Substrate-mediated Stabilization of a Tetrameric Drug Target Reveals Achilles Heel in Anthrax*DS

15 Pages·2010·3.63 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.285,NO.8,pp.5188–5195,February19,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Substrate-mediated Stabilization of a Tetrameric Drug Target
Reveals Achilles Heel in Anthrax*□S
Receivedforpublication,June25,2009,andinrevisedform,November13,2009 Published,JBCPapersinPress,November30,2009,DOI10.1074/jbc.M109.038166
JarrodE.Voss‡,StephenW.Scally‡,NicoleL.Taylor‡,SarahC.Atkinson‡,MichaelD.W.Griffin‡,CraigA.Hutton§,
MichaelW.Parker‡¶,MalcolmR.Alderton(cid:1),JulietA.Gerrard**,RenwickC.J.Dobson‡1,ConDogovski‡,
andMatthewA.Perugini‡2
Fromthe‡DepartmentofBiochemistryandMolecularBiologyandthe§SchoolofChemistry,Bio21MolecularScienceand
BiotechnologyInstitute,TheUniversityofMelbourne,Parkville,Victoria3010,Australia,the¶St.VincentsInstituteofMedical
Research,9PrincesStreet,Fitzroy,Victoria3065,Australia,the(cid:1)HumanProtectionandPerformanceDivision,DefenceScienceand
TechnologyOrganisation,FishermansBend,Victoria3207,Australia,andthe**SchoolofBiologicalSciences,Universityof
Canterbury,PrivateBag4800,Christchurch8020,NewZealand
Bacillus anthracis is a Gram-positive spore-forming bacte- that Bacillus anthracis is the causative agent of the disease.
riumthatcausesanthrax.Withtheincreasedthreatofanthrax Giventhetoleranceofitssporestoextremephysicalconditions
inbiowarfare,thereisanurgentneedtocharacterizenewanti- andtheireaseofdissemination,theuseofB.anthracisinbio-
microbialtargetsfromB.anthracis.Onesuchtargetisdihydro- terrorismandbiologicalwarfareisathreattodevelopedcoun-
dipicolinatesynthase(DHDPS),whichcatalyzesthecommitted triesworldwide.Forexample,in2001anumberofletterscon-
stepinthepathwayyieldingmeso-diaminopimelateandlysine. taining B. anthracis endospores were distributed via the U.S.
Inthisstudy,weemployedCDspectroscopytodemonstratethat postalsystem,whichresultedin11casesofrespiratoryanthrax.
thethermostabilityofDHDPSfromB.anthracis(Ba-DHDPS)is Sincethen,therehasbeenheightenedinterestinthedevelop-
significantly enhanced in the presence of the substrate, pyru- mentofanti-anthraxagents(2),especiallyconsideringthelim-
vate. Analytical ultracentrifugation studies show that the tet- itedrangeofcurrenteffectivetherapeuticsandtheabilityofB.
ramer-dimer dissociation constant of the enzyme is 3-fold anthracis to develop antibiotic resistance (3). One potential
tighterinthepresenceofpyruvatecomparedwiththeapoform. therapeutic target is dihydrodipicolinate synthase (DHDPS),3
Toexaminethesignificanceofthissubstrate-mediatedstabili- whichcatalyzesthefirstcommittedstepinthelysinebiosyn-
zationphenomenon,adimericmutantofBa-DHDPS(L170E/ theticpathway(Fig.1A).
G191E) was generated and shown to have markedly reduced Thebiosyntheticpathwayleadingtolysineanditsimmediate
activity compared with the wild-type tetramer. This demon- precursor,meso-diaminopimelate(meso-DAP),isknownasthe
stratesthatthesubstrate,pyruvate,stabilizestheactiveformof DAP pathway (4) (Fig. 1A). The first committed reaction in
theenzyme.Wenextdeterminedthehighresolution(2.15A˚) thepathwayisthecondensationofpyruvateand(S)-aspartate
crystalstructureofBa-DHDPSincomplexwithpyruvate(3HIJ) semi-aldehyde (ASA) to form hydroxytetrahydrodipicolinic
andcomparedthistotheapostructure(1XL9).Structuralanal- acid (5), which is catalyzed by DHDPS (Fig. 1, A and B). The
ysesshowthatthereisasignificant(91A˚2)increaseinburied DAPpathwayinbacterialeadstothedenovosynthesisoflysine,
surface area at the tetramerization interface of the pyruvate- essentialforproteinsynthesis,andmeso-DAP,whichisavital
boundstructure.Thisstudydescribesanewmechanismforsta- constituentofthepeptidoglycanlayerinthebacterialcellwall
bilizationoftheactiveoligomericformofanantibiotictarget (6)(Fig.1A).Thus,targetingtheDAPpathwaythroughdisrupt-
from B. anthracis and reveals an “Achilles heel” that can be ingthefirstcommittedstep,DHDPS,willyieldanovelclassof
exploitedinstructure-baseddrugdesign. therapeuticagentstotreatanthrax.Thisnotionissupportedby
recent work (7) that identified dapA, the gene encoding
DHDPSfromB.subtilis,asoneofonly271genesessentialfor
TheetiologyofanthraxwasfirstdescribedbyRobertKochin cell viability from a total of (cid:1)4,100 genes encoded by its
1876(1).Hisworkwasthefirsttodemonstrateunequivocally genome.Additionally,mammalsdonotsynthesizelysineand
therefore do not possess the DHDPS enzyme. This suggests
that specific inhibitors of DHDPS would have selective anti-
*ThisworkwassupportedinpartbytheDefenseThreatReductionAgency
bacterialactivitywithlowtoxicityinamammalianhost.
