.4e),indicatingtheimportanceofintactstructuralshapeoforthostericbindingpocketforreceptoractivity.

ActivationmechanismsofCysLT2RbyCysLTs
ComparisonanalysisindicatedthatLTC4andLTD4utilizeadistinctpropagationpathfromthatfoundinceramide-boundstructure.AnotableeventintheallostericpropagationpathisthereorganizationofICL2structure,whichispartiallydisorderedininactivestructures(Fig.5a).TheglycineheadgroupofLTC4formsapolarinteractionwithH1423.56,liftingICL2toremoveclashesbetweenthecytoplasmicendsofTM3andTM4,ICL2andGαq(Fig.5a),thusenablingsubsequentcoupling,andleadingtoF144ICL2projectingintothehydrophobicpocketonGαq(Fig.5a),afeaturealsofoundinBLT1activation39.AlaninesubstitutionofH1423.56,whichdisruptstheinteractionbetweenLTC4andICL2,decreasedthepotencyofLTC4inGqactivation(Fig.3b),highlightingtheimportanceoftheupwardshiftofICL2forCysLT2Ractivation.
ThesecondmostsignificantconformationarrangementoccurredatTM5andTM6(Fig.5b).ThecysteinylheadgroupofLTC4forcesTM5tomovetowardsTM6bydirectcontactclash(Fig.5b,SupplementaryFig.4c),leadingtothereorganizationoftheinterfacebetweenTM5andTM6toinitiateconformationalchange thatactivatethereceptor(Fig.5b).AmongthemovementsofTM5,Y2215.58exhibitsastriking‘induced-fit’conformationalchangebetweentheinactiveandactivestates,withitssidechainrotatingfromanoutwardtoacenterpositionoftheTMbundle.ThisinsertionofY2215.58createsadirectstericclashwithTM6,leadingtotheoutwardshiftofTM6.Moreover,Y2215.58,togetherwithR1363.50,formspolarinteractionnetworkwithY243ofGαqtoStabilizetheactivecomplex(SupplementaryFig.4d).Consistently,theY221AmutationabolishedLTC4andLTD4activity,indicatingtheyemployedthesamestrategytoactivateCysLT2R(Fig.5d,SupplementaryFig.4e).Interestingly,theY221Fmutantpartiallyrecoveredthefunctionbysubstitutingabulkysidechain,supportingthecrucialroleofbothY2215.58-R1363.50-GαqY243interactionsandtheTM5-TM6relativeconformationreplacementinreceptoractivation(Fig.5d,SupplementaryFig.4e).Additionally,F2145.51alsocontributestothereorganizationofTM5-TM6interface(Fig.5b,d,SupplementaryFig.4c,e).TheresidueY3057.53inNPxxYmotifpenetratestowardtothecenterofTMbundle(Fig.5c),acommonfeatureinclassAGPCRactivation.
Finally,consistentwiththeceramide-boundCysLT2Rstructure,activationbyCysLTsalsodisruptsapolarinteractionnetworkformedbyE3108.48andR1363.50andK2446.32(SupplementaryFig.4f),whichblocksthecytoplasmiccavitytomaintaintheinactivestatebyrestrainingthe7TMbundleconformation.
Overall,theseresultsstronglysupportthenoncanonicalactivationpropagationpaththattheCysLTs-bindingatICL2siterearrangestheintracellularregiontowardstheactiveconformationwithoutextracellularlyinducedsignalpropagation(Fig.5e).OurstructuralanalysisprovidedacomprehensiveunderstandingofallostericagonisminCysLT2Rsignaling.

SharedanddistinctstructuralfeaturesofCysLT2RinthecouplingofGq
AlthoughtheextracellularsideconformationofLTC4andceramide-boundCysLT2Rexhibitsremarkabledifferences(RMSD1.099Å),theconformationofintracellularsideissimilar(RMSD0.743Å)(Fig.4d).Thus,structuralcomparisonwithceramide-boundstructureshowedthatthereisasmallbutremarkableshiftoftheentireGqheterotrimerrelativetothereceptor(Fig.6a).GiventhesameC-terminalα5helixofmini-Gαqusedinthesetwostructures,theobserveddifferencesinGq-bindingmodesmayimplyanintrinsicdistinctionoforthostericandallostericactivation.However,wecannotruleoutthepossibilitythatthesestructuralvariationswerecausedbytheextrastabilizationofNb35boundtomini-Gαqinceramide-boundstructure.Nevertheless,theC-terminalα5helixofmini-Gαqinsertsintothecytoplasmiccavityofreceptor,leadingtoextensiveamphipathicinteractionswithreceptorsthatconstitutethemajorcouplinginterface.InourLTC4-CysLT2R-Gqstructure,CysLT2RresiduesR1363.50,L2386.26andS2416.29formpolarinteractionswithresiduesY243,Q237andV246ofmini-Gαq(Fig.6b),respectively.TheabsenceofdirectinteractionbetweenH8andGqsuggestsaninconsequentialroleinCysLT2R-Gqcomplexformation(Fig.6b),supportedbythepreviousfunctionalassaywithC-terminaltruncationatE30027.Importantly,mutationsoftheaminoacidsinvolvedinpolarinteractionstoalanine,significantlyimpairedagonist-inducedactivity(Fig.6c).Additionally,theresiduesM1403.54,I2245.61,L2285.65andA2456.33inCysLT2RformahydrophobiccorewithL236,L240andL245inmini-Gαq(Fig.6d).
Asmentionedabove,conformationchangesinICL2uponLTC4bindingplayanimportantroleinGqcoupling,andICL2contributesanotherregionthatinteractswithmini-Gαq.Notably,CysLT2R-GqcouplingisstabilizedbytheinsertionofF144ICL2intothehydrophobiccavityformedbyresiduesL34,V79andF228ofmini-Gαq(Fig.6e),whichwasalsofoundinBLT1-Gqstructure39andotherGPCR-Gqcomplexes31,40,41.ThesevereclashbetweenICL2regionandmini-Gαqisclearlyvisibleifwesuperimposetheinactive11a-boundtotheLTC4-boundR2(Fig.5b).UponGproteincoupling,theICL2regionisconformationalreplacedandstabilizedbytheinteractionbetweenthebackbonecarbonylgroupofV149ICL2andR31inmini-Gαqwithanintra-ICL2polarinteractionnetworkformedbyH148ICL2andbackboneofL146ICL2andV149ICL2(Fig.6e,f),leadingtotheunambiguousmodelingofL146ICL2-H148ICL2(SupplementaryFig.2h,p).
Thisdifferenceiscriticalbecause:Pre-SECligandsaddition.stabilizesthephysiologicalcomplex,allowingligandstoaccessmembrane-embeddedallostericsiteduringcomplexformationanddetergentsolubilizationsteps.Post-SECsupplementationofligandsapparentlycannotaccesstheallostericsite,whichremainssequesteredwithindetergentmicelles;accordingly,nointerpretabledensityofLTD4appearsatallostericsite(SupplementaryFig.3f).TheweaksignalformerlyassignedtoLTD4atorthostericsitemoreplausiblyrepresentsCHSintroducedinpurificationbuffer(SupplementaryFig.3g).Thisobserveddiscrepancieslikelystemfrommethodologicalvariations,underscoringhowpurificationstrategiesinfluencetheinterpretationofGPCR–ligandcomplexes.

