The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ Polymer-stabilizing Agent That ...

20 Pages·2010·10.93 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.285,NO.19,pp.14239–14246,May7,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
The Antibacterial Cell Division Inhibitor PC190723 Is an FtsZ
Polymer-stabilizing Agent That Induces Filament Assembly
and Condensation*□S
Receivedforpublication,December21,2009,andinrevisedform,February18,2010Published,JBCPapersinPress,March8,2010,DOI10.1074/jbc.M109.094722
Jose´ M.Andreu‡1,ClaudiaSchaffner-Barbero‡,SoniaHuecas‡,DulceAlonso§,MaríaL.Lopez-Rodriguez§,
LauraB.Ruiz-Avila‡,RafaelNu´n˜ez-Ramírez‡2,OscarLlorca‡,andAntonioJ.Martín-Galiano‡
Fromthe‡CentrodeInvestigacionesBiolo´gicas,ConsejoSuperiordeInvestigacionesCientificas,RamirodeMaeztu9and
§DepartamentodeQuímicaOrga´nica,FacultaddeCienciasQuímicas,UniversidadComplutenseMadrid,28040Madrid,Spain
CelldivisionproteinFtsZcanformsingle-strandedfilaments EssentialcelldivisionproteinFtsZassemblesintofilaments
withacooperativebehaviorbyself-switchingassembly.Subse- thatformthecytokineticringatthedivisionsite(1)andrecruits
quentcondensationandbendingofFtsZfilamentsareimpor- the accessory proteins of the bacterial divisome (2–4). FtsZ,
tantfortheformationandconstrictionofthecytokineticring. withanaddedmembraneanchor,canformconstrictingrings
PC190723isaneffectivebactericidalcelldivisioninhibitorthat in liposomes (5). FtsZ is a member of the tubulin family of
targetsFtsZinthepathogenStaphylococcusaureusandBacillus cytoskeletal GTPases, also including (cid:1)(cid:2)-tubulin, (cid:3)-tubulin,
subtilisanddoesnotaffectEscherichiacolicells,whichappar- bacterialtubulinBtubA/B(3,6),anddivergentTubZs(7).FtsZ
entlybindstoazoneequivalenttothebindingsiteoftheanti- andtubulinusethesamestructuralfoldandsimilarhead-to-tail
tumordrugtaxolintubulin(Haydon,D.J.,Stokes,N.R.,Ure,R., protofilamentswithGTPboundattheintersubunitassociation
Galbraith,G.,Bennett,J.M.,Brown,D.R.,Baker,P.J.,Barynin, interface(8),yettheyhaveevolvedtoformdifferentassemblies
V.V.,Rice,D.W.,Sedelnikova,S.E.,Heal,J.R.,Sheridan,J.M., with specialized functions. Their GTPase sites are completed
Aiwale,S.T.,Chauhan,P.K.,Srivastava,A.,Taneja,A.,Collins, uponcontactoftheapicalGTP-bindingdomainofonesubunit
I.,Errington,J.,andCzaplewski,L.G.(2008)Science312,1673– withthebasalGTPaseactivationdomainofthenextsubunit.
1675).WehavefoundthatthebenzamidederivativePC190723 GTPhydrolysisrendersthepolymersmetastableandreadyfor
isanFtsZpolymer-stabilizingagent.PC190723inducednucle- disassembly. There is evidence that unassembled tubulin,
atedassemblyofBs-FtsZintosingle-strandedcoiledprotofila- unlikeotherGTPases,ispredominantlyinaninactive(curved)
mentsandpolymorphiccondensates,includingbundles,coils, conformation regardless of which nucleotide is bound and
andtoroids,whoseformationcouldbemodulatedwithdifferent
switchesintotheactive(straight)conformationmainlythrough
solutionconditions.Underconditionsforreversibleassemblyof
the assembly contacts and that GTP binding lowers the free
Bs-FtsZ, PC190723 binding reduced the GTPase activity and
energybarrierbetweenbothconformations(6,9–11).Binding
inducedtheformationofstraightbundlesandribbons,which
ofthesuccessfulantitumordrugtaxoloverridestheGTP/GDP
wasalsoobservedwithSa-FtsZbutnotwithnonsusceptible
regulation,inducingmicrotubuleassemblyevenwithGDP(12,
Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide
13).Tubulinpromiscuouslyinteractswithsmallmoleculesof
alsoinducedBs-FtsZbundling.Weproposethatpolymersta-
natural origin that induce or inhibit its assembly. However,
bilization by PC190723 suppresses in vivo FtsZ polymer
therearefewerfunctionalinhibitorsforFtsZ.
dynamics and bacterial division. The biochemical action of
FtsZ is a relatively simple assembly machine capable of
PC190723onFtsZparallelsthatofthemicrotubule-stabiliz-
nucleatedlinearpolymerization.Suchcooperativebehaviorof
ingagenttaxolontheeukaryoticstructuralhomologuetubu-
alinearpolymerwasanticipatedbytheself-switchingassembly
lin.BothtaxolandPC190723stabilizepolymersagainstdis-
concept(14)andsuggestedtobepossibleforFtsZifdimeriza-
assemblybypreferentialbindingtoeachassembledprotein.
