Recognition of the Intrinsically Flexible Addiction Antidote MazE by a Dromedary Single Domain ...

12 Pages·2003·0.63 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY Vol.278,No.16,IssueofApril18,pp.14101–14111,2003
©2003byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedinU.S.A.
Recognition of the Intrinsically Flexible Addiction Antidote MazE
by a Dromedary Single Domain Antibody Fragment
STRUCTURE,THERMODYNAMICSOFBINDING,STABILITY,ANDINFLUENCEON
INTERACTIONSWITHDNA*
Receivedforpublication,September25,2002,andinrevisedform,January14,2003
Published,JBCPapersinPress,January17,2003,DOI10.1074/jbc.M209855200
JurijLah‡§¶,IrinaMarianovsky(cid:1),GadGlaser(cid:1),HannaEngelberg-Kulka**,Jo¨rgKinne‡‡,
LodeWyns‡,andRemyLoris‡§§
Fromthe‡DepartmentofUltrastructure,VrijeUniversiteitBrussel,Paardenstraat65,B-1640St.GenesiusRode,
Belgium,the§FacultyofChemistryandChemicalTechnology,UniversityofLjubljana,Askerceva5,1000Ljubljana,
Slovenia,theDepartmentsof(cid:1)CellularBiochemistryand**MolecularBiology,HebrewUniversity-HadassahMedical
School,EinKerem,Jerusalem,91120Israel,andthe‡‡CentralVeterinaryResearchLaboratories,P.O.Box591,
Dubai,UnitedArabEmirates
TheEscherichiacolimazEFoperondefinesachromo- encoding for a stable toxin and its labile antidote. Toxin and
somaladdictionmodulethatprogramscelldeathunder antidote are co-expressed. Their expression is auto-regulated
variousstressconditions.Itencodesthetoxicandlong- at the level of transcription either by a non-covalent complex
lived MazF and the labile antidote MazE. The denatur- formed between toxin and antidote or by the antidote alone
ation of MazE is a two-state reversible dimer-monomer (3–18). In the absence of co-expression the antidote is rapidly
transition.Atlowerconcentrationsthedenaturedstate degradedbyaspecificprotease,enablingthetoxintoattackits
issignificantlypopulated.Thisleadstoanewaspectof
target.Thetargetofthetoxinisknownonlyintheplasmidic
theregulationofMazEconcentration,whichmaydecide
ccdAB and kid-kis systems. In ccdAB, CcdB on plasmid F
aboutthelifeanddeathofthecell.InteractionsofMazE
attackstheAsubunitofgyrase,whereasinthekid-kissystem
withadromedaryantibodydomain,cAbMaz1(previous-
KidonplasmidR1targetsDnaB(19–21).
lyusedasacrystallizationaid),aswellaswithpromoter
Until recently, attention was paid mainly to the extrachro-
DNAwerestudiedusingmicrocalorimetricandspectro-
scopictechniques.UniquefeaturesofcAbMaz1enablea mosomal (plasmid) addiction modules, which are responsible
specificenthalpy-drivenrecognitionofMazEand,thus, for the death that occurs upon accidental plasmid loss (3–8).
a significant stabilization of its dimeric native confor- However,theE.colichromosomealsocontainsseveraloperons
mation.TheMazEdimerandtheMazEdimer-cAbMaz1 homologous to those found in plasmid addiction systems (22–
complexshowverysimilarbindingcharacteristicswith 25). The first discovered regulable prokaryotic chromosomal
promoter DNA, i.e. three binding sites with apparent addiction module is the mazEF system (or chpA), which en-
affinities in micromolar range and highly exothermic codesfortoxicandlong-livedMazFandanti-toxicandshort-lived
bindingaccompaniedbylargenegativeentropycontri- MazE(6,9,25).Incontrasttotheextra-chromosomaladdiction
butions.AworkingmodelfortheMazE-DNAassemblyis modules that are triggered by plasmid loss, death mediated by
proposed on the basis of the structural and binding
thechromosomalmazEFisachievedunderseveralstresscondi-
data.Bothbindingandstabilitystudiesleadtoapicture
tionsthatpreventmazEFexpression.Itwasinitiallyfoundthat
of MazE solution structure that is significantly more themazEFmoduleisunderthecontrolof3(cid:1),5(cid:1)-guanosinebispy-
unfoldedthanthestructureobservedinacrystalofthe
rophosphate (ppGpp) (25, 26), the amino acid starvation signal
MazE-cAbMaz1complex.
producedbyRelAprotein(27).OverproductionofppGppleadsto
inhibitionoftheexpressionofmazEFand,thereby,tocelldeath
(25, 26). However, inhibition of mazEF expression, and thus
Ingeneral,programmedcelldeathisrequiredfortheelimi-
induction of cell death, can also be achieved by using general
nationofthesuperfluousorpotentiallyharmfulcells(1,2).In
inhibitors of transcription and/or translation like antibiotics
Escherichiacoli,celldeathisprogrammedbygeneticelements
(rifampicin,chloramphenicolandspectinomycin)(28,29)andthe
called “addiction modules” (3–8). These consist of two genes
toxicproteinDoc(30).Ineachcase(ppGpp(25,26),antibiotics
(28,29)andtheDocprotein(30)),theinhibitionofgeneexpres-
*ThisworkwassupportedbytheVlaamsInteruniversitairInstituut sionleadstoalackofthelabileMazEand,thereby,allowsthe
voorBiotechnologie(VIB),theFondsvoorWetenschappelijkOnderzoek actionofthemorestableMazFtokillthecells.
Vlaanderen(FWO),andIsraelScienceFoundationGrants467/99-19(to To improve our understanding of addiction systems at the
G.G.)and215/99-2(toH.E.-K.),whichwereadministeredbytheIsrael
molecularlevel,structuralandalsothermodynamiccharacter-
Academy of Science and Humanities. The costs of publication of this
article were defrayed in part by the payment of page charges. This izationoftheproteinsinvolvedisneeded.Thelatterisdifficult
articlemustthereforebeherebymarked“advertisement”inaccordance toachievebecauseoftheproblemsofexpressionofthetoxins
with18U.S.C.Section1734solelytoindicatethisfact.
and the labile character of the antidotes. Therefore, for some
¶Recipientofashort-termfellowshipasvisitingscientistfromthe
timeonlyacrystalstructureofthetoxinCcdBhasbeenknown
FondsvoorWetenschappelijkOnderzoekVlaanderenandtowhomcor-
respondenceshouldbeaddressed:UniversityofLjubljana,Facultyof (31).Thethermodynamicinformationonaddictionproteinsis
Chemistry and Chemical Technology, Askerceva 5, 1000 Ljubljana, alsoratherscarce,andoftenonlypartialanswersareavailable
Slovenia.Tel.:[phone];Fax:[phone];E-mail:jurij.lah@
(10,32–34).
uni-lj.si
§§PostdoctoralfellowoftheFondsvoorWetenschappelijkOnderzoek Antibodies and their derivative fragments have long been
Vlaanderen. used as tools in a variety of applications in fundamental re-
Thispaperisavailableonlineathttp://www.jbc.org 14101
This is an Open Access article under the CC BY license.
14102 MazE-Dromedary Antibody and MazE-DNA Interactions
search,biotechnology,diagnosis,andhumantherapy(35,36). recordedbetween290and480nm((cid:2) (cid:4)280nm).InthecaseofDNA
ext
In contrast to conventional IgG molecules, one type of the titration with MazE at 25°C the emission spectrum was recorded
antibodiesgeneratedbycamels,dromedaries,andllamas(cam- between310and380nm((cid:2)ext(cid:4)300nm).The1.5(cid:1)MDNAoligomer
containingthepromotersequencewastitratedbya21(cid:1)Msolutionof
elids) is formed by two heavy chains but has no light chains
MazEdimer.
(37). Particularly interesting are camelid single variable do- Circular Dichroism (CD) Spectropolarimetry—CD spectra were re-
main antibody fragments (V H), which contain the smallest corded on J-715 spectropolarimeter (JASCO, Tokyo, Japan). MazE (cid:3)
H
antigen-binding unit with a molecular size of (cid:2)15 kDa. They cAbMaz1titrationswereperformedat25and45°Cbyincrementally
arecharacterizedbyhigh-yieldproduction,highsolubility,and injecting4–20(cid:1)laliquotsofcAbMaz1solutioninto2mlof(cid:5)1(cid:1)MMazE
dimersolution(1-cmcuvette)inthesamebuffer.Aftereachinjection,
highthermodynamicstability(38,39).
theellipticity,(cid:3),wasmonitoredbetween210and260nm(Fig.3).The
AsMazEalonehasalargefractionofunstructuredpolypep-
denaturation of MazE, cAbMaz1, and their tetrameric complex was
tide,aMazE-specificVHH(cAbMaz1)fragmenthasbeenused followedbyrecordingthefarUV(205–260nm)CDspectraatdifferent
asacrystallizationaid,leadingtothefirstcrystalstructureof temperatureswiththetemperaturestepof2or1°Catthetransition
an addiction antidote.1 In the current paper we focus on the region (Fig. 7). The temperature of the sample was controlled by a
thermodynamicsoftheMazE-cAbMaz1andMazE-DNA(MazE sensor built into the cuvette holder and connected to a Haake N3
(Gebrueder Haake, Karlsruhe, Germany) circulating bath which ad-
promoter)interactingsystemsandcorrelateitwiththestruc-
justedthetemperatureofthesamplewithanaccuracyof0.1°C.The
ture. The research described here is the first example of a
concentrationsofMazE,cAbMaz1,andtheirtetramericcomplexina
characterizationofMazEbindinganddenaturationenergetics. 0.1-or0.2-cmcuvettewereabout10(cid:1)M.