Methods
Celllines
Spodopterafrugiperda(Sf9,Expressionsystems)cellsweregowninsf-900TM SFM Ⅱ (ThermoFisher) medium at 27°C and 500g. The HEK293T(ATCC,CRL-11268)cellsweregrowninahumidified37°Cincubatorwith5%CO2andmaintainedinDulbecco’sModifiedEagleMedium(DMEM,Gibco)containing10%fetalbovineserum(FBS,Vivacell)and1%penicillin-streptomycin.

Constructs
DNAsequencesencodinghumanCysLT2Rwascodon-optimizedandsynthesizedforSf9insectcellexpressionandclonedintoapFastBac1(Invitrogen)vectorwithanN-terminalFLAGepitope(DYKDDDD)followedbyafragmentofβ2ARN-terminaltailregion(BN)48asfusionprotein,alongwithaC-terminal8×Histagtofacilitatetheproteinexpressionandpurification.TheCysLT2Rsequencehadnoadditionalmutationsorloopdeletions.ATEVcleavagesitewasinsertedbetweenBNandCysLT2Rgenesequences.TheprolactinsequencewasplacedintotheNterminusbeforetheFLAGtagassignalingpeptidetoincreaseCysLT2RcellmembranelocalizationandincreaseCysLT2Rexpression.
ANanoBiTtetheringstrategywasusedtoimprovesamplequalityasdescribedpreviously35.TheLgBiTsubunitwasdirectlyfusedattheC-terminusofthereceptors.miniGαqwereclonedintothepFastBac1vector.HumanGβ1andGγ2withanN-terminal6×HistagandhumanGγ2wereclonedintothepFastBac-Dualvector(Invitrogen).TheHiBiTsubunitwasfusedtoGβ1attheC-terminuswitha15-aminoacidlinker(GGSGGGGSGGSSSGG).
Forfunctionalstudy,CysLT2RwereclonedintothepcDNA3.1vector(Invitrogen),withaFlagtagaddedtothereceptor’sN-terminus.Receptormutantsweregeneratedbysite-directedmutagenesisusingtheMutExpressIIFastMutagenesisKitV2(Vazyme),andthesequencesofallconstructswereverified(SupplementaryTable4).

PreparationofscFv16
CodingsequenceofscFv16wasconstructedintopFastBac1vectorwithaGP67signalingpeptideinsertedintotheN-terminalandaTEVcleavage,8×HistagattheC-terminal.ThepurificationofscFv16wasconductedaspreviouslydescribed48.Inbrief,secretedscFv16fromSf9insectcellcultureinfectedbybaculoviruswaspurifiedusingNi-NTAandsizeexclusionchromatography.AfterbalancingthepHandremovingthechelatingagentsbyNi2+andCa2+,thecellculturesupernatantwasloadedintoNi-NTA(GenScript).TheNi-NTAresinwasfirstlywashedwith20mMHEPESpH7.5,150mMNaCl,50mMimidazolefor10columnvolumesandthenelutedinbuffercontaining250mMimidazole.Theelutedsamplewascollectedandconcentratedto1mlwitha30kDMWCOMilliporeconcentratorandthenappliedtoSuperdexTM200Increase10/300GLcolumn(Cytiva)withbuffercontaining20mMHEPESpH7.5and150mMNaCl.Themonomericpeakfractionswereconcentratedandfast-frozenbyliquidnitrogen.

Proteincomplexexpressionandpurification
CysLT2R,miniGαq,Gβ1γ2andscFv16wereco-expressedinSf9insectcellsusingtheBac-to-Bacbaculovirusexpressionsystem(ThermoFisher)asdescribedpreviously48.TheSf9cellsgrownatadensityof4×106 cells per ml were co-infected with separate recombinant baculoviruses at the ratio of 3:3:1:1 (receptor:miniGαq:Gβ1γ2:scFv16.). Forty-eight hours after infection, the cells were collected by centrifugation and stored at −80°C for further use.
For the purification of protein complex, we performed an anti-FLAG purification followed by size exclusion chromatography to avoid overnight incubation and minimized the time the protein complex spent in solution, thereby significantly reducing dissociation and aggregation, and completed cryo-EM grid preparation within a single day. In brief, cell pellets from 2 L culture were thawed at room temperature and resuspended in lysis buffer (20 mM Tris pH 8.0, 50 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.2 µg/ml leupeptin and 0.2 mg/ml benzamidine). The mixture was stirred at room temperature for 25 min, followed by centrifugation at 60,000×g for 30 min to collect the precipitate. Then, the mixture was resuspended in formation buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2). The complex was formed on membrane in the presence of 50 nM LTC4 or LTD4 (TargetMol) and treated with 25 mU/mL apyrase (Sigma), 2 mg scFv16, followed by incubation for 1.5 h at room temperature. The complex was then solubilized in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 20% glycerol, 0.75% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace), 0.1% (w/v) cholesteryl hemisuccinate TRIS salt (CHS, Anatrace), 50 nM LTC4 or LTD4, 25 mU/mL apyrase and 2 mg scFv16 for 1.5 h at 4°C. The supernatant was collected by centrifugation at 60,000×g for 30 min, and the solubilized complex was incubated with G1 anti-FLAG resin (GenScript) for 1 h at 4°C. The resin was collected and washed with 20 column volumes of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% (w/v) LMNG, 0.01% (w/v) CHS, 50 nM LTC4 or LTD4, 0.2 µg/ml leupeptin and 0.2 mg/ml benzamidine. The complex was then eluted with 20 mM HEPES pH 7.5, 150 mM NaCl, 0.05% (w/v) LMNG, 0.01% (w/v) CHS, 50 nM LTC4 or LTD4, 0.2 µg/ml leupeptin, 0.2 mg/ml benzamidine, and 0.2 mg/ml FLAG peptide. The complex was collected and concentrated to 0.5 ml with a 100 kD MWCO Millipore concentrator, then loaded onto a Superose 6 Increase 10/300 GL column (Cytiva) with buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.001% (w/v) LMNG, 0.0001% (w/v) CHS, 0.00025% (w/v) GDN and 50 nM LTC4 or LTD4. Fractions of the monomeric peak were analyzed by SDS-PAGE and Coomassie staining. Then the desired fractions of CysLT2R-miniGαq complex were pooled and concentrated for cryo-EMs studies. The sample was concentrated to ~10 mg/ml for making cryo grids.

EMsamplepreparationanddataacquisition
Forcryo-EMgridpreparations,4μLofthepurifiedsamplewasappliedtoglow-discharged(60s,SuProInstruments)QuantifoilR1.2/1.3grids(Au300;ElectronMicroscopySciences).Thegridswerethenplunge-frozeninliquidnitrogen-cooledliquidethaneusingaVitrobotMarkIV(ThermoFisherScientific).TheVitrobotwasoperatedat6°C,withablottingtimeof4–6s,usingablotforceof‘1’,andmaintaining100%humidity.MicrographswerecollectedwithTitanKriosmicroscopes(ThermoFisherScientific)operatingat300kV,equippedwithaK3Summitdetector(Gatan)incountingmode(pixel0.855Å).DetailsofalldatasetscanbefoundinSupplementaryTableS1.Allthedatasetsunderwentprocessingusingthesimilargeneralworkflows,asoutlinedbelow.

