Structural Insight into Archaic and Alternative Chaperone-Usher Pathways Reveals a Novel

22 Pages·2015·10.73 MB·English

RESEARCHARTICLE
Structural Insight into Archaic and
Alternative Chaperone-Usher Pathways
Reveals a Novel Mechanism of Pilus
Biogenesis
NataliaPakharukova1,JamesA.Garnett2¤a,MinnaTuittila1,SariPaavilainen1,
MamouDiallo2¤b,YingqiXu2,SteveJ.Matthews2,AntonV.Zavialov1*
a11111 1 DepartmentofChemistry,UniversityofTurku,Turku,JBL,Arcanum,Turku,Finland,2 Centrefor
StructuralBiology,DepartmentofLifeSciences,ImperialCollegeLondon,SouthKensington,London,United
Kingdom
¤a Currentaddress:SchoolofBiologicalandChemicalSciences,QueenMaryUniversityofLondon,
London,UnitedKingdom
¤b Currentaddress:WageningenUR,Food&BiobasedResearchDepartmentBioconversion,Wageningen,
TheNetherlands
*[email protected]
OPENACCESS
Citation:PakharukovaN,GarnettJA,TuittilaM,
PaavilainenS,DialloM,XuY,etal.(2015)Structural Abstract
InsightintoArchaicandAlternativeChaperone-Usher
PathwaysRevealsaNovelMechanismofPilus
Gram-negativepathogensexpressfibrousadhesiveorganellesthatmediatetargetingtosites
Biogenesis.PLoSPathog11(11):e1005269.
doi:10.1371/journal.ppat.1005269 ofinfection.Themajorclassoftheseorganellesisassembledviatheclassical,alternative
andarchaicchaperone-usherpathways.Althoughnon-classicalsystemsshareawiderphy-
Editor:JosephDMougous,Universityof
Washington,UNITEDSTATES logeneticdistributionandareassociatedwitharangeofdiseases,littleisknownabouttheir
assemblymechanisms.Herewereportatomic-resolutioninsightintothestructureandbio-
Received:June3,2015
genesisofAcinetobacterbaumanniiCsuandEscherichiacoliECPbiofilm-mediatingpili.We
Accepted:October22,2015
showthatthetwonon-classicalsystemsarestructurallyrelated,buttheirassemblymecha-
Published:November20,2015
nismisstrikinglydifferentfromtheclassicalassemblypathway.Non-classicalchaperones,
Copyright:©2015Pakharukovaetal.Thisisan unliketheirclassicalcounterparts,maintainsubunitsinasubstantiallydisorderedconforma-
openaccessarticledistributedunderthetermsofthe
tionalstate,akintoamoltenglobule.Thisisachievedbyauniquebindingmechanisminvolv-
CreativeCommonsAttributionLicense,whichpermits
ingtheregister-shifteddonorstrandcomplementationandadifferentsubunitcarboxylate
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare anchor.Thesubunitlackstheclassicalpre-foldedinitiationsitefordonorstrandexchange,
credited. suggestingthatrecognitionofitsexposedhydrophobiccorestartstheassemblyprocessand
DataAvailabilityStatement:Thecoordinatesand providesfreshinspirationforthedesignofinhibitorstargetingchaperone-ushersystems.
structurefactorshavebeendepositedintheProtein
DataBankunderaccessioncodes5D6Hand5DFK
fortheCsuC-CsuA/BcomplexandEcpBchaperone,
respectively.
