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Mechanism of selective nickel transfer from

HypB to HypA, Escherichia coli [NiFe]-hydrogenase accessory proteins

Michael J. Lacasse, Colin D. Douglas, and Deborah B. Zamble

Version Post-print/accepted manuscript

Citation

(published version)

M. J. Lacasse, C. D. Douglas, D. B. Zamble* (2016) “Mechanism of

selective nickel transfer from HypB to HypA, Escherichia coli [NiFe]-hydrogenase accessory proteins” Biochemistry, 55, 6821-6831.

Publisher’s Statement This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biochemistry, copyright ©

American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published

work see 10.1021/acs.biochem.6b00706.

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TheMechanismofSelectiveNickelTransferFrom

HypBtoHypA,E.coli[NiFe]-HydrogenaseAccessory

Proteins

B.FundingSourceStatement

ThisworkwassupportedinpartbyfundingfromtheNaturalScienceandEngineeringResearchCouncil

(Canada),includinganNSERCPostgraduateScholarship(MJL),andtheCanadianInstitutesofHealth

Research.

B.Byline

MichaelJ.Lacasse†1,ColinD.Douglas†1,DeborahB.Zamble*1,2

1Department of Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 3H6 and

2Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

*Correspondence to Deborah Zamble: [email protected], 416-978-3568

†Theseauthorscontributedequallytothiswork.

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AbbreviationsandTextualFootnotes

GDP:guanosine5ʹ-diphosphate,GppCp:βγ-methyleneguanosine5ʹ-triphosphate,GFC:gelfiltration

chromatography,DTT:dithiothreitol,EDTA:ethylenediaminetetraaceticacid,ESI-MS:electrospray-

ionizationmassspectrometry,HEPES:4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid,IPTG:

isopropylβ-D-1-thiogalactopyranoside,MF2:Mag-Fura-2,PAR:4-(2-pyridylazo)resorcinol,PMB:p-hydroxymercuribenzoate,PMSF:phenylmethanesulfonylfluoride,TCEP:tris(2-carboxyethyl)phosphine,

andTRIS:tris(hydroxymethyl)aminomethane.

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Abstract

[NiFe]-hydrogenaseenzymescatalyzethereversiblereductionofprotonstomolecularhydrogen

andserveasavitalcomponentoftheanaerobicmetabolismofmanypathogens.Thesynthesisofthe

bimetalliccatalyticcenterrequiresasuiteofaccessoryproteinsandthepenultimatestep,nickel

insertion,isfacilitatedbythemetallochaperonesHypAandHypB.InEscherichiacoli,nickelmovesfrom

asiteintheGTPasedomainofHypBtoHypAinaprocessacceleratedbyGDP.Todeterminehowthe

transferofnickeliscontrolled,theimpactofHypAandnucleotidesonthepropertiesofHypBwere

examined.IntegraltothisworkwasHis2GlnHypA,amutantwithattenuatednickelaffinitythatdoes

notsupporthydrogenaseproductioninE.coli.ThismutationinhibitsnickeltranslocationfromHypB.

H2Q-HypAdoesnotmodulatetheapparentmetalaffinityofHypB,butthestoichiometryandstabilityof

theHypB-nickelcomplexaremodulatedbynucleotide.Furthermore,theHypA-HypBinteractionwas

detectedbygelfiltrationchromatographyifHypBwasloadedwithGDP,butnottheGTPanalog,andthe

proteincomplexdissociateduponnickelbindingtoHis2ofHypA.Incontrast,nucleotidedoesnot

modulatezincbindingtoHypB,andloadingzincintotheGTPasedomainofHypBinhibitscomplex

formationwithHypA.TheseresultsdemonstratethatGTPhydrolysiscontrolsbothmetalbindingand

protein-proteininteractions,conferringselectiveanddirectionalnickeltransferduring[NiFe]-

hydrogenasebiosynthesis.

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Introduction

Metalloenzymesarevitalforthesurvivaloflivingorganisms,butthetransitionmetalsthatserveas

necessarycatalyticcofactorscanbetoxicifunregulated.1-3Tocontrolthedistributionandavailabilityof

thesecrucialnutrients,organismsdeploynetworksoftransporters,storageproteins,regulators,and

metallochaperones.1,4-6Thesesystemskeepmetalsappropriatelycompartmentalized,minimizingthe

amountsof"free"metalandensuringcorrectmetallationofmetalloenzymesinthefaceoftherelative

thermodynamicaffinitiesdictatedbytheIrving-Williamsseries.7Onestrategytoachievethesegoalsis

directedandselectivemetaltransferbetweenthemetallochaperonesthatfunnelthecognatemetalto

themetalloenzymeactivesites;however,themechanismsbehindtheseprocessesarenotwelldefined.

Hydrogenasesaremetalloenzymesthatcatalyzethereversibleinterconversionbetweenmolecular

hydrogenandprotonsandelectrons,andtheymakecrucialcontributionstothemetabolismofmany

microorganisms.8,9Canonically,hydrogenasesaredividedintothreecategoriesbasedonthemetalsat

theactivesite:[FeFe],[Fe],and[NiFe].9Thebiosynthesisandfunctionof[NiFe]-hydrogenasesareof

interestduetothedrivetounderstandfundamentalmetalmetabolism,theobligatoryroleofthese

systemsininfectionsbybacteriasuchasEscherichiacoliandHelicobacterpylori,aswellaspotential

applicationstothehydrogeneconomy.8,10-14MaturationoftheE.coli[NiFe]-hydrogenaseisoenzymes

involvesatleastsevenproteins,mostofwhichareencodedonthehypoperon.12,15-17Twoofthese

maturationproteins,HypA(orthehomologousHybF)andHypB,aremetallochaperonesrequiredfor

nickeldeliverytothehydrogenaseprecursorprotein.12,15

EscherichiacoliHypBisa31kDametal-bindingproteinwithGTPaseactivity.18,19Althoughithas

beenclearlydemonstratedthatGTPhydrolysisbyHypBisrequiredfornickeldeliverytothe

hydrogenaseaccessoryprotein,20thebiologicalpurposeofthisactivityisnotknown.Escherichiacoli

HypBhastwodistinctmetal-bindingsites.TheN-terminuscontainsthe“high-affinitysite”,aseven-

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residuesequencethatbindsnickelwithsub-picomolaraffinity.19,21,22Mutationofmetal-binding

residuesatthehigh-affinitysiteofE.coliHypBimpairsitshydrogenasematurationfunction,23butthis

siteisonlypartiallyconservedamongstHypBorthologs.16Asecondmetal-bindingsite,alsorequiredfor

hydrogenasematurationinE.coli,23islocatedintheC-terminalGTPasedomain(G-domain)ofHypBand

isconservedacrossvirtuallyallbacterialspeciescontainingthisgene.19,24-27Nickelbindstothissitewith

mid-micromolaraffinitywhilezincbindsanorderofmagnitudetighter.19MetalbindingtotheG-domain

ofHypBislinkedtotheGTPaseactivitybecausehydrolysisofGTPinE.coliHypBissignificantlyreduced

whennickelorzincisboundintheG-domain.25,27,28Thisrelationshipmaybebidirectional,as

biochemicalanalysisofH.pyloriHypBdemonstratedthatthenucleotide-loadedstateoftheprotein

impactsthemetal-bindingsite.25,26

HypAalsohastwometal-bindingdomains:azinc-bindingdomainandanickel-bindingdomain.The

zinc-bindingdomainiscomprisedofastructuraltetrathiolatesiteandisthoughttomediateprotein-

proteininteractions.29-32StudiesofHypAfromH.pylori,whereHypAandHypBalsomoonlightinthe

productionofthenickelenzymeurease,33suggestedthatthissitecanactasapHsensoranddirect

nickeltotheappropriatemetalloenzymebasedontheacidityofthecytosol.34,35Theotherdomain

bindsnickel,withmid-nanomolaraffinitymeasuredforE.coliHypA,36andincludesresiduesfromtheN-

terminus.29,30,32,37Biochemical,NMR,andX-rayabsorptionspectroscopydataindicatethatthehighly

conservedHis-2andGlu-3residuesinconjunctionwithbackbonenitrogensarelikelycoordinatingthe

nickel,butthecompletesiteiscurrentlyundefined.29,32,34,37

PurifiedE.coliHypAandHypBproteinsformacomplexinvitro,30aninteractionthatwasalso

observedbyusingpull-downexperimentsfromcrudeE.colicelllysates.38HypAandHypBproteinswere

detectedinacomplexwithHycE,thelargesubunitofhydrogenase3,butahypAknockoutprevented

