RESEARCHARTICLE Mechanism of Focal Adhesion Kinase Mechanosensing JingZhou1☯,CamiloAponte-Santamaría1☯,SebastianSturm2,JakobTómasBullerjahn2, AgnieszkaBronowska1,FraukeGräter1,3* 1HeidelbergInstituteforTheoreticalStudies,Heidelberg,Germany,2LeipzigUniversity,Institutefor TheoreticalPhysics,Leipzig,Germany,3InterdisciplinaryCenterforScientificComputing(IWR),Heidelberg University,Heidelberg,Germany ☯Theseauthorscontributedequallytothiswork. a11111 *[email protected] Abstract Mechanosensingatfocaladhesionsregulatesvitalcellularprocesses.Here,wepresent resultsfrommoleculardynamics(MD)andmechano-biochemicalnetworksimulationsthat OPENACCESS suggestadirectroleofFocalAdhesionKinase(FAK)asamechano-sensor.Tensileforces, Citation:ZhouJ,Aponte-SantamaríaC,SturmS, propagatingfromthemembranethroughthePIP bindingsiteoftheFERMdomainand 2 BullerjahnJT,BronowskaA,GräterF(2015) fromthecytoskeleton-anchoredFATdomain,activateFAKbyunlockingitscentralphos- MechanismofFocalAdhesionKinase phorylationsite(Tyr576/577)fromtheautoinhibitoryFERMdomain.Varyingloadingrates, Mechanosensing.PLoSComputBiol11(11): e1004593.doi:10.1371/journal.pcbi.1004593 pullingdirections,andmembranePIP concentrationscorroboratethespecificopeningof 2 theFERM-kinasedomaininterface,duetoitsremarkablylowermechanicalstabilitycom- Editor:PeterMKasson,UniversityofVirginia, UNITEDSTATES paredtotheindividualalpha-helicaldomainsandthePIP -FERMlink.Analyzingdown- 2 streamsignalingnetworksprovidesfurtherevidenceforanintrinsicmechano-signalingrole Received:June5,2015 ofFAKinbroadcastingforcesignalsthroughRastothenucleus.ThisdistinguishesFAK Accepted:October12,2015 fromhithertoidentifiedfocaladhesionmechano-responsivemolecules,allowinganew Published:November6,2015 interpretationofcellstretchingexperiments. Copyright:©2015Zhouetal.Thisisanopen accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninany AuthorSummary medium,providedtheoriginalauthorandsourceare credited. Focaladhesionsintegrateexternalmechanicalsignalsintobiochemicalcircuitsallowing DataAvailabilityStatement:Allrelevantdataare cellularmechanosensing.Althoughthezooofmechanosensingproteinsatfocaladhesions withinthepaperanditsSupportingInformationfiles. issteadilygrowing,force-inducedenzymaticmechanisms,asthoseuncoveredforautoin- Funding:FundingfromtheKlausTschiraFoundation hibitedkinasesinmuscle,remaintobeidentifiedforfocaladhesiondownstreamsignaling. (toJZandFG),theBMBFSYSTECprogramme(to Here,weprovideevidencethatfocaladhesionkinase(FAK)canactasadirectmechano- JZandFG),theDeutscheForschungsgemeinschaft enzymeatfocaladhesions,usingmoleculardynamicssimulationsandkineticmodelling. (DFG)researchgroupFOR1543(toCASandFG), WeshowthatanchorageofFAKtothemembraneviaPIP-2iscriticalforthismechanical andtheBIOMSprogrammeatHeidelbergUniversity activation.Ourresultssuggestsimilarmechanismstobeatplayforothermembrane- (toAB)isgratefullyacknowledged.JTBandSS acknowledgefinancialsupportfromtheEuropean boundautoinhibitedkinases. UnionandtheFreeStateofSaxony,andtheDFG throughFOR877andtheLeipzigSchoolofNatural Sciences-BuildingwithMoleculesandNano-objects (BuildMoNa).Thefundershadnoroleinstudy PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 1/16 FAKMechanosensing design,datacollectionandanalysis,decisionto Introduction publish,orpreparationofthemanuscript. Focaladhesions(FAs)actaskeycellularlocationsformechanosensingbyintegratingmechani- CompetingInterests:Theauthorshavedeclared calandbiochemicalsignalsbetweentheoutsideandinsideofthecell,therebyregulatingpro- thatnocompetinginterestsexist. cessessuchascellproliferation,motility,differentiation,andapoptosis[1–3].Theycontain numerousadapteroranchorproteins,whichestablishthemechanicallinkofthecytoskeleton withtheextracellularmatrix[4].Someoftheseproteinshavebeenidentifiedasmechano- responsiveelements[5–7]. FocalAdhesionKinase(FAK)centrallyregulatesFAsbyestablishingadhesiveinteractions