AuthorSummary
Funding:AVZissupportedbytheFinnishAcademy
(grants140959and273075;http://sciencenordic. Gram-negativepathogensdependonfibrousadhesiveorganellestoattachtotargettissues
com/partner/academy-finland)andSigridJuselius andestablishinfection.Themajorclassoftheseorganellesisassembledviatheclassical,
Foundation(grant2014;www.sigridjuselius.fi/
alternativeandarchaicchaperone-usher(CU)pathways.CUpathwaysarerecognizedas
foundation).SMissupportedbytheWellcomeTrust
promisingnewtargetsforthenextgenerationofantibacterialdrugs.Therecentlydiscov-
(SeniorInvestigatorAward100280,Programmegrant
eredarchaicandalternativesystemsareofparticularinterest,astheyareimplicatedin
079819;http://www.wellcome.ac.uk)Thefundershad
PLOSPathogens|DOI:10.1371/journal.ppat.1005269 November20,2015 1/22
Non-classicalChaperone-UsherPilusAssembly
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript. biofilmformationofantibioticresistantpathogens,haveawiderphylogeneticdistribution
andareassociatedwithabroaderrangeofdiseasesthantheclassicalsystems.Here,we
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. reportanatomic-resolutioninsightintothestructureandassemblymechanismoftwo
suchbiofilm-formingorganellesassembledviathearchaicandalternativepathways.We
showthatthearchaicandalternativesystemsarestructurallyrelated,buttheirassembly
mechanismisstrikinglydifferentfromtheclassicalassemblypathway.Whereastheclassi-
calchaperonesdeliverfoldedsubunitstotheusherassemblyplatform,non-classicalchap-
eronesapplyauniquebindingmechanismtomaintainsubunitsinsubstantiallyunfolded
state.Theopensubunitcoreallowsforanewmodeofstrandreplacementduringpolymer-
isation,andalsorepresentsanattractivetargetfortherationaldesignofantimicrobials.
Introduction
Allgram-negativebacteriaexpressfibrousadhesiveorganellesthatmediatetargetingtositesof
infection.Themajorclassoftheseadhesivepili(orfimbriae)isassembledviatheclassical,
alternativeandarchaicchaperone-usher(CU)pathways[1].CUpiliarelinearpolymersmade
ofsubunitscapableofeitherself-polymerisationorassemblywithothersubunits[2,3].TheCU
fibrecanpossessrichbindingproperties[3–5],whichfacilitatebindingtohostcellreceptors,
aswellasmediatebiofilmformationthroughself-association[6]andinteractionswithabiotic
surfaces[7].
ThebiogenesisofCUfibresrequiresaperiplasmicchaperoneandoutermembraneassem-
blyplatformtermedtheusher[2].Althoughtheseassemblyproteinsareconservedwithinthe
threeCUpathwayfamilies,littlesequencehomologyexistsbetweenthedifferentCUpathways,
whichsuggestsdistantphylogeneticrelationships[1].AmongthethreeCUsystems,the
archaic(alsotermedσ)pathwayassemblesthelargestclassofpili[1].Whereastheclassical
andalternateCUsystemsarerestrictedtoβ-andγ-proteobacteria,membersofthearchaicCU
familyarepresentinα-,β-,γ-,andδ-proteobacteria,whilstalsoinphylaCyanobacteriaand
Deinococcus-Thermus.Furthermore,archaicsystemsareassociatedwithbacteriathatcause
someofthemostseverediseasesinhumans,animals,andplants[1].ArchaicCsupilimediate
theformationofAcinetobacterbaumanniibiofilms,whichcontributetohighratesofnosoco-
mialinfections[7].Thispilusisformedfromfoursubunits,namelyCsuA/B,CsuA,CsuB,and
CsuE,andisassembledusingtheCsuC-CsuDchaperone-ushersecretionmachinery[7,8].The
alternativeorαCUpathwayisahighlydivergentfamilywithawidephylogeneticdistribution
[1].ThispathwayincludesCFA/I-likefimbriae,whicharetheprimaryadhesinsofhuman
enterotoxigenicEscherichiacoli,amajorcauseofmortalityinyoungchildrenfromdeveloping
countries.TheE.colicommonpilus(ECP)alsobelongstothealternativepathwayandisasso-
ciatedwithbothdisease-causingandcommensalstrains[9].ECPiscomposedoftheEcpAand
EcpDsubunits,whichareassembledusingtwoperiplasmicchaperones,EcpBandEcpE,and
theEcpCusher[10].TheclassicalCUpathways,namelyβ,γ,κandπ,arerelativelyconserved
andtheyassemblealargevarietyofstructuresthatareprimaryassociatedwiththevirulenceof
animalandhumanpathogens.