HypBfrombeingpulleddownwithHycE.38Takentogether,thepropertiesofHypAareconsistentwith

thehypothesisthatHypA/HybF(thelatterfunctionallyreplacingHypAformaturationofhydrogenases1

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and239)actas‘adaptors’thatdockthenickeldeliverycomplexontothetargethydrogenaseprecursor

proteinduringmetallocenterassembly.38

Aspredictedbythethermodynamicsofnickelbindingtotheseparateproteins,nickelwasobserved

tomovefromtheG-domainsiteofHypBtoHypAwithinminuteswhenthetwoproteinsweremixed

together.36WhenthesameexperimentwasperformedinthepresenceofGDP,thetransferofnickelto

HypAwasacceleratedbyseveralordersofmagnitude,butthetransferofnickelfromHypBtosmall

moleculechelatorswasslower.36Furthermore,mutationsinHypBthatdisruptedtheinteractionwith

HypAalsoretardednickeltransferinvitroandhydrogenaseproductioninvivo.36,40Theseobservations

supportthemodelinwhichtheHypB-HypAcomplexisakeycomponentofthenickeldeliverystageof

hydrogenasematuration,withnickelpassingfromoneproteintotheother.However,thespecific

molecularmechanismsbywhichthistransferiscontrolledwerenotknown.

Inthisstudy,weexaminedhownucleotideloadingandcomplexformationwithHypAimpactthe

metal-bindingpropertiesoftheG-domainsiteofE.coliHypB,allowingustoprobethechangesinHypB

duringthevariousstepsoftheGTPasecycle.WediscoveredthatGTPandGDPaffectnickelbindingto

HypBinamannerconsistentwiththeproteinfirstactingasanickelacceptorandthenasanickel

source.Furthermore,onlyGDPpromotescomplexformationwithHypA,andtheproteincomplex

dissociatesonceHypAisloadedwithnickel.Incontrast,zincappearstoinhibittheprotein-protein

interaction.Theseexperimentsdemonstratedirectionalandspecificnickeltranslocationandshedlight

onhowthisprocessiscontrolledbyGTPhydrolysisduringthematurationof[NiFe]-hydrogenaseinE.

coli.

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MaterialsandMethods

PAR(4-(2-pyridylazo)-resorcinol),PMB(para-mercury-benzenesulfonicacid),DTNB(5,5ʹ-dithiobis(2-

nitrobenzoicacid),GppCp(βγ-methyleneguanosine5ʹ-triphosphate),benzylviologen,sodiumformate,

arabinose,andGDP(Guanosinediphosphate)werepurchasedfromSigma-Aldrich.Metalsalts(NiSO4,

ZnSO4,andMgSO4)weretracemetalgradeandwerepurchasedfromSigma-Aldrich.MF2(Mag-Fura-2)

waspurchasedfromInvitrogen.AllotherchemicalswereobtainedfromBioShop,Canadaaseither

biologygradeorcertifiedACSreagents,unlessotherwisedescribed.Nitrogengasandhydrogengasmix

weresuppliedbyPraxair.SolutionsformetalassayswerepreparedwithMilli-Qwaterandtreatedwith

Chelex-100(Bio-Rad)tominimizetracemetalcontamination.Electronicabsorptionspectrawere

recordedonanAgilent8453spectrophotometeratroomtemperature.

Cloning,ExpressionandPurification

TheH2QmutationwasintroducedtobothstrA-pET24bandstrA-pBAD18-kanplasmids38inonestepby

usingQuikchangemutagenesistocreateH2QstrA-pET24bandH2QstrA-pBAD18-kanrespectively.

Forward(5'-GGAGATATACATATGCAGGAAATAACCCTCTGCCAACGGGCACTGG-3’)andreverse(5'-

CCAGTGCCCGTTGGCAGAGGGTTATTTCCTGCATATGTATATCTCC-3’)primerswereusedtocreatethe

mutationinpET24b.Forward(5'-GGAGGAATTCACCATGCAGGAAATAACCCTCTGCC-3’)andreverse(5'-

GGCAGAGGGTTATTTCCTGCATGGTGAATTCCTCC-3’)primerswereusedtocreatethemutationin

pBAD18-kan.PrimerswerepurchasedfromIntegratedDNATechnologies.Allmutationswereverified

bysequencing(ACGT,Toronto).HypAStrandH2Q-HypAStrwereexpressedinBL21(DE3)E.coliand

separatedfromcelllysatesaspreviouslydescribed,36usingStrep-TactinSepharoseaffinitypurification

(IBALifeSciences,Goettingen,Germany).AfterpurificationthroughStrepTactinSepharoseresin,

HypAStrandH2Q-HypAStrweredialyzedinto20mMTris,pH7.5,1mMTCEPandeitherstoredat-80°Cif

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thestoichiometriczincwasmeasured(PARassay)orfurtherpurifiedthroughaHiTrapQHPcolumn(GE

Healthcare)toremoveapo-protein.SDS-PAGEwithCoomassiebluestainwasusedtoscreenthe

chromatographyfractions.ThemolecularweightsofpurifiedproteinswereverifiedbyESI-MS,

performedbyAIMSLaboratory(UniversityofToronto,Toronto).HypAStrandH2Q-HypAStrwerepurified

with>90%zincasverifiedbynon-denaturingESI-MS(describedbelow)andPARassays.36

Wildtype(WT)E.coliHypBandmutantversionswerepreparedaspreviouslydescribed.19,36WT-HypB

waspurifiedwiththeN-terminalmetal-bidingsiteoccupiedwithnickel.TopreparemTM-HypB,a

combinationofmonomericHypB(L242A,L246A)andtriplemutantHypB(C2A,C5A,C7A),theplasmid,

mTmhypB-pET24bwasconstructedfromapreviouslypreparedpET24b-HypBC2A,C5A,C7Aplasmid.19

TheL242AandL246AmutationswereintroducedinonestepbyusingQuikChangemutagenesis.The

forward(5'-GCTCAACAAAGTTGACGCGTTGCCGTATGCCAACTTTGACG-3’)andreverse(5'-

CGTCAAAGTTGGCATACGGCAACGCGTCAACTTTGTTGAGC-3’)primerswereused.Allmutationswere

verifiedbysequencing(ACGT,Toronto).

Non-DenaturingElectrosprayMassSpectrometryAnalysis

HypAStrandH2Q-HypAStrproteinsampleswerebufferexchangedinto10mMammoniumacetate,

pH=7.5,usingAmiconUltra3kDamolecularweightcut-offcentrifugalfilters(Millipore)underan

anaerobicatmosphere(95%N2,5%H2),andthendilutedto5µMinthesamebuffer.Fornickelbinding

analysis,theproteinwasbriefly(<5min)incubatedwith50µMNiSO4priortoinjection.Themass

spectrometrydatawererecordedonanABSciexQStarXLmassspectrometerwithahotsource-induced

desolvation(HSID)interface(IonicsMassSpectrometryGroupInc.)aspreviouslydescribed.36

MetalBindingtoHypB

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Stoichiometryexperimentswereperformedbyincubating100μLof100μMTM-HypBin25mMHEPES,

pH7.5,100mMKCl,and5mMMgSO4(HKMbuffer)with2mMNiSO4or500μMZnSO4and500μMof

nucleotideovernightat4°C.Lowerconcentrationsofzincwereusedtoavoidproteinprecipitation.After

incubation,thefreemetalwasremovedfromtheproteinbyusingaPD-10desaltingcolumn(GE

Healthcare)thatwasequilibratedwithHKMbuffer.Onealiquotofthedesaltedproteinwasquantified

bybicinchoninicacid(BCA)proteinassay(Pierce)usingastandardcurvepreparedfromthesame

purifiedproteinatknownconcentrationsasdeterminedbyelectronicabsorptionat280nm.The

bicinchoninicacidassaywasusedinlieuofelectronicabsorptionspectroscopyforproteinquantification

becausethepresenceofnucleotidegeneratesignificantbackground.Asecondaliquotoftheprotein

wastreatedwith1mMp-hydroxymercuribenzoate(PMB)and100μM4-(2-pyridylazo)resorcinol(PAR),

whichallowedformetalquantificationbycomparingtheelectronicabsorbanceat494nmin

comparisontoastandardcurveofeithernickelorzinc.DesaltingstoichiometryexperimentswithHypB

wereperformedwithtriplemutantsproteins(TM-HypBandmTM-HypB)toavoidinterferencefrom

metalboundintheN-terminalmetalsite.