atthecellperiphery[8].Actingasasignalinghubbetweenintegrinandmultipleproteinspar- ticipatingindownstreamsignalingpathways,itcarriesoutdiversefunctionsinembryonic development,cellmigration,andsurvival,anditsmalfunctionisassociatedwithcancerpro- gressionandcardiovasculardiseases[9,10].FAKcomprisesacentraltyrosinekinasedomain flankedbytwolargenon-catalyticdomains:FERMandFAT(Fig1A).TheN-terminalthree- lobed4.1ezrinradixinmoesin(FERM)homologydomainisconnectedtothekinaseN-lobe Fig1.MechanicalactivationofFK-FAK.A)DomainorganizationofFAKanditsrelativepositionatthecell peripheryinthecytosol.Thekinasedomain(orange)containsthelobesNandCandtheFERMdomain (blue)consistsoflobesF1–3,withF2bindingtoPIP lipids(red)concentratedattheinnerleaflet(IL)ofthe 2 membrane(grey).ThemajorphosphorylationsiteTyr576/577(magentastar),locatedattheactivationloop (green)inthekinasedomain,isautoinhibitedbytheFERMdomain.TheautophosphorylationsiteTyr397 (greystar)ispositionedintheloopconnectingthekinaseandFERMdomains(grey).TheFATdomain(violet) isnotconsideredinourstudy.B)StretchingforceappliedtothebasicpatchinFERMandthekinaseC- terminalresidue(red),informofvirtualsprings,inducesFERM-kinasedissociation.Representative structuresofFAKinitsinitialautoinhibitedconformation(1),afterdissociationofthekinaseC-lobefromthe FERMF2lobe(2),andafterTyr576/577releaseandpartialC-terminalunfolding(3)areshown.Color-code andorientationoftheproteinasinA.C)Cumulativenumberofdissociationeventsasafunctionofthe distancebetweenthepulledelementsatthemomentofdissociation(De−e).Thisindicatestheextentof unfoldingpriordissociation.Twoeventsweremonitored:dissociationofthetheFERMF2lobefromthe kinaseC-lobe,F2-C(transitionfrom(1)to(2)inB),andseparationofFERMdomainfromtheTyr576–577 phosphorylationsite,F-YY(transitionfrom(2)to(3)inB).Totalnumberofsimulations(83)isindicatedwith thedashedline. doi:10.1371/journal.pcbi.1004593.g001 PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 2/16 FAKMechanosensing througha50-residuelinker.TheC-terminalFAT(focaladhesiontargeting)domainfollowsa 220-residuelongproline-richanddisorderedlinker,throughwhichitisconnectedtothe kinaseC-lobe.TheactivationofFAKfirstrequiresautophosphorylationofTyr397,which offersaSrchomology2(SH2)bindingsite.SrcbindingtoFAKincreasesSrckinaseactivity, inducingthephosphorylationofTyr576/577withinthekinasedomainactivationloop[11]. ThisisneededformaximalFAK-associatedactivityandleadstotheformationofaSrc-FAK complex,whichtriggerssubsequentphosphorylationsintheFATdomainandbindingof downstreamsignalingproteins[12].TheFERMdomainauto-inhibitsthekinasedomainby blockingtheTyr576/577phosphorylationsite[13,14].Exposureofthissiteisanessentialstep topermititsphosphorylation,andtherebyrendermaximumFAKactivity. FAKlocatesatsitesofintegrinclusteringthroughprotein-proteininteractionsofitsFAT domain,whichcontainsbindingsitesforintegrin-andactin-associatedproteins[4,15].Integ- rinsignalingandinteractionswithgrowthfactorreceptorsweredeterminedasFAKactivators [16,17].Recentstudiesprovidedevidencethatphosphoinositidephosphatidylinsositol- 4,5-bis-phosphate(PIP )iscriticalforefficientFAKactivationandautophosphorylation[18, 2 19].PIP ,aubiquitoussecondmessengerenrichedintheinnerleafletoftheplasmamembrane 2 andconcentratedatFAs,regulatestheinteractionofcytoskeletalproteinswiththemembrane [20,21].PIP interactsdirectlywiththebasicpatch(216KAKTLR221)intheFERMdomain[18, 2 19],whichinducesconformationalchangesinFAK.TheglobalconcentrationofPIP inthe 2 cellmembraneisonlyapproximately1%[22].However,PIP -proteininteractions[22,23]or 2 divalentions,suchasCa2+,[24,25]canleadtolocalPIP2accumulation.Localizedincrements ofCa2+werealsosuggestedtoincreasetheresidencyofFAKatFAs[26]. EvidenceforadecisiveroleofFAKinmechanotransductionissteadilygrowing[27].FAK isrecruitedtotheleadingedgeandphosphorylatedinmigratingcellsundershearstress[28, 