Differential Scanning Calorimetry (DSC)—The thermally induced
EXPERIMENTALPROCEDURES transitions of MazE, cAbMaz1, and their tetrameric complex were
Preparation of the Proteins and MazE Promoter DNA—Expression measuredusingaNano-IIDSCdifferentialscanningcalorimeter(Cal-
and purification of MazE and the cAbMaz1 fragment are described orimetrySciences,Provo,UT).Theheatingratewas1°C/min,andthe
elsewhere.1MazEandcAbMaz1solutionsforspectroscopicandcalori- concentrationofproteinsinthemeasuringcell(0.33ml)wasabout10
metricmeasurementswerepreparedbyextensivedialysisagainst50 (cid:1)M.Toobtainthepresentedthermograms((cid:6)CPversusTcurves,Fig.
mM sodium cacodylic buffer, pH 6.9, containing 150 mM NaCl and a 6a),theheatcapacityoftheproteininthenativestatewassubtracted
10-mindegassingofthesamplesolutionsbeforethemeasurements.For fromtherawsignal(correctedforbuffercontribution).Thetransition
DNAbindingstudies,thesamebufferwithoutadditionalsaltwasused. enthalpies(cid:6)H wereobtainedbyintegrationof(cid:6)C versusTcurves.
cal P
Concentrationsofa98-residue-long,Histag-fusedMazEproteinanda Two consecutive temperature scans were carried out to observe the
135-residue-longcAbMaz1antibody(Fig.1)weredeterminedspectro- extentofreversibility,whichwashigherthan0.8foralltheproteins.
photometricallybymeasuringabsorbanceat280nm.Thecorrespond- FL,CD,andCalorimetricTitrations—Changesinthespectralprop-
ing extinction coefficients were obtained from MazE and cAbMaz1 erties(Figs.3and4)suggestthatthecAbMaz1-MazEcomplexforma-
amino acid compositions by the method introduced by Gill and von tionisaccompaniedbystructuralalterationsofMazEor/andcAbMaz1.
Hippel(41)(www.expasy.ch). Moreover, binding of the first cAbMaz1 molecule to one MazE dimer
The50-basepair,double-strandedoligonucleotide(5(cid:1)-TGCTCGTAT- binding site influences the binding of the second one to the other
CTACAATGTAGATTGATATATACTGTATCTACATATGATAG-3(cid:1) and available site. Thus, the spectral changes (CD and FL) and the heat
3(cid:1)-ACGAGCATAGATGTTACATCTAACTATATATGACATAGATGTA- effects seen during titration experiments may contain contributions
TACTATC-5(cid:1)) containing the MazE promoter sequence (underlined) fromdirectbindingandstructuralchanges.TodescribethecAbMaz1-
was purchased from Invitrogen. To evaluate the specificity of MazE MazE association, a mass action model that includes the mentioned
bindingtothepromoterDNA,the107-bpcontrol(notrelatedtomazEF contributionsisproposed,asshownbelowinReaction1,
module) DNA was used. The same DNA was used as a control (not
related)DNAinthestudiesoftheccdABaddictionmoduleaswell(32). K1 K2
Lyophilizedsinglestrandsweredissolvedin50mMsodiumcacodylate, M2(cid:4)AO¢¡M(cid:1)2A(cid:1)(cid:4)AO¢¡M(cid:7)2A(cid:7)2
pH6.9,andextensivelydialyzedagainstthesamebuffer.Theconcen-
trationsofsinglestrandsweredeterminedbyUVabsorptionspectros- REACTION 1
copyat260nmbyusingextinctioncoefficientscalculatedonthebasis
ofthenearestneighborapproximation(42).Theduplexeswereobtained withK1andK2definedinEquations1and2,respectively,
bymixingthecorrespondingsinglestrandsinthe1:1molarratio.
Isothermal Titration Calorimetry (ITC)2—The heat accompanying K (cid:5)[M(cid:1)2A(cid:1)] (Eq.1)
MazE(cid:3)cAbMaz1,DNA(cid:3)MazE,andDNA(cid:3)MazE-cAbMaz1associ- 1 [M2][A]
ations was measured by an Omega isothermal titration calorimeter
(MicroCal,Northampton,MA).InMazE(cid:3)cAbMaz1experimentsat25, K (cid:5) [M(cid:7)2A(cid:7)2] (Eq.2)
35, 45, and 55°C the MazE dimer solution (1.33 ml) was titrated by 2 [M(cid:1)2A(cid:1)][A]
cAbMaz1solutioninthesamebufferusingamotor-driven250-(cid:1)lsy-
ringe. cAbMaz1 concentration was about 100 (cid:1)M, whereas the MazE and where M2, A, M(cid:1)2A(cid:1), and M(cid:7)2A(cid:7)2 represent the MazE dimer, the
dimerconcentrationinthetitrationcellwas4.8(cid:1)M.DNA(cid:3)MazEand cAbMaz1monomer,andtheirtrimericandtetramericcomplexesinthe
DNA (cid:3) MazE-cAbMaz1 experiments were performed at 25°C. MazE nativestate,respectively.Quantitiesinthesquarebracketsaretheequi-
dimerandMazE-cAbMaz1concentrationswerearound50(cid:1)M,whereas libriummolarconcentrations;K1andK2arethecorrespondingapparent
association constants. Overall change in the thermodynamic quantity
theDNAconcentrationinthetitrationcellwasabout50timeslower.
(standard Gibbs free energy, (cid:6)G°; standard enthalpy, (cid:6)H°; standard
Eachinjectiongeneratedaheatburst,withtheareaunderthecurve
entropy, (cid:6)S°) for the presented process can be expressed as a sum of
being proportional to the heat of interaction (Fig. 5a). The titration
contributions of the M(cid:7) (cid:3) 2A(cid:7) association as rigid bodies in the final
curves (Figs. 5b and 8) were constructed by subtraction of the heat 2
conformationandothercontributionswhichinvolvetheconformational
effectsthataccompanytheliganddilution.
changes,
FluorescenceSpectroscopy(FL)—FLspectrawererecordedusingan
AMINCO-BowmanSeries2luminescencespectrometer(SpectronicIn- (cid:6)G°(cid:5)(cid:6)G °(cid:4)(cid:6)G ° (Eq.3)
struments Rochester, NY) equipped with a thermally controlled cell rb other
holderandacuvetteof1cmpathlength.MazE(cid:3)cAbMaz1titrations (cid:6)G °(cid:5)(cid:8)RTln(cid:9)(cid:10)M(cid:7)A(cid:7)(cid:11)/(cid:10)M(cid:7)(cid:11)(cid:10)A(cid:7)(cid:11)2(cid:12) (Eq.4)
wereperformedat25°Cand45°Cbyincrementallyinjecting4–20-(cid:1)l rb 2 2 2
aliquotsofcAbMaz1solutioninto2mlof0.5–1(cid:1)MMazEdimersolution (cid:6)G °(cid:5)(cid:8)RTln([M(cid:7)]/[M])(cid:4)2ln([A(cid:7)]/[A])] (Eq.5)
in the same buffer. After each injection, FL emission spectrum was other 2 2
asshownaboveinEquations3,4,and5.Itfollowsthatthecontri-
butionsofconformationaleffects(other)canbeestimatedasadiffer-
1R.Loris,I.Marianovsky,J.Lah,T.Laermans,H.Engelberg-Kulka, ence between experimentally obtained(cid:6)G°,(cid:6)H°, andT (cid:6)S°and the
G.Glaser,S.Muyldermans,andL.Wyns,submittedforpublication. corresponding (cid:6)Grb°, (cid:6)Hrb°, andT (cid:6)Srb°, which were estimated by
2Theabbreviationsusedare:ITC,isothermaltitrationcalorimetry; M(cid:1)2(cid:1)A(cid:1)2(cid:1)-structure-basedcalculations.