Thestoichiometriesofthenickelcomplexesofnucleotide-loadedHypBwereconfirmedbytitratingWT-

HypBundersaturatingconditions.Nickelwasaddedto100μMWT-HypBin25mMHEPES,pH7.5,100

mMKCl,1mMTCEP,and5mMMgSO4andthesampleswereallowedtoreachequilibrium.The

fractionalsaturationofHypBwasdeterminedbymonitoringtheabsorbanceat340nm.

BenzylViologenHydrogenaseAssays

CulturesofE.coliweregrownanaerobicallyfor6hat37°CinmodifiedTYEPmediacontaining10g/L

tryptone,5g/Lyeastextract,69mMKH2PO4,supplementedwith30mMsodiumformate,0.8%glycerol,

and50mg/Lkanamycinwhereappropriate.Fourstrainswereusedinthisstudy:MC4100asawild-type

control,DPABF(hypAATG→TAA,ΔhybF),39DPABFcellstransformedwithstrA-pBAD18-kan,andDPABF

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cellstransformedwithH2QstrA-pBAD18-kan.InordertoensureconsistentexpressionofHypAStrand

H2Q-HypAStr,theamountofarabinosewastunedappropriatelybasedonwesternblotanalysisof

lysates,and25μMarabinosewasusedintheDPABFandMC4100controlswhereas10μMand50μM

wereusedforDPABFcellsbearingstrA-pBAD18-kanandH2QstrA-pBAD18-kan,respectively.Following

growth,cellswerewashedwithice-cold50mMpotassiumphosphateatpH7.6,andresuspendedinthe

samebuffercontaining200μMPMSFand1mMDTT.Cellswerelysedbysonication(BransonSonifier

185)oniceandcellulardebriswasremovedviacentrifugationat21,000g.Theproteincontentofcrude

celllysateswasdeterminedbyusingaBCAproteinassaywithbovineserumalbuminasastandard.The

totalhydrogenaseactivitiesoflysatesweredeterminedbymonitoringthehydrogen-dependent

reductionofbenzylviologen.Lysate(1-10μL)wasaddedtoa4mMsolutionofbenzylviologenin100

mMpotassiumphosphatebufferatpH=7.0under95%N2and5%H2atmosphere.41,42Thechangein

absorbanceat600nmwasmonitoredandconvertedtoactivity(unitsofμmolmin-1mg-1)byusingthe

benzylviologenextinctioncoefficientof7400M-1cm-1.

WesternBlotAnalysis

ToverifyHypAexpressionfromthepBADplasmidsunderourhydrogenaseassaygrowthconditions,the

presenceofHypAbywesternblotwasperformed.Duetotheexpressionlevelsandmethodsensitivity,

lysatesfromthreereplicateculturesforeachconditionwereusedforwesternblotanalysis.First,the

totalproteincontentofeachlysatewasdeterminedbyBCAproteinassay.Aconstantamountof

protein,basedonBCAproteinassay,wasprecipitatedbyadditionof2mLacetonefollowedby

overnightincubationat-30°C.Acetonewasremovedfollowingcentrifugationandtheprecipitated

proteinswereresuspendedin50μLSDS-PAGEloadingdyeand50μL4Murea.Proteinswereloadedon

15%SDS-polyacrylamidegelandtransferredtoImmobilon-PSQmembrane(Millipore)post-

electrophoresis.TheblotswereprobedwithRabbit-anti-HypApolyclonalantibodyata1:1000dilution.38

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The2°goatanti-rabbit(Bio-Rad)antibodieswereusedatadilutionof1:10,000.SuperSignalWestPico

ChemiluminescentSubstrate(ThermoScientific)wasusedfordetection.

CompetitionExperiments

TheaffinitiesoftheHypBG-domainsitefornickelandzincweredeterminedthroughcompetitionwith

themetal-sensitivedyeMag-Fura-2(MF2)atroomtemperature.Nickelcompetitionexperimentswere

performedbyincubating20μMWT-HypBand1.0μMMF2with0-1000μMNiSO4overnightunderan

anaerobicatmosphere(95%N2and5%H2)at4°Candwarmedtoroomtemperaturepriortoanalysis.

Thezinccompetitionexperimentswereperformedbyincubating10μMWT-HypBand1.0μMMF2with

0-1000μMZnSO4overnightunderananaerobicatmosphere(95%N2and5%H2)at4°Candwarmedto

roomtemperaturepriortoanalysis.Thebuffer(25mMHEPES,100mMKCl,5mMMgSO4atpH=7.5)

wastreatedwithChelex100resin(Bio-Rad)priortoadditionofMgSO4andstirredovernightunderthe

anaerobicatmospheretoensureremovalofdissolvedoxygen.Whereappropriatethebufferwas

supplementedwith500μMnucleotide,eitherGDPorGppCp.ThefluorescenceemissionofMF2at500

nm(λex=335nmforNiSO4titrationsandλex=390nmZnSO4titrations)wasrecordedonaCLARIOstar

platereader(BMGLabtech)andfractionalsaturationwascalculatedbasedonthemaximalandminimal

fluorescence.TheapparentdissociationconstantforMF2undertheseconditionswas1.7±0.6μMfor

nickeland1.0±0.2μMforzinc

AlltheHypBcompetitiondatawerefitbyusingcustomDynaFitscripts(supplementalinformation)to

eitheramonomer-bindingmodelforapo-HypBandnucleotide-loadedzincbindingoradimer-binding

modelfornucleotide-loadedHypBbindingnickelasperresultsfromstoichiometryexperiments.

TheaffinityofH2Q-HypAStrfornickelwasdeterminedthroughcompetitionwiththemetalsensitivedye

MF2.TheaffinityofMF2forNi(II)wasdeterminedbytitratingNiSO4into1μMMF2inbuffer(25mM

HEPES,100mMKCl,5mMMgSO4atpH=7.5)..Theintensityoffluorescenceemissionat500nm(λex=

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335nm)wasrecordedonaCLARIOstarplatereader(BMGLabtech)andfractionalsaturationwas

calculatedbasedonthemaximalandminimalfluorescence.TheresultantdatawerefittotheLangmuir

equationtoanapparentdissociationconstantof0.9±0.2μM.Subsequentcompetitionswere

performedbetween20μMH2Q-HypAStrand1μMMF2inbuffer(25mMHEPES,100mMKCl,5mM

MgSO4atpH=7.5)underananaerobicatmosphere(95%N2and5%H2).NiSO4wastitratedfrom0-1000

μMandthesolutionswereincubatedovernightat4°Candbroughttoroomtemperaturepriorto

analysis.TheresultingcompetitiondatawerefitusingDynaFitandamonomer-bindingmodel.The

affinityofHypAStrfornickelwascompletedaspreviouslydescribed.36

ThemetalaffinitiesofHypBinthepresenceofH2Q-HypAStrweredeterminedbycompetitionwithMF2.

NiSO4wastitratedintosolutionsof20μMH2Q-HypAStr,20μMWT-HypB,and1μMMF2in25mM

HEPES(pH7.5),100mMKCl,5mMMgSO4,pH7.5,andinsomecases500μMGDPorGppCpunder

anaerobicatmosphere(95%N2,5%H2)toavoidusingareducingagent.Solutionswereincubated

overnightat4°C,andthenbroughttoroomtemperature(RT)priortoanalysis.Thefluorescence

emissionofMF2at510nm(λex=335)wasmeasuredonaCLARIOstarplatereader(BMGLabtech)to

determinetheamountofmetalboundtothedye.Theresultingcompetitiondatawerecomparedto

simulations,generatedbyDynafit,basedonthetitrationsoftheindependentmoleculestoqualitatively

assessanysignificantchangeintheaffinityofHypBinthepresenceofH2Q-HypAStr.

MetalTransferExperiments

Nickeltransferexperimentswereperformedaspreviouslydescribed.36Briefly,50μMofWT-HypBwas

loadedwithoneadditionalequivalentofNiSO4,andthenmixedwitheither70μMHypAStr,H2Q-HypAStr

or1mMEDTAin25mMHEPES,100mMKCl,5mMMgSO4,and1mMTCEPbufferatpH7.5,and100

μMGDP.Theabsorbanceat340nmwasmeasuredoverthecourseoftenminutes.Adecreaseinthe

absorptionatthiswavelengthcorrespondswithalossofnickelfromtheG-domainsiteofHypB.