Cryo-EMstructuredetermination
SupplementaryFig.2showscryo-EMworkflows.ImageprocessingwasperformedinCryoSPARCv.450 and RELION 4.151. Moviestacks were gain-corrected, motion-corrected, and dose-weighted in MotionCor2.152. Contrast transfer function (CTF) estimates were performed using Patch CTF estimation in CryoSPARC v.4, and micrographs with CTF fit resolutions better than 4.0 Å were selected using Curate Exposures in CryoSPARC v.4. Particles were auto-picked using Blob picker in CryoSPARC v.450.
ForthedatasetofCysLT2R-LTC4-Gqcomplex,4,024,246particleswereextractedfrom5,327micrographsinCryoSPARCv.4usingaboxsizeof256withabinningfactorof3forabinitioreconstructionandheterogeneousrefinements.TheparticlesweresubjectedtothreeroundsofheterogeneousrefinementinCryoSPARCv.4toremoveerroneouslypickedparticlesandthoseparticlesthatdidnotyieldhigh-resolutionreconstructions.Aftertheheterogeneousrefinementprocedure,324,384particlesfromtheclassexhibitingclearstructuralfeatureswereselected,reextractedandsubjectedtonon-uniformrefinementwithC1symmetry,whichyieldedareconstructionat3.6Åoverallresolution.AfteroneroundofBayesianpolishinginRELION4.1,theparticleswereimportedintoCryoSPARCv.4forheterogeneousrefinementwhichfocusontransmembranedomain(TMD).111,836particlesfromtheclasswiththebestTMDdensitieswereselectedandsubjectedtoCTF(bothglobalandlocal)andnon-uniformrefinementsinCryoSPARCv.4,resultinginamapat3.3Åoverallresolution.
ForthedatasetofCysLT2R-LTD4-Gqcomplex,5,785,807particleswereextractedfrom6126micrographsinCryoSPARCv.4usingaboxsizeof256withabinningfactorof3forabinitioreconstructionandheterogeneousrefinements.TheparticlesweresubjectedtothreeroundsofheterogeneousrefinementinCryoSPARCv.4toremoveerroneouslypickedparticlesandthoseparticlesthatdidnotyieldhigh-resolutionreconstructions.Aftertheheterogeneousrefinementprocedure,344,699particlesfromtheclassexhibitingclearstructuralfeatureswereselected,reextractedandsubjectedtonon-uniformrefinementwithC1symmetry,whichyieldedareconstructionat3.8Åoverallresolution.AfteroneroundofBayesianpolishinginRELION4.1,theparticleswereimportedintoCryoSPARCv.4forheterogeneousrefinementwhichfocusontransmembranedomain(TMD).130,645particlesfromtheclasswiththebestTMDdensitieswereselectedandsubjectedtoCTF(bothglobalandlocal)andnon-uniformrefinementsinCryoSPARCv.4,resultinginamapat3.5Åoverallresolution.
ThecrystalstructuresofCysLT2R(PDBcode:6RZ6)27wereusedasinitialmodelformodelrebuildingandrefinementagainstEMdensitymap.GqtrimerandscFv16modelwastakenfromthe5-HT2AR–Gqcomplex(PDBID:6WHA)31.ThemodelwasdockedintotheEMdensitymapusingUCSFChimera53,thenrefinedinrealspaceinCOOT54,andfurtherrefinedinrealspaceusingPHENIX55.ThefinalmodelshavegoodstereochemistryandFouriershellcorrelations(FSC)withthecryo-EMmaps(SupplementaryFig.S2;SupplementaryTableS1).StructuralFigureswerepreparedwithPymol(pymol.org),ChimeraX56,Chimera53.

NanoBiT-basedGαq-recruitmentassay
TherecruitmentofGαqtoCysLT2RwasmeasuredusingthePromegaNanoBiTProtein-ProteinInteractionSystem. In brief, HEK293T cells seeded at 1.3×106 cells per well on 6-well plates were co-transfected with pcDNA3.1 plasmids encoding CysLT2R C terminus fusion of SmBiT (2.5 μg) and Gαq construct with N-terminal fusion of LgBiT (2.5 μg) using transfection reagent Lipofectamine 3000 (ThermoFisher). After 24 h, cells were harvested using trypsin and then seeded in the flat-bottom white 96-well plate at a density of 100,000 cells per well. This was followed by an overnight incubation, after which experimental assays were conducted.
The growth medium was removed, and NanoBiT assay buffer (1× Hank’s Balanced Salt Solution containing 20 mM HEPES at pH 7.5, and 10 μM Furimazine Promega) was added at a 90 μL per well volume. The plate underwent a 20 min dark incubation at 37°C, followed by a 5 min equilibration at room temperature before starting the readings. Bioluminescence was measured with Synergy H1 multimode reader (BioTek), baseline luminescence was measured 6 times at room temperature until signal stabilization. Subsequently, 10 μL of 10× ligand solution was added, followed by 6 s of vortex mixing. Chemiluminescence was then continuously monitored for 40 min using a multimode plate reader (gain=140). The data was subsequently analyzed using GraphPad Prism 10.1.1 with a nonlinear regression method.

Surfaceexpressionanalysis
Cell surface expression for WT-CysLT2R and various mutants was monitored by a fluorescence-activated cell sorting (FACS) assay (Supplementary Table 4). In brief, HEK293T cells expressing Flag-tagged CysLT2R were harvested 24 h after transfection. Cells were suspended by PBS buffer and blocked with 5% (w/v) BSA at room-temperature for 30 min, then followed by the incubation with mouse anti-Flag-FITC antibody (Proteintech) at a dilution of 1:200 for 30 min at 4°C, and then PBS buffer was added to cells. Finally, the surface expression of CysLT2R was monitored by detecting the fluorescent intensity of FITC with LSR Fortessa (BD). The FACS data were analyzed by FlowJo 10.