FL,fluorescencespectroscopy;CD,circulardichroism;DSC,diffferen- Byassuminglineardependenceofameasuredphysicalproperty(F)
tialscanningcalorimetry;ASA,accessiblesurfacearea. on the concentration of individual components in ideal solution, it is
MazE-Dromedary Antibody and MazE-DNA Interactions 14103
possible, by subtracting the contributions of M and A, to obtain the
2
differenceinphysicalproperty((cid:6)F),whichcanbeexpressedasshown
below(43)inEquation6,
(cid:2)
2
(cid:6)F(cid:5) (cid:6)fi[M(cid:1)2A(cid:1)i] where (cid:6)fi(cid:5)fM(cid:1)2A(cid:1)i (cid:6)fM2(cid:6)ifA
i(cid:4)1
and if i(cid:5)1 f (cid:1)(cid:5)(cid:1) and if i(cid:5)2 f (cid:1)(cid:5)(cid:7) (Eq.6)
wheref ,f ,andf areconcentration-independentphysicalprop-
ertiesofMM2 ,AAandMM(cid:1)(cid:1)2AA(cid:1)i(cid:1),respectively.Ineachofthetechniquesused
2 2 i
inthiswork,thedescribedpropertieshavespecificphysicalmeaningas
definedinthefollowing:(i)FL(Fig.3b),where(cid:6)Fisdifferencefluores-
cence and (cid:6)f depends on the optical path length, quantum yields,
i
intensityofincidentlight,andmolarextinctioncoefficientsofM ,A,
2
andM(cid:1) A(cid:1);(ii)CD,where(cid:6)F(cid:4)(cid:6)(cid:3)(cid:4)differenceellipticityand(cid:6)f isthe
2 i i
productbetweendifferencemolarellipticityofM(cid:1) A(cid:1) andopticalpath FIG.1.Aminoacidsequenceofcabmaz1alignedwithtypesof
2 i
length;(iii)ITC(Fig.8),where(cid:6)F(cid:4)Q(cid:4)cumulativeheateffectgiven othercamelidVHHdomainsofknownthree-dimensionalstruc-
per mole of added ligand at single injection and (cid:6)f is the product ture.Thelimitsoftheframeworkregionsweredeterminedbasedon
between(cid:6)Hi°(standardenthalpyofM(cid:1)2A(cid:1)icomplexforimation)andthe nthaelHsuispetarpilossaitrieonnootfsthhoewsntr.u(cid:9)c-tsutrraensdosfaarlleVinHdHicafrtaegdmbyenatrsr.oTwhse.RCe-steidrumeis-
volumeofsolutioninthemeasuringcelldividedbytheamountofadded
thatmakeupthespecificsignatureforaV Hdomainareindicated.
ligandperinjection.IntheITC,thedifferentialformofEquation6is H
usuallyusedwherethesignalisgivenas(cid:6)H(enthalpychangegiven
permoleofaddedligandatsingleinjection)and(cid:6)f as(cid:6)H°(Fig.5b). whereallquantitiesaredefinedbyReaction2andEquations7and8.
InthecaseofDNA(cid:3)M titration,themodelthaitassumiesbinding TakingintoaccountReaction2andEquations7and8,thetemperature
RofeMac2tioonni1deanntdicaElqsuiatetsioonnsD12NanAdw6asfoursie(cid:4)d.1T.hemodelwasderivedfrom pTrhoefiirlevcaalnuebsedweesrceriboebdtaiinnteedrmbysoffitptainrgamoeftethrse(cid:6)mHo°d(eTl1⁄2f)u,n(cid:6)cCtiPo°n,a(nCdDT1(cid:4)⁄2.
Equation 9; DSC (cid:4) Equation 10) to the experimental temperature
It follows from Reaction 1 and Equation 6 that spectroscopic and
profilesusingthepreviouslymentionednon-linear(cid:7)2regressionproce-
calorimetrictitrationcurves(Figs.3b,5b,and8)canbeatgiventotal
concentrationsofM andAdescribedonlyintermsofparameters(cid:6)f dure(44).
2 i Structure-based Thermodynamic Calculations—The non-polar
and K. Their values were obtained by fitting the model functions
i (ASA ) and polar accessible surface areas (ASA ) of proteins were
(Equation6)totheexperimentaltitrationcurvesusingthenon-linear N P
calculatedwiththeprogramNACCESS,version2.1(48).TheASAof
procedure based on Levenberg-Marquardt minimization of the non-
weighted(cid:7)2function(44). nativeproteinswasobtainedfromthecrystalstructureoftheMazE-
cAbMaz1complex(probesize(cid:4)1.4Å).ASAofthedenaturedproteins
Temperature-dependentCDandDSC—Ourresultsrevealthatallmon-
wasestimatedasthesumoftheaccessibilitiesoftheproteinresiduesin
itoreddenaturationprocessescanbeadequatelydescribedasreversible
anextendedAla-X-Alatripeptide.(cid:6)C °valueswerecalculatedfrom
twostatetransitions,aspresentedinReaction2andEquation7, P,rb
changes in non-polar and polar accessible areas from the equation
K introducedbyMurphyandFreire(49),
Nn ¢O¡ nD (cid:6)C °(cid:4)0.45[calmol(cid:8)1K(cid:8)1A˚(cid:8)2](cid:1)(cid:6)ASA
P,rb N
REACTION 2 (cid:8)0.26[calmol(cid:8)1K(cid:8)1Å(cid:8)2](cid:1)(cid:6)ASA (Eq.11)
(cid:3) (cid:4) P
(cid:6)G°(cid:5) (cid:6)RTlnK(cid:5) (cid:6)RTln (cid:9)n(cid:8)(cid:12)n(cid:10)P(cid:11)n(cid:8)1 (Eq.7) whichisshownaboveinEquation11.Forbindinganddenaturationof
1(cid:6)(cid:8) theproteinsstudiedhere,thecombinationofEquation11andsimilar
relationsintroducedbySpolarandRecord(50),Myersetal.(51),and
swthaeter,ewNhnirleepDrecsoernrtessMpo2n(dns(cid:4)to2)t,hAe(Mna(cid:4)zE1)aannddMcA(cid:7)b2MA(cid:7)a2z(1n(cid:4)in4t)hiendtheneantautrievde vMaalukehsaatasdtzheosaenodbtPairniveadlobvyE(5q2u)atrieosnu1lt1sailnonteh.eThseamenethaavlepryacghea(cid:6)ngCeP,frobr°
state. K is the equilibrium constant of denaturation, (cid:6)G° is the corre- M(cid:7) (cid:3)A(cid:7)(rigidbody)associationwascalculatedas(53,54)asshownin
spondingstandardGibbsfreeenergychange,nisthenumberofsubunits Eq2uation12,
towhicheachproteindissociatesupondenaturation,(cid:8)isthedegree
ofdenaturation,and[P]isthetotalproteinconcentrationgivenper (cid:6)H ° (cid:4) (cid:8)8.44[cal mol(cid:8)1A˚(cid:8)2](cid:1)(cid:6)ASA
moleofproteininitsnativestate.(cid:6)G°canalsobeexpressedbythe rb N
integratedGibbs-HelmholtzequationshownbelowinEquation8, (cid:3) 31.4[cal mol(cid:8)1A˚(cid:8)2](cid:1)(cid:6)ASA (cid:3) (cid:6)C °(T (cid:8) 333.15) (Eq.12)
P P,rb
(cid:5) (cid:6) (cid:7)
(cid:6)G°(cid:9)T (cid:12) 1 1 andtheentropychangeuponrigidbodyassociationwascalculatedasa
(cid:6)G°(cid:5)T 1⁄2 (cid:4)(cid:6)H°(cid:9)T (cid:12) (cid:6) sumofthreecontributions(53–55),
T 1⁄2 T T
1⁄2 1⁄2
(cid:6) (cid:3) (cid:4)(cid:7)(cid:8) (cid:6)S °(cid:5)(cid:6)S °(cid:4)(cid:6)S °(cid:4)(cid:6)S ° (Eq.13)
rb sol sc mix
(cid:4)(cid:6)CP° 1(cid:6)TT1⁄2(cid:6)ln TT1⁄2 (Eq.8) wobhtiacihneadreassh:o(cid:6)wCna,b°olvne(Tin/38E5q.u15a)tio(n551,35.6T)h.eTshoelvtaetriomnttheramt,r(cid:6)efSlesoclt°s,wthaes
Prb
riisnefetwhreheniccchoertr(cid:6)eemHsp°p(oeTnr1ad⁄2)tinuigrsesTtth1a⁄e2n(dtsrataarndnsdihatierodantetcenamtphpaaeclriptayytuocrfheadanetgn(cid:8)ea(cid:4)taus0rs.a5ut)mi,oanenddatt(cid:6)oCthbPee° sachulaamnnignoeev,einrpsreioadlcienhceh,aamainnindcoodnaifsocuirdlmfiadintei-obtnhoaneldepenrdotrtceoyipnsyt-,epi(cid:6)rnoSets)ec,i°n,swcainalistnecgrafalictcuselas(teiedxdeclaucshdtainhinge
independent of temperature. According to the model (Reaction 2 and conformationalentropybyitschangeinASAnormalizedtoitsASAin
Equation7),(cid:8)canbeexpressedasafunctionofellipticity,(cid:3)(measured Ala-X-Alatripeptide(54),i.e.(cid:13)((cid:6)ASAscssc°/ASAAla-X-Ala).ssc°wastaken
atsinglewavelength),asshowninEquation9, fromLeeetal.(57).(cid:6)Smix°wascalculatedasa“cratic”term(58)that
reflects the mixing of solvent and solute molecules and effectively ac-
(cid:3)(cid:8) (cid:3) countsfortheentropychangesduetochangesintranslational/rotational
(cid:8)(cid:4) (cid:3) (cid:8) (cid:3)N (Eq.9) degrees of freedom upon binding (54, 55). For the M(cid:7)2 (cid:3) A(cid:7) 7 M(cid:7)2A(cid:7)
D N reactionusinga1Mstandardstate,thisequalsRln(1/55.5).