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AnalyticalGelFiltrationChromatography

ASuperdex200Increase10/300column(GEHealthcare)wasusedforgelfiltrationexperiments,driven

byanAKTAfastproteinliquidchromatographysystem.Thecolumnwasequilibratedwithtwocolumn

volumesofSuperdexbuffer,whichconsistedof25mMHEPES,200mMKCl,5mMMgSO4atpH7.5.

Nucleotides(GDPorGppCp)wereaddedtoboththeproteinsample(at500μM)andrunningbuffer(at

100μM)tomaintainproteinloading.NiSO4orZnSO4wasaddedtotheproteinsamplesat500μM.

Proteinsampleswereincubatedovernightat4°C.ESI-MSexperimentsconfirmedthatthisincubation

withexcesszincdidnotdisplacethenickelintheN-terminalsiteofWT-HypB(datanotshown).Protein

elutionwasdetectedbyabsorbanceat280nm.PeakintegrationswereperformedinOriginPro8,fitting

aGaussiancurvetoeachexpectedpeak.PeakareacalculationsexcludetheHypAStrmonomerpeak

(elutionvolumeof17.4mL)forclarity.Thepeakswereassignedbasedoncomparisontomolecular

weightstandardsandthecomponentswereconfirmedbyusingSDS-PAGEanalysis.However,other

oligomericstatescannotberuledoutbyusingthismethod.

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Results

ImpactofNucleotideonMetalBindingtotheHypBG-domain

GiventhattheHypBmetalsiteintheG-domainisembeddedwithintheGTPasemotifs,itis

feasiblethatGNPnucleotidesmodulatethemetal-bindingcharacteristicsofthissite,andthatone

functionoftheGTPaseactivityistocontrolmetalbindingandtransferfromthismetallochaperone.A

linkbetweenthemetalandnucleotidebindingsitesofE.coliHypBwouldbeconsistentwiththe

observationthatmetaldecreasestherateofGTPhydrolysis,28andwiththenucleotide-dependent

changesinmetalbindingobservedwithH.pyloriHypB.25,27Totestthishypothesis,firsttheimpactof

nucleotideonthestoichiometryofmetalbindingtoE.coliHypBwasdetermined.Tosimplifythe

analysis,theseexperimentswereperformedwithaHypBvariantinwhichthecysteineligandsoftheN-

terminalhigh-affinitymetalsiteweremutatedtoalanine(Cys2/5/7toAla,termedtriplemutantHypBor

TM-HypB),leavingtheG-domainmetalsiteintact.TM-HypBwasincubatedwithexcessnickelorzincin

theabsenceofnucleotideorinthepresenceofGDPorGppCp(anon-hydrolyzableGTPanalogue).

Unboundmetalwasremovedbydesaltingandtheamountofboundmetalwasdeterminedbyusingthe

metallochromicindicator4-(2-pyridylazo)resorcinol(PAR)(Table1).Intheabsenceofnucleotide,TM-

HypBbindsoneequivalentofnickel,aspreviouslyobserved.19UpontheadditionofeitherGDPor

GppCp,onlyahalfanequivalentofnickelwasdetectedboundtoTM-HypB.Incontrast,ananalogous

nucleotide-dependentchangeinstoichiometrywasnotobservedwithzinc.Thenickelstoichiometryof

HypBloadedwithnucleotidewasconfirmedunderequilibriumconditionsbytitratinghigh

concentrationsofwild-type(WT)proteinwithnickel(SupplementalFig.1).Intheseandallsubsequent

experimentswithWT-HypB,theN-terminalsitewasloadedwithnickel.NickelbindingtotheG-domain

wasmonitoredbyusingelectronicabsorptionspectroscopy,andsaturateduponadditionofhalfan

equivalentofmetal.

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GiventhatnucleotideenhancestheformationofE.coliHypBhomodimers,24,28thequaternary

structureoftheproteinmaycontributetothechangeinnickelstoichiometry.Totestthispossibility,

thesameexperimentswereperformedwithaL242A/L246AmutantversionofTM-HypB.Previous

studiesdemonstratedthattheL242AandL246AmutationsintoE.coliHypBresultsinaproteinfor

whichdimersarenotdetectedinsolutioneveninthepresenceofnucleotide.24,28Theintroductionof

thesemutationsintoTM-HypBcreatedamonomerictriplemutantHypB,referredtoasmTM-HypB.The

stoichiometryofnickelboundtomTM-HypBwasnotobservedtochangeupontheadditionof

nucleotide(Table1),indicatingthatthedimerization,nickelbinding,andtheGTPasecycleare

connected.

CompetitionexperimentsbetweenWT-HypBandthefluorescentmetalindicatorMag-Fura-2

(MF2)wereperformedtoassesshownucleotidemodulatestheaffinityofmetalbindingtotheHypBG-

domain(Table2).Intheseexperiments,metal-dependentquenchingofMF2fluorescencewas

monitoredasmetalwastitratedintoamixtureofHypBandMF2,andtheresultantdatawere

interpretedusingacustomDynaFitscript(SupplementalFig.2).43Thedataandthecorrespondingfits

(SupplementalFig.3)revealthattheHypBnickelcomplexisabout10-foldstrongerinthepresenceof

GppCpcomparedtothecomplexformedinthepresenceofGDPorwithoutnucleotide.Thesame

experimentswereperformedwithzinc,whichbindstighterthannickelaspreviouslyreported,19,28but

HypBdidnotdisplayalargechangeinzincaffinityasafunctionofnucleotide.

HypAStr-HypBComplexFormation

Anotherwaythatcofactorscouldmodulatemetaltransferbetweenthemetallochaperonesisby

affectingcomplexformationbetweenHypAandHypB.Toprovidesomeinsightintothisissue,the

impactsofexcessmetalandnucleotidecofactorsoncomplexformationbetweenHypBandHypAStr

of 42

wereexaminedbyusinggelfiltrationchromatography(GFC)(Fig.1,Table3).TheHypAusedinthese

experimentswasmodifiedwithaC-terminalStrep-tagII,whichdoesnotaffecttheactivityofthe

protein.38BothGppCpandnickelpromotedtheappearanceofalargerformofHypBwhichwas

assignedasahomodimer,consistentwiththechangesinnickelstoichiometryaswellastheresultsof

previousstudies.24,28Incontrast,zincdidnotimpacttheelutionofHypB.Notably,thepresenceofGDP

wasrequiredtoobserveaHypAStr-HypBcomplex.ThestoichiometryoftheHypAStr-HypBcomplex

cannotbedeterminedwithGFC,whichislimitedbecauseitmeasureschangesinthehydrodynamic

radiusandnotabsolutemolecularweights,butthepresenceofbothproteinsinthepeakassignedasa

complexbetweenHypAandHypBwasconfirmedwithSDS-PAGE(datanotshown).Thisresultsupports

amodelinwhichGTPhydrolysisbyHypBpromotesnickeltransferbyregulatingtheinteractionwith

HypAwhilesimultaneouslyweakeningthenickelaffinityofHypB(Table2).However,theHypAStr-HypB

complexwasdestabilizedwhentheexperimentwasperformedinthepresenceofexcesszincand

completelyabolishedinthepresenceofexcessnickel,suggestingthatmetalbindingtooneofthe

metallochaperonesinhibitscomplexformation.

H2Q-HypAStrNickelAffinity

InordertodeterminehowmetalbindingtoHypAimpactscomplexformationwithHypB,andto

examinehowHypAcontributestometaltransferbetweentheproteins,itwasnecessarytogeneratea

HypAvariantwithimpairednickelaffinity.AlthoughtheexactcoordinationoftheHypAnickelsiteisnot

clear,studiesofE.coliHybFandH.pyloriHypArevealedthattheconservedresidueHis2iscriticalfor

nickelbindingaswellashydrogenasematurationbytheseproteins.29,37ToexamineifHis2hasasimilar

roleintheactivitiesofE.coliHypA,weintroducedaH2QmutationintotheHypAStrconstruct.Circular

of 42

dichroismspectroscopyandthermaldenaturationexperimentsdemonstratedthattheH2Q-HypAStr

proteinhasasimilarsecondarystructureandstabilityaswild-typeHypA(datanotshown).