Theclassicalsystemshavebeenstudiedforseveraldecadesandtheirbiogenesisisnow
understoodinexquisitedetail.Theperiplasmicchaperonesformabinarychaperone-subunit
complexbyoccupyingahydrophobiccleftcreatedbytheabsenceofaβ-strandfromthesub-
unitimmunoglobulin(Ig)likefold,inaprocessknownasdonorstrandcomplementation
(DSC)[11,12].Fibresubunitsaresubsequentlyassembledbydonorstrandexchange(DSE),in
whichtheN-terminalextensionfromanincomingsubunitdisplacesthechaperoneviaa"zip-
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Non-classicalChaperone-UsherPilusAssembly
in-zip-out"mechanism[13,14]andprovidesthenecessaryβ-strand[14,15].Thisprocess
occursattheentrancetotheusherporeandisfacilitated[16]byoptimalpositioningofthe
incomingchaperone-subunitcomplexbytheusher[17].
Althougharchaicandalternativesystems(groupedundertheterm‘non-classical’)havea
farwiderphylogeneticdistributionandareassociatedwithabroaderrangeofdiseasesthan
theirclassicalequivalents,littleisknownregardingtheirpreciseassemblymechanisms.Recent
structuralanalysisofthesubunitsfromtwoalternativesystemsconfirmthattheirbiogenesisis
governedbythegeneralprinciplesofDSCandDSE[6,18],althoughthelackofsequencesimi-
laritybetweenchaperonessuggeststhattheassemblyprocessforthenon-classicalpathways
coulddifferfromtheclassicalsystemssubstantially.Here,wereportatomic-resolutioninsight
intothestructureandbiogenesisofAcinetobacterbaumanniiCsuandEscherichiacoliECPpili
assembledviathearchaicandalternativepathways,respectively,whilstalsohighlightingsome
importantdeviationsfromtheclassicalassemblymechanism.Thenon-classicalchaperones,
unliketheirclassicalcounterparts,maintainsubunitsinasubstantiallyunfoldedstatebyutilis-
ingaregister-shiftedDSCandadistinctsubunitC-terminalcarboxylateanchor.Theextreme
dynamicnatureofthischaperone-boundsubunitarrangementallowsforamoreflexiblemode
ofDSEinitiationduringpolymerisation.Furthermore,thismechanisticdistinctionrepresents
anattractivetargetfortherationaldesignofnewantimicrobials.
Results
CsuA/BisthemajorCsupilussubunitandcapableofself-polymerisation
Thecsugeneclusterencodesfourdifferentpilussubunits(CsuA/B,CsuA,CsuB,CsuE)(Fig
1A).Basedonsize,positioningwithintheoperonandlevelsofexpression[7,8],wereasoned
thatCsuA/Bisthemajorshaft-formingsubunitandCsuEisatipsubunit.Inthisscenario,
CsuA/BshouldbecapableofpolymerisationwhilstCsuE,theproposedtipsubunit,should
not.Toverifythis,wepurifiedthesubunitsafterover-expressioninE.coliandexaminedtheir
abilitytopolymerizeinvitro.Intheabsenceofthechaperone,onlylowlevelsofexpression
weredetected,howeveruponco-expressionwithCsuC,theselevelsdramaticallyincreased.
Moreover,thesubunitsweresuccessfullyco-purifiedviatheHis-taggedCsuC(CsuC-His )by
6
Ni2+-affinitychromatography,suggestingthattheyformstablechaperonecomplexes(Fig1B).
SDS-PAGEofpurifiedCsuC-CsuA/Bcomplexesafterincubationatroomtemperature
revealedaladderofbandswithsizescorrespondingtoadimer,trimer,tetramerandlarger
multimersoftheCsuA/Bsubunit.Incontrast,electrophoresisoftheCsuC-CsuEcomplex
resultedinasinglebandoftheCsuEmonomer(Fig1B).BoilingtheCsuC-CsuA/Bsampledis-
ruptedtheorderedCsuA/Baggregation,resultinginasinglebandforCsuA/B(Fig1B).This
behaviourhasbeenpreviouslyobservedformajorsubunitsfromclassicalCUsystems[19]and
togetherthisconfirmsthatCsuA/Bisthemajorsubunitcapableofspontaneouspolymerisation
inpresenceofthechaperone.AlthoughsubunitsCsuAandCsuBarenotexpressedaseffi-
cientlyasCsuA/B[8],theyhavesimilarsizeandmightalsobecapableofpolymerisation,serv-
ingaseitheradaptorsorformingfinershaftstructures.