Quantificationandstatisticalanalysis
ForGαq-recruitmentassays,datawerenormalizedtothepercentageofthewild-typeresponseandanalyzedusingthe“log(agonist)vs.response”modelinGraphPadPrism10.1.1.Forcellsurfaceexpressionstudies,datawerenormalizedtothewild-typeresponse,andprocessedinGraphPadPrism10.1.1.AlldatainFiguresandtablesarepresentedasmean±standarderrorofthemean(s.e.m.),withthenumberofbiologicalandtechnicalreplicatesdetailedinthecorrespondingFigureandtablelegends,where“n”indicatesthenumberofbiologicalreplicates.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The study by Li et al. revealed cryo-EM structures of the human CysLT2R–Gq complex in complex with endogenous agonists LTC4 and LTD4. The authors identified a novel allosteric binding site for these ligands and characterized them as ago-PAMs that synergized with orthosteric ceramides. This work demonstrated that CysLTs were recognized within a lipid-facing pocket above ICL2 near the cytoplasmic side of the receptor. Furthermore, they proposed a noncanonical activation mechanism between this allosteric pocket and the Gq-binding site. The findings provided significant mechanistic insights into CysLT2R signaling and offered new perspectives for therapeutic targeting. Overall, I think this is a well-organized paper that advances our understanding of cysteinyl leukotriene receptor structure and function. However, I have several concerns that require clarification and revision before I can recommend publication.\n1. The manuscript highlights a significant discrepancy between your LTD4-bound structure (allosteric site) and the very recent structure by Jiang et al. (orthosteric site). While you propose the timing of ligand addition as a potential cause, this requires deeper discussion. Please provide a more detailed speculative analysis of the possible technical and biological reasons for this fundamental difference in the Discussion.\n2. While you conclusively show CHS is not a direct modulator, its consistent presence in the orthosteric pocket is intriguing. Could it be acting as a structural stabilizer that facilitates the capture of this specific active conformation? Please discuss the potential role of this detergent molecule in stabilizing the receptor structure observed in your complexes, especially considering the weak density for the extracellular regions.\n3. The proposed allosteric propagation path from the ICL2 site to the G protein interface is compelling. The Y221A/F mutagenesis is key evidence. Could you provide additional analysis or commentary on the conservation of this allosteric network (e.g., residues like Y221, R136) in other related lipid-activated GPCRs? This would help establish the broader significance of the proposed mechanism.\n4. The proposed activation path (Fig. 5) involves ICL2 reorganization and TM5-TM6 shifts, but some mutants (Y119A, Y123A and F260A) shows a significant loss of efficacy for LTC4 (lines 197–199 and Supplementary Fig. 4e), leaving ambiguity about how allosteric binding propagates signal without orthosteric involvement. Please briefly discuss the potential role of these residues in the allosteric activation pathway, why orthosteric pocket residues influence activity if they aren't part of the propagation path.\n5. The study focuses exclusively on Gq coupling. Given that CysLT2R is also reported to signal through Gi/o and other pathways, did you investigate whether this novel allosteric activation mechanism influences G protein coupling preference or bias? A comment on the potential implications of your structural findings for understanding biased signaling at CysLT2R would strengthen the biological relevance of the study.", "reviewer_2": "This manuscript presents a significant contribution to the field of G protein-coupled receptor (GPCR) biology, particularly focusing on the Cysteinyl leukotriene receptor 2 (CysLT2R). The study employs cryo-electron microscopy (cryo-EM) to elucidate the structural basis of ago-allosteric modulation by endogenous ligands LTC4 and LTD4, revealing a novel membrane-embedded binding site distinct from the orthosteric pocket. The findings challenge previous assumptions about CysLTs binding and activation mechanisms, positioning CysLTs as ago-positive allosteric modulators (ago-PAMs) that synergize with ceramides. The work is timely, given the therapeutic relevance of CysLTRs in inflammatory diseases, cardiovascular disorders, and cancers. The methodological rigor, including functional assays and mutagenesis, strengthens the conclusions. However, several areas could be refined to enhance clarity, impact, and accessibility for a broader audience. Below, I outline the strengths, followed by constructive suggestions for improvement.\nStrengths of the Manuscript\n1. Innovative Structural Insights: The cryo-EM structures of CysLT2R-Gq complexes bound to LTC4 and LTD4 (at 3.3 Å and 3.5 Å resolution) are a major highlight. They uncover an unexpected allosteric binding pocket involving TM3, TM4, TM5, and ICL2, which fundamentally shifts our understanding of ligand recognition.\n2. Comprehensive Functional Validation: The integration of NanoBiT-based assays with structural data robustly supports the ago-PAM mechanism. The dose-response curves in Fig. 2 and Fig. 3 quantitatively demonstrate how LTC4 enhances ceramide efficacy and potency, providing strong evidence for synergistic modulation. The mutagenesis data in Fig. 3d,e further validate key residues, reinforcing the structural observations.\n3. Therapeutic Implications: The discussion effectively links the findings to drug design, emphasizing how the allosteric site (e.g., \"ICL2 site\") could enable selective or biased modulators. This is clinically relevant, given the limitations of current CysLTR antagonists.\n4. Technical Excellence: The use of NanoBiT tethering and scFv16 stabilization for cryo-EM sample preparation is commendable, as detailed in the Methods. Supplementary Figures (e.g., Supplementary Fig. 2) provide transparent data processing workflows, enhancing reproducibility.\nConstructive Suggestions for Improvement\nTo elevate the manuscript's impact, consider the following revisions:\n1. Enhance Structural Interpretation and Clarity:\nWhile the cryo-EM structures are well-resolved, the description of conformational changes could be more intuitive. For example, in the Results section discussing Fig. 4, the comparison with ceramide-bound structures (PDB: 9J5H) is crucial but somewhat dense. Simplify this by adding a schematic or cartoon overlay in Fig. 4 to illustrate the \"noncanonical activation mechanism\" more vividly. Currently, Fig. 4 shows RMSD values, but an annotated version highlighting key movements (e.g., TM5-TM6 interface reorganization) would aid readability. Please include arrows or color-coding in the figure to denote specific shifts (e.g., TM6 outward movement).\nIn Fig. 5, the activation propagation paths are described textually, but the figure could benefit from labels or a flow diagram to map the \"distinct activation propagation paths by ceramide and CysLTs\". Please add panel (f) showing a simplified model of the pathways to complement the structural details.\n2. Expand Methodological Details for Reproducibility:\nThe Methods section is thorough but could provide more context on optimization steps. For instance, mention any challenges faced during complex purification (e.g., in Supplementary Fig. 1) and how they were overcome. Specifically, detail the criteria for selecting the final particle counts in cryo-EM processing (Supplementary Fig. 2), as this affects resolution claims.\nClarify the rationale for using miniGαq chimeras in functional assays. While referenced, a brief justification in the main text would preempt questions about potential artifacts in Gq coupling differences (e.g., in Fig. 6a).\n3. Strengthen the Discussion and Broader Implications:\nThe Discussion aptly notes the \"ICL2 site\" as a drug target but could delve deeper into why previous drug development failed (e.g., side effects of antagonists like montelukast). Connect this to the structural insights more explicitly—perhaps by proposing specific residue targets (e.g., H1423.56 or R145ICL2) for allosteric inhibitors.\nAddress the discrepancy with the recent LTD4-bound structure (PDB ID: 9IXX) more directly. In the Results, it's mentioned that LTD4 was added post-purification in that study, but a comparative analysis in the Discussion (e.g., using Supplementary Fig. 3) would strengthen the argument for the allosteric model.