TheimpactoftheH2QmutationonnickelbindingtoHypAwasexaminedfirstbyelectrospray-

ionizationmassspectrometry(ESI-MS).Thenickelcomplexofwild-typeHypAStrissufficientlyrobust

suchthatquantitativenickelbindingisdetectedwiththismethodwhenmicromolarprotein

concentrationsareused.36However,whensimilarexperimentswereperformedwithH2Q-HypAStr,

nickel-loadedproteinwasnotdetected(SupplementalFig.4),suggestingthatthemutationsignificantly

weakensthenickelcomplex.TodeterminethenickelaffinityofH2Q-HypAStr,competitionexperiments

withthemetallochromicindicatorMF2wereperformed(Table2),andthedataandthecorresponding

fits(SupplementalFig.5)revealthatthemutantproteincanbindnickelbutwithreducedaffinity.MF2

competitiondatawerefittoamonomer-bindingmodelwithanapparentaffinityfornickelof4±2μM.

Thisisadramaticreductionfromtheapparentaffinityofwild-typeHypAStrof40±30nM,whichis

comparabletoapreviouslyreportedvalue.36

H2Q-HypAStrinHydrogenaseMaturation

TotesttheimportanceofnickelbindingtoHypAduringhydrogenasematuration,hydrogenase

productionwasassessedinE.coliexpressingH2Q-HypAStr.HypAisrequiredfornickelinsertionintothe

activesiteofthelargesubunitof[NiFe]-hydrogenase3,sotheproteinwasexpressedfroman

arabinose-inducibleplasmidinDPABF(hypAATG→TAA,ΔhybF),39astrainofE.colithatalsolacks

backgroundactivityfromtheotherhydrogenaseisoenzymesmaturedviaHybF.Boththewild-type

HypAandtheH2QmutantproteinswereexpressedwithaC-terminalStrep-tagIIinordertoensurethat

thehydrogenaseassayswerecongruentwithinvitrocharacterizationandtoreaffirmthattheStrep-tag

of 42

IIdidnotimpacttheHypAmaturationfunction.Thehydrogenaseactivitiesincrudecelllysates

generatedfromcellsexpressingeithermutantorwild-typeHypAStrweremeasuredbymonitoringthe

reductionofthebenzylviologendyeinthepresenceofhydrogengas(Fig.2).41,42,44Theresults

demonstratethatwild-typeHypAStrisabletopartiallycomplementthehydrogenase-deficient

phenotypeoftheDPABFstrainaspreviouslyobserved,38whereasH2Q-HypAStrfailedtogenerateactive

hydrogenase.ComparableexpressionofHypAStrandH2Q-HypAStrwasconfirmedthroughWesternblot

analysis(SupplementalFig.6).TheobservationthatH2Q-HypAStrcannotsupporthydrogenase

maturationinE.colisuggeststhatthenickel-bindingactivityofHypAisessentialforthisprocess.

MetalTransferBetweenHypBandH2Q-HypAStr

WhenWT-HypBloadedwithnickelintheG-domainisincubatedwithHypA,nickelmoves

quantitativelyfromHypBtoHypAwithinseconds.36ToexaminehowtheH2QmutationinHypAaffects

thisprocess,nickeltransferfromHypBtoHypAStr,H2Q-HypAStrorEDTAwasexaminedinthepresence

ofGDP,whereEDTAservedasanonspecificsmallmoleculechelator(Fig.3).Electronicabsorption

spectroscopywasusedtomonitortheseexperiments,becauseaHypBG-domainnickelcomplex

producesaligand-to-metalchargetransferwithacharacteristicabsorbanceat340nmattributabletoa

cysteineligand.36,45NickelbindingtoHypAdoesnotresultinadetectablespectroscopicsignal.Inthe

presenceofwild-typeHypAStr,thenickelwasrapidlydepletedfromHypBwithsecondorderdecay

kinetics,aspreviouslyreported.36Incontrast,thefractionalsaturationofHypBdidnotchangeupon

incubationwithH2Q-HypAStr.Electrosprayionizationmassspectrometry(ESI-MS)onamixtureofthese

proteinswasalsoperformedtoconfirmtheseresults,andnickeltransferfromHypBtowild-typeHypAStr

wasobservedbutnickeltransfertoH2Q-HypAStrwasnotdetected(SupplementalFig.4).Overall,these

of 42

resultsdemonstratethatGDP-loadedHypBdoesnotrapidlytransfernickeltoH2Q-HypAStr,incontrast

towild-typeHypAStr.

ImpactofH2Q-HypAStronNickelBindingtotheHypBG-domain

Itispossiblethatthenickel-bindingsiteofHypBismodulatedbycomplexformationwithHypA

inanucleotide-dependentmanner,whichwouldacceleratenickeltransferbetweentheproteins.

Therefore,theaffinityofnickelbindingtotheWT-HypBG-domainwasexaminedinthepresenceof

H2Q-HypAStr.TheimpactofH2Q-HypAStronHypBnickelaffinitywasassessedbymonitoringthe

competitionbetweenMF2,HypB,andH2Q-HypAStrinthepresenceorabsenceofnucleotide(Fig.4).A

quantitativeanalysiswasnotperformedbecauseofthelargenumberofpossibleprotein-metal

complexes.Instead,theresultswerecomparedtosimulatedcompetitiondatageneratedfromacustom

Dynafitscript(SupplementalFig.6)bymodellingH2Q-HypAStrandHypBproteinsasindependentmetal

bindersusingtheaffinitiesmeasuredfortheseparateproteins.43Withthisapproach,theexperimental

resultscanbemodelledbythesimulateddata,suggestingthatH2Q-HypAStrdoesnotinduceachangein

theaffinityofHypBfornickel.

H2Q-HypAStr-HypBComplex

Totestifnickelbindingtoeitherproteincausesaconformationalchangethatdisruptsthe

HypA-HypBinteraction,thegelfiltrationchromatographyexperimentswererepeatedwiththeH2Q-

HypAStrmutant(Fig.1,Table4).Asobservedintheexperimentswiththewild-typeprotein,GDP

promotedcomplexformationbetweenH2Q-HypAStrandWT-HypB,whereasGppCpdidnot.However,in

thiscaseaH2Q-HypAStr–HypBcomplexwasstillobservedinthepresenceofexcessnickel,suggesting

of 42

thatnickelbindingtotheHypBG-domainsupportstheprotein-proteininteractionandthatitisnickel

bindingtoHis2inthewild-typeHypAproteinthatcausescomplexdissociation.Finally,theimpactof

zincbindingtoHypBwasexamined.Inthiscase,onlyasmallamountofH2Q-HypAStr–HypBcomplex

wasobservedinthepresenceofGDP,andincontrasttotheresultswithnickel,asimilaramountofthe

complexwasobservedforeitherwild-typeorH2Q-HypAStr.Altogether,theseexperimentsrevealthat

GDPenhancesHypA-HypBcomplexformation,whichispartiallyblockeduponloadingHypBwithzinc,

andthatHypAStrwillnotformacomplexwithHypBwhentheHis2siteisloadedwithnickel.

Discussion

Thematurationoffunctional[NiFe]-hydrogenase,anenzymevitalfortheanaerobicrespirationand

pathogenicityofmanymicroorganisms,9,14isaccomplishedbyasuiteofaccessoryproteinsthatput

togetherthemetalcenterinacoordinatedprocess.12,15,46Twoproteinsthatareinvolvedinnickel

deliverytohydrogenaseinE.coliareHypAandHypB,whicharebothcapableofbindingnickel.12,16

Previousworkdemonstratedthatthetwonickel-bindingsitesofHypBarerequiredforhydrogenase

production,23andthattheGTPaseactivityofHypBisanessentialelementofthepathway20asis

complexformationbetweenHypBandHypA.36,40However,itwasnotclearifthesepropertiesare

connectedorhowtheyareintegratedintothehydrogenasebiosyntheticpathway.Arecentstudyof

HypAandHypBfromthearcheaThermococcuskodakarensisdemonstratednucleotide-dependent

changesinproteinquaternarystructure,buttheT.kodakarensismaturationsystemisquitedifferent

fromthatofE.coli.47Inparticular,T.kodakarensisHypBisanATPasethatdoesnotbindmetal,

whereasE.coliHypBisaGTPaseandhastwonecessarynickel-bindingsites,includingoneintheG-

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domainthatappearstobeconservedacrossbacterialhomologs.19,24-27Therefore,itremainedunclear

howmetaltransferiscontrolledinpathogenicsystemssuchasinE.coli.Inthisstudy,wedemonstrated

thatnickelbindingtoHypAisrequiredforhydrogenaseproductioninE.coli.Complexformation

betweenthetwonickelaccessoryproteinsiscontrolledbytheGTPasecycle,asisthenickelaffinityof

HypB,suggestingthatthepurposeofGTPhydrolysisistoregulatenickeltransferbetweenHypBand

HypA.Furthermore,themetal-loadedstatesofeachproteinhaveanimpactontheinteraction,

resultinginunidirectionalandselectivenickeltransfer.

