Calorimetric and Crystallographic Analysis of the Oligomeric Structure of Escherichia coli GMP...

doi:10.1016/j.jmb.2005.07.042 J. Mol. Biol. (2005) 352, 1044–1059

Calorimetric and Crystallographic Analysis of theOligomeric Structure of Escherichia coli GMP Kinase

Guillaume Hible1†, Louis Renault1†, Francis Schaeffer2†Petya Christova1,3, Adriana Zoe Radulescu4, Cecile Evrin5

Anne-Marie Gilles5* and Jacqueline Cherfils1*

1Laboratoire d’Enzymologie etBiochimie Structurales, CNRSGif sur Yvette 91198, France

2Unite de Biochimie StructuraleURA2185 CNRS, InstitutPasteur, Paris 75724, France

3Institute of Organic ChemistryBulgarian Academy of SciencesSofia-1113, Bulgaria

4Laboratory of Enzymology andApplied MicrobiologyCantacuzino Institute, 70100Bucharest, Romania

5Unite de Genetique desGenomes Bacteriens, URA2171CNRS, Institut Pasteur, Paris75724, France

0022-2836/$ - see front matter q 2005 E

†G.H., L.R. and F.S. contributed eAbbreviations used: GMPK, guan

phate kinase; NMPK, nucleoside moecGMPK, Escherichia coli GMPK; mGyGMPK, yeast GMPK; apo-GMPK,GMPK; DSC, differential scanning cdynamic light-scattering; EGS, ethynimidyl) succinate; TS, transition stE-mail addresses of the correspon

[email protected]; [email protected]

Guanosine monophosphate kinases (GMPKs), which catalyze the phos-phorylation of GMP and dGMP to their diphosphate form, have beencharacterized as monomeric enzymes in eukaryotes and prokaryotes. Here,we report that GMPK from Escherichia coli (ecGMPK) assembles in solutionand in the crystal as several different oligomers. Thermodynamic analysisof ecGMPK using differential scanning calorimetry shows that the enzymeis in equilibrium between a dimer and higher order oligomers, whoserelative amounts depend on protein concentration, ionic strength, and thepresence of ATP. Crystallographic structures of ecGMPK in the apo, GMPand GDP-bound forms were solved at 3.2 A, 2.9 A and 2.4 A resolution,respectively. ecGMPK forms a hexamer with D3 symmetry in all crystalforms, in which the two nucleotide-binding domains are able to undergoclosure comparable to that of monomeric GMPKs. The 2-fold and 3-foldinterfaces involve a 20-residue C-terminal extension and a sequencesignature, respectively, that are missing from monomeric eukaryoticGMPKs, explaining why ecGMPK forms oligomers. These signatures arefound in GMPKs from proteobacteria, some of which are humanpathogens. GMPKs from these bacteria are thus likely to form the samequaternary structures. The shift of the thermodynamic equilibriumtowards the dimer at low ecGMPK concentration together with theobservation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be theactive species at physiological enzyme concentration.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: nucleoside monophosphate kinase; differential scanningcalorimetry; X-ray crystallography; guanosine monophosphate kinase;oligomerization
*Corresponding authors
Introduction

Guanosine monophosphate kinases (GMPK,ATP:GMP phosphotransferases, EC 2.7.4.8) belongto the nucleoside monophosphate kinase (NMPK)

lsevier Ltd. All rights reserve

qually to this work.osine monophos-nophosphate kinase;MPK, mouse GMPK;nucleotide-freealorimetry; DLS,lene glycol bis(succi-ate.ding authors:rs-gif.fr

superfamily, and are essential for maintaining theoverall concentration of GTP, for synthesis of RNAand DNA and for recycling the second messengercGMP.1,2 They catalyze the reversible phosphoryl-ation of GMP and dGMP to their diphosphateform using ATP as a preferred phosphate donor.GMPKs are ubiquitous in prokaryotic and eukary-otic cells as cytosolic enzymes and are of medicalimportance for the activation of guanosine analogpro-drugs used in cancer and viral infectiontherapies.3 Crystal structures have been solved forSaccharomyces cerevisiae GMPK in the nucleotide-free form and in complex with GMP (referred tohere as apo-yGMPK and yGMPK$GMP, respect-ively)4,5 and for mouse GMPK in an abortivecomplex with ADP and GMP (mGMPK$GMP$

d.