29].Ithasalsobeenshowntomediateforce-guidedcellmigration[30,31]aswellasstrain- inducedproliferation[32].Recently,themechano-sensitivityofFAKhasbeenascribedtothe force-sensingfibronectin-integrinlink[33].However,untilnow,theavailabledataon mechano-sensingthroughFAKisindirect.ItremainsunknownifFAKonlyliesdownstream ofmechano-sensingprocessessuchasthoseinvolvingintegrins,orifFAKisalsoperse exposedtoandactivatedbymechanicalforce. WeherehypothesizethatmechanicalforceactsasadirectstimulusofFAKactivity,indica- tionsforwhicharetwo-fold.First,FAKistetheredbetweenthePIP -enrichedmembraneand 2 thecytoskeleton,likelyactingasaforce-carryinglinkinFAs.Second,theFERM-kinasestruc- turesuggestsitselfasamechano-responsivescaffold,inwhichforcecouldspecificallydetach theautoinhibitoryFERMdomainfromtheactivesite.FAKwouldbethefirstmechanoenzyme ofFAs,allowingadirecttransductionofamechanicalsignalintoanenzymaticreactionand downstreameventsintothenucleus,whichwouldyieldamechanisticexplanationofFAK’s mechano-sensingrole[10].Indeed,twoanalogouscasesofmechanicallyactivatedenzymes havebeenpreviouslyidentified,bothofwhicharekinasesandfeatureforce-inducedactivation byremovalofanautoinhibitorydomain,namelytitinandtwitchinkinaseinmuscle[34–36]. IncontrasttotheFATdomain[37],theforceresponseoftheautoinhibitedFERM-kinasefrag- mentiscurrentlyunknown.TotestthehypothesisofFAKasaforce-sensor,weperformed extensiveequilibriummoleculardynamics(MD)andforce-probemoleculardynamics (FPMD)simulationsoftheFERM-kinasefragmentofFAKundervariousconditions.Force propagatingontoFAKfromaPIP -enrichedmembraneandthecytoskeletonspecifically 2 opensthehydrophobicFERM-kinaseinterface,preparingFAKforactivationviaphosphoryla- tionpriortotheunfoldingofthekinasedomain.Giventhelowstabilityofthelargelyα-helical kinaseandFERMdomains,thisisremarkable.Theenforcedactivationisrobustwithregardto PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 3/16 FAKMechanosensing alargerangeofpullingvelocities,butsensitivetothesiteofforceapplication.Ourforce- inducedactivationpathwaysuggestsadirectmechanoenzymaticfunctionofFAKinFAs. MaterialsandMethods EquilibriumMDsimulationsoftheFERM-kinasefragment(FK-FAK)fragment[14](PDB code:2J0J)intheapostate,bothintheabsenceandinthepresenceofamembranecontaining PIP andPOPElipids,aswellasofonlythemembrane,wereperformedusingtheGROMACS 2 package[38].FPMDsimulations[39,40]ofFK-FAKwithoutamembranewereperformedby subjectingtheC-terminalC-alphaatomandthecenter-of-massoftheC-alphaatomsofthe basicpatch216KAKTLR221toharmonic-springpotentialswhichweremovedawayfromeach otherwithconstantvelocity.FAThasbeensuggestedtointeractwithFERM,bindingtothe samesiteasPIP2does[41].However,PIP2bindingisrequiredforFAKactivation[18,19], thusexcludingthepossibilityofFAT-FERMstableinteractionsforPIP2-mediatedFAKactiva- tion.OtherinteractionsbetweenFATandtheFERM/Kinasecomplexarenotknownoratleast suggestedtobeverydynamicandweak[42],andtherebyeasiertobreakunderforcecondi- tions.Thissuggeststhatundertensileforce,FATismaintainedsufficientlyfarfromthecom- plex,andthattheforceistransducedtowardsthecomplexthroughthefully-stretched200 amino-acidproline-richdisorderedlinker.Inconsequence,inourFPMDsimulationsneither FATnorthelinkerwereconsidered. Theforceresponseofmembrane-boundFK-FAKwasinvestigatedbysubjectingitsC-ter- minustoaharmonicpotentialthatwasthenmovedawayfromthemembraneeithervertically ordiagonally,whilekeepingthemembranepositionatitsoriginalposition.PLS-FMA[43]was usedtodetectcollectivemotionsmaximallycorrelatedwiththeopeningoftheFERM-kinase interface.Theunderlyingfreeenergylandscapewascharacterizedbyanalyzingtherupture forcesasafunctionofloadingrate,usingboththeHSmodelbyHummer&Szabo[44]andthe BSKmodelbyBullerjahnetal.