GTPaseControl

ThepreviouslyreportedobservationthatmetalbindingtotheG-domainofHypBinhibitsGTP

hydrolysisindicatedthatthemetalcofactorhasanallostericeffectonthenucleotide-bindingsiteofthe

protein.26,28Thisreportestablishesthatthereverserelationshipisalsotrue,asthepresenceofGDPor

GppCpimpactstheaffinityandstoichiometryofnickelbindingtotheHypBG-domainsite.Specifically,

thestoichiometrydecreasesfroma1:1HypB:nickelratiointheabsenceofnucleotidetoa2:1ratioin

thepresenceofeitherGDPorGppCp.GiventheestimatedcellularGTP/GDPconcentrationsinE.coli

andtherelativenucleotideaffinities,itisexpectedthatthenucleotide-freestateofHypBisnotpresent

insignificantamountsinvivo.18,20,28,48ThisimpliesthatthefunctionalstateofHypBisadimer.

However,amonomericmutantversionofHypBwithnodetectabledimerizationinsolutionwasstillable

tosupportsomehydrogenaseproductioninE.coli,andthismutantstillexhibitednickelbindingandGTP

hydrolysisinvitro.28,40Furthermore,thepredominantHypA-HypBcomplexobservedbygelfiltration

chromatographywasassignedtobea1:1heterodimer(althoughotheroligomerizationstatescannotbe

ruledout),sotheroleoftheHypBdimerinthehydrogenasebiosyntheticpathwayremainsunclear.

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TheGTPasecyclehasanimpactontheaffinityofnickelboundtotheG-domain.Nickel

competitionexperimentsbetweenHypBandMF2revealedthatintheGTP-loadedstateHypBbinds

nickelmoretightlythanwhenloadedwithGDP.Theseresultsareconsistentwiththelinkbetweenthe

metalsiteandtheSwitchIIGTPasemotifhighlightedinthecrystalstructuresofseveralHypB

homologs.24,25TheresultsalsosuggesthowthedifferentstepsofHypBnickeltransfermaybecoupled

totheGTPasecycle.InitiallyHypBmustacquirenickel,andintheGTP-boundstatetheG-domainsite

couldmoreeffectivelycompetewithothermoleculesinthecytosol.Forinstance,HypBmighthaveto

competefornickelwithhistidine,whichispredictedtocoordinatethenickelionsthatareimported

throughtheperiplasmicmembranebytheNikABCDEtransporter.49,50OncenickelisboundtoHypB,the

metalinhibitstheGTPaseactivityoftheprotein.ItisspeculatedthatHypBGTPhydrolysisisaccelerated

onlywhenHypBisinacomplexwhereitcandelivernickeltothenextstageinhydrogenase

biosynthesis.26,28ThepreventionofwastefulGTPhydrolysismakesthisrationaleattractive,butthe

factor(s)thatacceleratetheGTPaseactivityarestillunknown.Preliminaryexperimentssuggestthat

complexformationwithHypAdoesnotaffectthisactivity(unpublisheddata).Athirdhydrogenase

accessoryprotein,SlyD,increasestherateofGTPhydrolysisseveralfold,51butitislikelythatadditional

factorsenhancetheactivitytoamuchlargerdegree.

OnceGTPhydrolysisistriggered,theswitchfromtheGTP-totheGDP-loadedstatereducesthe

affinityofHypBfornickel,facilitatingthepassageofmetaltothenextstepinthehydrogenase

biosyntheticpathway.RegulationofmetalbindingthroughtheGTPasecycleisanalogoustotheactivity

ofUreG,thenickel-bindingGTPaseaccessoryproteinrequiredforthematurationofurease.52,53As

observedforHypB,GTPhydrolysisbyUreGisrequiredforureaseactivationandalsoweakensnickel

binding,anditislikelythatthenickelpassesfromUreGthroughotheraccessoryproteinsbefore

insertionintotheenzymeactivesite.52,54InthecaseofHypB,notonlydoesGTPhydrolysisweaken

nickelbinding,butitalsoactivatescomplexformationwithnickel-freeHypA.Regulationofprotein

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interactionsbyGTPhydrolysisisacommonpropertyofGTPasesingeneral,55,56althoughitismore

typicalforGTPhydrolysistoturnoffaninteractioninsteadofswitchingiton,asinthecaseoftheHypB-

HypAcomplex.WhetherGTP-HypBispoisedtointeractwithadifferentpartnerprotein,perhapsa

nickeldonor,isyettobedetermined.Altogether,theeffectsofGTPhydrolysisonthepropertiesof

HypBresultinrapidandnickeltransferspecificallytoHypA.

HypAHis2inE.coliHydrogenaseMaturation

HypAisalsoavitalaccessoryproteinforhydrogenasematuration,andmutationofHis2to

glutamineweakensnickelaffinityanddidnotsupporthydrogenaseproduction,suggestingthatnickel

bindingtothissiteiscriticaltothematurationfunctionofHypA.InitialattemptsatHis2mutagenesisin

whichHis2wasreplacedwithalaninerevealedthatthepurifiedmutantproteinwasnotfoldedinthe

samemanneraswild-typeHypA(datanotshown),indicatingthateventhoughthisresidueisattheend

oftheproteinsequenceithasanimpactonproteinstructure.Switchingtoaglutaminesubstitution

affordedawell-foldedproteinthatpreservedtheabilityofHypAtobindzincandtointeractwithHypB,

suggestingthatonlythenickel-bindingactivitywasdisruptedwhileleavingtheoverallproteinstructure

undisturbed.

SolutionexperimentsdemonstratedthattheH2Qmutationcausesareductionintheapparent

affinityofHypAStrfornickelbyseveralordersofmagnitude,asevidencedbyanincreaseintheapparent

dissociationconstantfrom0.04±0.03µMto4±2µM.ThenickelaffinityofH2Q-HypAStrisattenuated

tothepointthatitissimilartothatoftheHypBG-domain.Thismutationreducesthethermodynamic

drivingforcefornickelrelocationfromHypBtoH2Q-HypAStrbutshouldtheoreticallyremaincompetitive

withHypBtosomedegree.However,nickeltransferbetweenHypBandH2Q-HypAStrwasnotobserved

intheshorttimeframeofthemetaltransferexperiments,incontrasttotheveryrapidnickeltransfer

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betweenHypBandwild-typeHypAStr.Furthermore,competitionexperimentsindicatedthatH2Q-

HypAStrdoesnotinduceadetectablechangeintheapparentnickelaffinityofHypBunderanyofthe

conditionstested,suggestingthatthatnickeltransferfromHypBtoHypAisnotsignificantlydrivenbya

HypA-inducedconformationalchangeinHypB.Takentogether,theseresultssupportamodelinwhich

nickeltransferoccursviaaligandexchangemechanismthatincludesHis2ofHypA.

DirectionalMetalTransfer

ComplexformationbetweenHypAandHypBiscontrollednotonlybythenucleotide-loaded

stateofHypB,butalsobythenickel-loadedstateofHypA.Dissociationofanickel-loadedHypAfrom

HypBisconsistentwithspectroscopicevidencethatnickelbindingmodulatestheconformationofthe

HypAprotein.32,34,47TheseobservationssupportamodelinwhichGDP-HypBwillonlyinteractwith

HypAifthenickelsiteonHypAisempty,andoncenickelhasbeentransferredtoHypA,theHypA-Ni(II)

complexwillnotinteractwithHypBregardlessofitsnucleotideloadedstate.Thisprocesswouldconfer

directionalitytonickeltransferbetweenthemetallochaperones,asnickelmovingintheopposite

directionisprevented.GiventhatHypAcandockontothehydrogenaseenzymeprecursorproteinHycE

bothwithandwithoutHypB,38itispossiblethatHypBcandelivernickeltoHypAwhileitisinacomplex

withHycE.FollowingnickeltransfertoHypA,HypBdisengagesfromthecomplex,leavingHypAtoinsert

themetalintotheenzymeactivesite.FutureworktocharacterizethecomplexbetweenHypAandHycE

willuncoverthedetailsofthisstageoftheprocess.