\nExplore therapeutic implications beyond structural biology. For example, how might these findings influence clinical trials for CysLT2R-related diseases? A short paragraph on future directions (e.g., in vivo validation) would add translational value.\n4. Improve Language and Presentation:\nSome sections, like the Introduction, are comprehensive but occasionally verbose (e.g., background on CysLTs could be condensed). Streamline for conciseness to maintain focus on the study's novelty.\nEnsure terminology consistency: For instance, use \"ago-PAM\" uniformly instead of alternating with \"ago-allosteric modulator.\" Also, define abbreviations like EC50 and Emax upon first use in the main text for clarity.\nProofread for minor grammatical errors (e.g., in Results: \"therefor hypothesized\" should be \"therefore hypothesized\").\n5. Enhance Data Visualization:\nIn Fig. 2e and Supplementary Table 3, the heatmap and ΔpEC50 values effectively show LTC4-ceramide synergy, but adding error bars or confidence intervals would bolster statistical rigor.\nFor mutagenesis data (Fig. 3d,e and Supplementary Table 2), consider plotting the surface expression levels alongside activity data to distinguish expression defects from functional impacts, as done in FACS assays.", "reviewer_3": "The manuscript entitled “Molecular insights into ago-allosteric modulation at Cysteinyl leukotriene receptor 2 ” (NCOMMS-25-59958) by Liu et al is interesting and the results will aid the field in searching for drug candidates targeting CysLT2R. Yet, the following comments should be addressed to make the manuscript suitable for publication in Nature Communications.\n1. Fig 1A-B are referred to in the introduction – the description of data, as shown in Fig1A-B, should be moved to the results section.\n2. The authors write “To understand the allosteric regulation mechanism of 127 ceramides by CysLTs, we conducted functional assays to study the cooperativity of these two 128 chemically distinct ligands (Fig. 1a, Supplementary Fig. 3c)”. It should be added that Fig. 1a, Supplementary Fig. 3c is referring to the chemical structures of some of these compounds and not the functional assays performed.\n3. The authors should include the EC50 and not just the pEC50 in Fig. 2B to aid the understanding to the result-text for the readers.\n4. Fig 4A, the manuscript text says “the 180 extracellular part of LTC4-bound CysLT2R resembles an inactive state (RMSD 0.899 Å)” but the figure shows RMSD relative to LTC4-bound conformation and shows that 11a-bound has an RMSD=0.899 Å – the authors should clarify how the manuscript text statement corresponds to what the fig. shows.\n5. Clarify if ND stands for not detected or not determined in supplementary Table 2, include some representative plots of the flow cytometry data showing the surface expression of CysLT1R mutants as compared to WT.\nMinor points:\n- Line 155, change “are distant form LTD4” to “are distant from LTD4”\n- The text includes a mixture of past and present tense, keep to past tense\n- The text includes a mixture of Gαq and Gq, use Gαq throughout the text"}, "author_response": "Point-by-point response (NCOMMS-25-59958)\nWe would like to thank all the reviewers for their thorough evaluation of our manuscript and\nfor their constructive suggestions, which helped us to improve the quality of our work. We\naddressed all reviewers' concerns by substantially revising the manuscript and adding new\nfigures and discussions. Please find a detailed response to each reviewer’s comments below,\nwhile all the changes in the manuscript are highlighted in yellow.\nReviewer #1 (Remarks to the Author):\nThe study by Li et al. revealed cryo-EM structures of the human CysLT2R–Gq complex in\ncomplex with endogenous agonists LTC4 and LTD4. The authors identified a novel allosteric\nbinding site for these ligands and characterized them as ago-PAMs that synergized with\northosteric ceramides. This work demonstrated that CysLTs were recognized within a lipid-\nfacing pocket above ICL2 near the cytoplasmic side of the receptor. Furthermore, they proposed\na noncanonical activation mechanism between this allosteric pocket and the Gq-binding site.\nThe findings provided significant mechanistic insights into CysLT2R signaling and offered new\nperspectives for therapeutic targeting. Overall, I think this is a well-organized paper that\nadvances our understanding of cysteinyl leukotriene receptor structure and function. However,\nI have several concerns that require clarification and revision before I can recommend\npublication.\nThank you very much for your positive comments on our study.\n1. The manuscript highlights a significant discrepancy between your LTD4-bound structure\n(allosteric site) and the very recent structure by Jiang et al. (orthosteric site). While you propose\nthe timing of ligand addition as a potential cause, this requires deeper discussion. Please provide\na more detailed speculative analysis of the possible technical and biological reasons for this\nfundamental difference in the Discussion.\nWe thank the Reviewer for your very helpful consideration and suggestions. Our speculative\nanalysis, based on our experimental approach and the known biochemistry of GPCR–ligand\ninteractions, is summarized below.\nOur cryo-EM structures (LTC4/LTD4-bound CysLT2R-G ) were solved with ligands\nq\npresent throughout purification, ensuring equilibrium binding to the native site. In contrast,\nJiang et al. added LTD4 after size-exclusion chromatography. This difference is critical because:\nPre-SEC ligand addition stabilizes the physiological complex, allowing ligands to access\nmembrane-embedded allosteric site during complex formation and detergent solubilization\nsteps. Post-SEC supplementation of LTD4 apparently cannot access the allosteric site, which\nremains sequestered within detergent micelles; accordingly, no interpretable density of LTD4\nappears at allosteric site (Supplementary Fig. 3f). Our functional data support allosteric binding\nas physiologically relevant: LTC4/LTD4 enhance ceramide efficacy (Fig. 2 c-e), implying\nsimultaneous occupancy of allosteric (CysLTs) and orthosteric (ceramide) sites.\nWe propose that our experimental approach more faithfully recreates the physiological\nbinding pathway of these lipid mediators. Converging evidence from structural, functional, and\nbiochemical analyses strongly supports the biological relevance of LTD4 allosteric binding.\nThe observed discrepancies likely stem from methodological variations, underscoring how\npurification strategies influence the interpretation of GPCR–ligand complexes. In response, we\nhave expanded the Discussion section in our revised manuscript to provide a more thorough\ntreatment of this issue.\n2. While you conclusively show CHS is not a direct modulator, its consistent presence in the\northosteric pocket is intriguing. Could it be acting as a structural stabilizer that facilitates the\ncapture of this specific active conformation? Please discuss the potential role of this detergent\nmolecule in stabilizing the receptor structure observed in your complexes, especially\nconsidering the weak density for the extracellular regions.\nWe thank the Reviewer for this insightful comment. The consistent presence of CHS in the\northosteric pocket across both structures is notable, and we agree it likely plays an important\nstructural role in stabilizing the observed receptor conformation. Our interpretation is\nsummarized below:\nAlthough functional assays confirmed that CHS does not directly modulate CysLT2R\nactivity as an agonist or antagonist, its well-defined density in the orthosteric pocket suggests\na structural stabilizer role. We propose that CHS acts as a placeholder that helps maintain the\narchitecture of extracellular and transmembrane domains during detergent solubilization and\ncryo-EM grid preparation. This is particularly relevant given the weak density in extracellular\nregions, suggesting inherent flexibility in the absence of a high-affinity orthosteric ligand.