[45].Kineticmodelswerebasedonpreviousbiochemicalnet- works[46–49]andsimulatedusingCOPASI[50].DetailsofthemethodsaregiveninS1Text. Results Force-inducedreleaseofFAKautoinhibition Invitro,phosphorylationoftheactivationloopofFAKisenhancedbyrelievingtheautoinhibi- tionthroughY180/M183mutation[14]orPIP -binding[18].Catalyticturnoverofwild-type 2 FAK,however,requiresanadditionalbiochemicalstimulus.Here,weaskifmechanicalforce couldpromotefulldomaindissociationofFAKasrequiredforauto-andSrc-phosphorylation– analogoustotheeffectoftheY180/M183mutation.Weexaminedtheeffectoftensileforceon theautoinhibitedFK-FAKusingFPMDsimulations.TetheringFAKbetweenthemembrane andthecytoskeletonresultsinforcetransmissionfromthemembraneontothebasicpatchof theFERMdomainandfromthepaxillin-interactingFATdomainthroughtheproline-rich linkerontothekinaseC-terminus(Fig1A).Accordingly,inoursimulations,apullingforcewas appliedtothebasicpatchofFERMandtheC-terminusofthekinasedomaininoppositedirec- tionswith13differentpullingvelocitiesfrom6×10−3nm/nsto1nm/ns(1inFig1B).Foreach pullingvelocity,multiplerunswerecarriedout(83runsintotal),yieldingaconcatenatedsimu- latedtimeofabout7μs,withtheslowestpullingsimulationcovering1μs.Weobservedthe autoinhibitoryFERMdomaintodissociatefromthekinasedomainin76outof83FPMDsimu- lations(morethan90%ofthecases).ConformationaldamageofeithertheFERMF2-lobeor kinaseC-lobeoccurredintheremaining7simulations.ReleaseofTyr576/577fromFERM occurredalwayslater,i.e.atlargerend-to-enddistances,thandissociationoftheF2-lobefrom theC-lobe(Fig1Band1C),suggestingtheF2-Cdetachmenttobearequirementformechanical PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 4/16 FAKMechanosensing FAKactivation.WeobservedpartialunfoldingatthekinaseC-terminuspriortoexposureof Tyr576/577toanonlyminorextentandmostlyathigherloadingrates,comprisingatmosta15 nmincreaseinend-to-endlength(Fig1Band1C),ornomorethan30residuesoftheC-termi- nalα-helix(S1Fig).Asthesecondhalfofthishelix(ormore)istypicallydisorderedinother kinases(e.g.inproteinkinaseAorSrc),itspartialunfoldingunderforceislikelynottoimpair FAKenzymaticfunction.Hence,ourdatasuggestdomain-domaindissociationtolargelythwart theunfoldingofthemoderatelystableα-helicaldomainstructures.However,moresubstantial unfoldingfromtheC-terminusofthekinasedomainwasthedominantpathwaywhenpulling FK-FAKfromitsNandC-terminus(S2Fig).Thus,weconcludethatforceactingspecifically betweentheFERMbasicpatchandthekinaseC-terminusremovestheinhibitoryFERMdomain andtherebyfacilitateskinaseactivation,insteadofdomainunfoldingandkinaseinactivation. FAK-membraneinteractionsunderforce AtFAs,thespecificinteractionoftheFERMbasicpatchwithPIP2isrequiredfortheanchor- ingofFK-FAKtothemembrane.Otherphospholipidsonlydisplaybackgroundlevelsofbind- ing[18].Inourpreviousstudy,weobservedanallostericchangeattheFERM-kinaseinterface uponPIP bindingtoFK-FAK,butnofullopening[18].Thisraisedthequestioniffulldomain 2 openingunderforce,asobservedforisolatedFK-FAKinsolution,alsooccurswhenFK-FAKis anchoredtoamembraneviaPIP .ThiswouldrequireboththePIP -containingmembraneas 2 2 wellasthePIP -FERMlinktobemechanicallymorerobustagainstrupturethantheFERM- 2 kinaseinteraction.Totestthis,wesetupapalmitoyloleoylphosphatidylethanolamine(POPE) membranecontaining15%(mol/mol)ofPIP intheinnerleafletofthemembrane,whichwas 2 surroundedbywaterandneutralizedbyCaCl .Within100nsofMDsimulationsstarting 2 fromindividualPIP moleculesinthemembrane,weobservedtheformationofsmallPIP 2 2 clustersinvolvingtwoormorelipidsandCa2+(S3Fig),accompaniedbyadecreaseofareaper lipidby*1Å2(S1Table),inagreementwithdivalent-cation-mediatedPIP -enrichmentin 2 membranes[18,24,51].FK-FAKwasanchoredtothemembraneandthedynamicsofthe resultingcomplexwasmonitoredover150nsofMD.Anchoragefurtherincreasedclustering. TheproteinremainedstablyboundtothemembranethroughtheFERM-PIP andadditional 2 interactionsbetweenthekinaseC-lobeandthemembrane,independentfromtheinitialorien- tationoftheproteinrelativetothemembraneplane(Fig2AleftandS3Fig).Thesamewas observedforamembranewith1%PIP ,which,however,showedlessclusteringandprovided 2 onlyasinglePIP lipidforanchorageofFK-FAK. 2 