CsuA/ButilizesitsN-terminaldonorstrandforpolymerisation
UponcloserinspectionofN-terminalsequencesofCsuA/B,CsuAandCsuB,aclearpatternof
alternatinghydrophilic-hydrophobicresiduesisobserved,characteristicforN-terminaldonor
strandsofclassicalpilins(Fig1A).TotestwhetherCsuA/BpolymerizesviaDSC,wesubsti-
tutedthefirst12residueswithaHis -tag(His -CsuA/B)andco-expresseditwithCsuC.Analy-
6 6
sisoftheco-purifiedcomplexbySDS-PAGEpriortoandafterboilingshownoladderof
CsuA/Bpolymers,indicatingthatthisN-terminalsegmentisresponsibleforassembly(Fig
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Non-classicalChaperone-UsherPilusAssembly
Fig1.CsuA/Bself-assemblydependsonitsN-terminaldonorstrandsequence.(A)AlignmentofsequencesofCsupilinsubunitsA/B,AandBandthe
pilindomainofsubunitCsuE.ClustalWwasusedtoalignthesequences.Residuesarecodedasfollows:identical(pinkshading);conservedcharacter(cyan
shading);pilinN-terminalresiduesproposedtotakepartindonorstrandcomplementationinthepilus(yellow);involvedinchaperonebinding(circlesabove
theresidue);invariantforarchaicpilindomaincysteines(starsaboveresidues).Limitsandnomenclatureforsecondarystructureelementsareshownabove
thesequence.Dashedlineindicatesunstructuredsequences.StructuraldatawerederivedfromthecrystalstructureofCsuC-CsuA/Bcomplex(thisstudy).
(B)CsuA/Biscapableofself-polymerisationandCsuEisnot.CsuA/BandCsuEwereco-expressedwithHis -taggedCsuCinE.coli,co-purifiedfrom
6
periplasmicextractsbyNi2+-affinitychromatography,andanalysedbySDS-PAGE.Complexeswereincubatedat22or100°Cpriortotheelectrophoresis.
(C)IdentificationofpolymerisationsequenceinCsuA/B.CsuA/Bmutantwith12N-terminalaminoacidresiduesreplacedbyaHis -tag(Δds)wasco-
6
expressedwithwildtypeCsuC.CsuA/BpointmutantsLeu10!AlaandIle12!Alaandatriplemutant(T)withVal8,Leu10andIle12substitutedtoalanines
wereco-expressedwithHis -taggedCsuC.CsuC-CsuA/BcomplexeswerepurifiedandanalysedbySDS-PAGEasin(B).
6
doi:10.1371/journal.ppat.1005269.g001
1C).Toconfirmthatthesequenceformsadonorstrandwepreparedsinglealaninesubstitu-
tionmutantsofthetwolargesthydrophobicresidues,Leu10andIle12,inataglessCsuA/B
constructandco-expressedthesewithCsuC-His .SDSPAGEanalysisrevealedasignificant
6
reductionofhighmolecularmasspolymers,particularlyfortheIle12Alamutant(Fig1C).
Moreover,simultaneousmutationofthreehydrophobicresidues(Val8,Leu10andIle12)abol-
ishedCsuA/Bpolymerisationcompletely.Theseresultsprovideconvincingevidencethatthe
assemblyofarchaicpiliisbasedonDSC.
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Non-classicalChaperone-UsherPilusAssembly
TheCsuCchaperoneadoptsthecanonicaltandemIgfold
TogaininsightintothestructureandassemblyofarchaicCUpili,wedeterminedthecrystal
structureofaCsuC-CsuA/Bpre-assemblycomplex,composedofHis -CsuA/BandCsuC(to
6
avoidpolymerisation).CrystalswerereadilyobtainedinspacegroupP6 22andthestructure
4
wassolvedusingSe-SADphasingtoaresolutionof2.4Å(Fig2).TheCsuCchaperonehasa
canonicalIg-likefoldwithtwo7-strandedβsandwichdomains(D1andD2)orientedat*90°
angle(Figs2and3A).Despitethelackofsequencesimilarity,comparisonofCsuCandclassical
chaperoneswithknownstructuresrevealedasignificantsimilarityinD1.Forexample,100
equivalentD1CαatomsofCsuCandtheYesiniapestisCaf1Mchaperonesuperimposewith
RMSDof1.9Å(Z-score=12.8,S2Table)(Fig3A).Thelargeststructuraldifferencesoccurat
theedgesoftheβsandwichdomain.CsuChasanadditionalβ-strandD (Fig3Aand3B).The
1
C -D hairpinprotrudesfromdomainD1towardsdomainD2,closingtheentrancetothe
1 1
inter-domaincleft.Thisadditionalsequenceispresentinallarchaicchaperones(S1Fig),sug-
gestingthatthisblockadeofinter-domaincleftplaysanimportantfunctionalrole.DomainD2
islesssimilartotheequivalentdomaininclassicalchaperones(Z-score=5.9,S2Table).The
principaldifferenceoccursinthepositionofβ-sheetD C F G (Fig3A);inCaf1Mthisis
2 2 2 2
rotatedwithrespecttoβ-sheetA B E by35–50°,whereasinCsuCthisis60–85°.Thenearly
2 2 2
orthogonalpackingofβ-sheetsrenderstheβ-barrelinCsuCmoreopenthanforCaf1M,
althoughtheβ-barreliscoveredbyanadditionalhelixfromtheE -F loop(helix2).