where(cid:3) and(cid:3) aretheellipticitiesofthenativeanddenaturedstate,
N D
respectively,whichareassumedtobelinearfunctionsoftemperature. RESULTS
InthecaseofDSC,themeasured(cid:6)C (nativestateisareferencestate) StructureofcAbMaz1andItsInteractionwithMazE
P
canbeexpressedas(45–47)asshowninEquation10,
Initially,weraisedanimmuneresponseagainstMazEina
(cid:8)(cid:9)1 (cid:6) (cid:8)(cid:12) ((cid:6)H°(cid:9)T )(cid:3)(cid:6)C °(T(cid:6)T ))2 dromedaryinordertoidentifyV Hdomainsthatcouldbeused
(cid:6)C (cid:5)(cid:8)(cid:6)C °(cid:4) 1⁄2 P 1⁄2 (Eq.10) H
P P n(cid:8)(cid:8)(n(cid:8)1) RT2 as aids for the crystallization of MazE. Cloning the V H rep-
H
14104 MazE-Dromedary Antibody and MazE-DNA Interactions
FIG.2.Panela,overallviewofthecrystalstructureoftheMazE-cAbMaz1complex.TwocAbMaz1molecules(green)areboundtoidenticalsites
onoppositesidesoftheMazEdimer(redandblue).Panelb,stereoviewoftheepitopeonMazErecognizedbycAbMaz1.Residuesinteractingwith
the antibody are shown as ball-and-stick figures. The epitope consists of two stretches (18–24 and 35–40) coming from two different MazE
monomers.Panelc,left,Trp-9(ingreen)ofMazE,whichisinvolvedincreatingtheMazEdimerviaplanarstackingwithitself.TheMazEdimer
consistsoftwomonomerspresentedinlightanddarktones,respectively.ThecAbMaz1isnotshownbecauseitdoesnotcomeclosetoTrp-9.Panel
c,right,Trp-102(ingreen)ofcAbMaz1(lighttone)interactingwithMazEdimer(darktone).ItinteractswithPro-18,Leu-21Ala-24,andThr-20
ofoneMazEmonomerandwithLeu-37oftheother.
ertoireinaphagedisplayvectorandisolatingtheMazEbind- cAbMaz1anddiscussedthestructurewithemphasisonMazE.1
ersresultedintheidentificationofasinglegenefragmentthat Here we analyze the complex in terms of its inter-protein
encodes a V H (cAbMaz1) that interacts with MazE with contacts. All three hypervariable regions (H1, H2, and H3) of
H
highaffinity. cAbMaz1areinvolvedintheinteractionwithMazE.However,
TheaminoacidsequenceofcAbMaz1isshowninFig.1and H1playsonlyamarginalrole(60Å2buried),whereasH2(190
contains all the V H-specific features that distinguish V H Å2 buried) and H3 (270 Å2 buried) have the largest contribu-
H H
domains from classic V domains. We have recently reported tion.IncontrasttomanyotherV Hdomains,theinteractions
H H
the successful crystallization of MazE in complex with involvemainlysidechainratherthanmainchainatoms.Itwas
MazE-Dromedary Antibody and MazE-DNA Interactions 14105
suggested previously (59) that the preferential use of main fromthehydrophobiccoreofMazE(Fig.2b).Theepitoperec-
chainatomsbyV Hdomainscouldcompensateforthelackof ognizedbycAbMaz1consistsoftwopolypeptidestretches.Most
H
contribution to binding from H1 as well as the missing V of the interactions involve the short (cid:8)-helix (18–24), of which
L
domain. about 340 Å2 gets buried upon complexation. The remaining
LikemostothercamelidV Hdomains,theH1conformation 180Å2ofburiedsurfacecomesfromresidues(35–40).Itshould
H
is unique, resembling neither the canonical structures ob- be noted that these two stretches come from two different
servedinclassicalV domainsnoranyoftheH1conformations monomers forming the MazE dimer. Thus, cAbMaz1 specifi-
H
of other camelid V Hs with the known three-dimensional callyrecognizesthedimerandis,assuch,expectedtostabilize
H
structure.TheH2conformation,ontheotherhand,isofcanon- thedimericfoldedconformationofMazE.
ical type 2A and closely resembles that of cabhcg (60) and
cabamb9 (61). This conformation is the one most commonly ThermodynamicsofcAbMaz1BindingtoMazE
observedforcamelidVHH’s. ItcanbeseenfromacrystalstructurethatMazEexistsasa
TheMazEdimercontainstwostructurallyidenticalbinding dimerinasolidstate.Itwasprovenbygelfiltrationin50mM
sites(Fig.2a)fortheantibodyfragment.cAbMaz1recognizesa sodium cacodylic buffer (pH 6.9) that MazE exists as a dimer
cluster of hydrophobic, mainly aromatic residues that extend also in solution and that its dimeric state does not change
under the experimental conditions applied in the binding
studies.
Fluorescence Spectroscopy—Fig. 3a shows raw FL emission
spectrathatcorrespondtothetitrationoftheMazEdimer(M )
2
by cAbMaz1 (A) at 45°C. It can be seen that the intensity
changes more rapidly when the A/M molar ratio (r) is(cid:10)1,
2
whereasatr(cid:14)1thechangesarelesspronounced.TheMazE(cid:3)
cAbMaz1 association is accompanied by a blue shift of FL
maximum. The shift is about 8 nm in comparison with the
corresponding sum of FL spectra of “total MazE” and “total
cAbMaz1”atr(cid:4)1(Fig3a).Atr(cid:4)2theblueshiftisreduced
to (cid:5)5 nm. The FL spectral changes (induced intensity, blue
shift)areaconsequenceofdifferentenvironmentsofTrpresi-
dues in the bound and free state of MazE and cAbMaz1. Ac-
cording to the crystal structure, the ordered part of MazE
contains a single tryptophane (Trp-9) that does not interact
withtheantibodybutisinvolvedincreatingtheMazEdimer
viaplanarstackingwithitself(Fig.2c).BytitrationofMazEto
thebuffersolution,weobservedlineardependenceofFLinten-
sity on MazE concentration with no spectral shift. This is an
additional proof that the oligomeric state of MazE stays the
same in the concentration range used in FL measurements.
cAbMaz1containsthreetryptophanesofwhichone,Trp-102,is
important for binding MazE. In the complex, it interacts hy-
drophobically with Pro-18, Leu-21, Ala-24, and Thr-20 of one
MazE monomer and with Leu-37 of the other (Fig 2c). There-
fore,theblueshiftandtheintensitychangescanbeinterpreted
asarisingfromtheshieldingofTrp-102fromitscontactwith
water. Because of the fact that FL spectral properties accom-
panyingbindingaredifferentatr(cid:15)1andr(cid:14)1andthatboth
cAbMaz1bindingsitesonMazEarestructurallyidentical,the
differencesaremostprobablycausedbystructuralchangesin
MazEand/orcAbMaz1.