Next,wemonitoredthemechanicalresponseofmembrane-anchoredFK-FAK.InFPMD simulations,wesubjectedtheproteintoforcebymovingaharmonicspringattachedtothe kinaseC-terminuswithconstantvelocityalongadirectionverticalordiagonaltothemem- brane,whilepositionrestrainingthecenter-of-massofthemembranebilayer(Fig2A).At15% PIP concentration,independentofthepullingdirection,weobservedalossofcontactsofthe 2 kinasedomainwiththemembraneandwiththeFERMdomain,whiletheFERM-membrane interactionremainedintact(Fig2B).Whilediagonalpullingledtoaconcurrentdissociationof thekinasefromthemembraneandtheFERMdomain,verticalpullingresultedinkinase-mem- branedissociationpriortokinase-FERMdissociation.Innoneofthesesimulations,we observedkinaseunfoldingpriortodissociation.Also,forbothpullingdirections,themem- braneandthePIP -FERMinteractionweremechanicallymorerobustthanthoseatthe 2 FERM-kinaseinterface.Thus,themembranesimulationsreproducedtheprocesspredomi- nantlyobservedforisolatedFK-FAKinsolution(compareFig2BwithFig1Band1C).Namely, theyallshowedforce-inducedremovaloftheautoinhibitoryFERMdomainandexposureof theactivationloopcarryingtheTyr576/577phosphorylationsite. PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 5/16 FAKMechanosensing Fig2.MechanicalactivationofFAKboundtothemembrane.A)ForcewasappliedtotheC-terminusof FK-FAK,inverticalordiagonaldirectionwithrespecttothemembrane,withacounter-forceactingonthe membrane,leadingtothereleaseofautoinhibition(lefttorighttransition).FK-FAKisshownasinFig1B,PIP 2 lipidsinthemembrane(hereat15%)incyan/redandPOPElipidsingrey.B-C)NumberofcontactsN betweentheFERMF2-lobe(F2)andthekinaseC-lobe(C)comparedtothenumberofcontactsbetween bothlobesandthemembrane(mem),at15%(B)and1%(C)PIP concentration.Numberofcontacts 2 betweenlobeswasdefinedasthenumberofatomsinoneofthelobescloserthan0.6nmtoatleastone atomoftheotherlobe.Upperpanelsshowresultsfordiagonalpullingwhilelowerpanelsforverticalpulling. DensitiesofN(forapullingvelocityof0.03nm/ns)areshownasagreygradient,withapolynomialfittothe datashownasasolidblackline.Thelabelsi,a,b,anducorrespondtotheinactive,active,boundand unboundstatesofFK-FAK,respectively,sketchedattherightside. doi:10.1371/journal.pcbi.1004593.g002 WhenweappliedforcetoFK-FAKanchoredtoamembranecontainingonly1%PIP ,i.e. 2 toasinglePIP molecule,detachmentofthekinasedomainfromthemembranewasfollowed 2 bythedetachmentofalsotheFERMdomain(Fig2C).Fulllossofmembraneanchoringnatu- rallystopsforcetransmissionandimpedesactivation.Thus,aninteractionoftheFERMbasic patchwithmultiplePIP ,whichislikelyinPIP -enrichedmembranes,isrequiredformechani- 2 2 calFK-FAKactivation.ThisisinlinewiththefactthatPIP5Koverexpressionincreasesand PIP5KknockdowndecreasestheopenFAKconformation[19].Thepullingdirection,instead, appearstobelessrelevant. Mechanismofforce-inducedFK-FAKopening Wenextanalyzedinfurtherdetailthedynamicsunderlyingtheforce-triggeredFK-FAK domain-domainrupture.Applyingpartialleastsquaresfunctionalmodeanalysis(PLS-FMA) [43]tothesimulationsofisolatedFK-FAKinsolution,weobtainedacollectiveopening PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 6/16 FAKMechanosensing motionthatmaximallycorrelateswiththeincreaseinminimaldistancebetweentheF2andC lobes(S4Fig).ThisopeningmotionalsostronglycorrelatedwiththeF2-Clobedistances obtainedforthetrajectoriesofmembrane-boundFK-FAK,suggestingthatitcapturesthe essentialopeningdynamicsofFK-FAKbothisolatedandboundtothemembrane.Thisimplies thesimplifiedsystemofisolatedFK-FAKinsolutiontofollowaFERM-kinasedissociation mechanism,whichishighlysimilartotheoneofthemorerealisticsystemincludingthemem- brane,eventhoughitlackseffectsfromFERM/kinase-membraneinteractions. WethenidentifiedthefirststepsalongtheopeningmotionofFK-FAKgivingrisetorup- tureforces.Fig3AshowstypicalforceprofilesandF2/C-lobeinteractionareasasafunctionof thespringlocationsrecoveredfromtheFPMDsimulations.ForbothFK-FAKinisolationand boundtothemembrane,andindependentoftheloadingrate,weobservedthattheinterface