\nIn our structures, CHS engages primarily with hydrophobic residues in the orthosteric pocket,\nwhich may pre-organize the binding site and reduce conformational heterogeneity. By\noccupying this cavity, CHS likely prevents collapse or distortion of extracellular regions,\nthereby facilitating resolution of key intracellular features, including the allosteric site and G -\nq\ncoupling interface. This role aligns with the known ability of CHS to stabilize other membrane\nproteins during purification and structural studies. Importantly, CHS did not impede binding of\nCysLTs to the allosteric site or receptor activation, confirming that its effect is structural rather\nthan functional.\nThus, CHS enhances the stability and conformational homogeneity of CysLT2R–G\nq\ncomplexes, enabling high-resolution structure determination. This does not diminish the\nbiological relevance of our structures, as the allosteric binding mode of CysLTs and the\nactivation mechanism are robustly supported by functional data.\n3. The proposed allosteric propagation path from the ICL2 site to the G protein interface is\ncompelling. The Y221A/F mutagenesis is key evidence. Could you provide additional analysis\nor commentary on the conservation of this allosteric network (e.g., residues like Y221, R136)\nin other related lipid-activated GPCRs? This would help establish the broader significance of\nthe proposed mechanism.\nWe thank the Reviewer for this helpful consideration and suggestion. We agree that examining\nthe conservation of key residues in the allosteric network (particularly Y2215.58and R1363.50)\nacross related lipid-activated GPCRs would strengthen the broader significance of our proposed\nmechanism. We have included this analysis in the manuscript, and our detailed response is\nbelow:\nIn our structure, Y2215.58 is a critical residue in the allosteric propagation path, forming a\npolar interaction with R1363.50 and sterically pushing TM6 outward to facilitate G coupling.\nq\nThis role is likely generalizable to other lipid GPCRs that couple to G . We examined its\nq\nconservation across lipid-activated GPCRs, it is conserved as Y2215.58and R1363.50 across many\nlipid GPCRs, including CysLT1R, BLT1, FFARs, and P2Y receptors.\nWhile the exact ligand-binding site differs (orthosteric and allosteric), the downstream\npropagation path involving ICL2 restructuring and TM5-TM6 repacking appears to be a\ncommon theme. Our structures now extend this mechanism to membrane-embedded allosteric\nagonists (CysLTs) that bypass the orthosteric site entirely.\n4. The proposed activation path (Fig. 5) involves ICL2 reorganization and TM5-TM6 shifts,\nbut some mutants (Y119A, Y123A and F260A) shows a significant loss of efficacy for LTC4\n(lines 197–199 and Supplementary Fig. 4e), leaving ambiguity about how allosteric binding\npropagates signal without orthosteric involvement. Please briefly discuss the potential role of\nthese residues in the allosteric activation pathway, why orthosteric pocket residues influence\nactivity if they aren't part of the propagation path.\nWe thank the Reviewer for raising this important point. It is indeed intriguing that mutations in\northosteric pocket residues (Y119A, Y123A, F260A) reduce signaling efficacy, even though\nthey are not directly involved in the allosteric propagation path. Our interpretation is\nsummarized below:\nAlthough CysLTs activate CysLT2R primarily through an allosteric pathway involving ICL2\nreorganization and TM5–TM6 rearrangement, the orthosteric pocket remains structurally\ncoupled to the global activation mechanism. We propose that residues such as Y119, Y123, and\nF260 contribute to the structural integrity and conformational stability of the receptor, rather\nthan directly participating in allosteric propagation.\nY119 and Y123 form a hydrogen-bond network with Y263 and H264 in the inactive state.\nAlthough this network remains largely intact in our LTC4-bound active structure, its disruption\nvia mutagenesis may destabilize the extracellular domain, indirectly altering the energy\nlandscape for activation. This is consistent with the general role of orthosteric residues in\nmaintaining GPCR fold and dynamics. F260, part of the conserved CWxP motif, serves as a\nhydrophobic toggle switch in many class A GPCRs. Its mutation likely perturbs the equilibrium\nbetween inactive and active states—even when activation is triggered allosterically.\nCritically, the allosteric pathway does not operate in isolation but is integrated within the\nreceptor’s broader conformational landscape. The orthosteric pocket may act as a structural\nscaffold that fine-tunes the activation energy barrier. This explains why its disruption\nattenuates—but does not abolish—signaling, as observed in our mutagenesis data.\nIn summary, although allosteric ligands (LTC4/LTD4) bypass the orthosteric site for binding,\nthe structural integrity of the orthosteric pocket remains essential for optimal signal\ntransmission.\n5. The study focuses exclusively on Gq coupling. Given that CysLT2R is also reported to signal\nthrough Gi/o and other pathways, did you investigate whether this novel allosteric activation\nmechanism influences G protein coupling preference or bias? A comment on the potential\nimplications of your structural findings for understanding biased signaling at CysLT2R would\nstrengthen the biological relevance of the study.\nWe thank the Reviewer for raising this important point about potential G protein coupling bias.\nWe agree that investigating whether the allosteric activation mechanism influences signaling\nbias would significantly enhance the biological relevance of our findings.\nOur structures reveal that allosteric binding of CysLTs induces a distinct conformational\nchange in ICL2 and TM5-TM6, which directly engages Gα . Notably, the rearrangement of\nq\nICL2 is a feature shared with other G -coupled GPCRs. This suggests that the allosteric site\nq\nmay be optimized for G coupling.\nq\nHowever, whether this mechanism preferentially promotes G over G coupling remains an\nq i/o\nopen question. As delineated in the Discussion section, “In LTC4-CysLT2R-miniG , besides\nq\nthe class 3 site occupied by LTC4, we also found a clear class 2 pocket (Supplementary Fig.\n6b), indicating a potential strategy of development selective or biased PAMs of CysLT2R that\ntarget this cavity, where accommodates various G protein selective and biased ligands in FFA2”.\nThe ICL2 site emerges as a critical determinant of G-protein selectivity, with therapeutic\nimplications for designing pathway-specific modulators.\nTherefore, to comprehensively investigate signaling bias, our future studies will employ\nNanoBiT-based assays or BRET biosensors to evaluate the activation of G and G as well as\nq i\narrestin recruitment mediated by wild-type and mutant CysLT2R in response to LTC4 and\nLTD4 stimulation. Additionally, structural studies of CysLT2R in complex with G protein\ni/o\nwill be conducted.\nReviewer #2 (Remarks to the Author):\nThis manuscript presents a significant contribution to the field of G protein-coupled receptor\n(GPCR) biology, particularly focusing on the Cysteinyl leukotriene receptor 2 (CysLT2R). The\nstudy employs cryo-electron microscopy (cryo-EM) to elucidate the structural basis of ago-\nallosteric modulation by endogenous ligands LTC4 and LTD4, revealing a novel membrane-\nembedded binding site distinct from the orthosteric pocket. The findings challenge previous\nassumptions about CysLTs binding and activation mechanisms, positioning CysLTs as ago-\npositive allosteric modulators (ago-PAMs) that synergize with ceramides. The work is timely,\ngiven the therapeutic relevance of CysLTRs in inflammatory diseases, cardiovascular disorders,\nand cancers. The methodological rigor, including functional assays and mutagenesis,\nstrengthens the conclusions. However, several areas could be refined to enhance clarity, impact,\nand accessibility for a broader audience. Below, I outline the strengths, followed by constructive\nsuggestions for improvement.\nWe thank the Reviewer for the positive comments and constructive suggestions on our study.\nStrengths of the Manuscript\n1. Innovative Structural Insights: The cryo-EM structures of CysLT2R-Gq complexes bound to\nLTC4 and LTD4 (at 3.3 Å and 3.5 Å resolution) are a major highlight. They uncover an\nunexpected allosteric binding pocket involving TM3, TM4, TM5, and ICL2, which\nfundamentally shifts our understanding of ligand recognition.