tioncausedaconformationalchangethatincreasestheaffinityof
Itisyettobeinvestigatedwhetherbothligandstargetstruc-
the next monomer to bind (3, 15). Cryoelectron microscopy of
turallyrelatedassemblyswitches.
unsupportedsampleshasshownthatFtsZfromEscherichiacoli
canformsemiflexiblesingle-strandedfilaments(16).Theircoop-
*ThisworkwassupportedbyGrantsMinisteriodeCiencia(MCINN)BFU2008- erativebehaviorisexplainedbyanunfavorablemonomerisomer-
00013(toJ.M.A.),ComunidadAuto´nomadeMadridS-BIO-0214-2006(to ization(activationswitch)betweenaninactive,assemblyincom-
J.M.A.andO.L.),MCINNSAF2007-67008(toM.L.L.-R.),S-SAL-249-2006 petent, and active conformation, which is coupled to assembly,
(toM.L.L.-R.),SAF2008-00451(toO.L.),RedTema´ticadeInvestigacio´n
creating a nucleation barrier (16–19). On the other hand, FtsZ
CooperativaenCa´ncerRD06-0020-1001(toO.L.),HumanFrontiersSci-
enceProgramRGP39-2008(toO.L.)andcontractsFormacio´ndePersonal from Methanococcus jannaschii forms characteristic double-
Investigatio´n(toC.S.-B.),ConsejoSuperiordeInvestigacionesCientificas strandedfilaments(20,21).AnumberofcrystalstructuresofFtsZ
i3p(toS.H.),andJuandelaCierva(toA.J.M.-G.).
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains did not reveal a nucleotide-induced activation switch (22). The
supplemental“ExperimentalProcedures,”TableS1,andFigs.S1–S6. structuralflexibilitychangescoupledtoassemblyareunknown,
1Towhomcorrespondenceshouldbeaddressed.Tel.:[phone](ext. requiringdeterminationofanFtsZfilamentstructure.
4381);Fax:[phone];E-mail:[email protected].
A cryoelectron tomography study of the Caulobacter cres-
2Presentaddress:InstitutodeEstructuradelaMateria,CSIC,Serrano113bis,
28006Madrid,Spain. centuscytoskeletonrevealedaputativeFtsZring,consistingof
MAY7,2010•VOLUME285•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 14239
This is an Open Access article under the CC BY license.
FtsZAssemblyInducedbySmallAntibacterialMolecule
afewshort(100nm)apparentlysingle-strandedFtsZfilaments EXPERIMENTALPROCEDURES
(5-nmwide)belowtheplasmamembranenearthedivisionsite, MethodsforBs-FtsZ,Ec-FtsZ,Sa-FtsZ,andtubulinpurifica-
andsuggestedthattheseFtsZpolymersgeneratetheforcethat tion, monitoring assembly with light scattering, electron
constrictsthemembranefordivisionthroughiterativecyclesof microscopy, polymer pelleting, binding measurements,
GTPhydrolysis,depolymerization,andrepolymerization(23). GTPase,andascreenofsolutionconditionsforassemblyare
Lateral FtsZ filament association is also important for Z-ring describedunderthesupplemental“ExperimentalProcedures.”
formationandconstriction.Severalproteinsareknowntobun- PC190723wasprovidedbyProlysis(32).Stocksolutions(1mM)
dleFtsZfilaments,includingZipA,ZapA,andSepF(4).Helical indeuterateddimethylsulfoxide(MerckUvasol)werekeptfro-
FtsZstructuresremodelbylateralassociationintotheZ-ringin zenanddry.Theywerediluteddirectlyintotheexperimental
Bacillus subtilis (24), and artificial FtsZ rings coalesce into samples.Residualdimethylsulfoxidewas2%(v/v)unlessindi-
brighterrings(5).SeveraltheoreticalmodelsfortheZ-ringhave cated.DFMBA,2,6-difluoro-3-hydroxybenzamide,andCTPM
proposed different roles for FtsZ filament condensation and were synthesized in this work (supplemental data). Buffers
bending(reviewedinRef.25). employed were as follows: Hepes50 (50 mM Hepes/KOH, 50
FtsZfilamentsaredynamic,withasubunithalf-lifeof(cid:1)10s, mMKCl,1mMEDTA,pH6.8),Hepes250(50mMHepes/KOH,
depending on the GTPase rate (26). Although FtsZ polymers 250mMKCl,1mMEDTA,pH6.8)(whereindicated,thesebuff-
can exchange nucleotides, GDP dissociation may be slow ersweremadeatpH7.5),andMes50(50mMMes/KOH,50mM
enoughforpolymerdisassemblytotakeplacefirst,resultingin KCl,1mMEDTA,pH6.5).
the subunits of FtsZ polymers recycling with GTP hydrolysis
RESULTS
(27,28).ThelengthdynamicsofthesmallindividualFtsZfila-
mentshavenotbeendetermined. PC190723 induced Bs-FtsZ assembly into bundles and iso-
Impairing FtsZ filament dynamics should block bacterial lated filaments. These processes could be dissected and ana-
division. FtsZ has been recognized as an attractive target for lyzed, employing different solution conditions, as described
newantibiotics(29)foremergingresistantpathogens.Expres- below.
sion of ftsZ is more stringently required for bacterial growth PCInducesBundlesofB.subtilisFtsZFilaments—Tocharac-
terizetheeffectsofPConFtsZassembly,full-length,untagged
than the established antibacterial targets murA and fabl (30).