2 2
TheCsuCchaperonemaintainstheCsuA/Binapartiallyorderedstate
WhilsttherefinementstatisticsforthestructureoftheCsuC-CsuA/Bcomplexaregood(S1
Table),approximately40%oftheCsuA/Bsequencewasnotevidentinelectrondensitymaps,
whilstanother7%hasverypoorelectrondensity(CαatomB-factorshigherthan80A2).Fur-
thermore,thisisalsoreflectedinthegradualincreaseinB-factorsforCsuA/Boutsideofthe
chaperoneinterface(Fig4A).Toprovidefurtherinsightintothestructuralheterogeneityin
CsuA/BwithintheCsuC-boundcomplex,weprepareda15N-labelledCsuC-CsuA/Bsample
andacquireda1H-15NTROSYNMRspectrum(Fig4B).Whilethespectrumshowsgood
chemicalshiftdispersionconsistentwithasignificantorderedstructure,alargerthanexpected
distributionofamidelinewidthsisobservedand~15%oftheexpectednon-prolineresonances
areabsentfromthespectrum.Thisstronglyimpliesthatwithinthecontextofapre-assembly
complexalargeportion,presumablywithinCsuA/B,displaysdynamicconformational
exchangeonanintermediatetimescale.Althoughourdatadoesnotruleoutthatthislarge
regionadoptsanalternativeconformationalstate,itmorelikelythatitexistsinmanydifferent
statesandexchangesbetweenthem.Additionalorderedconformationalstateswouldmanifest
asmultipleNMRresonancesforthesameresidue.Weobservenoevidenceforthiswithinthe
NMRspectrumforthechaperone-subunitcomplex.Infact,theNMRspectralpropertiesmore
akintothatforamoltenglobuleandisconsistentwithourcrystallographicdata,inwhichno
clearelectrondensitywasresolvedforalmosthalftheCsuA/Bsequence.
TheremainingstructureoftheCsuA/Bsubunitrevealsadoubleβ-sheetsandwichcompris-
ingstrandsA,B,andE(β-sheet1)andCandF(β-sheet2)andβ-strandD,whichswitches
betweenthesheets(Figs2and4C).Thethreestrandsofβ-sheet1(A,B,andE)areinterrupted
inthemiddlebyaperiodicregions.Conservedcysteines16and62formadisulphidebridge
linkingthebeginningofβ-strandAwiththeendofβ-strandB´.TheN-terminalsequenceup
toSer13isdisorderedandthelargeunstructuredsequencesarelocatedbetweenβ-strandsA
andA´,A´andB,CandD,D´andE,andE´andF.
ThemoststrikingstructuraldifferencebetweenCsuA/Bandclassicalsubunitsisinthe
degreetowhichsubunitsarefoldedwhenincomplexwiththechaperone.Inthemajorityof
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Fig2.StereodiagramofthecrystalstructureoftheCsuC:CsuA/Bcomplex.CsuCandCsuA/Bare
paintedincyanandmagenta,respectively.Donorresidues(Val110,Phe112,Met114,Tyr116)areshownas
balls-and-sticks.NandCterminiandβ-strandsarelabelled.Theasteriskinthe*N´labelindicatesthattheN-
terminaldonorsequenceofCsuA/BhasbeenreplacedbyaHis-tag.
doi:10.1371/journal.ppat.1005269.g002
availablestructuresofpreassemblycomplexesfromclassicalsystems,theentiresequence(99–
100%)ofthechaperone-boundsubunit(exceptfortheN-terminalextension)ishighlyordered
(S3Table,e.g.YersiniapestiscapsularsubunitCaf1incomplexwithCaf1MshowninFig4D).