\n2. Comprehensive Functional Validation: The integration of NanoBiT-based assays with\nstructural data robustly supports the ago-PAM mechanism. The dose-response curves in Fig. 2\nand Fig. 3 quantitatively demonstrate how LTC4 enhances ceramide efficacy and potency,\nproviding strong evidence for synergistic modulation. The mutagenesis data in Fig. 3d,e\nfurther validate key residues, reinforcing the structural observations.\n3. Therapeutic Implications: The discussion effectively links the findings to drug design,\nemphasizing how the allosteric site (e.g., \"ICL2 site\") could enable selective or biased\nmodulators. This is clinically relevant, given the limitations of current CysLTR antagonists.\n4. Technical Excellence: The use of NanoBiT tethering and scFv16 stabilization for cryo-EM\nsample preparation is commendable, as detailed in the Methods. Supplementary Figures (e.g.,\nSupplementary Fig. 2) provide transparent data processing workflows, enhancing\nreproducibility.\nWe thank the Reviewer for the appreciation on our study.\nConstructive Suggestions for Improvement\nTo elevate the manuscript's impact, consider the following revisions:\n1. Enhance Structural Interpretation and Clarity:\nWhile the cryo-EM structures are well-resolved, the description of conformational changes\ncould be more intuitive. For example, in the Results section discussing Fig. 4, the comparison\nwith ceramide-bound structures (PDB: 9J5H) is crucial but somewhat dense. Simplify this by\nadding a schematic or cartoon overlay in Fig. 4 to illustrate the \"noncanonical activation\nmechanism\" more vividly. Currently, Fig. 4 shows RMSD values, but an annotated version\nhighlighting key movements (e.g., TM5-TM6 interface reorganization) would aid readability.\nPlease include arrows or color-coding in the figure to denote specific shifts (e.g., TM6 outward\nmovement).\nFollowing the constructive suggestion of the Reviewer, we have included new subpanels Fig.\n4a and b to describe the overall conformational changes between our LTC4-CysLT2R-G and\nq\nceramide-bound structure (PDB: 9JH5). We have already highlighted the loops with large\nconformational changes by black dash lines. Additionally, we also used red arrows to show the\nreplacement of TM6 and TM7 during activation in Fig. 4d.\nIn Fig. 5, the activation propagation paths are described textually, but the figure could benefit\nfrom labels or a flow diagram to map the \"distinct activation propagation paths by ceramide\nand CysLTs\". Please add panel (f) showing a simplified model of the pathways to complement\nthe structural details.\nFollowing the constructive suggestion of the Reviewer, we have reorganized the Fig. 5 and\nincluded a new subpanel Fig. 5e to describe the distinct activation propagation paths by CysLTs\nand ceramide. In this new Fig. 5e, we represent the crucial structure determinants in both\nactivation paths.\n2. Expand Methodological Details for Reproducibility:\nThe Methods section is thorough but could provide more context on optimization steps. For\ninstance, mention any challenges faced during complex purification (e.g., in Supplementary\nFig. 1) and how they were overcome. Specifically, detail the criteria for selecting the final\nparticle counts in cryo-EM processing (Supplementary Fig. 2), as this affects resolution claims.\nWe thank the Reviewer for the constructive suggestion. Actually, we faced challenges in\npurifying the complex samples. The complex samples were not stable and tended to aggregate\nand dissociate when the purification lasted two days. So, we tried to streamline the protocol to\nfinish the protein purification and cryo-EM grid preparation within one day. Owing to the high\nquality of protein sample, we performed the cryo-EM experiment following standard\nprocedures without encountering any obstacles.\nFollowing the suggestion of this Reviewer, we have added the following to the text in\nMethods section:\n“For the purification of protein complex, we performed an anti-FLAG purification followed by\nsize exclusion chromatography to avoided overnight incubation and minimized the time the\nprotein complex spent in solution, thereby significantly reducing dissociation and aggregation,\nand completed cryo-EM grid preparation within a single day. In brief, cell pellets from 2 L\nculture were thawed at room temperature […]”\nWe also modified the description of cryo-EM data processing:\n“After the heterogeneous refinement procedure, 324,384 particles from the class exhibiting\nclear structural features were selected, […]. 111,836 particles from the class with the best TMD\ndensities were selected and subjected to CTF (both global and local) and non-uniform\nrefinements in CryoSPARC v.4, resulting in a map at 3.3 Å overall resolution.”\n“After the heterogeneous refinement procedure, 344,699 particles from the class exhibiting\nclear structural features were selected, […]. 130,645 particles from the class with the best TMD\ndensities were selected and subjected to CTF (both global and local) and non-uniform\nrefinements in CryoSPARC v.4, resulting in a map at 3.5 Å overall resolution.”\nClarify the rationale for using miniGαq chimeras in functional assays.\nWe apologize for not being clear enough in our Methods section. We used wild-type Gα in\nq\nNanoBiT-based Gα -recruitment assay and we have modified the description in Methods\nq\nsection:\n“In brief, HEK293T cells seeded at 1.3 × 106 cells per well on 6-well plates were co-transfected\nwith pcDNA3.1 plasmids encoding CysLT2R C terminus fusion of SmBiT (2.5 μg) and wild-\ntype Gα construct with N-terminal fusion of LgBiT (2.5 μg) using transfection reagent\nq\nLipofectamine 3000 (ThermoFisher).”\nWhile referenced, a brief justification in the main text would preempt questions about potential\nartifacts in Gq coupling differences (e.g., in Fig. 6a).\nFollowing the suggestion of this Reviewer, we have added the following to the text and cited a\npaper (ref 34) by Nehme, R. et al., in which they extended the family of mini-G proteins to\ndetermine the structure of relevant GPCRs in a fully active state:\n“[…], which has been successfully used to obtain cryo-EM structures of several other G -\nq\ncoupled GPCRs31-33. The engineered mini-Gα construct exhibits functional activity\nq\ncomparable to that of wild-type Gα 31. Owing to its enhanced tendency for forming stable\nq\ncomplexes with GPCRs, the mini-Gα variant, whose C-terminal α5 helix remains unchanged,\nq\neffectively mimics wild-type Gα in receptor coupling. Consequently, mini-G proteins have\nq\nbeen serving as ideal tools for biophysical investigations of GPCRs in their active state34. To\nimprove the stability of CysLT2R-G complexes for cryo-EM studies, […]”\nq\nIn addition, we propose that the structural variations of G coupling between our structure\nq\nand ceramide-bound structure (9JH5) may be due to two main reasons: First, the distinct\nactivation modes of receptor lead to the G -coupling differences. Second, compared to utilizing\nq\nscFV16 to stabilize the complex in our purification strategy, ceramide-bound structure was\nstabilized by scFV16 and Nb35 concurrently. It’s likely that the direct binding to Gα of Nb35\nq\ninduced the different conformation. We have modified the text accordingly:\n“Thus, structural comparison with ceramide-bound structure showed that there is a small but\nremarkable shift of the entire G heterotrimer relative to the receptor (Fig. 6a). Given the same\nq\nC-terminal α5 helix of different chimeric mini-Gα used in these two structures, the observed\nq\ndifferences in G -binding modes may imply an intrinsic distinction of orthosteric and allosteric\nq\nactivation. However, we cannot rule out the possibility that these structural variations were\ncaused by the extra stabilization of Nb35 bound to mini-Gα in ceramide-bound structure.”\nq\n3. Strengthen the Discussion and Broader Implications:\nThe Discussion aptly notes the \"ICL2 site\" as a drug target but could delve deeper into why\nprevious drug development failed (e.g., side effects of antagonists like montelukast). Connect\nthis to the structural insights more explicitly—perhaps by proposing specific residue targets\n(e.g., H1423.56 or R145ICL2) for allosteric inhibitors.\nWe thank the Reviewer for this constructive suggestion. We agree that connecting our structural\ninsights to the historical challenges in drug development significantly strengthens the impact\nof our work.