(ProjectIDAB07CBT004)andbyanAustralianResearchCouncilFuture
Fellowship(toM.A.P.)andaFederationFellowship(toM.W.P.). ThestructureofDHDPSintheabsenceofsubstratehasbeen
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains determinedforanumberofspeciesofbacteria,includingthat
supplementalFigs.1–4.
ofB.anthracis(8)(Fig.1B),Thermotogamaritima(9),Thermo-
Theatomiccoordinatesandstructurefactors(code3HIJ)havebeendepositedin
theProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics, anaerobacter tengcongensis (10), Escherichia coli (11), Myco-
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/). bacterium tuberculosis (12), Corynebacterium glutamicum
1SupportedbyaUniversityofMelbourneC.R.RoperResearchFellowship.
(13),andStaphylococcusaureus(14,15).Theenzymeusually
2Towhomcorrespondenceshouldbeaddressed:Dept.ofBiochemistryand
MolecularBiology,Bio21MolecularScienceandBiotechnologyInst.,The
UniversityofMelbourne,30FlemingtonRd.,Parkville,Victoria3010,Aus-
tralia.Tel.:[phone];Fax:[phone];E-mail:perugini@unimelb. 3Theabbreviationsusedare:DHDPS,dihydrodipicolinatesynthase;DAP,dia-
edu.au. minopimelate;ASA,(S)-aspartatesemi-aldehyde;Ba,B.anthracis.
5188 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010
This is an Open Access article under the CC BY license.
Pyruvate-boundB.anthracisDHDPS
wasamplifiedbyPCRandclonedintothepET11aexpression
vector as described elsewhere (22). Briefly, recombinant pro-
tein was produced in the host strain E.coli BL21-DE3 as fol-
lows. Cells harboring vector were cultured at 37°C in Luria
broth containing 100 (cid:1)g ml(cid:2)1 ampicillin to an A of 0.6.
600
Expression of recombinant Ba-DHDPS was induced by addi-
tionofisopropyl1-thio-(cid:2)-D-galactopyranosidetoafinalcon-
centrationof1.0mM.Cellswereharvested3hpost-induction
and resuspended in 20 mM Tris-HCl, pH 8.0, before lysis by
sonication.Ba-DHDPSwassubsequentlyisolatedbyanion-ex-
changeandhydrophobicinteractionliquidchromatographyas
describedelsewhere(22).
Generation of Dimeric Ba-DHDPS (L170E/G191E)—The
dimeric form of Ba-DHDPS was generated using the
QuikChange II XL site-directed mutagenesis kit (Stratagene).
Themutagenicoligonucleotideprimersets,5(cid:3)-GATGCAGGC-
GGCGATGTGGAAACAATGACAGAAATCATTG-3(cid:3) and 5(cid:3)-
CAATGATTTCTGTCATTGTTTCCACATCGCCGCCTGC-
ATC-3(cid:3), and 5(cid:3)-GTATACAGCGGTGATGACGAATTAACG-
CTACCAGCTATGG-3(cid:3)and5(cid:3)-CCATAGCTGGTAGCGTTA-
ATTCGTCATCACCGCTGTATAC-3(cid:3), were designed to
introduce amino acid substitutions L170E and G191E, respec-
tively.Mutagenesiswasperformedaccordingtothemanufactur-
er’s instructions. Briefly, 125 ng of primer and 10 ng double-
stranded DNA template utilized per reaction. Second strand
synthesiswasachievedthrough18cyclesofamplification.Follow-
ing a 1-h DpnI digestion, plasmid was transformed into E.coli
XL10-GoldUltracompetentcells.Theintroductionofpointmuta-
tionswasverifiedbyDNAsequencing.
