Conformation and dynamics of the kinase domain …...2020/07/13 · RCKW employing...

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ConformationanddynamicsofthekinasedomaindrivesubcellularlocationandactivationofLRRK2.SvenH.Schmidt1*,Jui-HungWeng2,3*,PhillipC.Aoto2,3*,DanielaBoassa4,5,SebastianMathea6,StevenSilletti3,JunruHu4,5,MaximilianWallbott1,ElizabethAKomives3,StefanKnapp6,FriedrichW.Herberg1a,SusanS.Taylor2,3a

1DepartmentofBiochemistry,UniversityofKassel,34132Kassel,Germany2DepartmentofPharmacology,UniversityofCalifornia,SanDiego,LaJolla,California92093,USA3DepartmentofChemistryandBiochemistry,UniversityofCalifornia,SanDiego,LaJolla,California92093,USA4NationalCenterforMicroscopyandImagingResearch,UniversityofCalifornia,SanDiego,LaJolla,California92093,USA5DepartmentofNeurosciences,UniversityofCalifornia,SanDiego,LaJolla,California92093,USA6 Institute for Pharmaceutical Chemistry, Goethe University Frankfurt, 60438 Frankfurt am Main,Germany*AuthorscontributedequallyaCo-correspondingauthors

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted July 14, 2020. ; https://doi.org/10.1101/2020.07.13.198069doi: bioRxiv preprint

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Abstract

Inamulti-tieredapproach,weexploredhowParkinson´sDisease-relatedmutationshijackthefinely

tunedactivationprocessofLeucine-RichRepeatKinase2(LRRK2)usingaconstructcontainingtheROC,

Cor,KinaseandWD40domains(LRRK2RCKW).WehypothesizedthattheN-terminaldomainsshieldthe

catalytic domains in an inactive state. PDmutations, type-I LRRK2 inhibitors, or physiological Rab

GTPases can unleash the catalytic domains while the active kinase conformation, but not kinase

activity, is essential for docking onto microtubules. Mapping solvent accessible regions of

LRRK2RCKW employing hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed how

inhibitorbindingissensedbytheentireprotein.MolecularDynamicssimulationsofthekinasedomain

elucidateddifferencesinconformationaldynamicsbetweenwtandmutantsoftheDYGψmotif.While

alldomainscontributetoregulatingkinaseactivityandspatialdistribution,thekinasedomain,driven

bytheDYGψmotif,coordinatesdomaincrosstalkandservesasanintrinsichubforLRRK2regulation.


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Introduction

Although Parkinson’s Disease (PD), first described in 1817 (1), is the second most common

neurodegenerativediseasetodate,therearestillnocuresavailable(2-4).Aprimaryreasonforthis

lackofprogressisthattheunderlyingmolecularprocessesthatdrivePDarestillbarleyunderstood.

Leucinerichrepeatkinase2(LRRK2)isagoodtargettoimproveourknowledgeoftheseunderlying

processesasmutationswithinthisproteinarethemostcommoncauseforgeneticallydrivenformsof

PD (5, 6). LRRK2 is a complex and multifunctional protein consisting of seven closely interacting

structuraldomains(7,8).LRRK2belongstotheproteinfamilyofROCOproteins,whicharedefinedby

containingaGTPasedomaincalledRasofComplex(Roc)followedbyaCORdomainincombination

withotherdomains.AspecialfeatureofLRRK2,aswellassomebutnotalloftheROC:CORproteins,

is that itcontainsboth,akinasedomainandaGTPasedomainsothattwocatalyticallyactivecore

domainsareembeddedwithinthesamepolypeptidechain(9-11).

Familial PD mutations in LRRK2 lead to altered cellular phenotypes such as microtubule (MT)

associatedfilamentformation,impededvesiculartrafficking,aswellaschangesinnuclearmorphology

(12-19).SomeofthesePDmutationsarefoundinthekinasedomain,forexampleG2019Sleadsto

increased kinase activity, and so kinase activity is thought to be a major component of LRRK2

regulation.Yet,somepotentkinaseinhibitorssuchastype-IinhibitorMLi-2(MerckLRRK2Inhibitor2)

aswellasotherkinasemutationssuchasI2020Twithunchangedorreducedcatalyticactivityalsolead

tothesamedisease-likecellularphenotypes(17,20,21).Thus,itisclearthatthekinasedomainplays

animportantpathogenicrolebutthatitsactivityaloneisinsufficienttodescribethecomplexitiesof

LRRK2 regulation.With five scaffoldingand twocatalyticallyactivedomains the interplayandhow

thesedomainseffectand regulateeachother isverycomplexandallows formultilayered intrinsic

cross-regulation between the kinase and the GTPase domains. However, due to the lack of high-

resolutionstructuraldatanotmuchwasknownaboutthese interactionsandhowthey intrinsically

regulateLRRK2.LRRK2-dimerization,andinteractionswithotherproteinslikeRaband14-3-3proteins

furtherincreasethecomplexityofLRRK2regulation(22-33).

TounravelthecomplexityofLRRK2regulationweusedamulti-tieredapproachbeginningwithacell-

basedassayforfilamentformation,aprocessthatcorrelateswithLRRK2dockingontomicrotubules

(MTs)(14).Specifically,weusedlivecellimagingtoexaminethespatialandtemporaldistributionof

full length wt and G2019S LRRK2 in HEK293T cells in the presence and absence of LRRK2 kinase

inhibitors.ToexploretheregulatoryfunctionsthatareembeddedinthecatalyticallyinertN-terminal

domains (NTDs)of LRRK2and todiscriminatebetween thecatalyticC-terminaldomains (CTDs)we

engineeredaconstruct,LRRK2RCKW,thatcontainsonlytheCTDs(ROC,COR,KinaseandWD40domains).


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Thisconstructcontainsbothcatalyticmoietiesof LRRK2andexhibits functionalkinaseandGTPase

activity embedded in the same protein. It was shown previously that kinase activity of LRRK2 is

dependentonthepresenceoftheROC:CORdomainandtheC-terminus(34,35).Ontheproteinlevel,

weinvestigatedthekinaseactivityofwtLRRK2RCKWandseveralvariantsusingLRRKtideandRab8aas

substrates.WefocusedinparticularontheDYGymotifinthekinasedomainwhichisahotspotfor

PDmutationsandwhereweshowedpreviouslythatthetyrosine(Y2018),conservedasaPheinmost

otherproteinkinases, isacriticalpartoftheswitchmechanismthatleadstoLRRK2activation(36).

Usinghydrogendeuteriumexchangemassspectrometry(HDXMS)wethenzoomedintothelevelof

LRRK2 peptides and demonstrated that LRRK2RCKW is a well-folded protein.We continued tomap

changes inthesolventexposureoftheLRRK2RCKWdomainsfollowingbindingofMLi-2tothekinase

domain,whichprovidedanallostericportraitof thekinasedomainasahub fordriving long-range

conformational changes. At yet another level, we used Gaussian molecular dynamics (GaMD)

simulationsasa computationalmicroscope toobserveatanatomistic levelhowsingleaminoacid

mutationsinthekinasedomaincontributetotheintrinsicdynamicregulationofLRRK2.

Ourmulti-scale approach allowed us to achieve a better understanding of the intrinsic regulation

processesofLRRK2andemphasizedthecrucialroleoftheDYGψmotifinregulatingLRRK2structure

andfunction.WehypothesizedthattheN-terminusofLRRK2(ARM:ANK:LRR)playsaregulatoryrole

actingasa“lid”,whichcanbe“unleashed”fromthecatalytic(RCKW)domainsbykinaseinhibitorsor

constitutively by some of the PD mutations. In addition to shielding the catalytic domains, the

N-terminallidmediatesinteractionswithotherproteinsthatcontributetoactivationandsubcellular

localization (26-29, 37, 38). Strikingly, the resulting LRRK2 model for activation and subcellular

localizationcloselyresemblestheactivationprocessofRafkinases,therebyunderliningtheplausibility

ofourmodelandthecentralconceptofkinasesservingasthehubfordrivingconformationalswitching

inmulti-domainsignalingproteins.

Methods

HEK293TCellcultureandTransfectionForexpressionofeachFlag-Strep-Strep-tagged (FSS) LRRK2RCKW construct cellson ten15 cmØcell

culture dishes were transfected. Therefore, 1.0x107 HEK293T cells (human embryonic kidney cells

carrying the temp sensitive mutant of the SV-40 large T-antigen, DSMZ, DSMZ-No:ACC635) were

seededperdishand incubated for24hat37°Cand5%CO2 inDulbecco'sModifiedEagleMedium

(DMEM)highglucose(w.L-Glutamine;w.o.SodiumPyruvate,biowest)supplementedwith10%fetal

bovineserum(FBS).Inthefollowing,transfectionswereperformedbyaddinga30minpreincubated


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mixture of 15 μg plasmid DNA (pCDNA3.0-FSS-LRRK2RCKW (aa1327-2527); NM_198578), 150 μl PEI

(Polyethylenimine)(1µg/µl)and1.5mlDMEMhighglucoseperdish.Mediumwasexchangedwith

freshDMEM(highglucose,+10%FBS)after24h.Afteranother24hcellswereharvestedandstored

at-20°Cbeforeuse.

LRRK2RCKWTransfectionandExpressioninSf9cells

ExponentiallygrowingTriExSf9insectcells(cellsarederivedfromahigh-yieldingcloneofSpodoptera

frugiperdacellIPLBSf21-AE(ATCCCRL-1711),Novagen,Prod.-No.:71023-3)weredilutedtoadensity

of2x107cells/mL.ToinitiateLRRK2RCKWexpression,ahigh-titervirussuspensionwasaddedintheratio

1:64.Theviruseshadbeengeneratedusing theBac-to-Bacexpression system (Invitrogen) and the

expressionvectorpFB-6HZB.Accordingly,theexpressedproteinwascomposedofanN-terminalHis6-

ZtagandtheLRRK2residues1327to2527.Theinfectedcellswereincubatedin800mLaliquotsin

shakerflasks(66h,gentleagitation,27°C),harvestedbycentrifugationandstoredat-20°C.