areabetweenthetwolobeswasreducedintwosteps,bothofwhichcoincidedwithnoticeable forcepeaks.Themaximalforcewasreachedwhenthefirstdecreaseininter-lobeareaoccurred Fig3.MechanismofFK-FAKmechanicalactivation.A)InterfacialareabetweentheF2-andC-lobe(grey) andaverageforceexertedbythetwosprings(blue)asafunctionofthedistancebetweensprings,D . spring ResultsfromsixindependentFPMDsimulationsareshown:(1–3)withoutthemembranepullingatV=0.006, 0.006and0.014nm/ns,respectively,and(4and5)pullingdiagonallyawayfromthemembraneatV=0.03and 0.05nm/ns,respectively.Theinterfacialareadropsfrominitialvaluesof3–4.5nm2tointermediatevaluesof 1.5–2.8nm2.Afterwardsitdecreasestozero.Ruptureforce(highestforcepeak)alwayscorrespondedtothe firstdropintheinterfacialarea(redline).Thepeakforceassociatedtotheseconddropintheareais highlightedwiththegreenline.B)DistributionofinterfaceareasreflectingthetwostatesofFK-FAKduringits force-inducedopening(highlightedwitharrows).AllFPMDsimulationswereconsideredtocomputethe distribution.C)Residuesinvolvedintherupturestepsarehighlightedassticks.FERMF2-andKinaseC-lobe areshowninsurfacerepresentation.Rupturestepsareassociatedtothedisruptionofhydrophobic interactions(green);saltbridges(blue)andotherelectrostaticinteractions(magenta),andinteractionswith otherpartners(cyan).ResidueswereidentifiedbyTRFDA(S5Fig).TheyarelistedinS2Table. doi:10.1371/journal.pcbi.1004593.g003 PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 7/16 FAKMechanosensing (from3–4.5nm2to1.5–2.8nm2).Thisledtoashort-livedintermediate,asreflectedbyasec- ondpeakinthedistributionoftheF2/C-lobeinterfacearea(Fig3B),beforethetwolobesfully dissociated.Wenotethattheintermediatebecomeslessevidentforfasterpullingvelocities. Topinpointtheload-carryingresidue-residueinteractionsacrosstheinterface,wecalcu- latedthepunctualstressofeachresidue,usingtimeresolvedforcedistributionanalysis (TRFDA)[52],andtherebydetectedthelossofinter-lobeinteractionsduringpulling(seeS5 Fig,S2Table,andS1Text).Inter-lobeinteractionswhichrupturedreproduciblyatoneofthe twodissociationstepsarehighlightedinFig3C.Thefirstmajorrupturesteprequiredthe breakupofahydrophobicclustercomposedofresiduesY180,M183,N193,V196,andF596, andofanadditionalsaltbridge(D200-R598).Ruptureoftheseinteractionsgaverisetothe maximalforce,thusstressingtheircriticalstabilizingrole.Ourresultsareinagreementwith theobservationthatmutationsY180A,M183A,andF596DresultinconstitutivelyactiveFAK withanopenFERM-kinaseinterface[14,18].Residuepairsrupturingatthesecondstep includedresiduesofmostlyelectrostaticnature(E182,R184,K190andN595,N628,N629, E636)andarelocatedfurtherawayfromthemembraneanchor.Thissecondrupturestepis immediatelyfollowedbytheopeningoftheremainingFERM-kinaseinterfaceestablished betweentheF1andtheN-lobe,includingtheexposureofTyr576/577.Thus,therupturepro- cessresemblesazipper-likemechanism,duringwhichtheFERMandkinaseinterfaceis sequentiallyopened.Herein,themembrane-proximalhydrophobicpatcharoundF596repre- sentsthemostrobustmechanicalclamptobeopenedfirst.OurPLS-FMAcalculationsfurther supportthissequentialmodeofopening(S4CFig). ForcerequiredforFAKactivation AretheforcespredictedbythesimulationstorelieveFAKautoinhibitionrelevanttoFAKat FAs?Atthirteendifferentloadingrates,coveringtwoordersofmagnitude,weobtainedmaxi- malruptureforcesforFK-FAKactivationbetween150and450pN(Fig4A).Thisforceregime issimilartotheoneobservedfortitinkinase(400pNat0.2pN/ps),akinaseknowntobe mechanicallyactivatedbyforcespresentinmuscle[35,36,53].Ourruptureforcesarealsosim- ilartoorslightlyhigherthanthosepredictedbyMDsimulationsofthefocaladhesionproteins talinandvinculin(250–400pNfornanosecondscaleactivationoftalin[54,55]and100pNfor sub-nanosecondactivationofvinculin[56],respectively).WethenusedboththeHSmodel [44]andtheBSKmodel[45]tofittheobservedruptureforcesasafunctionoftheloadingrate. ThisprovideduswithasetofcompatiblemodelparametersΔG,Dandx ,whereΔGdenotes b theactivationenergy,Dtheeffectivediffusivityandx theseparationbetweentheinactivestate b andthetransitionstate.Focusingonthoseparametercombinationsthatcorrespondtoaphysi- ologicallyplausiblespontaneousactivationrateofnomorethank =10−3Hz,weobtaineda 0 numberofbest-fitestimatesfromwhichwederivedtheforce-dependentactivationratek(F) usedinourkineticmodel(Fig4AandS6Fig).Wenotethatmodelparameterscorresponding toanunphysiologicallyhighspontaneousdissociationratecanimproveourfittotheobserved forcefluctuations(S7andS8Figs).Onthisbasisitmightbespeculatedthatthereexistsasec- ondenergybarrieratalargervalueofx thatguaranteesthermalstabilityatlowforces,but b vanishesunderthehighforcesusedinourMDsimulations.Nevertheless,thisdoesnotinvali- dateourqualitativefindingsonFK-FAKactivationasforcesensitivityincreasesexponentially withthebarrierlocationx (seetheS1Textforamoredetailedanalysis). b Discussion Wehereprovidecomputationalevidenceforaforce-inducedactivationmechanismofFAK,in whichtensileforcerelievestheblockageofitsactivesite,aswellasitscentralTyr576/577 PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 8/16 FAKMechanosensing Fig4.FAKmechano-signaling.A)RuptureforceFasafunctionoftheloadingrateF_,whereFisdefinedasthemaximalforceobservedduringFK-FAK activation(arrowintheinset).LightgreydotsrepresentindividualruptureforcesFobservedinourmembrane-freeFPMDsimulations.Darkgreydots representtheiraverages(foreachloadingrateF_).ThesolidlineshowsthemeanruptureforcehFipredictedbytheBSKmodel[45]forΔG=28.5k T,x = B b 0.86nm,andD=6.6×106nm2/s.AfitwiththeHS[44]modelyieldssimilarmodelparameters(notshown).Dashedlinesshowthevariationoftherupture forcespredictedbytheBSKmodel(2standarddeviations,seeS1Textforadetailedanalysis).Pullingmembrane-boundFK-FAKdiagonallyyieldedsimilarly largeruptureforces(greendots).Verticalpullingresultedinsignificantlylowerruptureforces(pinkdots),asthisdirectionpromotesthelessresistantzipper- likedissociationmechanismdescribedinS4BandS4CFig)Timeatwhich50%ofinactiveFAK(B)andGDP-boundRasprotein(C)areconsumed,under varyingexternalforce.Timesobtainedforthreesetsofparameters(1to3)correspondingtothethreefitspresentedinS6Fig. doi:10.1371/journal.pcbi.1004593.g004 phosphorylationsite,imposedbytheautoinhibitoryFERMdomain.Thereleaseofautoinhibi- tionislikelytomakethekinaseactivesiteaccessibleforitssubstrate,Tyr397ofthesameor anotherFAKmolecule[42,57],and/ortorenderTyr576/577accessibletoSrc.Itisnon-trivial thattheexertionofapullingforceatoppositesitesoftheFERMandkinasedomainsleadsto theirdissociation.OtherlikelyscenariosareproteinunfoldingandPIP -proteindissociation, 2 bothofwhichwouldinactivateFAK,becauseanintactkinasestructureandalsoPIP binding 2 [18]arerequiredforFAKactivity.Infact,α-helicalproteinsareknowntounfoldatforcestypi- callylowerthanβ-sheetproteins[58],andboththekinaseC-lobeandtheFERMF2domain featuremainlyα-helicalsecondarystructure.Inthisregard,force-inducedFAKunfolding wouldbeanexpectedresultandwasindeedpreferredoverFERM-kinasedissociationwhen pullingtheFERMF1-orF3-lobeawayfromthekinaseC-terminus.Instead,intheparticular– andphysiologicallyrelevant–casethatforceisappliedtothePIP bindingsiteandthekinase 2 C-terminus,wefounddomain-domaindissociationtobestronglypreferredoverunfoldingor membranedetachment,overalargerangeofpullingvelocities,androbustwithregardtothe pullingdirectionandpresenceofmembraneinteractions.Thus,theα-helicalregionssubjected tothepullingforcemostlyrefrainfromunfolding,andtheyinsteadtransducetheloadtothe F2/C-lobeinterface,whichreadilyopenspriortosubstantialkinaseunfolding.Wesuggestthat itisthezipper-liketopology,withtheforceapplicationsitesbothlocatedatthemembrane- proximalbasisofthetwodomains,thatmechanicallyweakensthedomaininterface,resulting inefficientFAKopeningandactivation.Forcetransductionthroughthetwotermini,incon- trast,resultsinshearingthetwodomainsrelativetoeachother,makingthemlesspronetodis- sociate.Thelowermechanicalresistanceofzipperversusshear-typetopologieshasbeen describedearlier(e.g.