FtsZ from B.subtilis was first expressed and purified, and its
Potentially druggable cavities in FtsZ structures (22) are the
apicalnucleotide-bindingsiteandalateralchannelbetweenthe reversible assembly was induced by 10 mM MgCl2 and 2 mM
GTPinHepes50,25°C(see“ExperimentalProcedures”)with-
N-andC-terminaldomains.Thelatteroverlapsthebindingsite
outotheradditives,whichwerechosenforconvenienceasref-
ofthemicrotubule-stabilizingantitumordrugtaxolineukary-
erenceconditions.AdditionofPCinducedadramaticincrease
otic tubulin. Several GTP analogues substituted at C-8 selec-
in FtsZ light scattering and stabilized polymers against disas-
tivelyinhibitFtsZpolymerizationbutsupporttubulinassembly
semblyinducedbyGDP(Fig.1A).ManylargePC-inducedbun-
into microtubules (31), indicating differences in nucleotide
dlesandribbons,inadditiontoFtsZfilaments,wereobserved
binding by each protein that may be exploited to selectively
bynegativestainelectronmicroscopy(Fig.1B).Thesebundles
inhibit bacterial FtsZ without poisoning eukaryotic tubulin. andribbonswereseveralmicronslong,hadavariable(cid:1)30to
FtsZ recently has been validated as the target of an effective (cid:1)60nmwidth,andwereapparentlymadeofassociatedFtsZ
antibacterialcompounddevelopedasacelldivisioninhibitorin
filaments. Computed diffraction patterns indicated an axial
B.subtilisandthepathogenStaphylococcusaureus,PC190723. spacing of 4.5 (cid:2) 0.5 nm between subunits (Fig. 1, C and D),
Its putative binding site, mapped with resistance mutations,
whichiscompatiblewiththe4.3-nmspacingofFtsZmonomers
correspondstothetaxol-bindingsiteintubulin(32).
associatedhead-to-tailintubulin-likeprotofilaments(20,21),
Weaimedtodeterminethemechanismoffunctionalinhibi- andashorterlateralspacingof3.8(cid:2)0.3nm,suggestingalateral
tion of FtsZ by the small antibacterial molecule PC190723, association of Bs-FtsZ molecules (Protein Data Bank code
3-[(6-chloro[1,3]thiazolo[5,4-b]pyridin-2-yl)methoxy]-2,6-dif- 2VXY; 32) in contact along their shortest dimension (that is,
luorobenzamide(abbreviatedhereasPC).3Wehavefoundthat rotated (cid:1)90° on the longitudinal axis with respect to tubulin
PC enhances the cooperative assembly of Bs-FtsZ into fila- protofilamentsinamicrotubule)(33).PC-inducedbundlesand
ments, bundles, and other condensates. PC also is a Sa-FtsZ ribbons also formed with the slowly hydrolyzable GTP ana-
polymer stabilizer. Stabilizing FtsZ polymers should reduce logue GMPCPP (0.1 mM). Most FtsZ above a small (cid:1)0.3 (cid:4)M
theirdynamics,whichexplainswhyPCblockscelldivisionin criticalconcentration(Cr)pelletedupon20minofcentrifuga-
bacteriaanalogouslytotheactionoftaxolontubulin. tion at 386,000 (cid:3) g (Fig. 1E). In the absence of PC, relatively
short, (cid:1)4-nm wide filaments were observed (Fig. 1F), which
sedimentedabovealargerCr(cid:1)5(cid:4)MFtsZ.Theseresultsindi-
cated that the small molecule PC stabilized Bs-FtsZ filament
3Theabbreviationsusedare:PC,3-[(6-chloro[1,3]thiazolo[5,4-b]pyridin-2-yl)-
bundlesbutdidnotdistinguishwhetherPCprimarilyenhances
methoxy]-2,6-difluorobenzamide;Mes,4-morpholineethanesulfonicacid;
DFMBA, 2,6-difluoro-3-methoxybenzamide; CTPM, (6-chloro[1,3]thia- filamentformationorbundling.
zolo[5.4-b]prydin-2-yl)methanol;Bs-FtsZ,FtsZfromB.subtilis;Sa-FtsZ,FtsZ EffectsofSolutionConditions,Mg2(cid:4)andNucleotide—Toelu-
fromS.aureus;Ec-FtsZ,FtsZfromEscherichiacoli;Cr,criticalproteincon-
cidatethemechanismofPC-inducedassemblyandtocharac-
centrationforpolymerization;HPLC,highpressureliquidchromatogra-
phy;GMPCPP,guanosine-5(cid:5)-[(2,(cid:2))-methylene]triphosphate. terize the effects of PC under more physiological conditions,
14240 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER19•MAY7,2010
FtsZAssemblyInducedbySmallAntibacterialMolecule
FIGURE1.AssemblyofbundlesofBs-FtsZ(10(cid:1)M)inducedbyPC(20(cid:1)M).A,lightscatteringtimecourseswithandwithoutPCinHepes50assemblybuffer,
10mMMgCl2,pH6.8,25°C.GTP(2mM)wasaddedattime0and2mMGDPasindicatedbythearrows.B,electronmicrographofBs-FtsZwithPC.Bar,200nm.