BysubtractionofthecontributionsofMazEdimer(M )and
FIG.3. FL spectra accompanying MazE (M2) (cid:1) cAbMaz1 (A) cAbMaz1(A)ateachtitrationpoint,thedifferencespectr2aand
titrationat45°CatA/M molarratiorvaryingbetween0and
3.5.Dottedlinesrepresentt2hespectrauptor(cid:4)1andfulllinesatr(cid:14) the corresponding titration curve at 350 nm (Fig. 3b) were
1.Theboldlinerepresentsasumofthespectraof“total”M and“total” constructed.Observationsofspectraandtitrationcurvessug-
A atr (cid:4) 1 (panel a). The corresponding titration curves2at 350 nm gestthatAbindstoM intwodistinctivebindingmodes.This
2
measuredat25°C((cid:2))and45°C(E)arepresentedinpanelb.Linesare hypothesiswastestedandprovedbyfittingofthemodelfunc-
graphsofthebestfittedmodelfunction(Equation6).Speciationdia-
tion(Equation6)totheexperimentaltitrationcurve.Itcanbe
gramsat25°C(dottedline)and45°C(fullline)calculatedfromthebest
seeninFig.3bthatmodelfunctionwithK andK asadjust-
fittedvaluesofapparentbindingconstants(Reaction1andEquations 1 2
1and2)arepresentedinpanelc.(cid:8)isthefractionofeachMazEspecies. able parameters correlates well with the experimental curves
TABLE I
ComparisonofthebindingparametersK andK
1 2
ApparentequilibriumconstantsK andK accompanyingbindingofthefirstandthesecondcAbMaz1moleculetoMazEdimerobtainedbymodel
1 2
analysisofcalorimetric(ITC),fluorimetric(FL),andspectropolarimetric(CD)titrationcurves.
ITC ITC ITC ITC FL FL CD
T(°C)
25 35 45 55 25 45 45
KK12((MM(cid:8)(cid:8)11))aa 94..99(cid:1)(cid:1)110087 23..77(cid:1)(cid:1)110087 62..81(cid:1)(cid:1)110077 15..79(cid:1)(cid:1)110076 61..75(cid:1)(cid:1)110087 86..39(cid:1)(cid:1)110076 51..91(cid:1)(cid:1)110077
aTherelativeparametererrorsare0.07–0.3forITCand0.15–0.5forFLandCD.
14106 MazE-Dromedary Antibody and MazE-DNA Interactions
FIG.4. CD spectra accompanying MazE (M2) (cid:1) cAbMaz1 (A)
titrationat25°CatA/M molarratiorvaryingbetween0and3.
Dottedlinesrepresentthe2spectrauptor(cid:4)1andsolidlinesatr(cid:14)1.
TheboldlineisthespectraoffreecAbMaz1(A)inits1(cid:1)Msolution.
measured at 25 and 45°C. The obtained K values are about
1
oneorderofmagnitudehigherthanthevaluesofK (TableI),
2
whichstronglysuggeststhatthebindingoftwoAmoleculesto
M isanti-cooperative.Thisallostericeffectcanbeseenmore
2
clearly in speciation diagram (Fig. 3c) calculated from the
fitted K and K values at 25 and 45°C. Up to r (cid:2) 0.5
1 2
lowering of free M concentration is a consequence of M(cid:1) A(cid:1)
2 2
formation, whereas at r (cid:14) 0.5 the M(cid:7) A(cid:7) complex starts to
2 2
form and finally becomes the predominating species in solu-
tionatr(cid:14)1.5(Fig.3c).InourFLtitrationexperimentsthe
initial concentrations of M2 were in the 0.5–1 (cid:1)M range,
whichenabledustoobtainreliableK andK incaseswhen
1 2
theirvalueswerelowerthan(cid:2)108M(cid:8)1.Asignificantdepend-
enceofthemodelfunction(Equation6)onK (K )islostatK
1 2 1
(K2) (cid:14)108 M(cid:8)1 and, therefore, the value of K1 determined at
25°C (Table I) should be considered only as a best lower
estimate of the apparent binding constant.
CDSpectropolarimetry—ThefarUVCDspectrathataccom-
panythetitrationofM byAat25°CarepresentedinFig.4.
2
TheintensityoftheCDsignalaround220nmdecreaseswithr
up to r (cid:2) 1 and increases at r (cid:14) 1. This fully supports the
cAontoclMus2iooncscumrsadineitnwtohdeifcfaesreenotfFbiLndspinegctmroosdcoeps.yTthhaetcbhianndginesgionf el Fa)IGa.n5d.TtyhpeiccaolrrceasloproinmdeitnrgicbMinadziEng(Mis2o)t(cid:3)hecrAmbMataz515(°AC)t(i(cid:2)tr)at(pioann(eplabn)-,
the far UV CD spectra induced by binding correspond to the wherethesolidlinerepresentsthebestfittedmodelfunction(Equation
changesinthesecondarystructureofM and/orAuponforma- 6).(cid:6)Hvaluesareexpressedinkcal/molofaddedA.Thecorresponding
tion of M(cid:1)2A(cid:1) and M(cid:7)2A(cid:7)2. Theoreticall2y, the changes of each wthheerrmeotdhyensaymmibcoplsroEfil(e(cid:6)Gof°M),2(cid:2)(cid:3)((cid:6)AHa°s)s,oacniadti(cid:6)on(Ti(cid:6)sSpr°e)sceonrtreedspinonpdatnoetlhce,
typeofthesecondarystructureuponbindingcanbeestimated bindingofthefirstAmol1ecule,and1(cid:1)((cid:6)G °),f((cid:6)1H °)andŒ(T(cid:6)S °)
fromthefarUVCDspectra.Fig.4showsthatCDspectrumof correspondtothebindingofthesecondon2e.Inthec2aseof(cid:6)H°sol2id
Ahasan“unusual”shape,whichhasoftenbeenobservedinthe llainteesd,awrehelirneeaasrinretghreescsaisoensloinfe(cid:6)sGfr°oamndwhTi(cid:6)cSh°(cid:6)tChPe,i°linvaelsuseesrvweerjueisctaalcsua-
antibody family but never explained in terms of secondary guidetotheeye.Thethermodynami icparameitersofM (cid:3)Aassociation
structure(38).ProteinswithsuchunusualCDspectraarenot asrigid-bodies((cid:6)G °,(cid:6)H °,andT(cid:6)S °)obtainedfro2mthestructure-
rb rb rb
involved in the databases of proteins (with known conforma- basedcalculations(Equations11–13)arepresentedasdottedlines.
tions and CD spectra) used for estimation of the secondary
structure.Therefore,acomparisonofthemeasuredCDspec- advantage over spectroscopic methods, the model function for
trawiththosecalculatedfromthedatabasewouldbemean- description of the ITC signal besides K and K (Table I) also
1 2
ingless. Raw CD spectra (Fig. 4) were analyzed in the same containstwootherthermodynamicparameters,(cid:6)H °and(cid:6)H °
1 2
wayasthecorrespondingFLspectra.Fromtheshapesofthe (apparentstandardenthalpiesofbinding).Thisenabledusto
corresponding titration curves we were able to detect two describethebindingofthefirstandthesecondAmoleculeto
differentbindingmodesofAbothat25and45°C.Becauseof M withathermodynamicprofile(Fig.5c)thatincludesappar-
2
high affinities of A, the starting concentration of M was ent standard Gibbs free energies of binding (cid:6)G ° and (cid:6)G °
2 1 2
chosen as low as possible to get reliable K and K values (Equation 8) and the corresponding apparent standard entro-
1 2
from the model analysis (Equation 6). The changes in CD piesofbinding(cid:6)S °and(cid:6)S °calculatedfromGibbsequation
1 2
signal upon titration were still well measurable at an M ((cid:6)G°(cid:4)(cid:6)H°(cid:8)T(cid:6)S°;i(cid:4) 1,2).Fromthetemperaturedepend-
2 i i i
concentration of (cid:5)1 (cid:1)M (Fig. 4). However, the quality of the ence of (cid:6)H1° and (cid:6)H2° (Fig. 5c) the standard heat capacity
titrationcurvesobtainedbysubtractionwastoolowfortrust- changes(cid:6)C °and(cid:6)C °werecalculatedastheslopesofthe
P,1 P,2
worthymodelanalysis.Therefore,wewereonlyabletoesti- linearregression(cid:6)H°versusTlines((cid:6)C °(cid:4)((cid:11)(cid:6)H°/(cid:11)T) ;i(cid:4)
i P,1 i P
mate K and K from CD titration at 45°C (Table I). 1, 2). Because of the very high affinity binding of the first A
1 2
Isothermal Titration Calorimetry—ITC experiments pre- moleculetoM at25,35,and45°Candtheappliedconcentra-
2
sented in Fig. 5 were performed at 25, 35, 45, and 55°C and tion conditions, almost all added A is bound at r (cid:15) 1 and,
analyzedintermsofthesamemodelasthecorrespondingFL therefore, the evaluation of K by a straightforward fitting
1
andCDtitrations(Reaction1andEquations1,2,and6).Asan procedure was not possible (see also FL spectroscopy). At all
MazE-Dromedary Antibody and MazE-DNA Interactions 14107
TABLE II
Comparisonofthermodynamicparametersofdenaturation
Thermodynamic parametersofMazE(M ),cAbMaz1(A),and their
tetramericcomplex(M(cid:7) A(cid:7) )denaturationob2tainedbymodelanalysisof
2 2
DSCthermogramsandspectropolarimetric(CD)temperatureprofiles.a
T1⁄2 (cid:6)H°(T1⁄2) (cid:6)CP°
DSC CD Calb DSC CD DSC (cid:6)ASAc
°C kcal/mol kcal/molK
M 77.7 76.7 67 68 69 1.5 2.1
2
A 75.5 75.0 110 114 103 2.0 1.7
M(cid:7) A(cid:7) 79.1 79.8 375 333 327 6.0 5.8
2 2
aTherelativeparametererrorsareestimatedtobeabout0.005for
T1⁄b2,M0.o0d5elfoirnd(cid:6)eHp°e(nTd1⁄e2)n,tan(cid:6)dH0.1vfaolru(cid:6)esCPd°etvearlmueins.edbyintegrationofthe
cal
correspondingDSCthermograms.
cThevalueswerecalculatedonthebasisofchangesinsolventacces-
sibleareas(cid:6)ASAfromEquation11.