Purification of overexpressed Flag-Strep-Strep-tagged LRRK2RCKW

constructsfromHEK293TCells

Eachpelletwasresuspendedin10mloffreshicecoldlysisbuffer(25mMTris-HClpH7.5,150mM

NaCl,10mMMgCl2,0.5%Tween20,500µMGTP,cOmplete™EDTAfreeproteaseinhibitorcocktail

[Roche],PhosSTOP™[Roche])andincubatedfor30minat4°Conarotatingwheeltolysecells.Inthe

following,celldebriswereremovedbycentrifugationat42,000xgand4°Cfor40minandafiltration

step(0.45µmsterilefilter).ThesupernatantwasloadedontoaStreptactinSuperflowcolumn(0.5mL

bedvolume,IBAGoettingen)andpurificationwasperformedaccordingtothemanufacturer’sprotocol

whileallbufferswereadditionallysupplementedwith500µMGTP(BiologLifeScienceInstitute)and

10mMMgCl2.ThepurifiedLRRK2RCKWconstructswerestoredat -80°Ccontaining10%Glyceroland

0.5mMTCEP.TheLRRK2RCKWconstructconcentrationsweredeterminedafterBradford(39).

PurificationofoverexpressedLRRK2RCKWconstructsfromSf9cells

TheLRRK2RCKWexpressionconstructcontainedanN-terminalHis6-ZtagandaTEVproteasecleavage

site.Forpurification,theSf9cellpelletswerewashedwithPBS,resuspendedinlysisbuffer(50mM

HEPESpH7.4,500mMNaCl,20mMimidazole,0.5mMTCEP,5%glycerol,5mMMgCl2,20μMGDP)

andlysedbysonication.ThelysatewasclearedbycentrifugationandloadedontoaNiNTAcolumn.

AftervigorousrinsingwithlysisbuffertheHis6-Ztaggedproteinwaselutedinlysisbuffercontaining

300mMimidazole.Immediatelythereafter,theeluatewasdilutedwithabuffercontainingnoNaCl,

inordertoreducetheNaCl-concentrationto250mMandloadedontoanSPsepharosecolumn.His6-


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ZTEV-LRRK2RCKWwaselutedwitha250mMto2.5MNaCl gradientand treatedwithTEVprotease

overnight to cleave the His6-Ztag. Contaminating proteins, the cleaved tag, uncleaved His6ZTEV-

LRRK2RCKWandTEVproteasewereremovedinanothercombinedSPsepharoseNiNTAstep.Finally,

LRRK2RCKWwasconcentratedandsubjectedtogelfiltrationinstoragebuffer(20mMHEPESpH7.4,

800mMNaCl,0.5mMTCEP,5%glycerol,2.5mMMgCl2,20μMGDP)usinganAKTAXpresssystem

combinedwithanS200gelfiltrationcolumn.

LiveCellImagingTime-lapse imaging was conducted using an Olympus FluoView1000 laser scanning confocal

microscopeequippedwithaCO2andtemperature-controlledchamber(at37°C)anda60X1.42NA

objectivelens.TheHEK293Tcells(CCLV,RRID:CVCL_0063)wereimagedinHBSSsupplementedwith

5%fetalbovineserumand10mMHEPESandmaintainedat37°Cthroughout theexperiment.YFP

fluorescenceandthedifferentialinterferencecontrastwerecollected,515nmexcitation/530–630nm

emission.Imageswererecordedevery5to11minforatotaldurationof2hor14hasspecifiedin

figurelegends,samplingspeed2μs/pixel,imagesize640X640pixelsin15-20zslices/area(stepsize1

μm). Cells were imaged before and after treatment with inhibitors MLi-2 (100 nM) or rebastinib

(100nM).Forwashoutexperiments,cellswerewashed5timeswithimagingmedia,2mineach,and

thentheimagingsessionwasresumedusingthesamesettingsasbeforewashout.Imageprocessing

suchas3Dvolumerenderingand4DmoviegenerationwasdoneusingtheImarisSoftware(Bitplane

AG,St.Paul,MN)andtheImageJsoftware(rsb.info.nih.gov/ij).

ImmunofluorescenceandlaserconfocalimagingHEK293Tcellswereseededonto6-welldishescontainingpoly-D-lysine-coatedglasscoverslipsoronto

35mm poly-D-lysine-coated glass bottom dishes (MatTek Corporation, Ashland, MA, USA). For

HEK293Tcelltransfection,1μgofFlag-Strep-Strep-(FSS)-taggedLRRK2RCKWcDNAandLipofectamine

2000reagent(ThermoFisherScientific,USA)wereusedaccordingtothemanufacturer’sprotocol.After

incubationfor48hat37°Ccellsweretreatedfor2hwiththeLRRK2inhibitorMLi-2.Subsequently

cellswerefixedwith4%paraformaldehydeinphosphate-bufferedsaline(PBS)for15minutesatroom

temperature.CellswerewashedinPBS,permeabilizedin0.1%TritonX-100,andblockedin1%BSA,

50mMglycineand2%normaldonkeyserum.Arabbitanti-Flagantibody(AbnovaCompany,Cat.No.

PAB0900)wasmixed1:200inblockingbufferdilutedfive-foldinPBS.Theprimaryantibodysolution

wasappliedto thecells for1hat roomtemperature.Thesecondaryantibody (donkey-anti-rabbit-

Alexa568,Invitrogen,Cat.No.A10042)wasalsodiluted(1:100)intheblockingbufferdilutedfive-fold

in PBS and applied for 1 h at room temperature. Samplesweremountedwith the antifade agent

ProLongGoldwithDAPI (ThermoFisherScientific,USA).TheOlympusFluoview1000 laserscanning


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confocalmicroscopeutilizinga60Xoilimmersionobjectivelenswithanumericalapertureof1.42was

usedforconfocalimaging.Z-stackimageswereacquiredwithastepsizeof0.3micronsandprocessed

using the Fiji software package (40). Cells expressing the differentmutantswere assessed for the

presenceofclearfilamentousstructuresandquantifiedintwoindependentexperimentsincaseofthe

untreatedcellsandinoneexperimentfortheMLi-2treatedcells.

Rab8a phosphorylation by LRRK2RCKW variants and pathogenic

mutations

PhosphorylationofT72ofRab8awasmeasuredviaWesternBlottingusingapT72specificantibody.

Priortotheblottingstepontoanitrocellulosemembraneaninvitrokinaseassayusingkinasebuffer

(25mMTRIS/HCl,pH7.5,50mMNaCl,10mMMgCl2,1mMATP,0.5mMGTP,0.1mg/mlBSAand

1mMDTT)andanSDS-PAGEwereperformed.Forthekinaseassay2.5µM(6xHis)-Rab8a(aa6-175)

wereusedassubstratefor200nMoftheLRRK2RCKWvariants.Rab8awasphosphorylatedat30°Cfor

7minat650rpmonashaker.Tostopthereaction1xNu-PAGELDSsamplebuffer(Invitrogen,Cat.No.

NP0007)supplementedwith250µMDTTwasaddedfollowedbyanincubationat80°Cfor5-10min.

AfterSDS-PAGEandwesternblotting,membraneswereblockedwith5%(w/V)BSAinTBS-T(1xTris-

buffered saline supplemented with 0.1% Tween20) for 1 h. Subsequently they were incubated

overnightat4°CwiththeprimaryantibodiesagainstpT72(MJF-R20,abcam,Cat.No.ab231706)and

theHis-tagofRab8a(anti-His-Antibody,GEHealthcare,mouse).Bothwerediluted(1:1000)inblocking

buffer.MembraneswerethenwashedthreetimeswithTBS-T.Aftersecondaryantibodyincubation

(anti-rabbitIRDye800andanti-mouseIRDye680,1:15000,LiCOR)for1hatRTsignalsweredetected

usingtheOdysseyFCimagingsystem(LiCOR).

MicrofluidicMobilityShiftKinaseAssay(MMSKA)

ToquantifythekinaseactivitiesoftheLRRK2RCKWvariantsMMSKAwereperformedusing1mMATP

and1mMLRRKtide(RLGRDKYKTLRQIRQ-amide,GeneCust)assubstrates.Fortheseassaystwostock

solutionswereprepared: a2x concentrated (conc.) LRRKtide solution (1900µMLRRKtide, 100µM

Fluorescein-LRRKtide,2mMATP)anda2xconc.LRRK2RCKWvariantsolution(100-200nMLRRK2RCKW

variant, 20mMMgCl2, 1mMGTP). All stock solutionswere preparedusing kinase buffer (25mM

TRIS/HCl,pH7.5,50mMNaCl,0.1mg/mlBSAand1mMDTT).Thereactionswerestartedbymixing

bothsolutionsina1:1ratioin384wellplates.Reactionswereperformedat30°Candmonitoredfor

60-90minusingaLabChipEZReader (PerkinElmer).Theslope (conversionrate, [m]=%/min)of the


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percentalconversionplottedagainstthetimewasdeterminedusingalinearfitmodelofGraphPad

Prism6andwasconvertedintoareactionvelocity([v0]=µmol/min).Foreveryconstructatleastthree

independentmeasurementswereperformed.

Titrationassaysusingthehigh-affinityinhibitorMLi-2(Merck,USA)wereperformedtodeterminethe

activeproteinconcentrationsoftheLRRK2RCKWvariants.Therefore,24µLofBufferA(25mMTRIS/HCl,

50mMNaCl,20mMMgCl2,1mMGTP,1mMDTT,0.5mg/mlBSA,52.1/104.2nMLRRK2RCKWvariant)

weremixedwith1µLofanMLi-2dilutionseries(50xconcentrated)preparedin100%DMSO.Tostart

thereaction10µLofthisreactionmixwasaddedto10µLofBufferB(25mMTRIS/HCl,50mMNaCl,

1 mM DTT, 0.5 mg/ml BSA, 1900 µM LRRKtide, 100 µM Fluorescein-LRRKtide, 360 µM ATP). The

resultingconversionrateswereplottedagainsttherespectiveMLi-2concentrationsandtoobtainthe

activeproteinconcentrationsthex-axesintersectionoftherespectivelinearfitwasdeterminedusing

GraphPadPrism6(assuminga1:1bindingofMLi-2).

Hydrogen-deuteriumexchangemassspectrometryHydrogen/deuteriumexchangemassspectrometry (HDXMS)wasperformedusingaWatersSynapt

G2SiequippedwithnanoACQUITYUPLCsystemwithH/DXtechnologyandaLEAPautosampler.The

sampleconcentrationwas5µM inLRRK2buffercontaining:20mMHEPES/NaOHpH7.4,800mM

NaCl,0.5mMTCEP,5%Glycerol,2.5mMMgCl2and20µMGDP.Thedeuteriumuptakewasmeasured

in LRRK2 buffer in the presence and absence of the kinase inhibitor MLi-2 (50 µM). For each

deuterationtime,4µLcomplexwasequilibratedto25°Cfor5minandthenmixedwith56µLD2O

LRRK2buffer for0, 0.5, 1or2min. Theexchangewasquenchedwithanequal volumeofquench

solution(3Mguanidine,0.1%formicacid,pH2.66).Thequenchedsample(50μL)wasinjectedinto

thesampleloop,followedbydigestiononanin-linepepsincolumn(immobilizedpepsin,Pierce,Inc.)

at 15 °C. The resulting peptideswere captured on a BEHC18Vanguard pre-column, separated by

analyticalchromatography(AcquityUPLCBEHC18,1.7μM,1.0X50mm,WatersCorporation)usinga

7-85% acetonitrile gradient in 0.1% formic acid over 7.5min, and electrosprayed into theWaters

SYNAPTG2Siquadrupoletime-of-flightmassspectrometer.Themassspectrometerwassettocollect

dataintheMobility,ESI+mode;massacquisitionrangeof200–2,000(m/z);scantime0.4s.Continuous

lockmasscorrectionwasaccomplishedwith infusionof leu-enkephalin (m/z=556.277)every30 s

(massaccuracyof1ppmforcalibrationstandard).Forpeptideidentification,themassspectrometer

wassettocollectdatainMSE,ESI+modeinstead.

ThepeptideswereidentifiedfromtriplicateMSEanalysesof10μMLRRK2RCKW,anddatawereanalyzed

usingPLGS3.0(WatersCorporation).Peptidemasseswereidentifiedusingaminimumnumberof250

ioncountsforlowenergypeptidesand50ioncountsfortheirfragmentions.Thepeptidesidentified


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inPLGSwerethenanalyzed inDynamX3.0 (WatersCorporation)usingacut-offscoreof6.5,error

toleranceof5ppmandrequiringthatthepeptidebepresentinatleast2ofthe3identificationruns.

Thepeptidesreportedonthecoveragemapsarethosefromwhichdatawereobtained.Therelative

deuteriumuptakeforeachpeptidewascalculatedbycomparingthecentroidsofthemassenvelopes

ofthedeuteratedsamplesvs.theundeuteratedcontrols(41).ForallHDX-MSdata,atleast2biological

replicateswereanalyzedeachwith3technicalreplicates.Dataarerepresentedasmeanvalues+/-

SEMof3technicalreplicatesduetoprocessingsoftwarelimitations,howevertheLEAProbotprovides

highly reproducible data for biological replicates. The deuterium uptake was corrected for back-

exchangeusingaglobalbackexchangecorrectionfactor(typically25%)determinedfromtheaverage

percentexchangemeasured indisorderedterminiofvariousproteins (42).Deuteriumuptakeplots

weregeneratedinDECA(github.com/komiveslab/DECA)andthedataarefittedwithanexponential

curveforeaseofviewing(43).

GaussianacceleratedMolecularDynamics(GaMD)simulationTheLRRK2kinasedomainconstructsforsimulationswerepreparedusingPhyre2(44)withthecrystal

structureofSrckinase(PDBID:1Y57)servingasan initialtemplate.Themodelwasseparatelythen

mutatedtoY2018F,G2019S, I2020TandphosphorylatedatS2032andT2035toformtheactivated

LRRK2kinase(45,46)andprocessedinMaestro(Schrodinger).TheProteinPreparationWizardwas

used to build missing sidechains and model charge states of ionizable residues at neutral pH.

Hydrogensandcounter ionswereaddedandthemodelsweresolvatedinacubicboxofTIP4P-EW

water(47)and150mMKClwitha10ÅbufferinAMBERtools(48).AMBER16(48)wasusedforenergy

minimization,heating,andequilibrationsteps,usingtheCPUcodeforminimizationandheatingand

GPUcode forequilibration.Parameters from theBryceAMBERparameterdatabasewereused for

phosphoserineandphosphothreonine(49).Systemswereminimizedby1000stepsofhydrogen-only

minimization,2000stepsofsolventminimization,2000stepsof ligandminimization,2000stepsof

side-chainminimization,and5000stepsofall-atomminimization.Systemswereheatedfrom0Kto

300 K linearly over 200 pswith 2 fs time-steps and 10.0 kcal×mol×Åposition restraints on protein.

TemperaturewasmaintainedbytheLangevinthermostat.Constantpressureequilibrationwithan8Å

non-bonded cut-off with particlemesh Ewald was performedwith 300 ps of protein and peptide

restraintsfollowedby900psofunrestrainedequilibration.GaussianacceleratedMD(GaMD)wasused

on GPU enabled AMBER16 to enhance conformational sampling (50). GaMD applies a Gaussian

distributedboostenergytothepotentialenergysurfacetoacceleratetransitionsbetweenmeta-stable

stateswhileallowingaccuratereweightingwithcumulantexpansion.Bothdihedralandtotalpotential

accelerationwereusedsimultaneously.Potentialstatisticswerecollectedfor2nsfollowedby2nsof

GaMDduringwhichboostparameterswereupdatedforeachsimulation.EachGaMDsimulationwas


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equilibratedfor10ns.Foreachconstruct10 independentreplicatesof200nsofGaMDsimulation

wererunintheNVTensemble,foranaggregateof2.0µsofacceleratedMD.Potentialenergysurfaces

weredeterminedalongeachreactioncoordinateusingacumulantexpansiontothesecondorder.

ResultsTocharacterizeanddissectthefunctionalpropertiesofthecatalyticdomainsofLRRK2,inparticular

the kinase domain, we used a multi-scale approach that begins with testing real time filament

formation in live cells to assessing the consequences of molecular dynamics simulations of PD

mutationsinthekinasedomain.Ofprimaryimportancewastocharacterizethebiochemicalproperties

ofLRRK2RCKWfollowingdeletionofthecatalyticallyinertN-terminallid.TonextconfirmthatLRRK2RCKW

wasawell-foldedproteinweusedhydrogendeuteriumexchangemassspectrometry(HDXMS)and

thentocapturetheallosteric featuresofthekinasedomainwemappedbyHDXMStheconformational

changesinLRRK2RCKWthatresultfromaddingMLi-2.

Capturingfilamentformationinrealtime.WeandotherspreviouslyshowedthattreatmentwithahighlyspecificLRRK2inhibitor(MLi-2)induces

filamentformationofwtLRRK2andtheG2019Smutantwhentheseproteinsaretransientlyexpressed

inmammaliancells(17,36).Tocapturethedynamicsofsuchredistributionweperformedtime-lapse

imagingofYFP-taggedG2019SandwtLRRK2.AsshowninFigure1andSupplementaryvideos,under

normalconditionsG2019SLRRK2ismostlydiffuseinthecytosol;however,15-30minfollowingMLi-2

treatmenttheproteinbeginstoconcentratefirstin‘satellite’structuresdiffusethroughoutthecells.

Itthenpolymerizestoformintricatethickerfilamentsby2.0-2.5haftertreatment.AlthoughwtLRRK2

followsasimilarredistributionupontreatmentwithMLi-2,ingeneralittakeslonger,approximately

30min-1h,beforethefirststructuresareobserved(Supplementarymovie).Inbothcasesthiseffect

isreadilyreversible:afterwashoutofMLi-2for2h,theproteinsgraduallydiffusebackintothecytosol.

To verify that this protein rearrangementwas truly dependent on the specificMLi-2 inhibitor,we

performed time-lapse imaging using a type 2 inhibitor, rebastinib (Figure 1B). Although rebastinib

stabilizesaKinase-WD40constructof LRRK2,basedona thermal shift assay (FigureS1), itdidnot

induce changes in the localization of G2019S proteins even after 8 h treatment, confirming the

predictionofDenistonetal.(2020)(13).


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Figure1.MLi-2,butnotRebastinib,affectsthelocalizationofthekinasehyperactiveLRRK2-G2019Smutant.A) Time-lapse imaging of HEK293T cells transiently expressing YFP-LRRK2-G2019S: confocal images (YFPfluorescencesignal,maximumintensityprojections)wereacquiredevery11min.Representativeimagesshowthetypicaldiffusecellularlocalizationoftheproteins(t=0h)priortotreatmentwith100nMMLi-2;followingMLi-2addition,proteinsrelocalizetoformcytoplasmicfilamentousstructures(yellowarrows,+MLi-2,t=2.5h). After washout of the inhibitor, the proteins gradually dissociate from the filaments into the cytosol(washout,t=2-3h).B)Time-lapseimagingofHEK293TcellstransientlyexpressingYFP-LRRK2-G2019Sbefore(t=0 h) and after treatment with 100 nM Rebastinib. No changes in the localization of the proteins areobserved.Scalebar,20microns.

LRRK2RCKW variants spontaneously form filaments around

microtubulesinanMLi-2independentmanner

Inourfilamentformationassay,flag-taggedvariantsoftheLRRK2RCKWconstructwereoverexpressed

andcellswereanalyzedafterfixationviaantibodystaininginaconfocal laser-scanningmicroscope.

The majority of the transfected cells, regardless of the mutation, displayed constitutive filament

formation (Figure 2). Most striking, in contrast to fl LRRK2, is that wt and G2019S are no longer

dependentonMLi-2 for dockingontoMTs. This supports thehypothesis that the inertN-terminal

scaffoldingdomainsarenotrequiredforthefilamentstoformbutinsteadareessentialforprotecting

orshieldingthecatalyticdomainstopreventthemfromdockingontoMTs.Inthiswaytheypromote

thecytosolicdistributionofLRRK2priortoactivation,whichis likelyfurtherfacilitatedbyphospho-

dependent interactions with specific 14-3-3 proteins (22, 27). The N-terminal domains are also

importantfordockingtoRabproteinssuchasRab29,whicharethoughttoinitiateactivationofLRRK2

(29).Thiswouldbeaphysiologicalmechanismwheremultiplebiologicalfunctionsareembeddedin


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thecatalyticallyinertN-terminaldomains,thisincludesactivationand/orlocalizationbyheterologous

proteinsaswellasinhibitionofthecatalyticdomains.WehypothesizethatmostofthePDmutations

circumventor“hijack”thisnormalprocess.

Of the mutants tested only LRRK2RCKW D2017A, a kinase dead mutant, showed strongly reduced

dockingontoMTs,andthisisconsistentwithourearlierfindingsshowingthatthefull-lengthD2017A

mutantdidnotdockontoMTseveninthepresenceofMLi2(Figure2).WeconfirmedherethatMLi-2

didnothaveanadditiveeffectonthepercentageofcellsshowingLRRK2RCKWfilamentsanddidnot

inducebindingoftheD2017Amutant(Figure2).WeconcludethatthehighaffinitybindingofMLi-2

tothekinasedomain issufficienttounleashtheN-terminalprotective lidthatnormallyshieldsthe

catalyticdomainsandpromoteslocalizationinthecytosol.WealsoshowthatsimplyremovingtheN-

terminal lid is inmostcases sufficient topromotedockingontoMTs.Theexception is theD2017A

mutant,whichcannotbindwellunderanyconditionseitherbecauseitlackstheabilitytoundergoa

subsequent essential auto-phosphorylation step or,most likely, because it is locked into an open

conformationsimilartowhatwesawwithrebastinib.WenextaskedwhetherLRRK2RCKWretainedits

full kinase catalytic activity even though the regulatory machinery embedded in the N-terminal

domainsisremoved.


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Figure 2. Filament formation of LRRK2RCKW constructs is independent of MLi-2 treatment butreduced in case of LRRK2RCKW D2017A. A) Schematic domain organization of LRRK2 full lengthprotein(bluebox)andtheLRRK2RCKWconstruct(redbox).B)Plasmids,encodingLRRK2RCKWvariants,were transfected into HEK293T cells for LRRK2RCKW overexpression. Transfected cells were thenanalyzedforthespatialdistributionofLRRK2RCKWbyimmunostaining.AlltestedLRRK2RCKWvariantsdisplayedahighlikelihood(80-90%)toformfilamentsinsidetheHEK293TcellsexceptforLRRK2RCKWD2017A (20-30%). Interestingly, in contrast to LRRK2 full length thepercentageof cells showingfilamentformationwasindependentofMLi-2treatmentoraspecificLRRK2RCKWmutation.Scalebar,30microns.


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Protein kinase activity is conserved and, in some cases, amplified in the LRRK2RCKW proteins.

Toassesskinaseactivity,weusedbothLRRKtide,asmallsyntheticpeptide,andRab8aassubstrates

for the LRRK2RCKW proteins. In addition towt LRRK2RCKWwemeasured the kinase activities of two

ROC:CORdomainmutations (R1441CandY1699C)and fourmutations in thekinasedomain,more

preciselyintheDYGψmotif(D2017A,Y2018F,G2019S,I2020T).R1441andY1699arelocatedinthe

ROCandCORdomains,respectively,and,basedonhomologymodels,arepredictedtobepartofthe

ROC:CORdomaininterface(11,51,52).Importantly,wefoundthatwtLRRK2RCKWhaskinaseactivity

that is comparable to full-length LRRK2 (36) although in the absenceof theN-terminal scaffolding

domainstheactivityisnolongerdependentonRabactivation.UsingLRRKtideasasubstratewefound

thatthepathogenicmutationR1441CslightlyincreasedthekinaseactivitywhileY1699Chadonlya

minor effect on LRRKtide phosphorylation (Figure 3). In contrast, when we used a physiological

substrate, Rab8a, Y1699C led to an enhanced pT72 phosphorylation in vitro, comparable to the

phosphorylationbyY2018F,whereasR1441Cbehavedlikewt(Figure3).Thefactthatkinaseactivity

isdependentonsubstratemayaccountforsomeoftheconfusionintheliteratureabouttheactivity

ofvariousLRRK2mutantsbutsuggeststhatsomeofthemutationsmaychangesubstratespecificity.

If these residues are indeed at a domain interface, as predicted, they could also introduce a

conformational change that would result in the unleashing of the N-terminal scaffolding domains

and/orpromotedimerizationwhichisassociatedwithmembranelocalizationandactivationofLRRK2

(29,53,54).

ThestrongesteffectsonkinaseactivityforLRRK2RCKWwereobservedformutationsembeddedwithin

the activation segment of the kinase domain, specifically in the DYGψmotif where ψ is typically

conservedasahydrophobicresidue.MostotherkinaseshaveaDFGψmotif,andY2018waspredicted

earlier,basedonactivationwhentheTyrisreplacedwithPhe,toserveasabrakethatkeepsLRRK2in

aninactivestate(36).Wemeasuredtheeffectofmutatingeachoftheseresiduesonkinaseactivity.

TheD2017A(DYG)mutantwasnotabletophosphorylateeitherLRRKtideorRab8a(Figure3)whichis

consistentwithotherkinases,sincethisresidue ispartoftheregulatorytriadand iscrucial forthe

correctcoordinationoftheMg2+-ionsandtheγ-phosphateofATPinthekinaseactivesitecleft(55).

Reintroducing theclassicalDFGmotif toLRRK2RCKW (DYG inLRRK2) increases thekinaseactivity for

LRRKtidebya factorof3-4,whereasRab8aphosphorylationwasonlyenhancedbya factorof1.7

(Figure 3). LRRKtide phosphorylation by LRRK2RCKW G2019S was comparable to LRRK2RCKW Y2018F.

WhenRab8aisusedasasubstrate,G2019SphosphorylationofT72wastwotimeshigherthanY2018F.

TheothertestedpathogenicDYGψmutation,I2020T,displayedareducedphosphorylationofLRRKtide


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aswellasRab8a(Figure3).ThisisalsoinaccordancewithourLRRK2full-lengthdataofI2020T,albeit

full-lengthI2020TRab8aphosphorylationwascomparabletowt.TheresultsfortheI2020Tmutation

in LRRK2 full length and LRRK2RCKW demonstrate that LRRK2 pathogenicity is not driven solely by

increased kinase activity but also by changed substrate preferences such as serine/threonine

specificityaswellaschangesinsubcellularlocalization.Welatershowthatthedynamicpropertiesare

alsoalteredbythesemutations.

Figure3.TheLRRK2RCKWvariantsY2018FandG2019SenhanceLRRKtidephosphorylation,whileRab8a phosphorylation is increased by Y1699C and G2019S but not by Y2018F. (A)Using thepeptide LRRKtide as a substrate for LRRK2RCKW variants revealed that although it is a deletionconstructitpreservesLRRK2fulllengthkinaseactivity.Additionally,theDYGψmutantstestedherealso resemble the results of their full-length counterparts. Interestingly, also the pathogenicmutationsR1441CandY1699CwhicharesituatedintheRocCORregionoftheLRRK2RCKWconstructdisplayamildincreaseinkinaseactivitycomparedtoLRRK2RCKWwt.ExperimentsforeachmutantwereperformedatleastinduplicatesofduplicatesfortwoindependentLRRK2expressions.Eachdotrepresentsthemeanofaduplicate,whilethedottedlinerepresentsthemeanofthemeasuredwtactivity.TodeterminesignificantdifferencesbetweenLRRK2RCKWwtandmutantactivityaone-wayANOVA testbasedon theDunnett’smultiple comparisons testwasperformed.Herebyoneasterisks(*)indicateaPvaluebetween0.05and0.01,twoasterisks(**)aPvaluebetween0.01and0.001andfourasterisks(****)aPvaluebelow0.0001.(B)BesidesLRRKtidealsoaphysiologicalsubstrateof LRRK2, Rab8a,was tested as a kinase substrateof theRCKWconstruct. The kinaseassayswereperformedfor7minat37°CandthenstoppedbyaddingSDS-samplebuffer.Westernblotting against the pT72 site of Rab8a and the His-tag of His-Rab8a revealed increasedphosphorylationofRab8abyLRRK2RCKWY2018F,G2019SandY1699C.MLi-2wasshowntoefficientlyblock phosphorylation of Rab8a which is also true for the kinase dead mutant D2017A.QuantificationwasperformedfortwoindependentWesternBlotsusingtwoindependentproteinpreparations.Foreachquantification,thepT72signalswerereferencedtothesignalfortheHis-tagof6xHis-Rab8aandthennormalizedtotheresultingwtsignal.Thedottedlinethereforerepresents100%ofthewtsignal.


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Mapping the conformational changes induced by MLi-2 using

HydrogenDeuteriumExchangeMassSpectrometry(HDX-MS).

TodefinetheglobalconformationalchangesinducedinLRRK2RCKWasaconsequenceofMLi-2binding

weusedHDX-MS,whichallowsustodeterminethesolventexposedregionsoftheproteinoveratime

courseof5minutes.TheexchangedatawasmappedontomodelsoftheROC:CORandkinasedomains

andonto the solved structureof thehumanWD40domain.Although this is a largeprotein (1200

residues),weobtainedexcellentcoverage (>96%),and thesolventexposedregionsareconsistent

withthepredictedfoldingofallfourdomains(FigureS2).Whilewefocushereprimarilyonthekinase

domain,thegraphsummarizingtheoverallsolventaccessibilityoftheentireproteinshowsnotonly

that the four domains are well-folded but also identifies several regions that are highly solvent

exposed.Ofparticularnoteistheactivationloopofthekinasedomainaswellasthesegmentthatlies

betweentheCOR-BdomainandthekinasedomainandthesegmentthatjoinstheGTPasedomainto

theCOR-Adomain.TheHDXMSdatasuggeststhattheseregionshavinghighdeuteriumuptakeare

highlyflexibleorunfolded.Conversely,therearealsoregionsonthesurfaceofeachdomainthatare

highlyprotectedfromsolvent,implyingthatthesearedomain-domaininterfacialsurfaces(FigureS2).

ItisimportanttoappreciatethattheHDXMSprofileisobtainedindependentofasolvedstructureand

canthusserveasvalidationofapredictedmodel.OverallLRRK2RCKW isawell-foldedproteinthat is

consistentwithacomplextopologicalmodelwithinter-domaininteractions.

UnderapoconditionstheN-lobeofthekinasedomainismoreshieldedfromsolventthantheC-lobe

(Figure4A).TheaC-b4loop,forexample,isalmostcompletelyshieldedfromsolvent.Thisissomewhat

unusualinthattheN-lobeintheabsenceofnucleotidetendstoberatherdynamicformanyprotein

kinases.TheorderedandstablestructureoftheLRRK2N-lobeispredictedtobeduetoconstraints

imposedbytheotherdomains.ThisisanalogoustothewaythatcyclinbindingorderstheN-lobeof

CDK2incontrasttotheisolatedkinasedomain(56).Mostkinasestructuresrepresentjustanisolated

kinasedomainsoonecannotappreciatehowotherdomainscontributetostabilizationand,inturn,

regulationoftheN-Lobe.OurHDXMSresultsalsohelptoexplainwhyithasnotbeenpossiblesofar

toexpressthekinasedomainindependentoftherestoftheprotein.Intheapoproteintheactivation

loop of the kinase domain in the C-lobe has the highest deuterium uptake suggesting it is highly

disorderedandexposedtosolvent(Figure4andS2).


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Figure4.ThedeuteriumuptakeoftheLRRK2RCKWkinasedomain.(A)Therelativedeuteriumuptakeafter2minofdeuteriumexposureoftheLRRK2RCKWkinasedomain isshowninacolor-codedhomologymodel.Greycolorindicatesnodeuteriumuptakeinformation.TheN-lobeofthekinasemostlyshowsbluetogreencolorsindicatinglowdeuteriumuptake.Ontheotherhand,theαD,activationloop,theendofαFtoαHhavehigherdeuteriumuptakesuggestingamoredynamic,solventaccessibleC-lobe.(B)Representativepeptidesthathavealmostnodeuteriumuptakearemappedonthekinasedomain. Insetsshowuptakefortheapokinase(black)andtheMLi-2boundstate(red).

Togain insight into theallosteric impactof inhibitorbindingwenext lookedat theconformational

changesinLRRK2RCKWfollowingtreatmentwithMLi-2.Theoverallchanges,capturedinthegraphin

Figure5,showthatthereissubtle,albeitimportant,protectioninregionsthatextendintotheGTPase

andCOR-A:COR-Bdomains,butbyfarthelargestchangesareconcentratedinthekinasedomainand

in the linker that precedes the kinase domain. There are no regions that show enhanced solvent

accessibilityinthepresenceofMLi-2.Wefocushereontheconformationalchangesthatarelocalized


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toourkinasedomainmodel.ThesechangeslienotonlyintheN-lobeandtheactivesitecleftwhere

theinhibitorisdirectlydockedbutalsointheC-lobeinregionsthatliefarfromtheactivesitecleft.

Figure5.BindingofMLi-2reducesthedeuteriumuptakeofLRRK2RCKW.(A)TherelativedeuteriumexchangeforeachpeptidedetectedfromtheNtoCterminusofLRRK2 inapo-kinase(Black)andMLi-2bound(red)conditionsat2min.ThearrowsindicatetheregionsofLRRK2thathavelessdeuteriumuptakewhenboundtoMLi-2 (B) Thedeuteriumuptakeof selectedpeptides isplottedandmappedon thekinasemodel. TheuptakeisreducedintheGlyrichloop,theαChelix,theactivationloop,theDYGmotif,theYRDmotifandthehingeregioninthepresenceofMLi-2.

Lookingmorecarefullyattheprotectedregions,wesawthatthebindingofMLi-2reducestheH-D

exchange in theATP-bindingsite, theactivation loop, theαChelixandthehingeregion (Figure5).

Theseregionsthatwouldbepredictedtocontacttheinhibitor(57,58)allshowsignificantlyreduced

deuteriumuptake.Peptides,forexample,inthehingeregion(aa1948-1958),includingtheaDhelix,

experiencedalargeincreaseinprotectionuponMLi-2binding(50%vs.20%).Thepeptidecoveringthe

catalyticloop(aa2013-2022)includingtheYRDmotifalsoexperiencedprotection(30%to<10%).The

Glycine-richloop(aa1884-1893)isalsohighlyprotected.Mostimportantlyweseethatthepeptide

containingtheDYGImotif (aa2013-2022) isalmostcompletelyshieldedasaconsequenceofMLi-2

binding;thedeuteriumexchangedroppedfrom70%tolessthan10%suggestingthatthisregion,highly

solventaccessibleintheabsenceofligand,becomesalmostcompletelyprotectedbythecoordination

oftheinhibitor(Figures5and6).Thisisquiteconsistentwiththepredictionthatthekinasedomain

assumesacompactandclosedconformationinthepresenceofMLi-2.TheC-terminusofthispeptide


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containsthebeginningoftheactivationloop,whichnowappearstobewell-foldedandshieldedfrom

solventincontrasttotheapostructure.Interestingly,theuptakespectraofthetwopeptidescovering

theactivationloopshowanEX1bimodaldistribution,whichindicatestwodifferentconformationsin

solution(59).Oneofthesepeptides(aa2028-2056)isshowninFigures5and6.Mostotherpeptides

showasinglepeakindicativeofthemoretypicalEX2exchangekinetics.Inaddition,MLi-2treatment

alsoinducedslow-exchangeintheDYGloopeventhoughitishighlyprotected.Thepeptidethatcovers

theN-terminusoftheaChelix(aa1915-1921)showssignificantslowexchangeevenintheabsenceof

MLi-2thatmostlikelycontinuesbeyond5min.Althoughtheexchangeisquenchedinthepresenceof

MLi-2,theslowexchangestillpersists.

TheprotectionoftheATP-bindingsiteandthehingeregionbyMLi-2areconsistentwithotherinhibitor

boundhomologkinasestructures(57,58)althoughourdataalsoreflectsthedynamicchangethatthe

bindingofMLi-2hasnotonlyonthekinasedomainbutalsoonLRRK2RCKWoverall.Essentiallyanyregion

inLRRK2RCKWthatinterfaceswiththekinasedomainwillsensebindingofnucleotideoraninhibitor.

ThisincludesalsotheLRRdomain,notincludedinourconstruct,butwhichispredictedtolieoverthe

kinasedomain(13,51,60)andwouldbedisplacedbythehighaffinitybindingofakinaseinhibitor.

HDX-MSshowsthatchangesinconformationanddynamicsofthekinasedomainarefeltthroughlong-

distancesinLRRK2RCKW,asflexibleregionsthroughouttheproteinexhibitincreasedprotectionupon

MLi-2binding(Figure5AandS2).

Figure6.ThedeuteriumuptakeandspectralplotofpeptidesintheDYG,activationloop,andaChelixrevealslowdynamics.IntheDYGpeptide(2013-2022)theapostate(black)plateauswithin2min.TheMLi-2boundstate (red) continues to slowly exchange at least up to 5minutes, suggesting thatwithMLi-2 this regionundergoesaslowdynamicprocess.Theapostateoftheactivationlooppeptide(2028-2056)againplateaus


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within2minwhiletheMLi-2boundstategraduallyincreasesafter2min.Fromthespectralplot,theuptakeoftheactivationlooppeptideintheMLi-2stateexhibitsbimodalbehavior.Oneprocesshasslowdeuteriumuptake (protected);and theotherprocesshas fastuptake (solventexposed)- similar to thesingleprocessobserved in the apo state. For theaC peptide (1915-1921), the deuterium increases without reaching aplateauover5minutesforbothstates.

GaMDsimulationsindicatethattheLRRK2kinasedomainmutations

Y2018F, G2019S and I2020T attenuate flexibility of the activation

segmentofthekinasecore

GaMD simulations were performed on the activated kinase domain of LRRK2 (1865-2135) to

investigatechangesintheconformationallandscapethatarecausedbytheD2017A,Y2018F,G2019S,

andI2020Tmutations.Duringall10replicateacceleratedsimulationsthewtkinasefavorsanopenand

inactiveactivecleftconformationasmeasuredbytherelativepositionoftheN-andC-lobesandthe

αC-helix(Figure7).Thefullyclosedandactiveconformation,inwhichtheN-andC-lobesarebrought

together in concert with an inward positioning of the αC helix to assemble the active site, is

infrequently sampledby thewtkinase. In contrast, Y2018F,G2019S, and I2020Tareall capableof

accessingaclosedandactiveconformation,whileD2017Asamplesamuchmoreopenandinactive

conformation(Figure7a).Thedegreeofstabilizationoftheclosedconformationroughlycorrelates

with the observed changes in MT association: D2017A<wt<G2019S<Y2018F <I2020T; it does not

correlatewithactivity.TheI2020Tmutantistrappedinamostlyclosedstatewithoutextensiveopen-

to-close transitions and αC in-to-out motion compared to wt, Y2018F and G2019S. This loss of

breathing dynamics may partially explain the reduced kinase activity of I2020T, where

substrate/productkineticsmaybeimpacted.Likewise,Y2018FandG2019Sbothpopulateawiderange

ofopenandalsoclosed-activeconformationslikelycontributingtotheirincreasedkinaseactivity.The

abilityofalloftheactivatingmutantstopopulateaclosedconformationmayplayaroleintheiraltered

MTassociationcomparedtowt,whereY2018FandI2020TspontaneouslyformfilamentsandG2019S

formsfilamentsfasterthanwtupontreatmentbythetype-IinhibitorMLi-2.

The hydroxyl moiety of Y2018 in the DYG motif forms persistent hydrogen bonds between the

backboneofbothI1933intheαC-β4loopandI2015(Figure7b).Thisinteractionstabilizesthetyrosine

sidechaininanorientationthatrestrictstheαChelixfromassemblingtheactivesiteduetostericclash

withL1924.ThissimulationagreeswellwitharecentCryo-EMstructureofaninactiveconformation

of LRRK2RCKW, (13). These authors identify the same hydrogen bond between Y2018 and the shell

residueI1933.OursimulationsprovidestrongindependentevidencethatY2018inwtLRRK2isakey

stabilizer of the inactive kinase conformation and may also act as a sensor of the αC-β4 loop

conformation,aconservedhotspotforkinaseallostericmodulation(61).AbsenceoftheOHhydrogen

bondsintheY2018FmutationleadstogreaterY2018FsidechaindynamicsandpackingwithL1924


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thatresemblesanactivekinaseconfiguration(oraproperlyformedR-spine).AnassembledR-spineis

thehallmarkforanactivekinase.StabilizationofthewtY2018sidechainleadstoa‘frustrated’DYG

backbonefree-energylandscapebypullingthemotifoutofideal f/yspace,whichislikelyimportant

forthekinase'sroleasaswitch(FigureS3).AconsequenceoffreeingthesidechainofY2018Fisthe

convergenceoftheDYGdihedralanglesintoacanonicallyactivekinase f/yregion(62)(FigureS3,S4).

The I2020Tmutation introduces a hydrogen bond between theOH group of the threonine to the

backbone of the catalytic YRDmotif (Y1992) (Figure 7c). The interactionwith the YRDmotif both

stabilizesthecatalyticloopandalsoleadstoaclosedkinaseactivesite(Figure7a,c).TheDYGmotifis

also stabilized in an active conformation, similarly to Y2018F, as measured by its ensemble DYG

dihedralangles(FigureS4).TheI2020Tequilibriumisshiftedtotheclosedconformationandactivity

may be reduced because the mechanism for opening is impaired. Finally, G2019S introduces a

hydrogenbondwiththesidechainofE1920intheαChelix,whichinturnformsahighlyconservedsalt-

bridgewithK1906ofβ3(Figure7d).TheinfluenceoftheG2019Smutationontheinteractionbetween

αCandβ3andtheDYGloopfavorstheclosedandactivekinaseconformation.TheG2019SDYGmotif

isstabilizedinanactiveconformationasdescribedbyitsdihedrals(FigureS4).


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Figure 7. Mutations in the DYGy loop alter kinase dynamics. (A) Kinase conformational free-energylandscape,representedby‘open-close’:thedistancefromthetopoftheactivesite(K1906/β3sheet)tothebottomoftheactivesite(D1994/YRDmotif),and‘αCin-out’:thedistancebetweenK1906andtheαChelix(E1920).Thewhitedashedlineshowstheclosed-activekinaseconformation.Theactivestateisinfrequentlysampledbythewtkinase,whereastheDYGymutantsmorereadilyaccesstheclosed-activeconformation.However,thekinasedeadD2017Amutantisdestabilizedtoamoreopenconformationrelativetowt.(B)InwtY2018(blackpanel)islockedinaninactiveorientationbyhydrogenbondswithI2015andI1933.Y2018F(redpanel)packswith L1924of theαChelix and releases theDYG loop froman inactive statehelping toassembletheactivesite.Y2018Fbreakstheinteractionleadingtoincreasedside-chaindynamics,measuredbythedistancebetweenthe2018ζ-carbonandthebackboneof I2015(wt:black,mutant:red). (C) I2020TmakesahydrogenbondwiththebackboneofY1992intheYRDmotif,couplingtheDYGandcatalyticloops,which results in decreased backbone dynamics. The mutation brings the DYG and YRD motifs together,measuredasthedistancefromtheCβof2020andthebackboneofY1992(wt:black,mutant:red).(D)G2019SbridgestheDYGlooptotheαChelixandβ3sheet,throughE1920andK1906.ThisstabilizestheDYGloop,shownbyRMSD(wt:black,mutant:red),andpromotestheclosedkinaseconformation.


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Discussion

LRRK2isahighlycomplexmulti-domainproteinandequallycomplex is itsregulation.Thesignaling

cascadesthatcontrolLRRK2stillremaintobeelucidatedandthemolecularmechanismsthatcontrol

itsintrinsicregulationarealsonotwellcharacterized.However,bothaspectsareofcriticalimportance

tounderstandthemechanisticconsequencesofthepathogenicmutationsandultimatelyfindacure

forParkinson’sDisease.Hereweinvestigatedafour-domainconstructofLRRK2consistingoftheROC,

Cor,KinaseandWD40domains(LRRK2RCKW).Thisconstructistheshortestfunctionalconstructtodate

thatmaintainskinaseaswellasGTPaseactivity.It isalsothesmallestconstructthatcandockonto

MTs. Inthecurrentworkweelucidatedifferentaspectsofthe intrinsicregulationofLRRK2usinga

multilayeredapproachfocusingontheimportanceofthekinasedomain.Ourfirstlayerconcentrated

onthespatialandtemporaldistributionoffull-lengthLRRK2inacellularcontextasafunctionofthe

highaffinitykinaseinhibitor,MLi-2,whichprovideduswithareal-timeassayforfilamentformationin

livecells.TheeffectsofremovingtheN-terminusoncellulardistributionwasthenexploredwithour

LRRK2RCKWvariants.ThesestudiesledtothepredictionthattheN-terminalscaffoldingdomainsshield

thecatalyticdomainsintheinactiverestingstateofLRRK2.The2ndlayerfocusedonthebiochemical

characterizationof LRRK2RCKW variantsbydemonstrating substrate-specific kinaseactivity.Herewe

showedthattheLRRK2RCKWproteinretainedthecatalyticmachineryformediatingphosphoryltransfer.

InthenextlayerweusedHDX-MSanalysisofLRRK2RCKWtoprovideaportraitoftheconformational

statesofLRRK2RCKWinthepresenceandabsenceofMLi2.Mappingthesolventaccessibleregionsina

modelof LRRK2RCKWnotonlyprovidesanallostericportraitof thekinasedomainbutalso suggests

multi-domain crosstalk. Finally, we performed GaMD calculations on the LRRK2 kinase domain to

elucidate at a molecular level the differences in breathing dynamics between LRRK2 wt and the

pathogenickinasedomainmutationsY2018F,G2019SandI2020T.Withthisapproachwewereableto

clearlydemonstratethatthekinaseactivityandthespatialdistributionofLRRK2isnotonlyregulated

bysingledomainsbutbyacomplexinterplayofalltheembeddedproteindomains.Thehighlydynamic

kinasedomain,nevertheless,seemstoplayacrucialroleincoordinatingtheoveralldomaincrosstalk

andservesasacentralregulatoryhubfortheintrinsicregulationofLRRK2.

Filament formation is dependent on unleashing the catalytic

domainsandontheconformationofthekinasedomain.AlthoughmultiplefunctionsareassociatedwiththemanydomainsofLRRK2,thesedomainscanbe

structurallyandfunctionallydividedintothecatalyticallyinertNTDsandthecatalyticCTDs,anddistinct

functionsareembeddedineach.Furthercomplexityisintroducedbyheterologousproteinssuchas

RabGTPasesand14-3-3proteins,whichalsocontributetotheactivationandsubcellularlocalization


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ofLRRK2.LRRK2alsoexistsinmultipleoligomericstateswherethemostactivestateisthoughttobe

adimerincontrasttothelessactivemonomer(53,54).Thestabilityofthemonomersanddimerscan

befurtherfacilitatedbyheterologousproteins,inparticular,the14-3-3proteins.PhysiologicallyLRRK2

isthoughttobeactivatedbyRabGTPasessuchasRab29,whichdockontotheNTDsandtargetLRRK2

toorganellessuchasthetransGolginetwork(29).OtherRabsmayalsoactivateLRRK2buttargetitto

different organelles (28, 63) while auto-phosphorylation on residues such as S1292 likely are

subsequentstepsintheactivationprocess(13,64).ManyofthePDmutations“hijack”thesefinely

tunedregulatorymechanisms.ConstitutivelocalizationtoMTsisaphenotypedisplayedbythreeof

the four common LRRK2 PDmutants (R1441C, Y1699C and I2020T)while the hyperactive G2019S

mutant,likewtLRRK2,remainscytosolic.UsingcryoElectronTomography(cryoET)Watanabeandco-

workers(60)wereabletocapturetheprecisewayinwhichaLRRK2mutant(I2020T)canpolymerize

anddockontoMTs.TheyshowspecificallyhowtheI2020TLRRK2mutantformsperiodicallyrepeating

dimers,whichthenpolymerizeinahelicalarrayontoMTs.Thecellularphenotypesassociatedwith

G2019S,I2020T,andR1441C/Y1699CandotherPDassociatedmutationsincludeperturbationofMT-

relatedprocessessuchasvesiculartrafficking,autophagy,ciliaformation,andnuclear/mitochondria

morphology,soitisverylikelythatLRRK2dysfunctionphysiologicallyinterferesgloballywithdynamic

crosstalkwithMTs(20,32,65-72).

Withlivecellimagingintheabsenceandpresenceofakinaseinhibitor,MLi-2,wewereabletocapture

at lowresolution inrealtimethere-localizationofcytoplasmicwtandG2019SLRRK2todecorated

MTs. MLi-2 binding to wt and G2019S thus introduces artificially the pathogenic phenotype

constitutivelyobservedforI2020T,R1441CorY1699C.IncontrasttoMLi-2andLRRK2-IN-1,whichare

typeIkinaseinhibitors,weshowthatinthepresenceofatypeIIkinaseinhibitor,rebastinib,G2019S

LRRK2 remained cytosolic confirming the predictions of Deniston and coworkers (2020) (13), that

dockingtoMTsisextremelysensitivetotheconformationalstateofthekinasedomain.Althoughthe

MLi-2complexiscatalyticallyinactive,MLi-2,whichisacompetitiveinhibitorofATP,neverthelesslocks

thekinaseintoanactive-likeconformation(58).Incontrast,rebastinibislikelytostabilizeaDYG/DFG-

out/openandinactiveconformationofthekinasedomain.IntheabsenceoftheNTDswtandG2019S

LRRK2RCKW spontaneously form filaments independent of MLi-2. Our HDX-MS data confirms that

deletionoftheNTDsinLRRK2RCKWdoesnotunfoldtheremainingCTDsassolventexchangeshowsthat

theproteinisproperlyfoldedwiththedomainswellpackedagainsteachother.Collectivelyourresults

supportamodelwherethecatalyticallyinertNTDsfunctionasa“lid”thatshieldstheactivesitesof

theCTDs.The lid canbeunleashedphysiologicallybyactivatingRabGTPasesorbymutations that

makeLRRK2ariskfactorforPD.TwoofthePDmutants,R1441C/G/HandY1699C,arelocalizedinthe

ROC and COR domains, respectively, and presumably disrupt a domain-domain interface which is

sufficienttounleashtheNTDs.Whiletargeting,activation,andinhibitionoftheCTDsareimportant


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functionsthatareembeddedintheNTDs,ourbiochemicalstudieshereshowthatallofthekinase

activity of the full length LRRK2 is embedded in LRRK2RCKW. The other two common PDmutations

(G2019Sand I2020T),althoughfunctionallydistinct,are inthekinasedomain.Our livecell imaging

together with our biochemical and simulation results, discussed below, and a recently published

cryoET structure (60), all support the hypothesis that unleashing the NTD lid as well as an active

conformation of the kinase domain, not necessarily kinase activity, are essential requirements for

dimerizationandMTbinding.

TheswitchmechanismforactivationofLRRK2 isembedded in the

DYGψmotifofthekinasedomainWithHDXMSweconfirmthatashiftinconformationisinducedbythebindingofMLi-2toLRRK2RCKW

and,significantly,wefindthatstabilizingtheactivekinaseconformationbyMLi-2drovechangesin

conformation and domain organization throughout LRRK2RCKW. The global decrease in backbone

deuteriumexchangemeasuredacrossallfourdomainsparticularlyinthelinkerbetweentheCORBand

kinasedomainsandinflexibleregionsthroughoutLRRK2RCKW(Figure5A)issuggestiveofchangesin

domain:domain packing as well as changes in global conformational dynamics. We propose that

changes in the CTDs organization and dynamics, driven by the stabilization of the active kinase

conformation, are likely coupledwith association and dissociation of the NTDs. This explains why

mutationssuchasR1441CandY1699C,thatliefarfromthekinaseactivesitebutatadomaininterface

arecapableofunleashingtheNTDs.UsingtheMLi-2boundLRRK2RCKWasaproxyforthe“frozen”closed

andactive-likeconformationofthekinasedomain,weexploredeachoftheactivatingDYGψmutants

usingMDsimulationsandaskedhoweachmutationperturbs the conformationalensembleof the

kinasedomain.

Our first hint that theDYGψmotif impacts the kinase conformationwith consequences on LRRK2

globalconformationandregulationcamefromourpreviousworkwiththeDYGψmutations,Y2018F

andI2020T,wherewewereabletocorrelatechangesintheinternalorganizationofthekinasedomain,

specificallyassemblyoftheRegulatorySpine,within-situMTassociation(36).Ourdatahere,shows

thatdeletionoftheNTDsinducesasimilarMTassociationphenotypeindependentofmutation.This

further confirms that filament formation of Y2018F and I2020T in full length LRRK2 is based on

unleashingoftheNTDscausedbyperturbationoftheconformationaldynamicsofthekinasedomain.

Although full-lengthG2019S, themost prevalent PDmutation, does not form significant filaments

spontaneously, we show that its kinetics ofMT association are increased relative to wt following

treatmentwithMLi-2(SupplementaryMovie),suggestingthatthismutationmayshareacommonde-

regulating mechanism through changes in the kinase domain conformation. Indeed, our GaMD

calculationsshowthatstabilizationoftheDYGψdynamicsbyallthreemutationsY2018F,G2019S,and


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I2020Tpromotestheactivekinaseconformation.Whereaspreviousmetadynamicstudiesshoweda

stabilizationofthe“DYG-in”overthe“DYG-out”conformation(73,74),weobserveinourunbiased

MDforY2018F,G2019S,andI2020TasubtlecoalescenceofDYGdihedralanglesintoanactive-DYG

loop conformation (Figure S4). However, each of the mutants favor the active conformation by

differentmechanisms. Y2018Freleasesthesidechain froman inactivenon-preferredrotamerwith

frustrated DYGψ backbone dynamics leading to closure by packingwith L1924 of the αC helix. In

contrast,G2019SconnectsandstabilizestheDYGmotifthroughhydrogenbondingtotheactivating

αC-β3salt-bridge(interactionofK1906andE1920),whileI2020Tattheψpositionstabilizesitselfwith

theYRD (Y1992,R1993andD1994)catalyticmotif throughhydrogenbonding.Significantly, thewt

kinaseinthesesimulationsstillfluctuatesbetweenbothinactiveandactivestateswhilefavoringthe

inactiveconformation.Thismeansinputfromexternalfactors,suchastheNTDs,arerequiredtofully

modulatetheconformationalequilibriumtoinhibitkinaseactivity.ActivatingDYGψmutationssubvert

thebuilt-inregulationbydistortingtheinherentconformationalequilibriumtoadegreethatbreaks

theselayersofcontrol.Ontheotherhand,thekinasedeadD2017Awhichhassignificantlyreduced

localization toMTs even in the absence of the NTDs cannot be stabilized in an active-like kinase

conformationbecauseitsactive-sitecleft isdynamicallydestabilized.Togetherwithourfindingsfor

rebastinibandMLi-2thisfurtheremphasizesthatfilamentformationisnotsolelydependentonthe

inhibitorylid-functionoftheNTDbutalsoonthekinasedomainintegrity/conformationalstate.

Our findings highlight that the activation of LRRK2, while simplistically represented by static

conformations(i.e.simpleactiveandinactiveconformations),ismoreaccuratelydefinedintermsofa

shift in conformational ensembles and associated dynamics. This is illustrated not just in theMD

simulationsbychangesinbulkconformationsduetoDYGψmutantsbutalsobychangesintime-scales

ofdynamicshighlightedbylivecellimagingandbytheinductionofbimodalHDXkineticsafterbinding

MLi-2.HDXshowsthattheactivationlooppeptidehastwodistinctconformationalpopulations.One

representsaminorpopulationofahighlysolventexposedspeciessimilartowhatwasseenintheapo

state and an additional highly protected species, which gradually becomesmore solvent exposed

(Figure 6). In MD simulations the DYGψ activating mutants mirror this behavior and shift the

equilibriumtowardsanorderedandlesssolventaccessibleactivationloop(FigureS5).MLi-2binding

appearstoleadtoanevengreatershiftinthisequilibriumandalargedecreaseinthekineticsofthe

exchangebetweenstates,effectivelytrappingamajorpopulationofaclosedandactive-likestateof

thekinasedomain.Extendingthisconceptofregulationbytuningofconformationaldynamicstothe

cellularlevel,ourworkimpliesthatchangesinthebalanceofLRRK2conformationalequilibriumwill

leadtoproportionalchangesinitscellulardistribution,i.e.thepopulationofLRRK2inthecytosolvs.

associatedwithMTshouldmirrortheconformationaldistributionofexpandedandclosedLRRK2.


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LRRK2andBRafsharethesameKinaseActivationMechanismBy comparing the resulting finely tunedmulti-layered regulationmechanism of LRRK2 with other

related homologs of the kinase tree we recognized that our model for LRRK2 regulation closely

resembles theactivationprocessofanothermulti-domainkinase:BRaf (75-77).LRRK2, likeBRaf, is

activatedbytheinteractionofitsN-terminalnon-catalyticdomainswithasmallGTPase:Rabvs.Ras.

In both cases autoinhibitory sites/domains (AI) in theNTDs become displacedwhen the activated

GTPasebinds,andthisunleashesthekinasedomain(Figure8).Thekinasedomains,nolongerlocked

intheirinactiveconformations,arefreetotogglebetweentheirinactiveandactivestates.Onlyinthe

activeconformationarethecatalyticdomainsabletodimerize.InBRafthispushesthekinasedomain

intotheactiveconformationandinducescis-autophosphorylationoftheactivationloop.Dimerization

of the catalytic domains of LRRK2, as seen in the cryoET structure (60), also requires an active

conformationofthekinasedomain,althoughinthecaseofLRRK2thedimerinterfacemostlikelydoes

notdirectly involvethekinasedomain.Inbothcasesthis laststepstabilizesthekinasedomainina

conformationwhere theR-spinesareassembled, rendering themready tobindandphosphorylate

theirsubstrates.WhenthisprocessisdisruptedbyamutationsuchasY2018ForI2020Tinthekinase

domainofLRRK2,kinaseactivationbecomesindependentofRabbinding,asthesemutationsshiftthe

equilibrium to amore active kinase conformationwhich also promotes displacement of theNTDs

(Figure9A).ThisrecapitulatesathemeobservedinBRaf,whereinthemostactivatingcancer-driving

mutation,V600F,rendersthekinaseactivewithouttheneedforheterologousactivationbyRas(78).


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Figure8.ComparisonoftheBRafandLRRK2activationmodels.UnderhealthyconditionsbothBRafandLRRK2areininactiveautoinhibitedstatesinthecytosol.ThekinaseactivationthenfollowsrecruitmentbyeitherGTPboundRas or Rab29, which are both small GTPases and closely related to each other. The recruitment induces aconformationalchangewhichdisplacestheN-terminaldomain(NTD)ofBRafortheautoinhibitionsite(AI)intheNTDsofLRRK2.Thisinturnallowsbothkinases,BRafandLRRK2,todimerize,resultinginstabilizationoftheactivekinasedomainconformations.Thereby,thekinasesbecomeactivatedascis-autophosphorylationoftheactivationloop is inducedforBRafand isalso likely tohappenforLRRK2.Finally, thisunleashesbothkinasesandresult inmaximalactivation,allowingforefficientphosphorylationoftheirsubstrates.


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Figure9.ImpairedLRRK2activationresultsinmicrotubuleassociation.A)InthemutatedorMLi-2inhibitedstatethe autoinhibitory site (AI) in theNTDsbecomes alreadydisplaced in the cytosol and the kinasedomain is in aconstitutivelyactiveconformation.ThisshortcircuitstheactivationprocesswhichdependsonRab29association.TheactivekinaseconformationissufficienttoinducedimerizationandtherebymultimerizationofLRRK2aroundMT.UsingMTsasscaffoldingstructuresforLRRK2multimers,whichareorderedinaperiodicfashionasshownbyWatanabeandcoworkers(2020),resultinthefilamentformationphenotype(60).EachLRRK2monomerprovidestwointeractionsurfacesnamelytheCORandtheWD40domainwhichallowseachLRRK2monomertointeractwithtwoadjacentmonomers.AnadditionalfindingwasthattheN-terminusformsbridginginteractionswiththeupperandthelowerturnoftheLRRK2filament.Furthermore,thenumberofLRRK2dimersneededforoneturncorrelateswellwiththenumberofMTprotofilaments.Therefore,webelievethatthisisapathogenicbutspecificinteractionwithMTsresultingfromimpairedregulationofLRRK2bystabilizinganactiveconformationofthekinasedomainofLRRK2eitherbymutationsorbybindingofMLi-2.B)TheimportanceoftheNTDsforstabilizingLRRK2inaninactivecytosolicallydistributedstatewas testedbydeleting theN-terminus.The resultingLRRK2RCKWdeletionconstructspontaneously forms filaments aroundMTs and is no longer able to become recruited by Rab29 as it lacks the


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interactionsitesintheArmand/orAnkdomain.WebelievethatthemissingAIdomain,whichwethinkispositionedintheNTDs,forcesthekinasedomainoftheRCKWconstructintoanactiveconformationresultinginmultimerizationanddockingtoMTs.

Othermutations that contribute to stabilizationof theassembledR-Spinealso lead to constitutive

activation that is independent of Ras (78, 79). Kinase inhibition was also shown to facilitate

downstream signaling as the inhibition of BRaf stabilized the active kinase conformation which is

sufficient to promote cis-autophosphorylation through heterodimerization (80, 81). This closely

resemblesthesituationweobservedforLRRK2whereMLi-2stabilizestheactivekinaseconformation

andtherebyinducesfilamentformation(Figure9A).AnotherfeaturecommontoLRRK2andBRafis

thatthedeletionoftheN-terminusofeitherproteingeneratesaconstitutivelyactivekinase(Figure

9B)(82-84).Finally,thekinasedeadmutationsinbothproteinsareunabletoformproductivedimers

(85,86).SincemanyaspectsofthekinasedomainregulationofLRRK2andBRafappeartobequite

similar,itcanbeconcludedthatactivationmechanisms,ingeneral,wherethekinasedomainswitching

between active and inactive conformations serves as a central hub are likely to be conserved

throughoutmuchofthekinome.Inaddition,fromtherecentstructuresoffulllengthBRafincomplex

withitssubstrateMEKand14-3-3proteinswecanseehowtheseauxiliaryproteinscanstabilizeeither

aninactiveoranactiveconformation(87).ThiswillcertainlyalsobetrueforLRRK2.Clearly,wecan

learnmuchaboutLRRK2activationbylookingatclosehomologsthathavebeenstudiedforalonger

time,andhopefullythiswillfacilitatethediscoveryofnewtherapeuticstrategiesforattackingLRRK2

asadriverofPD.

ConclusionAnalysis of multidomain kinases suggest that the conformation of the kinase domain might, as a

general principle, regulatemuchmore than just the activity of the kinase.Our resultswith LRRK2

demonstratethattheactivekinaseconformationnotonlyswitchesthekinaseintoan“on”statebut

also unleashes inhibitory domains, can promote dimerization, and can facilitate translocation to

anchoredsubstrates.InthecaseofLRRK2thisisacomplexandtightlyregulatedprocesswheremore

domainsandotherproteinsthanjustthekinasedomainareinvolved;however,theparadigmissimilar

forBRAFandprobablyformostkinasessuchasSrcandPKCthatarealsoembeddedinmulti-domain

proteins. Ineachcase,theconformationofthekinasedomainseemstoplayacrucialrole inthese

intrinsicregulatoryprocesses.Wealsofurtherconfirmherehowaswitchmechanismforactivation,

embedded in theDYGymotifof thekinasecore,allowsthekinasetotogglebetween inactiveand

activeconformationswhichisthencommunicatedtoallpartsoftheprotein.Wealsodemonstrate

herethatthenon-catalyticNTDsplayanimportantregulatoryrolebyshieldingthecatalyticCTDsin

theabsenceofphysiologicalactivators. Interactingproteins like14-3-3orRabproteinsare likelyto

furtherfine-tunethisregulationeitherpositivelyornegativelybystabilizingcertainconformationsof


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LRRK2.Thispreciselycontrolledsignalingprocesscanalsobehijackedbyavarietyofdiseasedriving

mutationssuchasthosethatleadtoPD.

Acknowledgments

We like toacknowledge theexcellent technicalassistanceofMichaelaHansch, IrmtraudHammerl-WitzelandtheassistanceofAlexandrKornevinpreparationofmodels.

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Supportinginformation:

Figure S1. The type-2 kinase inhibitor rebastinib binds to the LRRK2 kinase domain. Shown are the 1stderivativesoftheLRRK2meltingcurveswithorwithoutrebastinib.TheLRRK2proteincomprisedtwodomains(kinase andWD40). Accordingly, therewere twominima in the control curve. The addition of rebastinibstabilizedtheLRRK2kinasedomainandshifteditsmeltingtemperatureby14K,indicatingabindingconstant<1uM.


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FigureS2.The identifiedHDX-MSpeptidesof LRRK2RCKW . (A) The relativedeuteriumexchange foreachpeptidedetectedfromtheNtoCterminusofLRRK2at2min,apocondition.(B)Eachlineinthecoveragemaprepresentsan identifiedpeptide(exchanged2min)andthecolor indicatestherelativedeuteriumuptake.Locationofeachdomainis indicated.Themapidentifiesfoldedregionsaswellassolventexposedregionssuchastheactivationloopinthekinasedomain.Thecoverageis96.3%andtheredundancyis3.72,whichissuccessfulforaproteinthis large.(C)Theheatshowstherelativefractionaluptakebycolorat2min. It iscoloredbasedonthedifferenceofrelativedeuteriumuptakebetweenapoandMLi-2states.


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FigureS3.Y2018hasanon-idealsidechaintorsionanglethatintroducesdisordertotheDYGloop.(A)IdealTyrχ1/χ2gauche(-)torsionsfromtheDunbrackdatabaseisshownwithblue-to-redcolorindicatingincreasingtorsionanglepreference(88).TheaveragetorsionanglesforwtY2018(magentacircle)andY2018F(whitecircle)fromthesimulationareoverlaid,showingthatwtY2018’ssidechainisinanon-preferredconformation.Theχ1/χ2sidechaintorsionangledistributionforY2018andY2018Fduringthesimulationsareshown(rightpanels).Y2018FbreaksthehydrogenbondsthatlockthesidechainofY2018inwtandleadstoamoreidealside-chainconformation in themutant.Thewhitedashed line indicates thepreferredχ1 forTyr fromtheDunbrackdataset.(B)Theidealbackbonedependentconformationforthegauche(-)rotamerofTyr,derivedfromrotamerlibraries(88),isdepictedasthe probabilityofthebackboney-dihedralangle(iN-iCa-iC-i+1N)inblue. Thenon-ideal Y2018 rotamer inwt adds strain to thebackbone: theTyrbackboneexploresnon-preferred conformations (black area, arrows indicate a non-ideal backbone y-angle). The backboneconformationofY2018Fpopulatesidealdihedralspace(redarea).Theφ/ψdistributionofthebackboneofY/F2018duringthesimulationisshown(rightpanels).ThewtY2018backboneismoredynamicthanY2018F.InY2018Fthesidechainisfreetoaccessthepreferredrotamertorsionsandthebackboneconformationisstabilizedinthepreferredactivebackboneconformation.ThemutationstabilizestheentireDYGIbackboneasseenintheconformationalensemblefromMD(upperright).


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FigureS4.DYGIbackbonedihedralsfromMDsimulations.DYGψdihedralspacethatisassociatedwithanactivatedkinasearecircledincyan(62).ThewtkinaseandD2017AmutanthaveadynamicDYGloopthatsamplesconformationsassociatedwithinactivekinases.TheDYGloopofthemutantsconvergetoaφ/ψspacethatistypicalofactive/closedkinases.Asterisksindicatesitesofmutation.


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Figure S5. Activation loop stability from MD simulations. (A/B) The stability of the activation loop isrepresentedas thedistancebetween theYRD loop (R1993)andactivation loop (pS2032). Thesalt-bridgebetweenYRDandactivationloopcouplesthecatalyticloopwiththeN-andC-lobes.Theactivationloopofwt is the least stable, while activating DYGψ mutants increase stability. (C) The wt activation loopconformational ensemble is more widely distributed and more solvent accessible than (D) the I2020Tactivationloop.TheDYGImotifisshowninblue,YRDmotifingreen,andactivationloopinorange.

Supplementaryvideos:

Movie 1. Time-lapse imaging of HEK293T cells transiently expressing YFP-LRRK2-G2019S. The timeintervalis5minutes.MLi-2wasaddedrightafterthesecondframe.Images(640x640pixels)ofYFPfluorescence (515nmexcitation/530-630nmemission) represent 3D volume reconstructions fromconfocalimagestacksovertime.

Movie 2. Time-lapse imaging of HEK293T cells transiently expressing YFP-LRRK2-G2019S followingwash-outofMLi-2.Thetimeintervalis11minutes.Images(640x640pixels)ofYFPfluorescence(515nmexcitation/530-630nmemission)represent3Dvolumereconstructionsfromconfocalimagestacksovertime.


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Movie3.Time-lapseimagingofHEK293TcellstransientlyexpressingYFP-LRRK2-WT.Thetimeintervalis 5 minutes. MLi-2 was added right after the second frame. Images (640x640 pixels) of YFPfluorescence (515nmexcitation/530-630nmemission) represent 3D volume reconstructions fromconfocalimagestacksovertime.

Movie4.Time-lapseimagingofHEK293TcellstransientlyexpressingYFP-LRRK2-WTfollowingwash-outofMLi-2.Thetimeinterval is11minutes. Images(640x640pixels)ofYFPfluorescence(515nmexcitation/530-630nmemission) represent3Dvolume reconstructions fromconfocal image stacksovertime.


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Conformation and dynamics of the kinase domain …...2020/07/13 · RCKW employing...

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