DHDPS-DHDPR Coupled Enzyme Kinetic Assay—Enzyme
FIGURE1.Ba-DHDPS.A,DHDPScatalyzesthecondensationofpyruvateand
kineticanalysesofBa-DHDPSandBa-DHDPS(L170E/G191E)
ASAintohydroxytetrahydrodipicolinicacid,whichisthefirstcommittedstep
inthelysinebiosynthesispathwayinbacteria.B,crystalstructureofapo-Ba- were performed using the DHDPS-DHDPR coupled assay as
DHDPSshowingthepositionofoneactivesiteandtheself-associationinter-
describedinapreviousstudy(23).Assayswereroutinelyper-
faces.Twomonomerscometogetheratthetightdimerinterfacetoformthe
dimericunit,andthenthetwodimericunitsdockattheweakdimerinterface formed in duplicate at a constant temperature of 30°C with
toformatetramer. reactionmixturesallowedtoequilibrateinatemperature-con-
assemblesasatetramericprotein(16),bestdescribedasadimer trolled Cary 4000 UV-visible spectrophotometer for 12 min
of tight dimers (Fig. 1B). However, a native active dimer has beforeinitiatingthereactionwith60nMDHDPS.Priortothe
recently been described (14), which makes the quaternary experiment,pyruvateandASAconcentrationswereroutinely
structure of this enzyme of particular interest. DHDPS from quantifiedbytheadditionoflimitingamountsofsubstrateby
plants(17,18)andsomeGram-negativebacterialspecies(19)is measuringtheconsumptionofNADPHat340nmintheCary
feedback-inhibitedby(S)-lysine,actingasanallostericmodu- 4000UV-visiblespectrophotometer.Initialvelocitydatawere
lator through partial inhibition of catalytic activity. However, bestfittedtoaPingPongModelusingENZFITTER.4Ratever-
allosteric regulation by lysine at biologically relevant concen- susenzymeconcentrationassayswerealsoconductedwithBa-
trationsdoesnotoccurinDHDPSfromGram-positivespecies DHDPSconcentrationsrangingfrom2.5to120nM.
(14,20),includingB.anthracis(21). CDSpectroscopy—CDspectrawererecordedusinganAVIV
Recentworkinourlaboratoryhasshownthatthepurifica- 410-SF CD spectrometer. Wavelength scans were performed
tionofrecombinantDHDPSfromB.anthracis(Ba-DHDPS)in between190and250nmin20mMTris,150mMNaClwith0.15
the presence of its substrate pyruvate increases the yield and mgml(cid:2)1Ba-DHDPSandBa-DHDPS(L170E/G191E)in1-mm
specificactivityofthefinalproduct(22).Therefore,theaimof quartz cuvettes. Data were analyzed using the CONTINLL
thisstudywastothoroughlycharacterizetheeffectofpyruvate algorithmfromtheCDProsoftwarepackage(25)andtheSP29
onthesolutionpropertiesandstructureofBa-DHDPS.Here, proteindatabase.Forthermaldenaturationscans,ellipticityat
we report solution and structural studies that unravel the 222nmwasmonitoredbetween20and90°Cin1°Csteps.
mechanism for substrate-mediated stabilization of the active Analytical Ultracentrifugation—Absorbance-based sedi-
quaternarystructureofBa-DHDPS. mentation velocity and equilibrium experiments were per-
formed in a Beckman model XL-I analytical ultracentrifuge
EXPERIMENTALPROCEDURES
Cloning, Expression, and Purification of Ba-DHDPS—The 4R.J.Leatherbarrow(1987)ENZFITTER,ImperialCollegeofScienceandTech-
dapAgeneencodingDHDPSfromB.anthracis(Sternestrain) nology,London.
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5189
Pyruvate-boundB.anthracisDHDPS
WINCOOT. A round of simulated
annealing was performed with a
starting temperature of 5000 K to
assign the R set of reflections
free
from the apo structure (PDB ID:
1XL9) that shared the same crystal
properties with the newly solved
pyruvate-boundstructure(PDBID:
3HIJ).Thefinalmodelwaschecked
withPROCHECK(37).
RESULTS
Effect of Pyruvate on the Second-
ary Structure Stability of Ba-
FIGURE2.SecondarystructureandthermostabilitymeasuredbyCDspectroscopy.A,wavelengthscanof
DHDPS—Werecentlyreportedthat
Ba-DHDPSalone(F)andinthepresenceof2.0mMpyruvate(ƒ)or0.5mMASA(E).Ellipticity(mdeg)hasbeen
normalizedtomeanresidueellipticity(degcm2dmol(cid:2)1residue(cid:2)1).B,thermaldenaturationofBa-DHDPS therawenzymeactivityandyieldof
alone(F)andinthepresenceofitssubstrates,2.0mMpyruvate(ƒ),and0.5mMASA(E). recombinantBa-DHDPSaresignif-
icantlyincreasedwhentheenzyme
usinga4-holeAn-60Tioran8-holeAn-50Tirotor.Double- is purified in the presence of its substrate, pyruvate (22). To
sectorquartzcellswereloadedwith380(cid:1)lofsampleand400(cid:1)l examinetheeffectofpyruvateonthestabilityofBa-DHDPSin
ofreference(20mMTris,150mMNaCl,pH8.0)forsedimenta- aqueous solution, thermal denaturation experiments moni-
tion velocity, or a 100-(cid:1)l sample and a 120-(cid:1)l reference for toredbyCDspectroscopywereconductedinthepresenceand
sedimentation equilibrium. Experiments were conducted at absenceofthesubstratespyruvateorASAoverthetemperature
4°Cusingarotorspeedof40,000rpm(sedimentationvelocity) rangeof20–90°C.Initially,wavelengthscanswereperformed
or 12,000 and 18,000 rpm (sedimentation equilibrium) with at20°Ctomonitorglobalsecondarystructureinthepresence
absorbance measured at 227 nm (1.6 (cid:1)M enzyme) or 235 nm of substrates, which revealed no change in response to either
(4.8 (cid:1)M enzyme). Solvent density, solvent viscosity, and esti- pyruvate or ASA (Fig. 2A). Ba-DHDPS appears to follow a
matesofthepartialspecificvolumeofBa-DHDPSandBa-DH- three-statemechanismforthermalunfoldingintheabsenceof
DPS (L170E/G191E) were calculated using SEDNTERP (26). pyruvateandinthepresenceofASA,withapotentialinterme-
Initialscanswerecarriedoutat3,000rpmtodetermineopti- diatepersistingattemperaturesof(cid:4)50–60°C(Fig.2B).How-
mum wavelength and radial positions for the experiments. ever,inthepresenceof2.0mMpyruvate,thethermaldenatur-
Samples monitored in the presence of 0.6 mM pyruvate con- ationofBa-DHDPSisdelayedwithrespecttotemperatureand
tained pyruvate in both the reference and sample channels. unfoldingappearstooccurviaatwo-statemechanismwithout
Sedimentationvelocitydatawerefittedtoasinglediscretespe- the propagation of an intermediate, indicating that pyruvate
ciesoracontinuoussedimentationcoefficient[c(s)]model(27– stabilizesthefoldedstateoftheenzyme.Accordingly,theeffect
29)usingtheprogramSEDFIT.Additionally,vanHolde-Weis- ofpyruvateonthequaternaryandtertiarystructureofBa-DH-
chet analysis (30) was performed using the ULTRASCAN DPSwassought.
softwarepackage(31),whereassedimentationequilibriumdata EffectofPyruvateontheQuaternaryStructureofBa-DHDPS—
werefittedtovariousself-associatingequilibriummodelsusing TocharacterizethequaternarystructureofBa-DHDPSinsolu-
theprogramSEDPHAT(32). tionintheabsenceandpresenceofpyruvate,absorbance-de-
Crystallization of Ba-DHDPS and X-ray Diffraction Data tected sedimentation velocity and equilibrium analyses were
Collection—Ba-DHDPSwascrystallizedaccordingtomethods conducted in the analytical ultracentrifuge. The absorbance
described previously (22) using sitting- and hanging-drop, versus radial position profiles of Ba-DHDPS (1.6 (cid:1)M) during
vapordiffusion.Thecrystalsusedfordiffractionanalysiswere sedimentationvelocityintheabsenceofpyruvateareshownin
soakedwith20mMpyruvateovernighttofacilitateligandbind- Fig.3A,whereasFig.3Bshowstheequivalentdatasetsinthe
ing.Forx-raydatacollection,crystalsweretransferredtores- presence of pyruvate. Two predominant boundaries were
ervoirsolutioncontaining20%(v/v)glycerolwith20mMpyru- observedforBa-DHDPSintheabsenceofsubstrate(Fig.3A).
vateanddirectlyflashfrozeninliquidnitrogen.Intensitydata Bycontrast,theradialabsorbanceprofilesshowedasinglepre-
were collected at the Australian synchrotron using the MX1 dominantboundaryinthepresenceofpyruvate(Fig.3B).These
beamlineasdescribedbefore(22). data were analyzed initially using the enhanced van Holde-
Phasing and Model Refinement—Diffraction data sets were Weischet method (30), which is a model-independent
processed and scaled using the package MOSFLM (33) and approachtoanalyzingsedimentationvelocitydata.Theresult-
SCALA(34).Initialphaseestimatesweresolvedbymolecular ingintegraldistributionforBa-DHDPSintheabsenceofpyru-
replacementusingPHASERwiththeligand-unboundstructure vate(Fig.3C,whitecircles)suggestsitexistsinareversibleself-
(PDBID:1XKY)asthesearchmodel.Structuralrefinementwas association with sedimentation coefficients ranging from 2 S
performedusingREFMAC5(35)withiterativemodelbuilding through to 6 S. This is consistent with previous reports of
using WINCOOT (36). Water, glycerol, sodium ion, and the DHDPSfromE.coli,whichwasdeterminedtoexistinequilib-
pyruvate-boundlysineatomswereaddedatlaterstagesusing rium between a dimer and tetramer (38). In the presence of
5190 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010
Pyruvate-boundB.anthracisDHDPS
ramer, compared with the dimer,
was observed (Fig. 3D, solid line).
The resulting c(s) distributions
shown in Fig. 3D therefore agree
well with the van Holde-Weischet
analyses (Fig. 3C). This effect was
subsequentlyquantifiedusingsedi-
mentation equilibrium analysis of
Ba-DHDPSintheabsenceandpres-
ence of 0.6 mM pyruvate. Samples
containing0.80,1.6,and3.2(cid:1)MBa-
DHDPSwerecentrifugedat12,000
and18,000rpmuntilsedimentation
equilibrium was attained at each
speed (supplemental Fig. 1). Con-
sistent with previous studies of
DHDPS (38), the global nonlinear
leastsquaresbestfitofthedata(glo-
bal (cid:3)2 (cid:5) 0.6) was obtained from a
dimer-tetramer association model
and revealed that the dissociation
constant (K 4–2) ofBa-DHDPS in
D
the absence of pyruvate was 1.90
(cid:1)M, compared with K 4–2 of 0.66
D
(cid:1)Minthepresenceofthesubstrate
(Table 1 and supplemental Fig. 1).
That is, the tetramerization con-
FIGURE3.Analyticalultracentrifugationanalyses.Absorbanceat227nmmeasuredasafunctionofradial
stantwas3-foldtighterinthepres-
positionfromtheaxisofrotation(cm)forBa-DHDPS(1.6(cid:1)M)centrifugedat40,000rpmintheabsence(A)and
presence(B)ofpyruvate.Therawdataarepresentedasopensymbols(E)plottedattimeintervalsof10min enceofpyruvatecomparedwiththe
overlaidwiththenon-linearleastsquaresbest-fit(solidline)toacontinuoussedimentationcoefficientdistri- unliganded enzyme. Together, the
butionmodel(c(s))(27–29).C,vanHolde-Weischetintegraldistributionplotfromextrapolationofrawdatain
resultsofCDthermostability,sedi-
AandB.Thecorrectedsedimentationcoefficientisplottedagainsttheboundaryfractionforthesamplesinthe
absence(E)andpresence(F)ofpyruvate.D,c(s)plottedasafunctionofs (S)forBa-DHDPSintheabsence mentationvelocity,andsedimenta-
20,w
(dashedline)andpresence(solidline)ofpyruvate. tion equilibrium analyses demon-
strate that the secondary and
quaternarystructureofBa-DHDPSwassignificantlystabilized
TABLE1
Summaryofhydrodynamicpropertiesofwild-typeBa-DHDPS in the presence of pyruvate. To gain further insight into the
K 4–2dminus K 4–2dplus importanceofthetetramericstructureofBa-DHDPS,wesub-
Species Massa s b f/fc D D
20,w 0 pyruvate pyruvate sequentlysetouttogenerateadimericmutantoftheenzymeto
kDa S (cid:1)M (cid:1)M assesstheactivityofthedimerincomparisontothewild-type
Dimer 62 4.0 1.17 1.9 0.66 tetramer.
Tetramer 124 6.5 1.27 1.9 0.66
Dimeric Mutant of Ba-DHDPS Shows Significantly Attenu-
aRelativemolecularmasscalculatedfromaminoacidsequence.
bStandardizedsedimentationcoefficienttakenfromtheordinatemaximumofthe ated Activity—To highlight the importance of the homo-tet-
c(s)distribution(Fig.3D). ramericstructureofBa-DHDPS,adoublemutant,Ba-DHDPS
cFrictionalratiocalculatedusingthev(cid:1)methodfromSEDNTERP(26).
dDissociationconstantfortetramertodimer.Notethattherateofdissociation(k ) (L170E/G191E),wasdesignedtobreakapartthe“weakdimer”
is10(cid:2)5.3s(cid:2)1intheabsenceofpyruvate(supplementalFig.4A)and10(cid:2)5.1s(cid:2)1oiffn interface.Leu170formsanimportanthydrophobicinteraction
thepresenceofpyruvate(supplementalFig.4B).
withGly191fromitsneighboringchainattheweakdimerinter-
face. Therefore, by mutating both these residues to glutamic
pyruvate(Fig.3C,blackcircles),theequilibriumappearstobe acid,itwasthoughtthattheresultingchargerepulsionatthis
shiftedgreatlyinfavorofthelargerspecies.Todefinetheoli- site would stabilize the dimer. The purified recombinant Ba-
gomericspeciesofBa-DHDPSinsolution,thedataweresub- DHDPS (L170E/G191E) product (supplemental Fig. 2, A–C)
sequentlyfittedtoacontinuoussedimentationcoefficient(c(s)) retainednativesecondarystructure(supplementalFig.2D)and
distribution model (27–29) (Fig. 3D). In the absence of pyru- notsurprisinglywasshowntobedimericinsolutionbysedi-
vate,thec(s)distributionshowstwonon-baselineresolvedspe- mentationvelocityanalysis(Fig.4A,dashedline).Furthermore,
cies, in approximately equal proportions, with standardized enzymekineticanalysisofthedimericmutantrevealedthatit
sedimentationcoefficients(s )of4.0and6.5S(Fig.3Dand retainedonly1.8%ofthetotalcatalyticactivityofthewild-type
20,w
Table 1). These values correspond to the DHDPS dimer and tetramericenzyme(Fig.4BandTable2).Moreover,wild-type
tetramer(38),respectively(Table1).Bycontrast,inthepres- Ba-DHDPShadamaximumcatalyticturnover(k )of92s(cid:2)1,
cat
enceofpyruvate,asignificantlygreaterproportionofthetet- K ASAof0.25mM,andK PYRof1.2mMcomparedwith1.7s(cid:2)1,
m m
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5191
Pyruvate-boundB.anthracisDHDPS
FIGURE5.X-raycrystalstructureofBa-DHDPSincomplexwithpyruvate.
A,theasymmetricunitcontainedfourmonomersassembledasahomotet-
ramerofBa-DHDPSboundtopyruvate.B,activesiteresiduesoftheunbound
(darkgray)(8)overlaidwiththepyruvate-bound(lightgray)structureofBa-
DHDPS.Pyruvate(PYR)isshowninblackandindicatedwithanarrow.C,weak
dimer interface of Ba-DHDPS showing inter-chain interactions that are
uniquetothepyruvate-boundcrystalstructure(3HIJ)relativetotheunbound
structure(1XL9).AnalysiswasperformedusingSTINGMILLENIUM(40).
CrystalStructureofBa-DHDPSBoundtoPyruvate—Toelu-
cidate the structural mechanism behind the substrate-medi-
atedstabilizationphenomenonobservedinsolution,wesolved
thecrystalstructureofBa-DHDPSincomplexwithpyruvate.
Interestingly, the newly solved pyruvate-bound crystal struc-
ture (PDB ID: 3HIJ) shared the same space group, unit cell
FIGURE4.Quaternarystructureandenzymekineticpropertiesofdimeric
parameters, and a similar resolution to the structure of sub-
Ba-DHDPS.A,thec(s)distributionisplottedasafunctionofstandardized
sedimentationcoefficientforBa-DHDPS(solidline)andLG-DHDPS(dashed strate unbound Ba-DHDPS (PDB ID: 1XL9). Crystallization
line) at 4.8 (cid:1)M. Top, residuals resulting from thec(s) distribution best fits and preliminary diffraction analysis were reported recently
showninpanelAplottedasafunctionofradiusfromtheaxisofrotation.B,the
initialvelocityisplottedasafunctionof(S)-ASAconcentrationforBa-DHDPS (22).Theinitialstructurerevealedfourmonomersintheasym-
(circles)andLG-DHDPS(triangles).Thenonlinearbestfittotheping-pong metricunit,arrangedinthebiologicallyrelevanttetramer(Fig.
modelispresentedassolidlinesandresultsinthekineticparameterssumma-
5A).ThefirstroundofrefinementgaveanR of21.4%(R
rizedinTable2. cryst free
of23.2%),whichalsorevealedelectiondensityassociatedwith
TABLE2 Lys163 in the active site of all four monomers that wasn’t
EnzymekineticpropertiesofBa-DHDPS accountedforbythesearchmodel.Thisisthesitewherepyru-
Enzyme Km K (ASA) k vate binds DHDPS (39). The pyruvate-bound molecule was
(pyruvate) m cat
modeledmanuallyatLys163(Fig.5B),andlaterroundsofstruc-
mM mM s(cid:2)1 turalrefinementwereperformedusingREFMAC5.Theitera-
Ba-DHDPS 1.2(cid:7)0.09 0.25(cid:7)0.02 92
Ba-DHDPS(L170E/G191E) 3.7(cid:7)0.1 0.05(cid:7)0.01 1.7 tive model-building tool, WINCOOT, was used to model in
E.coli-DHDPSa 0.16(cid:7)0.03 0.13(cid:7)0.03 78 waters, Na(cid:6) ions, and glycerol molecules. To make a direct
aDatawereobtainedfromRef.23forcomparison. comparison to the Ba-DHDPS substrate-unbound structure
0.05mM,and3.7mMforBa-DHDPS(L170E/G191E)(Table2). (PDBID:1XL9),aroundofsimulatedannealingwasperformed
TheseresultsshowthattheBa-DHDPStetramerissignificantly onthesubstrate-boundstructure(PDBID:3HIJ)withastarting
moreactivethanthedimericformoftheenzyme,whichhigh- temperatureof5000Ktoensurebothstructurespossessedthe
lights the significance of the substrate-mediated stabilization sameR setofreflections.Thefinalmoleculehadanoverall
free
phenomenon described above. To support this we show that R of15.3%(R of21.0%)to2.15-Åresolution.Theresult-
cryst free
therateversusenzymeconcentrationprofileofwild-typeBa- ing model was examined using PROCHECK, which revealed
DHDPS was nonlinear at low enzyme concentrations, which that99.2%ofresiduesinBa-DHDPSboundtopyruvatewerein
indicatesthatthenativedimerwassignificantlylessactivethan thefavoredregionsofaRamachandranplot.The8residuesin
thetetramericspecies(supplementalFig.2E). the“disallowed”regionswereTyr109andIle142fromchainsA,
5192 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER8•FEBRUARY19,2010
Pyruvate-boundB.anthracisDHDPS
TABLE3 TABLE4
Structureandrefinementstatistics Interfaceanalysisofselfassociationinterfaces
R a 0.153(0.170)b Buried
cryst Hydrophobic Electrostatic
R c 0.207(0.253)b Interface H-bondsa surface
free interactionsa interactionsa
areab
Numberofatoms 10,075
Protein 8,894 Å2
Na(cid:6)ions 4 Tightinterface
Glycerol 24 Pyruvate-unboundc 2 28 0 2780
Water 1,153 Pyruvate-boundd 8 28 0 2782
Rootmeansquaredeviationd Weakinterface
Bonds(Å) 0.024 Pyruvate-unbound 2 26 6 1735
Angles 1.87° Pyruvate-bound 8 28 4 1826
AverageBfactors aAnalysiswasperformedusingSTINGMILLENIUM(40).
Protein 17.8 bAnalysiswasperformedusingPISA(44).
Na(cid:6)ions 22.1 cpdbID:1XL9.
Glycerol 53.8 dCrystalstructureofBa-DHDPSwassolvedboundtopyruvate.
Water 28.8
Ramachandranplot,no.ofresiduesin:
Favoredregion 918(91.4%) suchasB.anthracis.Therefore,weexaminedtheeffectofpyru-
Allowedregion 78(7.8%) vateinstabilizingtheBa-DHDPStetramer.
Disallowedregion 8(0.8%)
ThesolutionstabilityofBa-DHDPSwasinitiallyinvestigated
aR (R-factor)(cid:5)(cid:8)(cid:1)F(cid:2)(cid:2)(cid:2)F(cid:1)/(cid:8)(cid:2)F(cid:2),whereF andF aretheobservedandcalcu-
lacrtyesdtstructurefactoraomplitucdes,roespectivelyo. c atthesecondarystructurelevelusingCDspectroscopy.Neither
bValuesinparenthesesareforthehighestresolutionshell. substrate of DHDPS, pyruvate nor ASA, had an effect on the
cR istheR-factorcalculatedwith5%ofthereflectionschosenatrandomand
ofmreeittedfromrefinement. overall secondary structure of Ba-DHDPS at 20°C (Fig. 2A).
dThe root mean square deviation of bond lengths or bond angles from ideal Thisagreeswithpreviousx-raycrystallographicstudiesofapo-
geometry.
andsubstrate-boundintermediatestatesofE.coliDHDPS(41).
However, the thermostability of Ba-DHDPS was significantly
B, C, and D, which is consistent with the crystal structure of stabilizedinthepresenceofpyruvatecomparedwiththeunli-
substrate-unboundBa-DHDPS(8).Tyr109isanimperativecat- ganded enzyme (Fig. 2B). By contrast, the presence of ASA,
alyticsiteresidue,andIle142interactswithitsequivalentneigh- whichisthesecondsubstratetobind,showednoeffectonthe
boratthetightdimerinterfaceprovidingstructuralstability.A stabilizationoftheenzyme(Fig.2B).Interestingly,theunfold-
fulltableofstatisticsisprovided(Table3).Boththepyruvate- ingofligand-freeBa-DHDPSrevealedthepresenceofaninter-
bound and the previously reported ligand-free structures are mediate state that persisted over a temperature range of
very similar (Fig. 5). The two tetramers align at alpha carbon 50–60°C(Fig.2B).Theintermediatecouldpotentiallyrepre-
atomswitharootmeansquaredeviationof0.50Å,andlittle sent a collapsed molten globule state of Ba-DHDPS that has
difference could be observed in active site residues (Fig. 5B). native-like secondary structure, but a more dynamic tertiary
However,the“weak”dimerinterfaceismoreextensiveinterms structure,aphenomenonthathasbeenpreviouslyobservedin
of buried surface area in the presence of pyruvate (Table 4). otherbacterialenzymes(42).Thisintermediatestatewasnot
Thisistheinterfacewherethetwotightdimersdocktoform propagated during thermal denaturation in the presence of
the tetramer (Fig. 1A). At this interface the buried surface pyruvate(Fig.2B).Infact,thepresenceofpyruvatesignificantly
area increases from 1735 Å2 in the absence of pyruvate to stabilizedthefoldedstateoftherecombinantenzymeforafur-
1826Å2whenboundtopyruvate.Bycomparison,theburied ther 20°C relative to the thermal denaturation profile in the
surface area at the “tight” dimer interface has only changed absenceofpyruvate(Fig.2B).Conversely,ASAhadnoeffecton
from2780Å2intheapostructureto2782Å2inthepresenceof the thermostability of the enzyme with respect to secondary
pyruvate. Additionally, the number of interactions is signifi- structure (Fig. 2B), which demonstrates that the stabilization
cantly greater at the weak dimer interfaces as calculated by phenomenon is specific to pyruvate and thus associated with
STINGMILLENIUM(40)(Table4).Thereisalsoanincreasein Schiff-baseformationattheactivesitelysine(Lys163).
hydrogen bonds at the tight dimer interface in the pyruvate- Given the observed secondary structure stabilization phe-
bound structure (Table 4). In total, there were twelve more nomenoninducedbypyruvate,thepropensityforthesubstrate
hydrogen bond interactions at both interfaces in the crystal tostabilizethequaternarystructureinsolutionwastherefore
structureofpyruvate-boundBa-DHDPS(PDBID:3HIJ)rela- investigated. Absorbance-based analytical ultracentrifugation
tivetotheapostructure(PDBID:1XL9). experiments revealed that Ba-DHDPS existed in an apparent
equilibriumbetweentwooligomericstates,thedimerandtet-
DISCUSSION
ramer,atlowmicromolarconcentrationsat4°C(Fig.3).Sedi-
TherecentlydescribedDHDPSstructurefrommethicillin- mentationvelocityanalysisshowedthatpyruvatesignificantly
resistant S. aureus, the first reported native dimeric DHDPS shifted the apparent equilibrium in favor of the tetramer in
enzyme,wasobservedtohavegreatersolutionstabilityinthe solution(Fig.3,CandD),therebyenhancingtetramerizationin
presenceofpyruvate(14).Itisthusofinteresttoprobetherole solution.Thestabilizationofquaternarystructurewasquanti-
ofthissubstrateinstabilizingtheactivequaternarystructureof fied using sedimentation equilibrium analysis (supplemental
DHDPS. Disruption of quaternary structure offers a new Fig.1),showingthatthetetramerwas3-foldtighterinthepres-
approach for inhibitor design of active oligomeric enzymes, enceofpyruvate(Table1).Theimportanceofthetetrameric
revealing an “Achilles heel” to target in pathogenic bacteria quaternary structure of Ba-DHDPS is highlighted by kinetic
FEBRUARY19,2010•VOLUME285•NUMBER8 JOURNALOFBIOLOGICALCHEMISTRY 5193
Pyruvate-boundB.anthracisDHDPS
and solution studies of the dimeric mutant, Ba-DHDPS potentialantibiotictarget(16,24),whichhasrecentlybeenval-
(L170E/G191E).Ourresultsshowthatthedimericmutantwas idatedbyknock-outstudiesdemonstratingthatDHDPSisthe
significantly less active than the wild-type tetramer (Table 2 productofoneofonly271essentialgenesinBacillusspecies
andFig.4B).Thisisconsistentwithrecentstudiesdemonstrat- (7).Accordingly,ourstudyprovidessignificantstructure-func-
ing that the enzymatic activity of dimeric mutants of E.coli tion knowledge that can be applied to rationale drug design
DHDPSpossess(cid:9)2.5%ofthecatalyticactivityofthewild-type strategies in the pipeline to generating novel anti-anthrax
tetramer(43). agents.Thisstudyhasthusidentifiedan“Achillesheel”inan
Toprobethestructuralmechanismgoverningthepyruvate- essentialenzymeandantibiotictargetfromasignificanthuman
mediatedstabilizationoftheactivetetramericform,x-raydif- pathogen.
fractionstudiesofBa-DHDPSinthepresenceofpyruvatewere
undertaken. Recombinant native Ba-DHDPS, purified as Acknowledgments—Wefirstacknowledgethesupportandassistance
describedpreviously(22),crystallizedinidenticalconditionsto ofthefriendlystaffattheBio21CollaborativeCrystallographicCen-
thosepublishedfortheunligandedenzyme(PDBID:1XL9)(8). treatCommonwealthScientificandIndustrialResearchOrganisa-
ThefinalstructureofBa-DHDPSincomplexwithpyruvatewas tionMolecularandHealthTechnologies,Parkville,Melbourne,and
solved to a resolution of 2.15 Å, and initially no significant thebeamlinescientistsattheAustralianSynchrotron.Wealsothank
changescouldbeobservedincomparisontothepyruvate-un- allmembersofthePeruginilaboratoryforhelpfuldiscussionsduring
thepreparationofthemanuscript.
boundstructure.However,uponcloseinspectionoftheweak
dimerinterfaceusingPISAanalysis(44),significantdifferences
wereobserved.Moreover,anincreaseinburiedsurfaceareaof
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