StabilityofMazE,cAbMaz1,andTheirComplexes
The thermodynamic stability of MazE, cAbMaz1, and their
tetrameric complex was studied by DSC and temperature-de-
pendent CD spectropolarimetry. Usually it is described in
terms of the standard Gibbs free energy change, (cid:6)G°, which
corresponds to the reversible denaturation of a given protein.
(cid:6)G°asafunctionoftemperature,T,wascalculatedfromEqua-
tion8.
DifferentialScanningCalorimetry(DSC)—Thermogramsfor
M , A, and M(cid:7) A(cid:7) (Fig. 6a) were described in terms of the
2 2 2
equilibriumtwo-statemodel(Reaction2andEquation7).For
M and A, the model function (Equation 10) correlates well
2
with the experimental data. Moreover, model-independent
transition enthalpy ((cid:6)H ) values for M and A show good
cal 2
agreementwith(cid:6)H°(T ),indicatingthatthetwo-stateapprox-
1⁄2
imationisapplicablefordescriptionoftheirdenaturationpro-
cesses(TableII).Therefore,theparametersT ,(cid:6)H°(T ),and
1⁄2 1⁄2
(cid:6)C °(TableII)derivedfrommodelanalysisofDSCdatawere
P
used in interpretation of the thermodynamic stability of M
theFirIGt.e6t.raPmaneerlica,coDmSpCletxhe(Mrm(cid:7)2oAg(cid:7)r2a)m. (cid:6)sCoPfMisaezxEp(rMes2s)e,dcAinbMkacaz1l/K(Am)oalnodf and A (Fig. 6b). In the case of M(cid:7)2A(cid:7)2, (cid:6)H°(T1⁄2) is somewha2t
monomeric unit. Full lines represent graphs of the best fitted model lower than (cid:6)H , indicating that the model that takes into
cal
function (Equation 10). Panel b, comparison of the thermodynamic account only native M(cid:7) A(cid:7) and denatured M and A is too
stabilityofM ,AandM(cid:7) A(cid:7) .ThestandardGibbsfreeenergychange 2 2
2 2 2 simple for description of the DSC thermogram. Namely, be-
upondenaturationversustemperaturediagramsisdeterminedonthe
basis of parameters obtained by the analysis of the DSC transition cause of high affinities of A to M2, at applied concentrations
curves (Equations 7, 8 and 10). The dotted line is a sum of stability and lower T M(cid:7) A(cid:7) is practically the only protein species in
curves,M2(cid:3)2A.Panelc,thetransitiontemperature,T1⁄2,asafunction solution.Howev2er,f2romextrapolationofbindingconstantsby
of decadic logarithm of protein concentration, log[P], calculated for
reversibletwo-statedenaturationofM andM(cid:7) A(cid:7) (Equation7). Equation8,itfollowsthatatT,wherethedenaturationoccurs
2 2 2 (Fig.6a),thepresenceofM(cid:1) A(cid:1)aswellasfreeM andAmay
2 2
notbeneglectedanymore.Becausethethermogramisthesum
appliedtemperaturesthefittingwassuccessfulinobtainingall ofthecontributionsofallthespeciesinsolutionandonlythe
othermodelparameters,i.e.K2,(cid:6)H1°,and(cid:6)H2°.At55°Cthe Ademnaotluecrualteiosn,tohfeM(cid:6)(cid:7)H2A°((cid:7)T2in)vooflMve(cid:7)sAth(cid:7)eiisntheirgahcetriotnhaenntphraeldpiyctoefdtwbyo
mvailnueedobfyKt1h(eTamboledeIl)awnaaslylsoiws.eTnhoeugohbttaoinbeedaKccuarantdel(cid:6)yHde°teart- thedescribedanalysis(1E⁄2quatio2n72).Asthedenaturationtran-
55°C in combination with (cid:6)C ° enabled us t1o calculat1e K sitionsofallspeciesoccurinthesameTinterval,thedeconvo-
P,1 1 lutionofthethermogrambasedonmultiplecontributionsisnot
valuesat25,35,and45°CfromEquation8.TableIshowsthat
possible.However,becausetheenthalpyofAbindingtoM was
K valuesarehigherthanK .Theyareingoodagreementwith 2
1 2 determined by ITC, we were able to estimate that the inte-
the values determined by the fitting of FL and CD titration
grated (cid:6)H is (cid:15)5% (experimental error) different from the
curveswiththesamemodel.Thissupportstheconclusionmade denaturatiocanlenthalpyofM(cid:7) A(cid:7) .Apparently,themultiplespe-
in the case of FL and CD that the binding of A to M is 2 2
2 cies contributions affect the shape of the thermogram
anti-cooperative.Furthermore,theanti-cooperativebindingef- ((cid:6)H°(T )) more than the corresponding integrated area
fect is lowered when the temperature rises (the difference ((cid:6)H ).1⁄2Therefore, (cid:6)H instead of (cid:6)H°(T ) was used in the
betweenK1andK2isreduced;TableI).ByextrapolationofK1 calcucallationofthethermcaoldynamicstability1o⁄2fM(cid:7)2A(cid:7)2(Fig.6b).
andK2viaEquation8itcanbeshownthatthisallostericeffect Temperature-dependent CD Spectropolarimetry—The dena-
is lost above 80°C. The binding of both A molecules is highly turation of M , A, and M(cid:7) A(cid:7) (Fig. 7a) was monitored by
2 2 2
exothermicwith(cid:6)H °being1–2kcal/mollowerthan(cid:6)H °.The measuringfarUVCDspectraatdifferenttemperatures.Atthe
1 2
(cid:6)H °and(cid:6)H °valuesbecomemoreexothermicathighertem- end of the M(cid:7) A(cid:7) transition, the corresponding CD spectrum
1 2 2 2
peratures, resulting in negative (cid:6)C ° and (cid:6)C ° values of (Fig.7a)isequaltothesumofindividualCDspectraofdena-
P,1 P,2
(cid:8)0.25(cid:16)0.04and(cid:8)0.18(cid:16)0.01kcal/mol,respectively. tured M and A, suggesting that two M and two A domains
14108 MazE-Dromedary Antibody and MazE-DNA Interactions
dissociate from M(cid:7) A(cid:7) upon melting. The denaturation tem-
2 2
peratureprofiles(Fig.7b)constructedfromthecorresponding
meltingcurvesatasinglewavelengthweredescribedinterms
of an appropriate two state model (Equations 7–9) to obtain
(cid:6)H°(T ) and T values. They are very close to those derived
1⁄2 1⁄2
from DSC (Table II). Despite the fact that the fitted model
functions (Equation 9) display good agreement with experi-
mentalmeltingcurves(Fig.7b)wewereabletoestimate(cid:6)C °
P
onlyfordenaturationofM ((cid:6)C °(cid:4)(1.9(cid:16)0.5)kcal/molK).It
2 P
can be shown by simulation of the model melting curves that
theirshapesdonotdependsignificantlyonparameter(cid:6)C °in
P
thecaseofsharptransitions(high(cid:6)H°(T )),so(cid:6)C °cannotbe
1⁄2 P
obtainedbythemodelanalysisofAandM(cid:7) A(cid:7) meltingcurves.
2 2
BindingofMazEtoItsPromoterDNAandthe
InfluenceofcAbMaz1
To characterize the binding of M to its promoter DNA at
2
25°C, FL spectroscopic titrations and ITC were employed. In
addition,asignificantdifferencebetweenthefarUVCDspec-
tra of free M and M bound to DNA in the 230–220 nm
2 2
wavelengthrangewasobserved.However,duetohighabsorp-
tionofDNA,thespectrabelow220nmwerenotgoodenough
even for qualitative analysis. The M promoter consists two
2
alternatingpalindromesequences(Fig.9a),whichwereprevi-
ously found to be responsible for binding of a MazE-MazF FIG.7. Panel a, far UV CD spectra of tetrameric MazE-cAbMaz1
complexaswellasMazE(M2)alone(9). cthomespulemxo(Mft2(cid:7)hAe2(cid:7)C)Dmesapseuctrreadoffrdomena4t0urtoed9M5°aCzE.Tahned(cid:2)cAsbyMmabzo1ls(2reMp(cid:3)res2eAn)t.
Fluorescence Spectroscopy—M binding to promoter DNA
2 Inset,thecorrespondingmeltingcurveconstructedatsinglewavelength
was characterized by a significant quenching of Trp fluores- (332nm).Panelb,degreeofdenaturation(cid:8)obtainedfromCDmelting
cenceaccompaniedbyasmallblueshiftofFLmaximum(2nm curvesasafunctionofTforM (f),A((cid:2)),andM(cid:7) A(cid:7) ((cid:1)).Fulllinesare
2 2 2
atM /DNAratioof3).TheemissionFLspectrawereanalyzed graphsofthebestfittedmodelfunction(Equation9).
2
inthesamewayasinM (cid:3)Atitrations(Fig.3).Theresulting
2
titrationcurveispresentedinFig.8.Themodelfunctionbased mational effects. Thus, the thermodynamic parameters ob-
onM dimerbindingtotheindependentsitesonDNA(Equa- tainedfrommodelanalysisofFL,CD,andITCsignalscontain
2
tion 6) gave the best agreement for the value of apparent contributionsfrombothdirectbinding(rigidbodyassociation)
binding constant, K (cid:4) (2.8 (cid:16) 0.8)(cid:1)106 M(cid:8)1, and the number of andconformationalchangesofMazEand/orcAbMaz1.Inthis
bindingsitesonDNA,n(cid:4)3.1(cid:16)0.1.Wehavetriedtoemploy studyanattemptwasmadetoseparatethetwocontributions.
morecomplicatedmodels(includingdifferenttypesofbinding It was shown that the structure-based thermodynamic calcu-
sites and cooperativity) for description of FL titration curves. lationsgivereliableestimatesofthermodynamicparametersof
However,thereciprocalcorrelationbetweenadjustableparam- binding in the systems where the conformational effects are
eterswastoohigh,meaningthatthedatacouldbeequallywell negligible (54, 62). Therefore, they were used to describe the
fittedbydifferentsetsofmodelparameters.Becausethephys- ‘‘rigid body’’ association of cAbMaz1 with MazE. In the calcu-
ical meaning of parameters obtained by these models is com- lations,theconformationalstatesofcAbMaz1andMazEwere
pletelylost,westicktoamodelofindependentidenticalsites. definedbythecrystalstructureoftheircomplex.Becauseboth
Thetitrationofthe‘‘control’’(notmazEFrelated)DNAbyM binding sites on MazE dimer are structurally identical, the
2
resulted in the induced FL intensities that are negligible in calculatedparametersarethesameforbindingofthefirstand
comparison to those resulted from the binding of M to the the second cAbMaz1 molecule. Fig. 5c shows that the MazE
promoterDNA(Fig.8). 2 dimer (cid:3) cAbMaz1 rigid body association is an entropy-driven
Isothermal Titration Calorimetry—Calorimetric titration processcharacterizedbyasmallnegative(cid:6)Hrb°andanegative
curvesdescribingthebindingofM2andthecomplexM(cid:7)2A(cid:7)2to (cid:6)CP,rb°.Suchenergeticsmaybeconsideredashydrophobicin
DNAarepresentedinFig.8.ForthesamereasonasFLdata, nature.However,interpretationofrigidbodyassociationsolely
ITCmeasurementswereanalyzedonlyintermsofM (M(cid:7) A(cid:7) ) in terms of hydrophobic effect may be misleading, because
binding to the independent sites on DNA (Equatio2n 6).2Th2e (cid:6)Hrb°,(cid:6)Srb°,and(cid:6)CP,rb°areproducedfromlargecompensat-
modelanalysisdescribesM bindingashighlyexothermicwith ingeffects(Equations11–13).InMazE,thehydrophobiccoreis
2
the enthalpy of binding (cid:6)H° (cid:4) (cid:8)71 (cid:16) 4 kcal/mol, apparent smallbutextendstothesurfaceontwosidesofthedimer(Fig.
bindingconstantK(cid:4)(2.5(cid:16)0.6)(cid:1)106M(cid:8)1,andthenumberof 2b). It is exactly this hydrophobic patch that is recognized by
binding sites for an oligomer, n (cid:4) 3.0 (cid:16) 0.1. Binding of the two cAbMaz1 molecules. In addition, we have also found sev-
M(cid:7) A(cid:7) complex (Fig. 8) is characterized by (cid:6)H° (cid:4) (cid:8)80 (cid:16) 9 eral hydrogen-bonding contacts between MazE and cAbMaz1.
kca2l/m2ol, K (cid:4) (1.9 (cid:16) 0.6)(cid:1)106 M(cid:8)1 and n (cid:4) 3.2 (cid:16) 0.3, values Thesmallexothermic(cid:6)Hrb°(Equation12)valueshowsthatits
which are very similar to those obtained in the case of free partthatisassociatedwiththehydrogen-bondingpolargroups
M . The heat effects accompanying the M (cid:3) ‘‘control DNA’’ is only slightly dominant over the unfavorable (endothermic)
2 2
titrationarenegligibleincomparisontotheeffectsthatcor- non-polar contribution. The largest contribution that makes
respond to the M2 (cid:3) promoter DNA titration (Fig. 8). rTi(cid:6)giSdrbb°o(dEyqcuoamtipolnex13c)omtheesmfraojmorhdyrdivrionpghfoobricceeifnfesctta(b(cid:6)iSlizin°g).tOhne
sol
DISCUSSION the other hand, the ‘‘freezing’’ of side chains ((cid:6)S °) and the
sc
MazE-cAbMaz1 Interactions—The observed changes in FL loweringoftranslational/rotationaldegreesoffreedom((cid:6)S °)
mix
and CD spectra upon MazE (cid:3) cAbMaz1 titrations strongly uponbindingcausetheentropiclossthatreducesthe(cid:6)S °to
rb
suggest that their association is accompanied also by confor- nearly half of the (cid:6)S ° value. In some studies, much higher
sol
MazE-Dromedary Antibody and MazE-DNA Interactions 14109
FIG.8.FL(E)andcalorimetric((cid:2))titrationcurvesaccompa-
nyingDNA(cid:1)MazE(M )associationat25°Cincomparisonwith
correspondingcontrol2(unrelated)DNA(cid:1)M FL(‚)andITC(Œ)
curves.ITCcurveforDNA(cid:3)MazE-cAbMaz1(M2(cid:7) A(cid:7) )bindingatthe
sametemperatureispresentedbyblacksquares(f2).2Dottedlinesare
graphsofthebestfittedmodelfunction(Equation6).ThedifferenceFL
at360nm((cid:6)F )asafunctionofM /DNAmolarratio,r,wasobtained
360 2
fromthecorrespondingemissionspectra.Qisacumulativeheateffect
ateachtitrationpointexpressedinkcal/molofaddedM (M(cid:7) A(cid:7) )per
2 2 2
injection.
values for the loss of translational/rotational entropy were
takenintoaccount(63,50);however,ithasbeensuggestedthat
they are overestimates (54, 64, 65). It was shown that when
(cid:6)S ° and (cid:6)S ° are taken into account the cratic correction
sol sc
((cid:6)S °) accounts well for the loss of translational/rotational
mix
entropyuponrigidbodyassociation(54,55).
By contrast, the overall thermodynamic profiles obtained
frommodelanalysisofFL,CD,andITCdata(Fig.5c)indicate
thatthebindingofbothcAbMaz1moleculestoMazEdimeris
an enthalpy-driven process accompanied by unfavorable en-
tropiccontributions.Fig.5showsthattheoverall(cid:6)G°isinthe FIG.9.Panela,45bpofidealB-DNAshowingthealternatingpalin-
samerangewith(cid:6)G °.However,(cid:6)H°ismuchmorei exother- drome structure (9) found in the MazE promoter. Panel b, model for
rb i MazEbindingtoitspromoterDNA.BecauseofthelengthoftheDNA,
micthan(cid:6)Hrb°,indicatingthatadditionalenergeticallyfavor- largespacesremainbetweentheindividuallyboundMazEdimers,in
ablecontacts(duetostructuralalternations)areformedupon agreementwithindependentbinding.Sufficientroomisavailablefor
cAbMaz1bindingtoMazE.Thefavorableenthalpydifferenceis positioningMazFand/orthedisorderedpartofMazE.Panelc,equiva-
compensated by a large conformational entropy loss (T(cid:6)S° (cid:15) lentmodelofthebindingoftheMazE-cAbMaz1complextothemazEF
i promoter DNA. In this model, the antibody fragments touch neither
T(cid:6)S °). Thermodynamic studies of biomolecular associations
rb eachotherortheDNA.
and protein unfolding have demonstrated that (cid:6)C ° may be
P,i
considered as the most reliable distinctive feature of site-spe-
cific binding (49–52, 66–68). For cAbMaz1 binding to MazE MazEpromotercomplexes)mayberatherlow.Asloweringthe
(cid:6)C °(cid:3)(cid:6)C °,thevalueof(cid:8)0.43kcal/molKobtainedbyITC concentrationshiftstheT (Fig.6c)tolowervalues,itfollows
P,1 P,2 1⁄2
issignificantlylowerthanthecorresponding2(cid:6)C °valueof that, under physiological temperatures, the free MazE might
P,rb
(cid:8)0.28kcal/molK,whichisinaccordancewiththeremovalof bepartiallydissociatedandunfolded.Thenativedimer-dena-
large amounts of nonpolar surface from water and with the tured monomer equilibrium may therefore play an important
structural alternations MazE or/and cAbMaz1. The observed roleinregulationofthemazEFsystemasawhole,becauseitis
bindinganti-cooperativity,duetothestructuralalterationsof known that fluctuations in the concentrations of the system
the involved proteins, is also visible from the thermodynamic products may result in cell death (28). The thermodynamic
parameters. ITC results show that (cid:6)H ° (cid:14) (cid:6)H ° (more ener- stability of the cAbMaz1 was also compared with the corre-
2 1
getically favorable contacts or/and less unfavorable contacts sponding (cid:6)G° values of some other members of the camelid
are formed upon binding of the first cAbMaz1 molecule) and V Hantibodyfamilydeterminedat25°C.The(cid:6)G°valueofour
H
(cid:6)C ° (cid:14) (cid:6)C ° (larger part of a non-polar surface is buried cAbMaz1at25°Cis8.7kcal/mol,whichplacesitsomewherein
P,2 P,1
or/andmorepolarsurfaceisexposeduponbindingofthefirst the middle of the list of V H stabilities (38, 39). However, its
H
cAbMaz1molecule). transition temperature T (Table II) is among the highest
1⁄2
Thermodynamic Stability of MazE, cAbMaz1, and Their observedforanyV H(38,39).ThehighT andhighdegreeof
H 1⁄2
Complexes—The stability curves ((cid:6)G° versus T) of MazE, reversibility (0.85) are the properties of V H that may be
H
cAbMaz1, and their tetrameric complex are presented in Fig. highly appreciated in processes where transient heating may
6b. The comparison of the stability curve of MazE to that of takeplace.Fig.6bdisplaystheextenttowhichMazEdimeris
anotheraddictionantidote,CcdA(32),showsthatbothproteins stabilized by the binding of two cAbMaz1 molecules. The dif-
haveverysimilarthermodynamicstability.BecauseMazEand ference between the curve obtained as a sum of (cid:6)G° versus T
thetoxinMazFareco-expressed,andtheirexpressionisauto- curves for MazE and cAbMaz1, and the curve of their tet-
regulatedontheleveloftranscription(6,9),thetotalconcen- rameric complex is a measure of apparent binding affinity of
trationofMazEinthecellwillneverbehigherthanacertain thetwocAbMaz1molecules((cid:6)G °(cid:3)(cid:6)G °).(cid:6)G °(cid:3)(cid:6)G °values
1 2 1 2
levelsetbyequilibriumconstantsofMazE-MazFpromoterand obtainedfromDSCareingoodagreementwiththoseobtained
MazE promoter complex formation and the concentration of by model analysis of the ITC, FL, and CD titration curves
promoterDNA.Consequently,theconcentrationoffreeMazE (TableI,andFig.5c).AscAbMaz1isalsorelativelysmalland
(not bound in the MazE-MazF, MazE-MazF promoter, and wellsoluble,itprovedtobeanidealcrystallizationandphasing
14110 MazE-Dromedary Antibody and MazE-DNA Interactions
aidforMazE.1ItfollowsthatV Hsmaybeapplicablealsofor unit, we see that all MazE molecules are too far apart from
H
stabilization of other proteins with short shelf life and rela- each other to directly interact. This is in full agreement with
tivelylowthermodynamicstability. our model of three non-interacting, quasi-equivalent binding
Thegeneralarchitectureofglobularproteinsissuchthata sites(Fig.9b).Sufficientspaceisavailablebetweenthebound
hydrophobiccoreissurroundedbyhydrophilicshell.Thiscom- MazE dimers to position the otherwise unstructured MazE
partmentalizationisthemaindrivingforceoftheproteinfold- domain (which we assume to become more structured upon
ing.Toqualitativelydescribetowhatextentburiedhydrophilic DNAbinding).ThespacingbetweentheboundMazEdimersis
groups as well as exposed hydrophobics destabilize the folded also of the right magnitude to allow them to be bridged by
conformationofthestudiedproteins,wecomparedtheexperi- MazFdimers.InthepresenceofboundcAbMaz1,noadditional
mental(cid:6)C °valueswiththosecalculatedfromthechangesin protein-DNA contacts or steric hindrance of any sort is ob-
P
the non-polar and polar solvent accessible surface areas upon servedinourstructuralmodel(Fig.9c),whichisalsoinagree-
denaturation(Equation11).Thecalculated(cid:6)C °valuesforthe mentwiththeexperimentalresults.
P
denaturation of the tetrameric MazE-cAbMaz1 complex and Thegeneralpropertyofautoregulationoftranscriptioninall
cAbMaz1 agree well with the experimental ones, whereas in described addiction systems is that the antidote is the main
the case of MazE, the calculated (cid:6)C ° is significantly higher DNA-binding protein of which affinity to promoter DNA is
P
(Table II). As the opposite effect was observed upon MazE- significantly enhanced in the presence of the toxin. On the
cAbMaz1binding,thisdiscrepancymaybeascribedtothemore otherhand,thedetailssuchasthenumberofpalindromeDNA
non-polar(or/andlesspolar)residuesexposedtosolventinthe sequences involved in binding, the protection of DNA against
MazEnativestateinthesolutionthanispredictedbyMazE- DNaseIbythecomplexandalsobytheantidotealone,andthe
cAbMaz1crystalstructure. lengthoftheprotectedregiondifferfromsystemtosystem.In
Binding of MazE to the Promoter DNA—It was reported the case of phd-doc, under physiological conditions unfolded
recently, that two alternating palindrome sequences in the Phd monomers are stabilized by binding to promoter DNA, a
promoter (Fig. 9) are crucial for DNA binding of the MazE- process that is accompanied by dimer formation (10–11). The
additional stabilization of Phd by dimer formation is most
MazFcomplexandthusfortheregulationofMazEandMazF
probablythereasonwhyPhdbindingtoeachofthetwodistinct
expression (9). Footprint analysis of the promoter revealed
palindromesismoreenergeticallyfavorablethanthedescribed
protection of DNA against DNase I by the MazE-MazF com-
binding of MazE, where the dimers already exist in solution.
plex. On the other hand, it was suggested that the binding of
CcdA and ParD (the antidote from parDE system) are, like
MazE to DNA is weaker than the binding of the MazE-MazF
MazE,dimericproteinsinthemicromolarconcentrationrange
complex (9). In the present work, we were able to estimate
at physiological temperatures. Under these conditions they
thermodynamicparametersofMazEassociationonthebasisof
bind to DNA as dimers. However, the binding affinities and
a simple model (Reaction 1 and Equations 1 and 6), which
other thermodynamic parameters have not been reported
showsthatuptothreeMazEdimersareboundatthepromoter
sequenceandthatthebindingishighlyexothermic((cid:6)H°(cid:4)(cid:8)71 (32–34).
kcal/mol) with the apparent binding constant in the micro- Acknowledgments—We acknowledge the use of synchrotron beam
molarrange((cid:6)G°(cid:4)(cid:8)RTlnK(cid:4)(cid:8)8.7kcal/mol).MazE-promoter timeattheEuropeanMolecularBiologicalLaboratory(EMBL)beam-
DNAinteractionsmaybecharacterizedasspecific,becausethe lines at the DORIS storage ring (Hamburg, Germany) and the Euro-
inducedITCandFLsignalsuponMazE(cid:3)controlDNAtitra- pean Synchrotron Radiation Facility (ESRF; Grenoble, France). We
thankJorisMessensandKarolienVanBelleforpurifyingtheMazE-
tion were negligible in comparison to those resulted from the cAbMaz1 complex and performing quantitative gel filtration experi-
MazE binding to the promoter DNA. Rather low K can be ments with MazE. Professor Gorazd Vesnaver from University of
ascribed to the highly unfavorable entropic contribution Ljubljana(wheretheDSCmeasurementswereperformed)andPatrick
(T(cid:6)S°(cid:4)(cid:6)H°(cid:8)(cid:6)G°(cid:4)(cid:8)62kcal/mol).AsDNAisrelativelyrigid Van Gelder are gratefully acknowledged for critical reading of the
manuscriptandusefulsuggestions.
and the displacement of water from interacting surfaces is
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