[53]),andFAKforce-induceddomain-domainruptureandactivation appearstobeanothervariationofthistheme. PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 9/16 FAKMechanosensing Ourfindingsnotonlydecidedlyargueforamechano-sensingfunctionofFAK,butalso emphasizethecrucialroleofthemembraneinmechanotransduction.First,membranebinding allowsforcetopropagatetotheF2-lobeofFAK.Second,theFAK-membraneinteractionwith- stoodtheexternalloadonlyinthecaseofPIP -enrichedmembranes(15%PIP ).Incontrast, 2 2 FAKdetachedfromlowPIP -contentmembranes.ThissupportsthenotionofPIP clustering 2 2 asarequirementforFAKactivationatFAs[18,51].WenotethatFAKcanbeactivatedinvitro bythesoleactionofPIP andSrc,i.e.intheabsenceoftensileforcesactingonmembrane- 2 boundFAKatFAsinstretchedcells.However,ithasbecomeclearthatFAKactivationcan proceedalongdifferentroutes,dependingonthecellularenvironment,andpotentiallycanalso involvepHchanges[59],and/orgrowthfactorreceptors[16].Wehereproposeforcetosubsti- tuteorcomplementsomeoftheseactivatorsshapingthemulti-dimensionallandscapeofFAK activity. UsingTRFDA,werecoveredthestabilizingroleofY180,M183,V196andF596,ahydro- phobiccorepreviouslyshownbymutagenesistostabilizetheautoinhibitedstate[14],validat- ingoursimulationdata.Inaddition,wefoundD200andR598tocontributetotherupture force,andpredicttheirmutationtoresultinincreasedFAKactivity. Thequestionarises,howforcefeedsintoFAK-mediatedRasGDP/GTPexchangeandregu- latesERK-dependentgeneexpression,analogoustothechemicalstimulationofgrowthfactor receptor-dependentRassignaling(S9Fig)[27].ToassesshowFAKasamechanosensorcou- plesmechanicalsignalsintothedownstreambiochemicalnetwork,wedefinedforce-dependent FAKactivationastheinitialstepofakineticmodelfortheRassignalingpathway(S10Fig) [46–49].BothFAKopeningandGDP/GTPexchangeinRasareacceleratedbyexternalforces, asexpected(Fig4Band4CandS11AFig).Intriguingly,whileFAKforce-inducedactivation showsanearlylineardependencyonforceonthelogarithmicscale(Fig4B),Ras-GTPproduc- tionshowsahighlynon-lineardependencyandsaturatesbeyondacriticalforce(Fig4C).The reasonisthatactivatedFAKincomplexwithitspartners,Grb2andSOSviac-SrcandSHC, actsasanenzymeforRasactivation.Asadirectconsequence,Rasactivationfollowsmechano- enzymatickineticsreminiscentofaninhibitoryMichaelis-Mentenmechanisms(S11BFig) [60],inwhichforceregulatestheenzymeconcentration. Inconclusion,ourcomputationalstudyprovidesdirectevidenceatthemolecularlevelfora mechano-sensoryroleplayedbyFAKatPIP -enrichedmembranesofFAs.Throughaspecific 2 domainopeningmechanismregulatedbyforce,FAKcanintegratemechanicalandchemical stimuliintodownstreamsignalingtothenucleus.Wesuggestthemechano-enzymaticsofFAK andRastoprovideacaponthecell’smechano-response.Ourresults,onFAKactivationand signaling,aredirectlytestableamongothersbymolecularforcesensors[61,62]andcell stretchingexperiments.Howotherputativelymechano-activatedkinases,suchastherelated Srckinase,followsimilarmechanisms,atfocaladhesionsorelsewhere,remainstobeshown. SupportingInformation S1Fig.Partialunfoldingofthekinasedomain.A)Distancebetweenthepulledgroupsatthe momentofdissociation(De−e)asafunctionoftheappliedloadingrate.Dissociationwasmoni- toredbetweentheFERMF2-lobeandthekinaseC-lobe(F2-C)andbetweentheFERMdomain andtheTyr576–577phosphorylationsite(F-YY).Eachdotcorrespondstoonesimulationrun andthelineisapolynomialfittoallthepointsasaguidetotheeye,indicatingaslightaugment inDe−ewithincreasingloadingrate.B)NumberofunstructuredresiduesNUNasafunctionof thetime,forasimulationdisplayingalargeDe−evalue(circleinA).Timetraceisshownin blackandapolynomialfitasaguidetotheeyeinblue.Residualunfoldingofaround30resi- dueswasobservedattheC-terminus.Thenumberofunstructuredresidueswasobtainedusing PLOSComputationalBiology|DOI:10.1371/journal.pcbi.1004593 November6,2015 10/16
Description: