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ASpring-LoadedMechanismGovernstheClamp-Like1
DynamicsoftheSkpChaperone2
3
DanielA.Holdbrook1,BjörnM.Burmann2,RolandG.Huber1,MaximV.4
Petoukhov4,DmitriI.Svergun4,SebastianHiller2,*,PeterJ.Bond1,3,*5
6
7
1BioinformaticsInstitute(A*STAR),30BiopolisStr,#07-01Matrix,1386718
Singapore9
2Biozentrum,UniversityofBasel,Klingelbergstrasse70,4056Basel,Switzerland10
3DepartmentofBiologicalSciences,NationalUniversityofSingapore,14Science11
Drive4,117543Singapore12
4EuropeanMolecularBiologyLaboratoryHamburg,c/oDESY,Notkestrasse85,13
22607Hamburg,Germany14
15
16
*Correspondence17
Dr.PeterJ.Bond18
Prof.Dr.SebastianHiller20
22
23
2
Abstract24
ThetrimericperiplasmicholdasechaperoneSkpbindsandstabilizesunfolded25
outermembraneproteins(OMPs)aspartofbacterialOMPbiogenesis.Skpbinds26
clientproteinsinitscentralcavity,therebyreducingitsbackbonedynamics,but27
itisunknownwhichmolecularmechanismsgovernSkpdynamicsandhowthe28
chaperoneadaptstodifferentlysizedclients.Here,weemployacombinationof29
microsecond-timescalemoleculardynamics(MD)simulation,small-angleX-ray30
scattering(SAXS)andNMRspectroscopytorevealthatSkpisremarkably31
flexible,andfeaturesamolecularspring-loadedmechanisminits“tentacle”arms32
thatenablesswitchingbetweentwodistinctconformationsonsub-millisecond33
timescales.Theconformationalswitchisexecutedaroundaconservedpivot34
elementwithinthecoiledcoilstructuresofthetentacles,allowingexpansionof35
thecavityandthusaccommodationofdifferentlysizedclients.Thespring-loaded36
mechanismshowshowachaperonecanefficientlymodulateitsstructureand37
functioninanATP-independentmanner.38
39
40
3
Theoutermembrane(OM)ofGram-negativebacteriafunctionsasageneral41
selectivitybarrier.Itprovidesthecellwithprotectionfrompotentiallyharmful42
agentsintheenvironment,whileallowingusefulmoleculestoenter.Inorderto43
fulfillthisfunction,transmembraneβ-barrelOMPsactasselectiveandnon-44
selectiveporins.Theirabsenceseverelyinhibitsnutrientcollectionandtherefore45
growth.OMPsaresynthesizedinthecytosolofthebacterialcell,andassuch46
theyfollowacomplexbiogenesispathway[1].UnfoldedOMPstraverseboththe47
hydrophobicinnermembraneandthehydrophilicperiplasmbeforetheyare48
foldedandintegratedintotheOM[2–4].Duetotheirarchitectureas49
transmembraneproteins,OMPsarepronetomisfoldingandaggregationinthe50
aqueous,butcrowdedperiplasmiccompartment.Undesirableintra-andinter-51
molecularinteractionsintheperiplasmaretypicallypreventedbyanetworkof52
periplasmicchaperonesincludingSurA,DegPandSkp[5,6].Theseaidinthe53
propertransport,foldingandinsertionofOMPsintotheOM.Skpbindsto54
unfoldedOMPsastheyemergefromtheSectransloconattheinnermembrane.55
ThebindingofSkppreventsprematurefoldingofOMPs,andpromotestheir56
releasefromtheSeccomplexoncetranslocationiscomplete.SkpholdstheOMP57
inafoldingcompetentstate[7],priortoarrivalandreleaseattheOM[8].58
59
Skpisatrimerwitha“jellyfish”-likearchitecture[9,10].Asmallβ-sheet“head”60
domainisthemajorsiteofassociationbetweenthemonomers,whilethreelong,61
hairpin-shapedα-helical“tentacles”or“arms”definetheouterboundariesofa62
centralcavity,characteristicofaclamp-likebindingsitetypicallyobservedin63
chaperones[11].Unlikethestructureofprefoldin[12],aeukaryoticchaperone64
withrelatedarchitecture,inthestructuresofSkptheα-helicalextensionsmay65
4
interactwithoneanotheratthetips.Eachtentacleiscomposedofashortα-helix66
(α1)thatleadsfromtheβ-sheetheadintotwoextendedantiparallelα-helices67
(α2andα3).TheOMPsubstratesofSkparediverseinshapeandsize.The68
smallestoftheseOMPs,suchasOmpA,havean8-strandedβ-barrelwith69
diameterof3.0nm,whereasthelargest,LptD,is24-stranded[13].Studiesof70
Skp–OmpXconformationanddynamicshaveshownhowSkpcanbind8-71
strandedOmpXandtOmpA[14],butitisyetunclearhowSkpisabletoadapt72
sufficientlytoaccommodatedifferentlargersubstrates.NMRstudieshave73
identifiedahingeregionaroundVal42/Phe50betweenα-helix1and2thatmay74
allowthetipsofthetentaclestomoveawayfromoneanother,thereby75
expandingthesizeofthecentralcavitytoallowlargersubstratestoenter[14].76
Suchsubstantialflexibilityintheα-helicalregionsofSkpisalsoconsistentwith77
absenceofelectrondensityforpartsofthetentaclesinbothoftheX-ray78
structures,andwithstructuraldisparitiesobservedtowardsthetipswhen79
comparingtheresolvedsubunits.80
81
Here,weutilizeextended,microsecond-timescale,atomic-resolutionMD82
simulationsofsubstrate-free(apo)trimericSkptoexploreitsconformational83
landscape,andcomparetheresultswithdatafromsmall-angleX-rayscattering84
(SAXS)andamide–amidedistancesobtainedfromNMRspectroscopy.Unbiased85
simulationsamplingisusedtodemonstratethattheSkptrimerisextremely86
flexible,revealingthefullrangeofpossiblemotionofthetentacles.Basedon87
theseobservations,weproposehypotheticalclosed-andopen-statemodelsand88
identifyaswitchingmechanismthatleadstolargevariationsinthevolumeofthe89
Skpcentralcavityviatheexchangeofahelicalkinkbetweenhelixα2andα3.90
5
ThisallowsustodefinethelimitsofSkp’sconformationalmobility,which91
explainsitscapacityforbindingsubstratesofvariablesize.Subsequentfittingof92
simulatedconformationalensemblestodatafromSAXSmeasurementssupports93
thenotionthatapoSkpexistsinadynamicequilibriumbetweenopenandclosed94
statesinsolution.Finally,comparisonofthesimulationswithNOESY95
experimentaldataconfirmsthatapoconformationsobservedspectroscopically96
arewellrepresentedinthesimulationtrajectories,andthattheyaredistinct97
fromthesubstrate-boundformofSkp.98
99
Results100
OpeningoftheSkpCavityviaSeparationoftheTentacles101
TheX-raystructureofSkp(PDBID:1SG2)representsa“closed”stateofthe102
protein,withatip-to-tipdistanceof<0.78nmmeasuredbetweentheCα103
carbonsofAla76[9].Complementedbyapartiallymodeledthirdsubunit,the104
1SG2structureservedastheinitialseedfor15independentMDsimulation105
replicasofatleast100nsinlength.Thesesimulationsservedtosearchfor106
possibleopenstates.Startingfromthe“closed”state,dissociationofthehelical107
tips,asidentifiedbyatip-to-tipdistance>1.4nm,wasobservedinthreeofthe108
15simulations,inamannerthatwasinsensitivetoinitialconditions[8].These109
separationsofthetwosubunitstowardsan“open”stateoccurredinallthree110
casesafter>80nsofsimulation.111
112
Followingtheobservationthatthetentacle-likearmswereabletoseparatefrom113
oneanotheronthenanosecondtimescale,thedynamicsofSkpwere114
investigatedfurtherintwosignificantlyextendedsimulations,of1μsinlength115
6
each,beginninginthe“open”conformation.Additionally,wemeasuredSAXS116
profilesofasampleofhighlypureapoSkpatasynchrotronbeamline.Duringthe117
two1μsMDsimulations,thetipswereobservedtospontaneouslyre-associate118
anddisassociateatthreedifferenttimepoints.There-associationofthetipswas119
apparentinthemeasuredradiusofgyration(Rgyr)ofSkp(Figure1A),which120
droppedbelowtheRgyroftheX-raystructure(3.0nm)at~250nsinone121
simulationandat~80nsand~850nsintheother.TheaverageRgyrof3.28nm122
observedinthe1μsMDsimulations(Figure1B)agreeswellwithexperimentally123
measuredRgyrof3.3nmfortheapo-Skptrimerinsolution,determined124
previouslybyneutronscattering[15].ItthusalsoagreeswellwiththeRgyrof3.6125
nmdeterminedbySAXS,consideringthatthescatteringofthehydrationshell126
likelyadds~0.6nmtothetriaxialdimensionsoftheproteininSAXS127
measurements[16,17].128
129
TheexperimentaldeterminationsofRgyrbySAXSandneutronscatteringare130
ensembleaveragesandthusmaskindividual,short-livedconformationalstates.131
Indeed,thestructuresdeterminedbyMDsimulationfeatureawiderangeof132
individualRgyrvalues,rangingbetween2.9and3.7nm(Fig.1A).Thereby,the133
largervaluesofRgyrcorrespondedtoconformationsofSkpwherethehelical134
armsareprojectedawayfromthetrimericaxisofsymmetry,insomecases135
dramaticallyexposingthelargecentralcavity(Figure1C–I).Previous136
simulationsthatconstrainedSkptoexploreaparticularRgyrhintedatsuch137
expansionofthecentralcavity[15].Ontheotherhand,thesmallervaluesofthe138
RgyrcorrespondtoconformationalstatesofSkpwherethetipsofallthreehelical139
armsarere-associatedwithoneanother(Figure1C–II).Inthedistributionoftip-140
7
to-tipdistances,theseclosedconformationspopulateadistinctstateat2nm,141
wellseparatedfromtheconformationalensemblecontinuumofthepartiallyand142
fullyopenstate(Figure1D).WhentheSkparmsareseparated,thetip-to-tip143
distanceistypicallybetween3to7nm(Figure1D),withthedistributionof144
distancespeakingat6nm.Themaximaldistancebetweenthetipsoftwo145
subunitsencounteredinfreesimulationwas7.2nm.Overall,thesedatathus146
revealanextraordinarydegreeofflexibilityoftheSkparmsintheapostate,in147
agreementwithsolutionNMRdynamicsmeasurements[14].Thesemotions148
averageoutonthesub-mstimescale,resultinginNMR-spectroscopically149
equivalentresonancesfortheentiretrimer.150
151
“Spring-Loaded”DynamicsCharacterizetheHelicalArms152
Ithasbeensuggestedthatapivotelementexistsaroundahighlyconserved153
phenylalanineresidue(Phe50)inhelixα2,allowingforaconformationalchange154
andincreasingthevolumeofthecentralcavity[8].Inagreementwiththis155
hypothesis,multipleobservationsofaspontaneousconformationalchangeand156
rotationaroundPhe50wereobservedduringsimulation.Thischangeinvolved157
theexchangeofahelicalkinkfrominitiallybenthelixα2(Figure2A-I)to158
initiallystraighthelixα3(Figure2A-II),andresultedinlateralprojectionofthe159
tipofthehelicalarmfromthethree-foldaxisofsymmetry.Anexampleofthis160
transitionisillustratedinSupplementaryMovie1.Thekinkinhelixα3wasmost161
oftenaccommodatedfromAla100toAsp105.Intotal,theconformational162
transitionatthehelicalkinkwasobservedtwelvetimesduringthesimulation163
trajectories(FiguresS1),butneveroccurredsimultaneouslyintwoorthree164
subunits.Inallobservedcases,theexchangeofthekinkfromhelixα2toα3165
8
exhibitedall-or-nothingmechanics,andhadamaximumlifetimeof~60ns166
(FigureS1).Thekinkexchangehadasimultaneouseffectonthephysical167
dimensionsoftheSkptrimer,withadirectlycoupledincreaseintheRgyrfrom168
0.15to0.30nm.Thisopenedthecavityandmaythuswellplayarolein169
substratebinding.170
171
DefiningtheMaximalCapacityoftheSkpCavityintheOpenState172
Toisolatethebiologicallyrelevant,dominantconcertedmotionsofSkp,we173
removedthehigh-frequencybackgroundnoisebyperformingprincipal174
componentanalysis(PCA)onthecombined(2x1μs)MDtrajectoriesofthe175
entire,trimericSkpassembly(Figure3).Theexchangeofthehelicalkink176
betweenhelixα2andα3didnotappearinthelowestfrequencymodes(Figure177
3A,FigureS2),indicatingthatthislarge,switchableinter-subunitmotionofan178
armfromSkpoccursindependentlyoftheotherlocalsubunitdynamics.This179
findingimpliesthatallostericcommunicationbetweenSkpsubunitsisabsent.In180
lightofthis,wethereforeinvestigatedtheinternalarmdomainmotionsby181
performingPCAonan“artificial”6µstrajectorycomposedoftheindividual182
subunittrajectoriesfromeachsimulation(i.e.3subunitsx2simulationsx1μs183
each).Principalcomponent1(PC1),thedominantmotionoftheSkpsubunit184
(Figure3Bi),accountedforoverhalfofthetotalstructuralvariance.This185
componentinvolvedoutwardprojectionofthetipsofthetentacles,awayfrom186
thethree-foldsymmetryaxisofSkp,withaconcurrentexchangeofthehelical187
kinkfromhelixα2toα3,asdescribedabove,whilstmaintainingastablehead188
domainconformation(FigureS3).Thespring-loadedmovementisthusakey189
elementofSkptentacledynamics.190
9
191
Inordertoestimatethepossiblerangeinsizeofthecentralcavity,two192
structuralstates,termed“extremeclosed”and“extremeopen”,wereconstructed193
byapplyingC3symmetrytothetwoextremestructuresofPC1(Figure4A).In194
bothcases,aleast-squares-fittotheβ-sheetheaddomainoftheX-raystructure195
wasperformedinordertopositioneachsubunitintheextrememodels.The196
transitionbetweenthesestatesisillustratedinSupplementaryMovie2.While197
thetipsoftheheliceswereobservedtoextendupto7.2nmfromoneanother198
duringfreesimulation,thesymmetric“extremeopen”modelfeaturesadistance199
betweentipsof12nm.ThedimensionsoftheresultingcavityenclosedbySkp200
wereestimatedbyexpandingavirtualsphereatintervalsalongtheaxisof201
symmetry.Inthe“extremeclosed”model,amaximumradiusof1.75nmforthe202
expandingspherewasachievedatadepthof~2.0nmbeneaththeheaddomain203
(Figure4B).Thispositioncorrespondspreciselytotheheightofthekinkinhelix204
α2.Fromthispointonwards,theradiustapers,decreasingtowardsthetipsof205
thehelicalarms.Inthe“extremeopen”model,theupperpartofthecentral206
cavity,immediatelybelowtheheaddomain,hassimilardimensionstotheclosed207
model.However,theradiusofthespherecontinuestoincreaselinearly,208
reachingamaximumradiusof3.2nmata4.0nmdistancefromtheheaddomain.209
UsingNMR-baseddistancesmeasurements,itwaspreviouslyshownthatwhen210
boundtoSkp,8-strandedOmpX,oneofthesmallestpossiblesubstrates,adopts211
toafirstorderapproximationasphericalensembleofconformerswitharadius212
of2.1nm.Thisfluidglobulestatecanalreadybenearlyaccommodatedwithin213
the“extremeclosed”stateofapoSkp,whereaslargersubstratesmightbe214
accommodatedintheSkpcavitybygradualopeningofthearmstowardsthe215
10
“extremeopen”state.Extrapolatingundertheassumptionofequalmassdensity216
fromunfoldedOmpXtothelarge22-strandedsubstrateFhuA,thelatter217
polypeptidewouldadoptasphericalvolumewith3.5nmradius.Thislarge218
substratemighteventhereforebeaccommodatedatthelowerendofthecavity219
intheextremeopenstate;conceivably,anadditionalSkptrimermayalsobe220
recruitedforbinding[7].221
222
SAXSRevealsaDynamicEnsembleofOpenandClosedStates223
ForadescriptionoftheSkpapostate,theexperimentallydeterminedSAXS224
intensitywascomparedwiththeoreticalcalculations(Figure5).Becausethe225
individualconformersofthesimulationtrajectoriesfeaturesubstantial226
variabilityintheconformationsadopted,theircalculatedintensitiesvary(see227
FigureS4fortherangeoftheoreticalscatteringintensitiesandindicative228
structures),andnointensitycomputedfromasinglestructuredescribedthe229
experimentalSAXSdatawithinexperimentalerror.Likewise,thescattering230
computedfromtheX-raycrystalstructuredidnotagreewiththeSAXSdata,as231
evidencedbyadiscrepancyinthegoodnessoffit,withχ2=5.9.Abetter232
agreementwiththeexperimentaldatawasobtainedbyallowingformixturesof233
individualconformers,i.e.linearcombinationsofthecomputedpatterns.The234
ensembleoptimizationmethod(EOM)[18,19]usesageneticalgorithmto235
recombinemodelsfromapoolofstructures,untilanoptimalfittothe236
experimentalSAXScurveisobtained.Inordertoincludeasmanymaximally237
openstatesinthedataset,thepoolofSkpstructuresfromthesimulation238
trajectorieswasenhancedwithanequalnumberofhypotheticalsymmetrical239
structures,createdbyapplyingC3symmetrytoarandomselectionofindividual240
11
subunitconformations.ThecombinedpoolcontainedstructuresthathadRgyr241
valuesintherange2.75to4.25nm(Figure5A,greencurve).Thesestructures242
generatedafittotheexperimentalcurvewithaχ2=1.0(Figure5B).243
Representativestructuresobtainedfromthefitweresimilartothe“extreme244
open”and“extremeclosed”models(Figure5i,Figure5ii).Thus,theSAXS245
experimentsdirectlyindicatethatapoSkpinsolutionadoptsaconformational246
ensemble,composedofvariablyopenedandclosedstructuresandtheir247
intermediatestates.248
249
Whilethefittotheexperimentaldatawasimprovedconsiderablybyconsidering250
anensembleofstructures,thereremained,however,adeviationatthelow251
anglesoftheSAXSmeasurements,indicatingadearthoflargerstructuresinthe252
dataset.Inordertoincreasethenumberofstructuresinthepoolwithalarger253
Rgyr,anew,independentrandompool(Figure5A,redcurve)wasgenerated254
usingRANCH(RANdomCHain)withinEOM[18,19],allowingthepositionsofthe255
helical“tentacles”tovarywithrespecttothefixed“head”domain.EOMwas256
againthenemployedtoselectanoptimizedsetofmodelsconsistentwiththe257
data(Figure5A,pink/cyancurve).ThisapproachyieldedafittotheSAXSprofile258
withaχ2=1.5(Figure5B),wherebytherepresentativestructuresalsoincluded259
openandclosedconformations(Figure5iii,Figure5iv,Figure5v)further260
confirmingthestructuralvariabilityofSkpinsolution.Inthiscase,theselection261
frequencyofstructuresindicatedabimodaldistribution(Figure5A,pink/cyan262
curve).ThefirstpeakcorrespondedtoaRgyrof~3.2nm,similartothatinthe263
MD-generatedpool(Figure5A,greencurve).TheRgyrforthesecondpeakranged264
12
between3.8and4.1nm(Figure5A),andwasthuscomparabletotheRgyrof4.1265
nmobtainedforthe“extremeopen”modelofSkpgeneratedviaPCA.266
267
NOESYExperimentsConfirmDistinctDynamicsofApoSkp268
ThestructuralconfigurationofSkpinitsapostatewasinvestigatedfurtherby269
thecalculationofasubsetofinter-backboneamidehydrogendistancesforeach270
subunitconfigurationobservedinthesimulationtrajectories(Figure6A,Table271
S1).Thesedistanceswerecomparedtodistanceassignmentsderivedfrom272
NOESYspectroscopyforbothapo-SkpandSkpboundtoanOmpXsubstrate273
(Figure6B,C).Anarrow,unimodaldistributionwasobtainedforthemean274
deviationofbackbonedistancesofthetentacledomainacrosssimulatedSkp275
comparedtotheapoSkpNOEs,peakingat<0.8Å.Incontrast,thedeviationfrom276
theOmpX-boundSkpNOEdataexhibitedabroaderbimodaldistribution,which277
onlypartiallyoverlappedwiththeapodata,andextendedfurtherout,upto~1.3278
Å.MeasurementoftheequivalentdistancesfortheX-raycrystalstructure279
revealedthatthethreesubunitsofSkpdeviatetovaryingdegreesfromboththe280
apo-andsubstrate-boundNOEdata.However,inallcases,thecrystallographic281
deviationsareincreasedcomparedtothoseforthemostfrequentconformations282
observedinthesimulations.Thus,thesimulatedensemble,characterizedbya283
rangeofopenandclosedstates,isbestrepresentedbyligand-free,apoSkpin284
solution.ItsconformationislikelymodulatedbythepresenceofOmpX,possibly285
biasingtheensembletowardscollapsedstatesthatprotectthesubstrate.286
287
288
13
Discussion289
Viaacombinationofsimulationandexperiment,wehaveshownherethat290
ligand-free,apoSkpexistsasadynamicensembleofmultipleconformational291
states,withvariableaccesstothecentralcavity.Interconversionbetweenopen292
andclosedstatesisfast,withdramaticchangesalreadyonthemicrosecond293
timescale,asreflectedintheobservedfrequenciesoftip-to-tipseparation,294
helicalexchangebetweenhelixα2andα3,andconcomitantchangesinprotein295
Rgyrandcavityvolume.ThisfindingisinfullagreementwithpreviousNMR296
measurementsofSkpbackbonedynamics,showingthatcomplete297
conformationalaveragingisobtainedinatmost1ms[14].Thesimulationsthus298
revealanovelmechanismforchaperonecavityexpansion,involvingspring-299
loadeddynamicsofthetentacles,wherebyahelicalkinkisexchangedbetween300
helicesα2andα3.Thismechanismservestoallowthetipsofeachsubunitto301
projectawayfromthecentralaxisofsymmetry,whilstensuringminimal302
disruptionoftheproteinstructuralfold.Itwillbeofgreatinteresttoassess303
whetherarchitecturallysimilar(butevolutionarilydistinct)eukaryotic304
chaperonessuchasTim9/10orprefoldinhaverelatedfunctionalmechanisms305
[20,21].306
307
TheabilityofSkptoadapttoavarietyofdifferentlysizedsubstratesiskeytoits308
roleinperiplasmicOMPtrafficking.Thisfunctionalrequirementismetby309
variabilityintheshapecreatedbythehelicaltentacles.Thereby,thephysical310
limitoftheexpansionoftheSkpcavityisfargreaterinsizethandisplayedin311
availableX-raycrystallographicstructures,andthussufficienttoaccommodate312
Skpsubstratesabove8strands.Skpsubstantiallyaltersitsconformationupon313
14
substratebindingbyreducingbackbonedynamicsandflexibilityinthepivot314
region[14].Thesubstrate-boundstateofSkpwaspreviouslyfoundtobe315
structurallydistinctfromtheconformationsexploredbyapoSkp,indicatinga316
substrate-inducedconformationalchange.Thelarge-scalemovementsofthe317
subunitsrelativetooneanotheroccurindependentlyofboththeexchangeofthe318
helicalkinkandofoneanother.Suchloose,dynamicassociationoftheSkpcavity319
withsubstrateensuresthattheOMPisheldinafoldingcompetentstate,without320
over-stabilizingnon-nativecontactsoftheOMPbackbone[7].TheabilityofSkp321
toexpanditscavityinthiswaylikelyhasfunctionalimpactbothinthecapture322
andreleaseofdiverseOMPsubstrates,andmightalsoenableaccesstothe323
centralcavityfortheOMP-insertingBAMcomplex[22–24].324
325
326
15
Acknowledgements327
ThisworkwasfundedbytheSwissNationalScienceFoundationandA*STAR.328
ExperimentalSAXSintensityofSkpandthestructuralmodelsgeneratedbyEOM329
aredepositedinSASBDBdatabase(entriesarebeingassigned).330
331
AuthorContributions332
DAHandPJBperformedthecomputationalexperiments;BMB,MVP,andDISand333
SHperformedtheSAXSandNMRexperiments;allauthorsanalyzedthedata;PJB,334
DAH,andSHwrotethepaperwiththecontributionsfromBMB,RGH,MXP,and335
DIS;PJBandSHdesignedthestudy.336
337
Competingfinancialinterests.Theauthorsdeclarenocompetingfinancial338
interests.339
340
Materials&Correspondence.Correspondenceandrequestsformaterialsmay341
bedirectedtoProf.Dr.SebastianHiller([email protected])andDr.342
PeterJ.Bond([email protected]).343
344
345
16
Methods346
SimulationSystemConfiguration347
AcrystalstructureofSkp(PDBID:1SG2)[9]wasusedasaninitialconfiguration.348
TheresiduenumberingconventionusedhereinisbasedonUniProtentry349
sp|P0AEU7|21-161,wherethefirstresidue,Ala21,isthestartofthemature350
proteinchain.Thehelicaltipresiduesofthethirdsubunit,fromMet60toAla95,351
areunresolvedintheX-raystructure.Themissingresidues,therefore,were352
modeledwithMODELLER[25],usingthetworesolvedsubunitsastemplates.353
Theinitialtip-to-tipdistanceofthemodeledsubunitwithregardstotheother354
twosubunitswas2.53and1.74nm,respectively.15independentsimulations,355
eachof100nsinlength,wereinitiallyperformedofSkp,eitherinisolation,orin356
thepresenceoflipidAorlipidA+KDOboundattheputativeLPSbindingsiteon357
eachsubunitoftheSkphomotrimer,asdescribedinapreviousstudy[8].In358
accordancewiththeconclusionsofthispreviousstudy,thesimulationsoflipid-359
boundSkpshowedbroadlysimilardynamicstothelipidfreesimulations.360
Snapshotsofthe“openstate”ofSkpdefinedfromthesepreliminaryshorter361
simulationswereusedtoinitialize2x1µssimulations.Thiswasdefinedasthe362
largestdistancebetweentheCαofAla76residues(locatedatthetipofeach363
subunit).TheopenSkpstructuresweresolvatedinarhombicdodecahedronbox,364
containing~110,000watermoleculesanda0.15MNaClsolution.Position365
restraintsof1,000kJmol-1nm-2wereappliedfor5nstotheCαatomsofthe366
protein,priortoperforming1μsproductionsimulations.367
368
SimulationParametersandAnalysis369
17
SimulationswereperformedwiththeGROMACSsimulationpackage[26,27],370
usingtheCHARMM22forcefieldparameterset,incorporatingtheCMAP371
potentialcorrections[28,29],asdescribedpreviously[8].Allsimulationswere372
performedintheNPTensembleatatemperatureof298Kandpressureof1atm.373
Thetemperaturewascontrolledwiththevelocity-rescalethermostat[30],and374
pressurebytheParrinello-Rahmanbarostatusingisotropiccoupling[31,32].A2375
fstimestepwasusedtointegratetheequationsofmotion,andtheLINCS376
algorithmwasusedtoconstrainallbondlengths[33].A1.2nmcut-offwasused377
forLennard-Jonesinteractions,withthepotentialsmoothlyswitchedoff378
between1.0and1.2nm.Electrostaticinteractionswerecalculatedusingthe379
Particle-Mesh-Ewaldalgorithmwithareal-spacecut-offof1.2nm[34].VMDwas380
usedforvisualizationandcreatingimages[35].TheVMDplugin,Bendix,was381
usedtocalculatethedegreeofbendinginthehelicesandforthegenerationof382
imagesofkinkedhelices[36].OtheranalyseswereperformedusingGROMACS383
toolsandin-housescriptsthatutilizedthecapabilitiesofMDAnalysis[37]and384
MODELLER[25].385
386
SAXSMeasurementsandPrimaryProcessing387
SAXSmeasurementswereperformedattheP12beamlineofEMBL(DESY388
Hamburg)[38]coveringtherangeofmomentumtransfer0.01<q<0.44Å-1(q=389
4πsin(θ)/λ,where2θisthescatteringangleandλ=1.2ÅistheX-ray390
wavelength).SamplesofSkpwerepreparedinthebuffercontaining25mM391
Hepes(pH7.5),150mMNaCl,1mMDTT.Datawasacquiredinarangeof392
proteinconcentrationsfrom5.8to0.6mg/mlandanalyzedusingtheATSAS393
softwarepackage[39].Theprimarydataprocessingwasperformedusing394
18
PRIMUS[40].TheforwardscatteringI(0)andtheradiiofgyrationRgwere395
evaluatedusingtheGuinierapproximation[41],assumingthatatverysmall396
angles(s<1.3/Rg),theintensityisrepresentedasI(s)=I(0)exp(−(sRg)2/3).397
ThemaximumdimensionsDmaxwerecomputedusingtheindirecttransform398
packageGNOM[42],whichalsoprovidesthedistancedistributionfunctionp(r).399
400
ComparisonofMD-DerivedConformerstoSAXS401
CRYSOL[43]wasusedtocalculatetheformfactorsforthestructuresderived402
fromMDtrajectories.Defaultvalueswereusedforthesolventdensity(0.334403
e/Å3).Alltheoreticalscatteringcurvesderivedfromsimulationstructureswere404
fittedtotheexperimentaldata.405
406
FlexibilityAssessmentbyEnsembleOptimizationMethod407
EnsembleOptimizationMethod(EOM)[18,19]hasbeenappliedtocharacterize408
conformationalvariabilityofSkpinsolution.Thepositionsandorientationsof409
thethreehelicalarms(Asn20–Ala115)wererandomizedwithrespecttothe410
restofthestructuretogenerateapoolof10,000models.Thegeneticalgorithm411
wasappliedtoselectfromthepoolanoptimizedensemblethatbestfitthe412
experimentalSAXSdata.413
414
ProteinExpression,PurificationandIsotopelabeling415
SkpcontaininganN-terminalhexa-histidinetagandlackingitssignalsequence,416
andOmpXobtainedfrominclusionbodies,wereexpressedandpurifiedas417
describedpreviously[14,44].[U–2H,15N,13C]-labeledSkpwasobtainedby418
growingtheexpressioncellsinM9-minimalmedia[45]supplementedwith419
19
(15NH4)Cl,D–[2H,13C]–glucoseandD2O.[U–2H]-labeledOmpXwasobtainedby420
theadditionofD–[2H]–glucoseandD2OtoM9minimalmedium.Isotopeswere421
purchasedfromSigma-AldrichorCambridgeIsotopeLabs.422
423
NMRSpectroscopy424
NMRexperimentsofhumanSkpandSkp/OmpXwereperformedinNMRbuffer425
containing25mMMES,150mMNaClpH6.5.Themeasurementswererecorded426
at304KonaBrukerAscendII700MHzspectrometerequippedwitha427
cryogenicallycooledtriple-resonanceprobe.The3D[1H,1H]-NOESY-15N-TROSY428
experiments[46–48]wererecordedwithamixingtimeof100msresultingina429
totalexperimenttimeof141.5hforSkpinitsapoformandfor112.5hforSkp-430
OmpX.Theinterscandelaywassetto0.95s.Inthedirectdimension,1024431
complexpointswererecordedinanacquisitiontimeof91ms,multipliedwitha432
75°-shiftedsinebell,zero-filledto2048pointsandFouriertransformed.Inthe433
nitrogenindirectdimension,90complexpointsweremeasuredwithamaximal434
evolutiontimeof40ms,multipliedwitha75°-shiftedsinebell,zero-filledto256435
pointsandFouriertransformed.Intheprotonindirectdimension,150complex436
pointsweremeasuredwithamaximalevolutiontimeof18msforSkpinitsapo437
form,multipliedwitha75°-shiftedsinebell,zero-filledto512pointsandFourier438
transformed.Forallspectraapolynomialbaselinecorrectionwasappliedinall439
dimensions.NMRdatawereprocessedusingPROSA[49]andanalyzedwith440
CARA[50]andXEASY[51].441
442
ComparisonofSimulationandNMRobserveddistances443
Asubsetofamidehydrogen-hydrogeninteractionswaschosenforcomparisonof444
20
simulationandNOESYdata.Thesubsetwaschosenbycomparingthedistances445
oftheextremestructuresofPC1andPC2totheX-raystructure.Twoamide446
hydrogenswereselectedtobeinthissubsetiftheywere<6Åinanystructure,447
andiftheirdistancedifferedby>1ÅcomparedtotheX-raystructure.Onlythose448
residuepairsforwhichaclearcrosspeak/diagonalpeakquotientforeither449
apoSkporSkp-OmpXcouldbemeasuredwereusedinthefinalanalysis.The450
distance,𝑑,betweentwoamidehydrogens,𝑖and𝑗,wasdeterminedusingthe451
followingEquation: 𝑑!" = 𝑐 ∙ 𝑃!"!!!,where𝑃isthecrosspeak/diagonalpeak452
quotientand,𝑐isaglobalconstantobtainedbyminimizingtheaveragedistance453
betweentheNMR(bothapoSkpandSkp/OmpX)andsimulationdata,withthe454
additionalconstraintthatnoNOEdistancecouldbe>6Å.Inanygivensimulation455
frame,thedeviationfromexperimentforanatompairwasmeasuredasthe456
absolutedifferencebetweenthesimulationdistanceandthedistanceestimated457
fromtheNOEsignal.ForamidehydrogenpairswithoutanNOEsignal,the458
deviationwas0ifthesimulateddistancewas>6Å,andotherwisewascalculated459
astheabsolutedifferencefrom6Å.460
461
21
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Figures628
629
Figure1.Openingandclosingofapo-Skpinsolution.A)Theradiusof630gyrationtimelinesforSkpintwo1µstrajectoriesareshowninredandblue.The631linesindicatedtotherightofthegraphrepresent:a*,the“extremeopen”model632(shownlaterinFigure4);b*,thelargestradiusofgyrationachievedduringfree633simulation;c*,theSAXSestimateoftheradiusofgyrationdescribedherein(after634removingtheinfluenceofthehydrationshell),alsoequaltotheestimate635providedin[15];andd*,theradiusofgyrationcalculatedforthecrystal636structure(PDB1SG2).B)DistributionoftheradiiofgyrationforSkpfromboth6371µstrajectories.Thepinklineisthecombineddistribution.Theothercolored638linescorrespondtothosedescribedinA.C)ThestructurescorrespondingtoI)639themaximumandII)theminimumradiusofgyrationachievedinthe640simulations.D)Thetip-to-tipdistancebetweenallpairsofsubunitsintheSkp641trimer.Thedistributioniscombinedforbothtrajectories.Thetip-to-tipdistance642wascalculatedasthedistancebetweentheCαatomsofAla76inallsubunitpairs.643Theline,d*,correspondstothevaluefromthecrystalstructure.644645
646
29
647648Figure2.Dynamicexchangeofakinkfromhelixα2toα3inthetentacle649domain.A)CylinderrepresentationofthesubunitinA,displayingthekinkin650helixα2(stateI),andhelixα3(stateII).TheothertwosubunitsofSkpare651displayedingreyforclarity.B)Estimatedkinkangleforhelixα2andα3forone652subunitoverthecourseofa1µssimulation.Thedegreeofbendingisshownin653thetopleftcorner.Anexchangeofkinkbetweenhelicesmaybeclearlyobserved654betweenthe400-500nstimeperiod.655656
30
657658Figure3.Large-scaledominantmotionsofboththetrimericcomplexand659individualsubunitsofSkp.A)TheextremeconformationsoftheentireSkp660trimeralongprincipalcomponentsi)PC1andii)PC2.PCAwasperformedforthe661combined(2x1μs)“openSkp”trajectories.Whencombined,PC1toPC4662explainedalmost90%ofthetotalvarianceinthedata.Betweenthetwo663individual1μstrajectories,therewassubstantialsimilarityinthesemodes664(covarianceoverlap,0.68),indicatingconvergenceinthesubspaceexplored.The665exchangeofthehelicalkinkbetweenhelixα2andα3(Figure2)appearedin666noneofthefirstfourlowfrequencymodes,andwasinsteadobservedinthe667higherfrequencymodes,PC5toPC9.B)Theextremeconformationsofthe668isolatedsubunit,alongprincipalcomponentsi)PC1andii)PC2.PCAwas669performedforthecombinedtrajectoriesofeachindividualproteinsubunit(6x1670μs),afterleast-squaresfittingtotherigidheaddomain.PC1representsamotion671
31
involvingexchangeofthehelicalkinkandoutwardprojectionofthetipsofthe672tentacle,andaccountedfor>50%ofthetotalvarianceinthedata.PC2accounted673foronly10%ofthedata,andinvolvesarotationofthehelicaltentaclesrelative674totheβ-sheetheadinanorthogonaldirectiontoPC1.InbothA)andB),the675arrowsindicatethedirectionandmagnitudeofmotion,andthepercentageof676thetotalvarianceexplainedbytheprincipalcomponentisshownbesideeach677figure.Notethedifferenceindistancescaleineachfigure.678679
680
32
681682Figure4.Hypotheticalmodelofthe“extremeopen”and“extremeclosed”683statesofSkp.A)The“extremeopen”and“extremeclosed”modelscreatedby684assumingtheextremestatesofprincipalcomponent1(PC1)ofthesubunit685dynamicsareappliedtoeachsubunitoftheSkptrimersimultaneously.B)The686largestsphereradius,alongthecavityaxis,calculatedforthe“extremeopen”and687“extremeclosed”statesshowninA).Theradiusofgyrationforthe“extreme688open”modelisindicatedinFigure1A(a*).689690
691
33
692693Figure5.EnsemblefittingtotheexperimentalSAXSintensities.A)Relative694frequencyofstructureswithaparticularRgyrintheMD(green)andRANCH(red)695pools.ThefrequencyofmodelsselectedfrombyEOMisshownfortheRANCH696pool(pink/cyan).B)TheoptimizedensembleofRANCH(redline)andMD697(green)generatedstructuresfittedtotheexperimentalSAXSprofile.698RepresentativestructuresselectedbyEOMareshownforboththeMD(iandii)699andRANCH(iii,ivandv)generatedmodels.700701
702
34
703704Figure6.NMRobservedbackboneamide-amidehydrogendistancesfor705apo-andOmpX-boundSkp,comparedtosimulatedapoSkp.A)The706backbonestructureofSkp,withresiduesinblueindicatingthosebetweenwhich707amidehydrogendistancesweremeasuredforcomparisonwithNOE708assignments.Ahydrogen-hydrogeninteractionwasselectedforanalysisifthe709pairofatomswere<6ÅfromoneanotherintheX-raystructureortheextreme710PC1andPC2structures.Asubsetofthesewasselectedfromthoseinteractions711thatdifferedby>1ÅbetweenthePCstructuresandtheX-raystructure.Ofthese,712onlythoseresiduepairsforwhichaclearcrosspeak/diagonalpeakquotientfor713eitherapoSkporSkp/OmpXcouldbemeasuredwereusedinthefinalanalysis.714B)Distributionacrossallsimulationtrajectoriesofmeandeviationsfromamide715hydrogendistancesfromNOESYspectraofapo-andOmpX-boundSkp.Deviation716fromexperimentforanatompairwasmeasuredastheabsolutedifference717betweenthesimulationdistanceandthedistanceestimatedfromtheNOEsignal.718ForamidehydrogenpairswithoutanNOEsignal,thedeviationwas0ifthe719simulateddistancewas>6Å,andotherwisewascalculatedastheabsolute720differencefrom6Å.DeviationsforeachsubunitoftheX-raystructureare721indicatedwithdashedlines.C)Assigned2Dstripsfrom3D15N-edited-[15N,1H]-722NOESYspectratakenattheindicatedpositionsof250µMSkpinitsapoform723(cyan)and250µMSkpwithboundOmpX(holoform,purple)inNMRbufferat72437°C.SpectrawererecordedwithaNOESYmixingtimeof100ms.Brokenlines725indicateNOEconnections.BrokencirclesindicatemissingNOEcross-peaksin726eithertheholoortheapo-state.**-denotescrosspeaksfromadjacentplanes.727728