C,anenlargedbundle.D,itscomputeddiffractogram.Thecorrespondingspacingofthemainequatorialspotandthefirstlayerlineareindicatedinnm.
E,Bs-FtsZpolymerpelletingassaysatvaryingpHandionicstrength.F,Bs-FtsZwithoutPC,magnifiedasinB.
pHwasincreasedto7.5,orionicstrengthincreasedto250mM
KCl,whichreducedtheextentofspontaneousBs-FtsZassem-
bly with nucleotide and Mg2(cid:4). Assembly was efficiently
inducedbyPCinthesecases(Fig.1E),althoughbundleforma-
tion was reduced. Assembly was markedly sensitive to the
bufferanion(supplemental“ExperimentalProcedures”).Disas-
semblycouldbemonitoredbylightscatteringfollowingassem-
blyof10(cid:4)MBs-FtsZwith25(cid:4)MGMPCPP(expectedtostabi-
lize polymers) and 10 mM MgCl in 250 mM KCl (scattering
2
with GTP was very small). This was attributed to GMPCPP
hydrolysisandwasnotobservedinthepresenceofPC.GTPor
GMPCPPwasrequiredforPC-enhancedassembly.Mg2(cid:4)with-
outnucleotidewasineffective.Interestingly,GTPorGMPCPP
without Mg2(cid:4) induced assembly of Bs-FtsZ filaments, which
were not formed with GDP or GMPCP. Mg2(cid:4) plus GDP or
FIGURE2.PCandmagnesium-inducedBs-FtsZassemblies.Electronmicro-
GMPCPandPCgaveaweakpolymerformation.Theseresults graphofnegativelystainedpolymersformedbyBs-FtsZ(10(cid:4)M)inHepes250
confirmedthatPCstabilizesBs-FtsZpolymersandshowedthe assemblybufferwith0.1mMGMPCPP(A),0.1mMGMPCPPand20(cid:4)MPC(B),
requirementofthenucleotide(cid:3)-phosphate. a0n.1dm2M0G(cid:4)MMPPCCP(PD)a.nTdhe10bamrMinMdigcCatl2es(C2)0,a0nndm0..1mMGMPCPPand10mMMgCl2
It was possible to separate PC-induced filament assembly
from bundle formation by using 250 mM KCl and GMPCPP.
Bs-FtsZ assembly was only incipient without Mg2(cid:4) (Fig. 2A). IncipientassemblyofBs-FtsZwasobservedwithnucleotidein
AddingonlyPCinducedcharacteristicfilamentcoils(Fig.2B), the absence of PC (no nucleotide-induced light scattering
whereasMg2(cid:4)aloneinducedtheformationoffilaments(Fig. increase or pelleting). However, addition of PC under these
2C).BothwereinhibitedwithGMPCP.PCandMg2(cid:4)together conditions resulted in Bs-FtsZ polymerization. There was a
inducedtheformationoffilamentsandbundles(Fig.2D).Sim- smalllightscatteringincreasewithPCandGMPCPP(Fig.3A).
ilar results were obtained with GTP, but with PC and Mg2(cid:4), These PC-induced FtsZ filaments sedimented in 80 min at
dense coils and no bundles were observed (supplemental 386,000(cid:3)g,whichallowedtheirquantification.Practicallyall
Fig.S1). Bs-FtsZwaspelletedwithPCandGTPorGMPCPPaboveaCr
PC Induces Bs-FtsZ Single-stranded Filament Assembly— (cid:1)1(cid:4)M(Fig.3B;GTP,0.8(cid:2)0.1(cid:4)M;GMPCPP,1.3(cid:2)0.3(cid:4)M).
Mg2(cid:4)-less Hepes250 buffer permitted PC-induced nucleated InsignificantGTPhydrolysiswasdetectedunderthesecondi-
assembly of single-stranded Bs-FtsZ filaments. Our divalent tions. These results indicated a nucleated polymerization
cation-free solutions typically contain (cid:1)1 (cid:4)M residual Mg2(cid:4) mechanismforthePC-inducedassemblyofBs-FtsZfilaments,
(34)thatwith1mMEDTAinbufferresultsin(cid:1)4nMfreeMg2(cid:4). with an apparent free energy change for polymer elongation
MAY7,2010•VOLUME285•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 14241
FtsZAssemblyInducedbySmallAntibacterialMolecule
between0.5and1PCperFtsZ(Fig.
4A), compatible with PC inducing
polymerization by binding to one
site in FtsZ. For a ligand to induce
protein association, it has to bind
more to the protein polymer than
the monomer. As a first approach,
PCbindingtoBs-FtsZpolymerswas
determined by extraction of the
ligandfromthepelletsandsuperna-
tantsfollowedbyHPLC.PCcosedi-
mentedwiththepolymersandinhi-
bition of polymerization with GDP
alsoinhibitedthePCpulldown.The
resultswere0.5PCboundperFtsZ
at (cid:1)10 (cid:4)M free PC, irrespective of
ionicstrengthandmagnesiumlevel
(0.46(cid:2)0.14inHepes50with10mM
MgCl , 0.40 (cid:2) 0.10 in Hepes250
2
without MgCl , and 0.54 (cid:2) 0.10 in
2
Hepes250with10mMMgCl ).FtsZ
2
was active in polymer formation
(Fig.3B),andsaturatingthebinding
to an expected 1 PC per FtsZ was
precludedbythefreeligandsolubil-
ity ((cid:1)10 (cid:4)M in our buffers). The
simplest interpretation of these
results is one PC binding site per
polymer FtsZ molecule with an
apparentaffinity(cid:1)105M(cid:8)1.