Incontrasttotheclassicalsystems,nearlyhalfofCsuA/Bisdisorderedordisplaysverypoor
electrondensity.
Nevertheless,theorderedpartofCsuA/Bprovidessufficientinformationtoconcludethat,
aswithclassicalsystemsthispilinhastheincompleteIg-likefoldinasix-strandedβ-sandwich,
inwhichtheabsent7thstrand(G)leavesalargehydrophobiccleft.TheorderedpartofCsuA/B
showslimitedstructuralsimilaritytoCaf1withaZ-scoreof3.7(Fig4D).
ThedonorstrandregisterintheCsuC-CsuA/Bcomplexisshiftedrelative
tothatinclassicalchaperone-subunitcomplexes
CsuA/BinteractspredominantlywithD1ofthechaperoneCsuCviaedgestrandstoforma
closed“super-barrel”withacommoncore.ThechaperoneA andG strandsarehydrogen
1 1
bondedtothesubunitAandFstrands,respectively.Fourlargehydrophobicresiduesfromthe
chaperoneG strand(Val110,Phe112,Met114,Tyr116)aredonatedtothesubunittocompen-
1
sateforthemissingGstrand.Inaddition,strandA providesseveralhydrophobicresiduessta-
1
bilisingthesuper-barrel(S2Fig).
SuperpositionofCsuC-CsuA/BandCaf1M-Caf1complexesrevealedthatCsuA/Bissitu-
atedclosertothechaperoneD2thanCaf1(Fig5A).Toexplorethisglobaldifference,wecom-
paredthepositionofdonorstrandresiduesinCsuCandCaf1M.Intheclassicalchaperones
thedonorstrandmotifcanvaryinlength(from3to5hydrophobicdonorresiduesasin
Caf1M),butitstartsfromthesameposition1intheclassicaldonorstrandregister,corre-
spondingtoIle134inCaf1M.However,inCsuC,thedonorstrandmotifisshiftedtowardsthe
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Fig3.Structuralcomparisonofarchaicandclassicalchaperones.(A)SuperpositionofCsuCandclassicalchaperoneCaf1M(stereodiagram).CsuC
andCaf1Marecyanandyellow,respectively,exceptdonorstrandsegments,whichareblueandorangeinCsuCandCaf1M,respectively,andtheC -D
1 1
hairpininCsuC,whichisviolet.(B)StructuralalignmentofCsuCandCaf1M.Structurallyequivalentandnon-equivalentresiduesareshowninupperand
lowercases,respectively.Thenumberofresiduesinunstructuredsegmentsisindicated.Aminoacididentitiesareindicatedbybackgroundshadingincyan.
Donorandsubunitcarboxylateanchoringresiduesareshownonyellowandgreenbackgrounds,respectively.Theusher-bindingresiduesinCaf1Mand
correspondingresiduesinCsuCareunderlined.Theproline,whichisinvariantfortheentirechaperonesuperfamily(classical,archaic,andalternative
chaperones)isshowninred.Thedisulphidebond-linkedcysteinesinCaf1Mareshowninbold.Positionsofdonorresiduesinacceptorpocketsareindicated
(0–5).Secondarystructure(cylinder,α-helix;arrow,β-strand;line,coil)isshownaboveandbelowtheaminoacidsequenceofCsuCandCaf1M,
respectively.
doi:10.1371/journal.ppat.1005269.g003
C-terminalendofβ-strandG bytworesidues(oronedonorresidue).Itstartswiththehighly
1
conservedTyr116andcorrespondstoposition0usingtheclassicaldonorstrandregister(Figs
5B,3B,andS1).Thesubunitanddonorresiduemotifinthechaperoneareshiftedinthesame
directionalongstrandG ,arguingthatthedonorstranddeterminesthepositionofthe
1
subunit.
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Fig4.Chaperone-boundCsuA/Bispartiallyfolded.(A)DiagramofthecrystalstructureoftheCsuC:CsuA/BcomplexcolouredbyB-factorofCαatoms
withthecolourrangingfrombluetoredandcorrespondstoaB-factorrangefrom12.39to113.65Å2.(B)SolutionNMRanalysisoftheCsuC-CsuA/B
complex.1H-15NTROSYHSQCspectrumofCsuC-CsuA/B.(C)TopologydiagramsofCsuA/B.Arrowsindicateβstrands.CsuA/Bismagenta;CsuCA and
1
G βstrandsarecyan.ThewavydashedlineattheN-terminusofCsuA/BindicatestheN-terminalpolymerisationsequence.Otherdashedlinesindicate
1
unstructuredsequencesinthecorestructure.(D)SuperpositionofCsuA/B(magenta)andCaf1(green)(stereodiagram).ThedisulphidebridgeinCsuA/Bis
shownasballs-on-sticks.NotethatalargepartofCsuA/Bisunstructured.
doi:10.1371/journal.ppat.1005269.g004
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Non-classicalChaperone-UsherPilusAssembly
Fig5.DifferencesintheregisterofdonorresiduesandpositionofthesubunitinCsuC:CsuA/Bandcomplexesfromclassicalsystems.(A)
SuperpositionofthearchaicCsuC:CsuA/BandclassicalCaf1M:Caf1chaperone-subunitcomplexes(stereodiagram).CsuC,CsuA/B,Caf1M,andCaf1are
paintedincyan,magenta,yellowandgreen,respectively.StrandG isshownascartoondiagramanddonorstrandsegmentsinCsuCandCaf1Mareshown
1
inblueandorange,respectively,asinFig3AandpanelB.(B)DetailedalignmentofG donorstrandsinCsuCandCaf1M.Hydrophobicdonorresiduesin
1
positionsP0-5areshowninballs-and-sticksandlabelled.
doi:10.1371/journal.ppat.1005269.g005
CsuCutilisesauniqueC-terminalcarboxylatebindingmechanism
StrandFisoneresidueshorterinCsuA/BthaninclassicalsubunitsandtheC-terminalPhe152
issituatedinthecentreofthecomplex,withintheinterdomaincleftofthechaperone(Fig2A).
TheC-terminalcarboxylateofPhe152interactswithtwohighlyconservedresiduesofCsuC
(Fig6Aand6B).Oneoxygenatomofthecarboxylateformsanionicinteractionwithinvariant
Arg89(S1Fig)inβ-strandFofD1.Theotheroxygenatomofthecarboxylateishydrogen
bondedtothehydroxylgroupofthehighlyconservedTyr196fromβ-strandFofD2.Interest-
ingly,thehydroxylgroupofTyr196isalsohydrogenbondedtotheconservedArg174.This
basicresidueservesasanacceptorofelectrons,strengtheningthehydrogenbondbetweenthe
hydroxylgroupofTyr196andC-terminalcarboxylate(Fig6B);therefore,Arg174alsocontrib-
utesasapartofthecarboxylateanchoringmechanism.
Tostudythecontributionofthisnetworktothesubunitbinding,weconstructedpoint
mutantsofCsuC,namelyArg89Ala,Arg174Ala,andTyr196Phe.WildtypeCsuCandthe
mutantversionswereco-expressedwithHis -CsuA/BandassessedwithNi2+-affinitypull-
6
downs(Fig6C).Arg89AlaandTyr196Phemutationsreducedthesubunitrecoveryequally,
suggestingthatbothionic(carboxylate-Arg89)andhydrogen(carboxylate-hydroxylgroupof
Tyr196)bondscontributetocomplexformation.Furthermore,combiningthesemutationsled
tonearzerorecoveryofthesubunit.TheArg174Alamutationhadameasureableeffecton
periplasmiclevelsofCsuA/B,butwaslessdramaticandsupportsasecondaryroleforArg174
inpositioningthecarboxylate-Tyr196.SubunitC-terminalcarboxylatebindinginarchaic
chaperonesisnotablydifferentfromthatoftheclassicalchaperones.Classicalchaperonesbind
thecarboxylateviatwohighlyconservedbasicresidues(Arg20andLys139inCaf1M),which
havenoanaloguesinarchaicchaperones.Furthermore,theseresiduesarelocatedinD1,
whereasinarchaicsystemsonlyoneanchoringresidueisprovidedbyD1andtwoarelocated
inD2(Fig3B).
PLOSPathogens|DOI:10.1371/journal.ppat.1005269 November20,2015 9/22
Non-classicalChaperone-UsherPilusAssembly
Fig6.Networkofionic-hydrogenbondsanchoringtheC-terminalcarboxylateofCsuA/Bintheinter-domaincleftofCsuCisessentialforthe
complexformation.(A)Close-upoftheCsuC:CsuA/Bstructure,demonstratinginteractionsbetweenC-terminalcarboxylateofCsuA/BandCsuC(cartoon
diagram).C-terminalPhe152inCsuA/BandArg89,Tyr196,andArg174inCsuCareshownasballs-and-sticks.Hydrogenbondsareshownwithdashesand
theirlengthisindicated.Thecolor-codingissameasinFig2.(B)InteractionmapbetweentheC-terminalcarboxylateandCsuC.(C)ContributionofCsuA/B
C-terminalcarboxylatebindingresiduestoCsuC-CsuA/Bassociation.CsuA/Bwasco-expressedwithwildtypeandmutatedCsuCinE.colicells,followed
bytheperiplasmextractionandNi2+-columnfractionation.SamplesofperiplasmicextractsandpurifiedcomplexeswereseparatedbyaSDS-polyacrylamide
electrophoresisandopticaldensityofbandsofCsuA/BandCsuCwasmeasuredandintegrated.Segmentsofgelsofperiplasmicextracts(top)andpurified
complexes(bottom)showbandsofCsuA/BandCsuC.Integratedopticaldensity(IOD)ofthebandsnormalizedagainstwildtypevaluesisshownonbar
plots.Theresultsarerepresentativeofthreeindependentexperiments.
doi:10.1371/journal.ppat.1005269.g006
Chaperonesfromarchaicandalternativesystemsarestructurally
related
Subunitpolymerisationwithinthealternativeassemblysystem,ECP,cannotbeexplainedfully
bytheclassicalCUmechanism.Thisisbecauseitisnecessarytoinsertalargetryptophanside
chainfromthemiddleoftheEcpAN-terminalextensiondeeplywithinthecoreofanadjacent
subunitduringassembly[6].Ourobservationsthatarchaicchaperonesmaintaintheirsubunits
inapartiallyfoldedpreassemblystateviaadistinctbindingmode,ledustosuggestthatalter-
nativesystemscouldemployasimilarmechanism.Furthermore,archaicandalternativeCU
assemblysystemspre-datetheevolutionoftheclassicalpathways,suggestingthattheyshare
similarnon-classicalCUfeatures.
TotestthiswefirstdemonstratedaninteractionbetweenthemajorpilinsubunitEcpAand
itscognatechaperone,presumedtobeEcpB.Weco-expressedEcpA(withaTrp11Alamuta-
tiontoabrogateself-polymerisation:EcpAW11A)intheE.coliperiplasmwithHis-taggedEcpB
(EcpB-His ),followedbypurificationviaNi2+-affinitychromatography.Subsequentgelfiltra-
6
tionandSDSpageanalysisrevealedthatatightcomplexisformedbetweenEcpBandEcpA
witha1:1stoichiometry(S3Fig)andthiswasalsoconfirmedbysubsequentNMRanalyses
(seelater).WenextcrystallizedfreeEcpBanddetermineditsstructureusingI-SIRASphasing
to2.4Åresolution(S1Table).EcpBconsistsofthetwocharacteristicIg-likedomainsasseen
inallotherchaperonesandhasanoverallboomerang-likeshape(Fig7A).Surprisingly,it
superimposespoorlywithanysubunitfreeandsubunitboundchaperonestructuressolved
thusfar(S2Table).However,whenwecomparedtheindividualdomains,considerablestruc-
turalsimilaritycouldbeidentified,particularlyforD1.Asexpected,thesuperpositionrevealed
thatEcpBisstructurallymorerelatedtoCsuC(Z-score=14.2)thanclassicalchaperones(e.g.
Caf1M,Z-score=12.0)(S2Table).NotopologicaldifferenceswerefoundinD1betweenEcpB
andCsuC,butbothpossessanadditionalstrandD thatisabsentintheclassicalchaperones.
1
PLOSPathogens|DOI:10.1371/journal.ppat.1005269 November20,2015 10/22