\nWe propose that the failure of previous orthosteric-based drug development may be due to\ntwo main reasons: (1), Incomplete inhibition: Orthosteric antagonists cannot fully inhibit\nreceptor activation driven by the synergistic action of ceramides (orthosteric) and CysLTs\n(allosteric), as revealed by our functional data. (2), Lack of selectivity: The orthosteric pocket\nis structurally conserved among related receptors, potentially leading to off-target effects.\nOur structures now provide a clear solution to these challenges. We explicitly highlight the\nallosteric pocket residues H142 and R145—validated by our mutagenesis studies as critical for\nagonist function—as prime targets for a new class of drugs. We suggest that\ndeveloping negative allosteric modulators (NAMs) aimed at this pocket could achieve more\ncomplete and selective inhibition of CysLT2R signaling. This structural insight offers a direct\nand actionable path for rational drug design, moving beyond the limitations of traditional\northosteric antagonists.\nWe have addressed this point in the third paragraph of the Discussion section.\nAddress the discrepancy with the recent LTD4-bound structure (PDB ID: 9IXX) more directly.\nIn the Results, it's mentioned that LTD4 was added post-purification in that study, but a\ncomparative analysis in the Discussion (e.g., using Supplementary Fig. 3) would strengthen the\nargument for the allosteric model.\nWe thank the Reviewer for your very helpful consideration and suggestion. We contend that\nour experimental strategy more faithfully recapitulates the physiological binding pathway of\nthese lipid mediators. The observed discrepancies most likely originate from methodological\ndifferences, highlighting the decisive influence of purification schemes on the interpretation of\nGPCR–ligand complexes.\nWe have addressed this point with Supplementary Fig. 3f and g in the second paragraph of\nthe Discussion section.\nExplore therapeutic implications beyond structural biology. For example, how might these\nfindings influence clinical trials for CysLT2R-related diseases? A short paragraph on future\ndirections (e.g., in vivo validation) would add translational value.\nWe thank the Reviewer for raising this critical point regarding the translational impact of our\nwork. We have now added a dedicated paragraph to address the therapeutic implications and\nfuture directions in the fourth paragraph of Discussion section.\n4. Improve Language and Presentation:\nSome sections, like the Introduction, are comprehensive but occasionally verbose (e.g.,\nbackground on CysLTs could be condensed). Streamline for conciseness to maintain focus on\nthe study's novelty.\nFollowing the suggestion of the Reviewer, we have condensed the background on CysLTs and\nstart the Introduction section with the description of receptors.\nEnsure terminology consistency: For instance, use \"ago-PAM\" uniformly instead of alternating\nwith \"ago-allosteric modulator.\" Also, define abbreviations like EC50 and Emax upon first use\nin the main text for clarity.\nWe thank the Reviewer for reminding us of this point, as we have checked the consistency and\ndefined all abbreviations.\nProofread for minor grammatical errors (e.g., in Results: \"therefor hypothesized\" should be\n\"therefore hypothesized\").\nWe appreciate this point as well and have corrected this error. We have proofread the entire\nmanuscript and corrected all grammatical errors.\n5. Enhance Data Visualization:\nIn Fig. 2e and Supplementary Table 3, the heatmap and ΔpEC50 values effectively show LTC4-\nceramide synergy, but adding error bars or confidence intervals would bolster statistical rigor.\nFollowing the suggestion of this Reviewer, we have performed statistical significance testing\nin Fig. 2e and Supplementary Table 3.\nFor mutagenesis data (Fig. 3d, e and Supplementary Table 2), consider plotting the surface\nexpression levels alongside activity data to distinguish expression defects from functional\nimpacts, as done in FACS assays.\nWe thank the Reviewer for the constructive suggestion. We have added the surface expression\nlevels plots in Supplementary Fig. 4g.\nReviewer #3 (Remarks to the Author):\nThe manuscript entitled “Molecular insights into ago-allosteric modulation at Cysteinyl\nleukotriene receptor 2” (NCOMMS-25-59958) by Liu et al is interesting and the results will aid\nthe field in searching for drug candidates targeting CysLT2R. Yet, the following comments\nshould be addressed to make the manuscript suitable for publication in Nature Communications.\nWe thank this Reviewer for the detailed and positive evaluation of our work.\n1, Fig 1A-B are referred to in the introduction – the description of data, as shown in Fig1A-B,\nshould be moved to the results section.\nWe thank the Reviewer for reminding us of this point, as we have removed Fig. 1a, b and the\ndescription to Results section.\n2, The authors write “To understand the allosteric regulation mechanism of 127 ceramides by\nCysLTs, we conducted functional assays to study the cooperativity of these two 128 chemically\ndistinct ligands (Fig. 1a, Supplementary Fig. 3c)”. It should be added that Fig. 1a,\nSupplementary Fig. 3c is referring to the chemical structures of some of these compounds and\nnot the functional assays performed.\nWe agree with the Reviewer that the description was not clear, and we have modified our\ndescription:\n“Furthermore, our CysLTs-bound CysLT2R-G structures suggest that the CysLT2R-G\nq q\nsignaling pathway is synergistically regulated by ceramides and CysLTs through both\northosteric and allosteric mechanisms, which is also implied by their distinct chemical\nstructures (Fig. 1a, Supplementary Fig. 3c). To understand the allosteric regulation mechanism\nof ceramides by CysLTs, we conducted functional assays to study the cooperativity of these two\nkinds of ligands. Firstly, […]”\n3, The authors should include the EC50 and not just the pEC50 in Fig. 2B to aid the\nunderstanding to the result-text for the readers.\nWe thank the Reviewer for the suggestion. As requested, we have added EC in Fig. 2b.\n50\n4, Fig 4A, the manuscript text says “the 180 extracellular part of LTC4-bound CysLT2R\nresembles an inactive state (RMSD 0.899 Å)” but the figure shows RMSD relative to LTC4-\nbound conformation and shows that 11a-bound has an RMSD=0.899 Å – the authors should\nclarify how the manuscript text statement corresponds to what the fig. shows.\nFollowing the suggestion of this Reviewer and Reviewer #2, we have reorganized Fig. 4 and\nmodified our description to avoid any confusion:\n“Therefore, comparison analysis revealed that the extracellular part of LTC4-bound CysLT2R\nresembles the 11a-bound inactive state with a higher structural similarity than active state in\nthe ceramide-bound structure exhibited by residue mean square distance (RMSD) value (0.899\nÅ and 1.099 Å, respectively) (Fig. 4d). The prominent conformational changes upon LTC4\nactivation occurred at cytoplasmic side, […]”\n5. Clarify if ND stands for not detected or not determined in supplementary Table 2, include\nsome representative plots of the flow cytometry data showing the surface expression of\nCysLT1R mutants as compared to WT.\nND stands for “not detected” in supplementary Table 2, as the activation level is too low to\ndetect.\nWe thank the Reviewer for the constructive suggestion. We have added the surface expression\nlevels plots in Supplementary Fig. 4g.\nMinor points:\n- Line 155, change “are distant form LTD4” to “are distant from LTD4”\nWe have changed it.\n- The text includes a mixture of past and present tense, keep to past tense\nThank you for pointing out the inconsistent use of tenses. We have now gone through the entire\nmanuscript and made corrections.\n- The text includes a mixture of Gαq and Gq, use Gαq throughout the text\nWe have checked the use of Gα and G in entire manuscript, and made changes to avoid any\nq q\nconfusion."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed my concerns in the revised ms.", "reviewer_2": "I really appreciate the authors have well addressed all my concerns, and I have no more further comments.", "reviewer_3": "The authors have made appropriate changes and responded satisfactory to my comments, and I now believe that the revised manuscript ““Molecular insights into ago-allosteric modulation at Cysteinyl leukotriene receptor 2 ” (NCOMMS-25-59958) by Liu et al is suitable for publication in the Nature Communications."}, "author_response": ""}]

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