FIGURE3.Bs-FtsZfilamentassemblyinducedbyPC.A,solidline,lightscatteringchangesinducedonBs-FtsZ Interestingly, the PC fragment
D(1a0s(cid:4)heMd)wliniteh,sPaCm(e20w(cid:4)itMho)buytPaCd.dBit,ieoxnasmopfGleMoPfsCePdPim(2e5n(cid:4)taMt)ioanndofMBgs-CFlt2s(Z1p0omlyMm)(earrsrofowrms)eindHweiptheos2u5t0M,gp2H(cid:4)6(.t8o,p2)5an°Cd. DFMBA(1–4mM;Fig.5,AandC),
2,6-difluoro-3-hydroxybenzamide
pelletquantification(bottom).Filledcircles,PCandGMPCPP(0.1mM);filledsquares,PCandGTP(2mM);open
symbols,withoutPC;triangle,PCandGDP;invertedtriangle,PCandGMPCP.C,cryoelectronmicrographof (1–4 mM) and to a lesser extent
Bs-FtsZfilamentsassembledwithPCandGTPunderthesameconditionswithoutMg2(cid:4).Inset,negatively 3-methoxybenzamide (20 mM),
stainedfilaments.Bar,200nm.D,cryoelectronmicroscopyfilamentwidthdistribution,indicatingthatthe
filamentsareoneBs-FtsZmolecule-wide. werefoundtoinducelargeBs-FtsZ
bundles similar to those obtained
(cid:6)G0 (cid:7) RTlnCr (cid:7) (cid:8)8.2 kcal mol(cid:8)1 (16). With GTP or with PC (20 (cid:4)M), thus following their relative antibacterial
app
GMPCPPandnoPC,abackgroundsimilartothatofPCand activities(35).TheotherfragmentofthePCmolecule,CTPM
GDPorGMPCPwasobserved(Fig.3B).Crdecreasedtoinsig- (1mM)didnotinduceBs-FtsZbundling(Fig.5,BandC).These
nificant values in experiments made in parallel with 10 mM resultssuggestedspecificbutweakbindingofDFMBAtoBs-
MgCl (supplementalFig.S2),indicatingalargereffectiveelon- FtsZpolymers,whichcouldnotbeaccuratelymeasuredbypel-
2
gationaffinity,althoughthesystemwasnotatchemicalequi- leting HPLC. DFMBA has an (cid:1)102-fold higher effective con-
libriumduetonucleotidehydrolysis.Interestingly,thePC-in- centrationthanPC,suggestingatwoorderofmagnitudelower
duced Bs-FtsZ filaments without Mg2(cid:4) typically had an affinity.NotethatifPCwereanonself-associatingidealbifunc-
(cid:1)100-nm curvature radius and appeared single-stranded. To tionalligand(36),therestofthemolecule,ifisolated,wouldbe
confirmthatthesefilamentsaresingle-stranded,weperformed expected to bind more weakly than DFMBA and be inactive
cryoelectron microscopy under these conditions, using holey below(cid:1)0.5M.
carbon film. The sample trapped within the small holes was Modulation of the GTPase Activity of Bs-FtsZ by PC—The
quicklyvitrifiedandkeptunderliquidnitrogentemperaturesto effects of PC on the GTPase activity of polymerizing Bs-FtsZ
ruleoutanyeffectofthestainingagentortheadsorptiontoa solutionswasexaminedwith10mMMgCl inthreebuffersthat
2
supportfilm(Fig.3C).Filamentwidthmeasurementsfromthe give quite different degrees of PC-induced bundling (see
cryomicrographshadadistributionwithanaverage3.5(cid:2)0.7 above).ControlGTPasewithoutPCwasdependentontheKCl
nm (Fig. 3D), indicating that these filaments are one Bs-FtsZ concentration: 0.61 (cid:2) 0.09 min(cid:8)1 in Hepes250, 0.22 (cid:2) 0.02
moleculewide. min(cid:8)1inHepes50,0.15(cid:2)0.08min(cid:8)1inMes50.Apartialinhi-
BindingofPCandItsFragmentDFMBAtoBs-FtsZPolymers— bition of the GTPase rate was observed with PC, to a similar
Titrations of Bs-FtsZ polymer formation with PC showed an extent(Fig.6)irrespectiveofthebuffer.Theinhibitionbetween
increasing fraction of sedimented FtsZ, which saturated (cid:1)1and(cid:1)10(cid:4)MPCroughlyparalleledtheformationofpellet-
14242 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER19•MAY7,2010
FtsZAssemblyInducedbySmallAntibacterialMolecule
FIGURE5.Bs-FtsZpolymersinducedbyPCfragmentsinMes50assembly
bufferwith10mMMgCl2and2mMGTP.A,1mMfragmentofDFMBA.B,1
mMfragmentofanalogueCTPM(similartocontrolwithoutfragment,asin
Fig.1F).Bar,200nm.C,chemicalstructureofPCandlowspeedFtsZpolymers
pelleting(15,000(cid:3)g,20min)underthesameconditions(PC,20(cid:4)M).DFMBA
also induced a FtsZ light scattering increase that was not observed with
CTPM.TheCrforassemblyinHepes50wasreducedfrom(cid:1)5(cid:4)Mto(cid:1)1(cid:4)M
FtsZwithDFMBA(4mM).
FIGURE4.BindingofPCtoBs-FtsZpolymers.A,Bs-FtsZ(10(cid:4)M)sedimenta-
tionassaysinHepes250,pH6.8,25°C,andquantificationofpolymerwith
increasingconcentrationsofPC.Opencircles,with2mMGTP;filledcircles,with
2mMGTPandMg2(cid:4);voidsquares,with0.1mMGMPCPP.Triangles,corre-
spondingdatawithGDP,GDP,andMg2(cid:4)orGMPCP.B,HPLCanalysisofPC(10
(cid:4)Mtotal)co-sedimentationwithBs-FtsZpolymers.Solidlinesandarrow,with
GTPandMg2(cid:4);dashedgraylinesandarrow,controlwithGDPandMg2(cid:4).Taxol
isaninternalstandardhere.Inthisexperiment,thepelletedPCandFtsZwere FIGURE6.EffectsofPContheGTPaseactivityofFtsZ(10(cid:1)M),with1mM
4.8and10(cid:4)M,respectively.mAU,milliabsorbanceunits. GTPand10mMMgCl2,pH6.8at25°C.Bs-FtsZ:solidcircles,Hepes50;trian-
gles,Hepes250;squares,Mes50.Ec-FtsZ:opencircles,Hepes50(averageof
threedifferentproteinpreparations).Thelinesaredrawnsolelytoshowthe
able polymer (Fig. 4A). Low PC concentrations variably
trendofthedata.TheincreaseinGTPaseactivityofEc-FtsZwasconfirmedby
enhancedtherelativeGTPaseactivityinHepes250.Thismay measurementsatvaryingproteinconcentrationswithexcess15(cid:4)MPC(not
be a simple consequence of a large increase in FtsZ GTPase shown).
uponpolymerizationrelativetotheweaklypolymerizingcon-
trolwithoutPC(Fig.1E)(sincetheratemeasuredispertotal
FtsZ,notperassembledFtsZ).Controlmeasurementsshowed Formation of Polymorphic Bs-FtsZ Filament Condensates
that3H[GTP]bindingtoBs-FtsZandtoEc-FtsZwasnotmod- with Different Curvatures—Bs-FtsZ filaments frequently
ifiedbyPC(supplementalFig.S3). adopted bow shapes and easily coalesced into coils or associ-
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FtsZAssemblyInducedbySmallAntibacterialMolecule
FIGURE7.PolymorphicassembliesofBs-FtsZ,inadditiontobundlesand
ribbons(seeFig.1B).A,filamentsobservedinHepes50,pH6.8,withGTPand
PC. B, toroids in another field of the same sample. C, helical bundles in
Hepes50atpH7.5withGTP,10mMMgCl2,andPC.D,coiledfilamentsand
toroidsformedwithoutPCordimethylsulfoxideinMes50withMgCl and
2
GMPCPP.SimilarpolymerswereobservedinHepes50,pH6.8,orwithGTP.
Bar,200nm.
atedintostraightbundles(seeabove).OtherBs-FtsZfilament
condensatesalsoobservedincludetoroidsandhelicalbundles
(seesupplementalTableS1foralistofpolymorphsandcondi-
tions).Briefly,alongwithfilamentsformedwithGTPandPC
(Fig. 7A) and filament coils (similar to Fig. 2B), toroids(cid:1)300
nmindiameterwereobservedonthesamegrids(Fig.7B).Large
helical bundles were observed at pH 7.5 with PC, GTP, and
Mg2(cid:4)(Fig.7C).Interestingly,Bs-FtsZcondensatescouldalso
form in the absence of PC with GMPCPP and Mg2(cid:4) as coils,
and smaller toroids (cid:1)150 nm in diameter were observed
(Fig.7D).
Specificity of FtsZ-PC Interactions—We next compared the
effectsofPContheassemblyofFtsZfromsusceptible(B.sub-
tilis and S.aureus) and nonsusceptible (E.coli) bacteria in
Hepes50(Fig.8A).Theweakformationofpelletablepolymerby
Sa-FtsZ was markedly enhanced by PC, at concentrations
higherthanwithBs-FtsZ.AlargePC-inducedincreaseinSa-
FtsZlightscatteringwasobservedwithnucleotideandMg2(cid:4)
(supplementalFig.S4).Electronmicrographsshowedatrans-
formationofSa-FtsZoligomers(Fig.8B)intocoils,toroids,and FIGURE8.EffectsofPConthepolymerizationofFtsZ(10(cid:1)M)fromsus-
ceptible(B.subtilisandS.aureus)andresistant(E.coli)bacteria.A,FtsZ
straightbundleswithPC(Fig.8,CandD).DFMBAhadsimilar
polymerversusPCconcentration.Circles,Bs-FtsZ;squares,Sa-FtsZ;triangles,
effects at higher concentrations. On the other hand, PC Ec-FtsZ.Thegraysymbolsarecorrespondingcontrolswithoutmagnesium.
induced bundling was not observed with Ec-FtsZ. Filament TheconditionswereHepes50bufferwith10mMMgCl2and1mMGTP,pH6.8,
25°C.AGTPregeneratingsystem(1unit/mlacetatekinaseand15mMacetyl
morphologywasnotmodified(Fig.8E),andchangesinpoly- phosphate)wasaddedforthemeasurementsinA.Thiswasrequiredtorepro-
merizationwereinsignificantwhenemployingaGTP-regener- duciblypellettheEc-FtsZpolymersintheexperimenttimeanddidnotmod-
ifythepelletingofBs-FtsZandSa-FtsZ.B–D,electronmicrographsofSa-FtsZ
atingsystem(Fig.8A).Unexpectedly,PCinducedafasterdis- withGTP(B),withGTPand20(cid:4)MPCintwofieldsfromthesamesample(C)
assembly without a GTP regenerating system (supplemental and with 0.1 mM GMPCPP and 20 (cid:4)M PC (D, upper panel displayed with
Fig.S4C)anda2-foldenhancementofEc-FtsZGTPase(Fig.6). reducedcontrast).E,Ec-FtsZfilamentswithGTPand20(cid:4)MPC.Controlswith-
outPCgavesimilarimages.Bar,200nm.
HPLCmeasurements(with10(cid:4)MEc-FtsZand20(cid:4)MPC)indi-
cated binding of 0.4 PC per polymerized Ec-FtsZ, although it
didnotinducebundling.Finally,PC(20(cid:4)M)orDFMBA(4mM) DISCUSSION
did not affect the assembly of the more distant eukaryotic Wehaveshowninthisstudythatbindingofthesmallanti-
homologue(cid:1)(cid:2)-tubulin(15(cid:4)M)inlightscatteringtests,orthe bacterialmoleculePC190723enhancestheassemblyofpurified
grossmorphology(inelectronmicroscopy)ofthemicrotubular cell division protein Bs-FtsZ into filaments and bundles. Pre-
polymersformed(notshown). ventingdisassemblyofFtsZpolymersshouldinhibittheirfunc-
14244 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER19•MAY7,2010
FtsZAssemblyInducedbySmallAntibacterialMolecule
tionaldynamicsinvivo,whichweproposeexplainscytokinesis WehaveemployedBs-FtsZasamodelandconfirmedthat
inhibition. This is supported by the observation of FtsZ foci PC also induces bundling of Sa-FtsZ, but does not induce
alongPC-treatedbacilliinsteadofZ-rings(32). assembly or condensation of Ec-FtsZ filaments. This is in
Our results also give insight into the mechanisms of FtsZ agreementwiththeantibacterialspectrumofPC,whichhas
assemblyandfacilitatetheuseofPCasatooltoinvestigatethe beenrelatedtothepresenceofVal307inFtsZfromsuscep-
biological role of FtsZ. PC binding induces Bs-FtsZ filament tibleorganisms(32).MutationsconferringresistancetoPC
formationunderincipientassemblyconditions(250mMKCl, clusteratthelongchannelbetweenthetwodomainsofthe
noMg2(cid:4)).Thesefilamentsaresinglestranded,yettheyassem- FtsZmoleculewherePChasbeendocked(32).Thiszoneis
blewithacooperativecriticalconcentrationbehavior,typicalof differentfromtheGTPbindingsiteanditoverlapsthetaxol
multi-stranded polymers. This may be explained by Bs-FtsZ binding site in (cid:2)-tubulin. In agreement with the large
switching between inactive and active states similarly to Ec- sequencedivergencebetweenbothproteinsnomarkedcross
FtsZ(16),indicatingthatPCinducesthepolymerBs-FtsZcon- reactions have been observed (32; Results). In order to sta-
formation,possiblybydruggingitsassemblyswitch.However, bilize susceptible FtsZ polymers PC should bind more to
itcannotberuledoutthatthecooperativebehaviorofBs-FtsZ them than to monomers. It could be thought that PC does
with PC might be caused by some filament cyclization in the notbindtoFtsZmonomers,norfitswellintothebindingsite
coilsobservedorbyfilamentnetworkformation.Directmea- ofnon-susceptibleFtsZ(32).However,theGTPaseenhance-
surementsofPCbindingtoFtsZpolymerssuggestpartialsat- ment and HPLC measurements indicate PC binding to Ec-
urationofonePCbindingsiteinFtsZ,limitedbythesolubility FtsZ.GiventhatPCdidnotmodifysteady-statepolymeriza-
of the ligand. PC-induced Bs-FtsZ filament assembly in the tion,itshouldbindsimilarlytothemonomersandpolymers
absenceofmagnesiumproceedswithoutGTPhydrolysis,con- of this non-susceptible FtsZ. This binding could occur in a
stitutinganequilibriummodelsystemofligand-inducednucle- different manner (ineffectively or at another site) than in
ated assembly, analogous to the taxol-induced assembly of susceptibleBs-FtsZandSa-FtsZpolymers.Theasyetunre-
GDP-tubulin(12).Itwouldbeworthstudyinginmoredetailthe solved atomic structure of an FtsZ-PC complex could give
linkageofPCbindingtoFtsZfilamentassembly. insightintothedetailedstructuralmechanismofPCaction,
TheFtsZGTPasesiteisattheaxialinterfacebetweencon- possiblytheFtsZactivationswitch,andwouldfacilitatethe
secutiveFtsZmoleculesinaprotofilament(8,37),sothatGTP optimizationofPC-deriveddrugstargetingFtsZinbacterial
hydrolysistakesplaceuponFtsZassembly.PCmodulatesthe pathogens. The major determinant of PC binding to FtsZ
GTPaseactivityofsteady-statepolymerizingBs-FtsZsolutions. (Results) and antibacterial activity (35) appears to be its
TheGTPaseactivationatearlystagesofassemblyremainstobe DFMBAmoiety.
studied. Focusing on the effects of the higher PC concentra- Anincreasingnumberofproteinsareknowntomodulate
tions,thepartialinhibitionofBs-FtsZGTPaseandthelackof FtsZassembly(4),inhibitingorenhancingFtsZpolymeriza-
PCeffectofGTPbindingargueagainstbindingtothesamesite. tionandbundling.SmallmoleculesreportedtostabilizeFtsZ
This GTPase inhibition is difficult to explain by a bundling polymers include zantrins 3 and 5 (39), ruthenium red (40)
effect,duetothecoincidentextentofinhibitionunderdifferent andDAPI(41),andveryrecentlyFtsZbundlingbytheben-
bundlingconditions(Fig.6).Mechanistically,PCbindingata zoicacidderivativeOTBA(42)hasbeenproposedasastrat-
sitedifferentfromtheGTPsitemayeitherdecreasetheFtsZ egy to inhibit bacterial division. It would be of interest to
subunitturnoverinstabilizedfilamentsandthereforetheGTP know whether these molecules share the PC site or bind
hydrolysisturnover(27)orallostericallymodulate(32)theGTP elsewhere in FtsZ. It is known that Ca2(cid:4) ions induce FtsZ
hydrolysisandexchangeinFtsZfilaments. filament bundling (43, 44) and that FtsZ condensates can
Depending on the solution conditions, the assembly of forminanonspecificmannerbymacromolecularcrowding
Bs-FtsZ varies and so does the effect of PC, with a similar (45)orbyinteractionswithpolarmoleculesatrelativelyhigh
global assembly enhancement. PC binding under Bs-FtsZ concentrations (46). Volume exclusion in the bacterial
filamentassemblyconditions(50mMKCl,Mg2(cid:4))resultsin cytosolshouldfavortheformationofFtsZcondensates.An
the formation of large condensates of Bs-FtsZ filaments. importantquestioniswhetherthereareendogenousregula-
Thismaybeaconsequenceoffilamentaccumulation,orPC torssharingthePCbindingsiteofFtsZ.
might induce the condensation of existing filaments, by ThebiochemicalactionofPConFtsZpolymersparallels
bindingmoretocondensedthantoisolatedFtsZfilaments. that of taxol on tubulin (12), since each drug stabilizes its
PCinduces,inadditiontocharacteristicallycurvedBs-FtsZ respectiveproteinpolymersagainstdisassembly,bybinding
filamentsandstraightbundles(Fig.1),toroidsatpH6.8and moretopolymersthantomonomers.Taxolwasthefirst(47)
helicalbundlesatpH7.5(Fig.7),underscoringtheflexibility among antitumor Microtubule Stabilizing Agents, which
of FtsZ filaments. The observation of Bs-FtsZ coils and impair cellular microtubule dynamics and mitosis, trigger-
toroidsformedwithnucleotideandMg2(cid:4)intheabsenceof ingcelldeath(48).MicrotubuleStabilizingAgentsworkby
PC(Fig.7)indicatethattheabilitytoformcurvedconden- preferential binding to assembled tubulin (49). The struc-
sates is an intrinsic property of Bs-FtsZ filaments, which is tural mechanism coupling taxol binding and microtubule
important for the formation of the Z-ring (24), and is assemblyiscurrentlyunderinvestigation(50).Inthecaseof
enhanced by PC binding. Curved FtsZ filaments coalescing thePC-inducedFtsZfilamentstheproblemmaybesimpler
intotheZ-ringhaverecentlybeenproposedtogeneratethe due to the lack of lateral interactions. At a cellular level,
constrictionforce(38). multiplemicrotubuleastersformintumorcellstreatedwith
MAY7,2010•VOLUME285•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 14245
FtsZAssemblyInducedbySmallAntibacterialMolecule
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S5; 51). Multiple mislocalized FtsZ foci, instead of a func- 1004–1017
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Natl.Acad.Sci.U.S.A.99,3171–3175
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27. Huecas,S.,Schaffner-Barbero,C.,García,W.,Ye´benes,H.,Palacios,J.M.,
classofsyntheticantibacterials,theFtsZPolymerStabilizing
Díaz,J.F.,Mene´ndez,M.,andAndreu,J.M.(2007)J.Biol.Chem.282,
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PChavetotallydifferentstructuralmechanismsofactionor, 28. Chen,Y.,andErickson,H.P.(2009)Biochemistry48,6664–6673
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