Incontrasttonickel,zincloadingoftheHypBG-domainreducestheextentofcomplex

formationwithHypA.TheavailablezincconcentrationsinanE.colicytoplasmaremaintainedat

extremelylowlevelsinaerobicallygrowingcells,57,58butitisnotclearhowmuchzincisavailablein

anaerobicbacteriawhennickelisbeingactivelyimportedtosupplythehydrogenaseactivesite,59or

of 42

howthezinclevelscomparewiththeamountofbioavailablenickel.GiventhattheHypBG-domain

bindszincwithtighteraffinitiesthannickel,19cellularconditionsmayexistsuchthatsomeHypBis

loadedwithzincinsteadofnickel.ThereductionincomplexformationwithZn(II)-HypBversusNi(II)-

HypBwouldpreventHypAfrombeingsiphonedoffintoadead-endzinccomplexandprovidesanadded

layerofmetalselectivity.

HypAandHypBplaycrucialrolesinthedeliveryofnickeltothe[NiFe]-hydrogenaseprecursor

protein,sotheseproteinsmakeasignificantcontributiontoanaerobicmicrobialmetabolismand

pathogenicityeventhoughtheyarenotresponsibleforanysynthetictransformation.12,39,60

Furthermore,thedeficienthydrogenaseproductioninthehypAorhypBknock-outstrainsofE.colican

bepartiallyrestoredbygrowthinmediasupplementedwithhighconcentrationsofextranickel.20,29,33,39

Thisobservationsupportsthemodelthatinahealthycellthereislittlefreelyavailablenickel.Inthis

context,controlledpassageofthenickelionthroughtheaccessoryproteinsiscriticaltofunnelnickelto

theactivesiteoftheenzyme.TheproposedmechanismofnickeltransferfromtheHypBG-domainto

HypAandthentoHycEissummarisedinFigure5.TriggeringE.coliHypBGTPaseactivityactsasswitch

andpromptsachangeinHypBproteinstructure.InthepivotalGDP-loadedstateofHypB,complex

formationwithHypAoccursandfacilitatesjudiciousnickeltransferfromHypBtoHypAinaprocessthat

involvesHis2ofHypA.ThedatasuggestthatonefunctionofthecomplexistoensurethatHypAis

correctlymetallatedbeforeHypBdissociation,leavingNi(II)-HypAatlibertyforthesubsequentinsertion

ofnickelintothelargesubunitof[NiFe]-hydrogenase3.ThisdirectednickeltransferfromHypBtoHypA

providesaGTPase-controlledmechanismtospecificallytrafficnickeltothehydrogenaseprecursor

proteininanefficientmanner.Severaloutstandingquestionsaboutthisintricateprocessofnickel

deliverywillbeaddressedinfuturework,suchashowHypA/HypFdelivernickeltothehydrogenase

activesite,theroleofHypBdimerization,andhowHypBacquiresnickel.

of 42

Acknowledgements

WethankAriCuperfainforpreparationoftheH2QstrA-pET24bplasmid,membersoftheZamblelabfor

supportandsuggestions,andNSERCforaPostgraduateScholarship(MJL).

SupportingInformationParagraph

SupplementalInformationcanbefoundattheACShttpaddress(http://pubs.acs.org)andcontainsthe

followinginformation:

SupplementalFigure1.NickelTitrationsforStoichiometryDetermination.

SupplementalFig.2.CustomDynaFitscriptforusedfitting.

SupplementalFig3.HypB-MF2CompetitionFits

SupplementalFig.4.Reconstructedmassspectrafromnon-denaturingESI-MS.

SupplementalFig5.H2Q-HypAMF2CompetitionFits.

SupplementalFig.6.WesternBlotAnalysisforHypA.

SupplementalFig.7.CustomDynaFitscriptforsimulatingMF2fractionalsaturation.

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Tables

Table1.HypBmetalstoichiometries.a

Proteins Metal Ligands Metal/HypB

TM-HypB

Ni(II)

- 0.99±0.06

500μMGDP 0.48±0.01

500μMGppCp 0.42±0.05

Zn(II)

- 1.00±0.09

500μMGDP 0.96±0.08

500μMGppCp 0.99±0.07

mTM-HypB

Ni(II)

- 0.90±0.09

500μMGDP 0.99±0.07

500μMGppCp 1.00±0.05

Zn(II)

- 0.90±0.09

500μMGDP 0.99±0.07

500μMGppCp 1.00±0.05

aHypBwasincubatedovernightwithanexcessofmetalintheabsenceorpresenceofnucleotide.

Unboundmetalwasremovedbygelfiltrationchromatographyfollowedbymetalanalysis.The

experimentswereperformedwithHypBlackingtheN-terminalmetal-bindingsite(TM-HypB)ormTM-

HypB,whichhasadditionalmutationssuchthattheproteindoesnotdimerizeinsolution.Thevalues

areaveragesfromthreeexperiments±onestandarddeviation.

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Table2.HypBandH2Q-HypAStrapparentmetaldissociationconstants.a

Protein Metal Cofactors KD(μM)

HypB

Ni(II)

- 12.6±0.8

500μMGDP 8±4

500μMGppCp 0.6±0.5

Zn(II)

- 0.17±0.14

500μMGDP

0.13±0.08

500μMGppCp 0.12±0.02

H2Q-HypA Ni(II) - 4±2

HypA Ni(II) - 0.04±0.03aApparentdissociationconstantswerecalculatedbyusingcompetitionexperimentswithafluorescent

metalsensingdyefollowedbyanalysisofthedatabyusingDynaFit.Bindingmodelswereselected

basedonthemetalstoichiometries(Table1).Thevaluesarepresentedasaveragesfromthree

experiments±onestandarddeviation.

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Table3.OligomericstatesofHypA-HypBobservedbygelfiltrationchromatography.

Proteins Metal Nucleotide Est.MW(kDa)

Fraction,TotalPeakArea† Assignment*

HypB+

HypAStr

-

-37±2 0.92±0.03 HypB

81±3 0.09±0.04 (HypB)2

GDP52±3 0.88±0.11 HypA-HypB

89±6 0.12±0.04 (HypB)2±HypA‡

GppCp39±3 0.53±0.08 HypB

76±3 0.47±0.11 (HypB)2

Ni

-35±3 0.21±0.04 HypB

79±3 0.80±0.10 (HypB)2

GDP37±2 0.19±0.03 HypB

80±3 0.81±0.10 (HypB)2

GppCp39±2 0.23±0.04 HypB

76±3 0.77±0.07 (HypB)2

Zn

-34±3 0.89±0.05 HypB

79±2 0.11±0.03 (HypB)2

GDP34±2 0.84±0.05 HypB

49±3 0.17±0.06 HypA-HypB

GppCp33±2 0.68±0.07 HypB

78±3 0.32±0.07 (HypB)2

*Assignmentsweremadebasedontheelutiontimesrelativetomolecularweightproteinstandards,butother

oligomericstatesoftheproteincomponentsarepossible.‡Becauseofthelargesizeoftheobservedcomplex,itis

difficulttoresolve(HypB)2and(HypB)2-HypA.†PeakareacalculationsexcludetheHypAmonomerpeak(elution

volumeof17.4mL)forclarity.Thevaluesareaveragesfromthreeexperiments±onestandarddeviation.

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Table4.OligomericstatesofH2Q-HypA-HypBobservedbygelfiltrationchromatography.

Proteins Metal Nucleotide Est.MW(kDa)

Fraction,TotalPeakArea† Assignment*

HypB+

H2Q-HypAStr

-

-37±3 0.91±0.09 HypB

80±3 0.09±0.04 (HypB)2


93±3 0.09±0.03 (HypB)2±HypA‡

GppCp38±2 0.53±0.03 HypB

79±3 0.47±0.05 (HypB)2

Ni

-37±2 0.25±0.06 HypB

80±3 0.75±0.11 (HypB)2


92±3 0.11±0.07 (HypB)2±HypA‡

GppCp40±3 0.20±0.10 HypB

79±3 0.77±0.03 (HypB)2

Zn

-35±3 0.90±0.07 HypB

75±2 0.09±0.05 (HypB)2

GDP34±3 0.89±0.03 HypB

49±2 0.12±0.08 HypA-HypB

GppCp34±2 0.66±0.04 HypB

77±4 0.35±0.09 (HypB)2

*Assignmentsweremadebasedontheelutiontimesrelativetomolecularweightproteinstandards,butother

oligomericstatesoftheproteincomponentsarepossible.‡Becauseofthelargesizeoftheobservedcomplex,itis

difficulttoresolve(HypB)2and(HypB)2-HypA.†PeakareacalculationsexcludetheHypAmonomerpeak(elution

volumeof17.4mL)forclarity.Thevaluesareaveragesfromthreeexperiments±onestandarddeviation.

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FigureLegends

Figure1.GelFiltrationAnalysisofHypA/HypBQuaternaryStructure.

HypBandeitherHypAStr(top)orH2Q-HypAStr(bottom)wereincubatedtogetherwithoutcofactors(solid

lines),with500μMGDP(dashedlines),orwith500μMGDPand500μMNiSO4(dottedlines),and

analyzedbygelfiltrationchromatography.AHypAStr-HypBcomplexwasdetectedinthepresenceofGDP

butnocomplexwasobserveduponnickeladdition.Incontrast,nickeldidnotdisruptcomplex

formationwithH2Q-HypAStr.SomeHypAformsaggregatesandelutesatanearliervolume,thispeak

wasomittedforclarity.

Figure2.Hydrogenaseactivities.

Hydrogenaseactivitywasdeterminedbymonitoringthehydrogen-dependentreductionofbenzyl

viologenincelllysatesofwild-typeE.coli(MC4100),theDPABFstrain(hypAATG→TAA,ΔhybF),or

DPABFtransformedwithanarabinose-inducibleplasmidbearingthegeneforHypAStr(strA-pBAD18-kan)

orH2Q-HypAStr(H2QstrA-pBAD18-kan).**Activitywasbelowtheassaydetectionlimit.Thevaluesare

averagesfromthreeexperiments±onestandarddeviation.

Figure3.NickeltransferfromGDP-loadedHypBtoacceptors.

ThefractionalsaturationofnickelboundtotheG-domainofHypBwasdeterminedbymonitoringthe

electronicabsorptionat340nm.HypB(50µM)wasincubatedwith70μMHypAStr,70μMH2Q-HypAStr,

or1mMEDTAinthepresenceofGDP.Thevaluesaretheaveragesfromthreeexperiments±one

standarddeviation.

Figure4.H2Q-HypAStr-HypB-MF2Ni(II)competitionversussimulations.

NickelwastitratedintosolutionscontainingWT-HypB,H2Q-HypAStr,andMF2eitherintheabsenceof

nucleotideorinthepresenceofGDPorGPPCP.Thefluorescenceintensityat510nm(λex=335nm)of

MF2wasrecordedandconvertedintofractionalsaturation(filledcircles).Dynafitsimulations(solid

of 42

lines)weregeneratedbasedondissociationconstantsderivedfromexperimentsperformedunder

identicalconditionswithHypBorH2Q-HypAStrseparately.Theproteinsweretreatedasindependent

nickelchelatorsinthesesimulations.Thevaluesaretheaveragesfromthreeexperiments±one

standarddeviation.

Figure5.HypA-HypBNi(II)transfermodel.

HypBbindsGTPandiscapableofcompetingfornickelwithotherspeciesinthecytosol.UponGTPase

activationbyanunidentifiedfactor,GTPhydrolysisweakensHypBnickelaffinityandpromotescomplex

formationwithHypA.ThisinteractionresultsinrapidandspecifictransferofnickelfromHypBtoHypA.

Atthispointnickel-loadedHypAisliberatedtoinsertnickelintothehydrogenaseprecursorprotein.If

zincbindstoHypBinsteadofnickel,thepathwayisdisconnectedbecausetheinteractionwithHypAand

subsequentmetaltransferisprohibited

of 42

Figures

Fig.1.GelFiltrationAnalysisofHypA/HypBQuaternaryStructure.

0

20

40

60

12 14 16 18

Abso

rban

ce (m

AU)

Elution Volume (mL)

0

20

40

60

12 14 16 18

Abso

rban

ce (m

AU)

Elution Volume (mL)

of 42

Fig.2.Hydrogenaseactivities.

0.00

0.10

0.20

0.30

0.40

MC4100 no plasmid **

strA pBAD18-kan

H2QstrA pBAD18-kan

**

DPABF (hypA ATG→TAA, ΔhybF)

H2a

se A

ctiv

ity (µ

mol

min

-1 m

g-1)

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Fig.3.NickeltransferfromGDP-loadedHypBtoacceptors.

0.00

0.25

0.50

0.75

1.00

0 100 200 300 400 500

Hyp

B F

ract

iona

l Sat

urat

ion

Time (sec)

+HypAWT +HypA H2Q +EDTA

40

Fig.4.H2Q-HypAStr-HypB-MF2Ni(II)competitionversussimulations.

0.00

0.25

0.50

0.75

1.00

0.1 1 10 100 1000 [NiSO₄] (μM)

HypB + HypA-H2Q + GDP

0.00

0.25

0.50

0.75

1.00

0.1 1 10 100 1000 [NiSO₄] (μM)

HypB + HypA-H2Q + GppCp

0.00

0.25

0.50

0.75

1.00

0.1 1 10 100 1000

Frac

tiona

l Sat

urat

ion

[NiSO₄] (μM)

HypB + HypA-H2Q

41

Fig.5.HypA-HypBNi(II)transfermodel.

42

GraphicfortheTableofContents

of 7

Supplemental Information for

The Mechanism of Selective Nickel Transfer From HypB to HypA, E. coli [NiFe]-Hydrogenase Accessory Proteins

Michael J. Lacasse, Colin D. Douglas, Deborah B. Zamble

Supplemental Figure 1. Nickel Titrations for Stoichiometry Determination. Nickel was titrated into 100 μM WT-HypB. The electronic absorbance at 340 nm was recorded and converted into fractional saturation of the HypB G-domain site. Saturation of the G-domain site occurs at 0.5 equivalents of nickel.

of 7

Supplemental Fig. 2. Custom DynaFit script for used fitting. Note: concentrations and constants were

changed as appropriate based on experimental conditions. This script was used to calculate the

dissociation constants for the competition experiments (Table 2).

of 7

Supplemental Fig 3. HypB-MF2 Competition Fits. Nickel and zinc competition between WT-HypB and MF2 with and without nucleotide. The fluorescence intensity of MF2 was converted to fractional saturation of MF2 and the dissociation constants were calculated using DYNAFIT and reported in Table 2. Fractional saturation from the average HypB dissociation constants from three independent experiments are shown in the solid grey line and fractional saturation from tenfold weaker and tenfold tighter dissociation constants are shown in the black dashed lines.

of 7

Supplemental Fig. 4. Reconstructed mass spectra from non-denaturing ESI-MS.

HypAStr (5 µM, A-C) or H2Q-HypAStr (5 µM, D-F) were incubated with 50 µM NiSO4 (B, E), or 10 µM nickel-

loaded HypB (C, F) in 10 mM ammonium acetate at pH=7.5 followed by analysis with ESI-MS. A 58 Da

change in reconstructed molecular weight of HypAStr was observed upon incubation with nickel or nickel-

loaded HypB, corresponding to a Ni(II)-HypAStr complex. No change in reconstructed molecular weight

was observed for H2Q-HypAStr. Upon incubation of either HypA variant with zinc, no change in

reconstructed molecular weight was observed (data not shown).

of 7

Supplemental Fig 5. H2Q-HypA MF2 Competition Fits. Nickel competition between H2Q-HypAStr and MF2. The fluorescence intensity of MF2 was converted to fractional saturation and the dissociation constants were calculated using DYNAFIT and reported in Table 2. Fractional saturation from the average HypB dissociation constants from three independent experiments are shown in the solid grey line and fractional saturation from tenfold weaker and tenfold tighter dissociation constants are shown in the black dashed lines.

of 7

Supplemental Fig. 6. Western Blot Analysis for HypA.

Western blot analysis of cell lysates with an anti-HypA antibody confirms that HypAStr and H2Q-HypAStr

were expressed at similar levels by the pBAD18-kan plasmids.

of 7

Supplemental Fig.7. Custom DynaFit script for simulating MF2 fractional saturation. Note:

concentrations and constants were changed as appropriate based on experimental conditions. This

script was used to simulate fractional saturation using the measured dissociation constants or constants

10-fold larger or smaller. Dimerization of protein was assumed to take place independently of a metal

binding event.

mechanism of selective nickel transfer from hypb to hypa ... · protein during metallocenter...

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