Oligomeric E. coli GMP Kinase 1045

ADP).6 The enzymes from both species have thecommon fold of NMPKs, comprised of threedomains termed the CORE, NMP-binding andLID domains.7,8 The catalytic site assembles uponmotion of the LID and NMP-binding domains ontothe CORE domain, which is induced by the bindingof ATP and GMP, and allows the substrates to comeinto contact and the phosphoryl transfer reaction totake place.5,6

Like most NMPKs, GMPKs from eukaryotes andprokaryotes that have been studied so far aremonomeric enzymes, including the yeast,4,5

mouse6 and Mycobacterium tuberculosis9 enzymes.In contrast, Escherichia coli GMPK (ecGMPK) wasreported to form oligomers based on sedimentationequilibrium experiments10 and gel-filtration chro-matography.11 This oligomer was predicted to be atetramer and proposed to dissociate into dimers byincreasing the ionic strength.11 Here, we report athermodynamic and crystallographic analysis ofthe quaternary structure of ecGMPK. The thermalstability of ecGMPK was determined in solution bydifferential scanning calorimetry (DSC), suggestingthat the protein is in equilibrium between dimeric,tetrameric and hexameric species. At higher proteinconcentrations, both dynamic light-scattering (DLS)and chemical cross-linking were consistent with ahexameric species. Crystal structures of ecGMPKwere solved in its nucleotide-free, GMP-bound andGDP-bound forms from three different crystalforms. The enzyme is a hexamer with D3 symmetryin all crystal forms, in which substrate-induceddomain motions can take place. Taken together, thesolution and crystal data point to the dimeric formas the folding unit of ecGMPK, the dimeric interfacebeing mediated by a C-terminal extension unique toecGMPK and GMPKs from related bacteria.

Results

The oligomeric structure of ecGMPK in solution

Recombinant ecGMPK was stable for severalmonths at K20 8C after purification by BlueSepharose and gel-permeation chromatography.The molecular mass of the monomer determinedby electrospray ionization-mass spectrometry(ESI-MS) (23,462(G1.41) Da) corresponded to thatcalculated from the sequence, assuming that theN-terminal methionine residue is cleaved. GMPkinase activity after gel-permeation chromato-graphy was recovered in fractions correspondingto different molecular masses depending on theionic strength of the elution buffer (data not shown).At low ionic strength (!0.1 M NaCl or KCl), theactivity was recovered in fractions predicted tocontain a tetramer based on standard molecularmass markers. At higher ionic strength (O0.25 MNaCl or KCl), fractions were predicted to contain adimer based on standard molecular mass markersand on their co-migration with dimeric thymidinemonophosphate kinase (TMP kinase) from E. coli

K-12 whose monomer has a molecular mass similarto that of the ecGMPK monomer. The catalytic ratesof purified ecGMPK for the forward and reversereactions are 188 sK1 and 55 sK1, respectively,which is in the same range as that of other bacterialand eukaryotic GMPKs.9 We also observed that theenzyme does not display cooperativity towards itsGMP substrate (data not shown).The oligomeric structure of ecGMPK was inves-

tigated by analyzing the enzyme thermal stability indilute solution by DSC. Experiments were per-formed at ecGMPK monomer concentrations in the2–18 mM range at low and high ionic strength. At0.1–0.25 M NaCl (pH 7.4), the Cp curve wasasymmetric with a gradual increase in heat uptakeat low temperature followed by a sharp transitionpeak at high temperature, thus revealing a complexunfolding process (Figure 1). The reversibility of theunfolding process was checked by reheating theprotein sample after it had been cooled to roomtemperature in the calorimeter. Two typical succes-sive scans at 4.3 mM (100 mg/ml) are shown inFigure 1(a). The unfolding was found to bequantitatively reversible in the protein concen-tration range studied provided that the samplewas quickly cooled to 20 8C immediately after thetemperature of 70 8C was reached and left forseveral hours at this temperature prior to reheating.Reversibility of 80% is indicated by the percentagechange in the area under the curves (i.e. DHcal;Figure 1(a)). Protein unfolding reactions that dis-play such small changes are generally consideredreversible,12 thus validating our application ofequilibrium thermodynamics to analyze the data.The construction of the chemical baseline is

illustrated in Figure 1(a). The pre and post-transition baselines were evaluated by linear least-squares treatment of the data obtained at the native(below 40 8C) and the unfolded (above 62 8C) states.The post-transition baseline has a negative slope inall experiments, indicating that the apparentspecific heat decreases with increasing the tem-perature. This unusual behavior may result fromaggregation of unfolded species. However, theslope of the post-transition baseline is even morenegative when decreasing the protein concentration(not shown), indicating that this explanation isunlikely. A negative slope for the post-transitionbaseline has been observed for other proteins, buttheir origin is not understood.13,14 Since theunfolding process in Figure 1(a) presented a smallchange in the apparent specific heat, the excessspecific heat absorption of GMPK at 0.25 M NaClwas analyzed after subtraction of the chemicalbaseline.15,16 The Cp,ex curve could be preciselycalculated by fitting the data to a model ofindependent non-two-state transitions as describedin Materials and Methods. The best estimates of Tm,DHcal and DHvH, obtained from fitting the DSC datain Figure 1(b) by three-parameter (Tm, DHcal andDHvH) non-linear regression, are given in Table 1.The Cp,ex profile presented two unfolding transitions(Figure 1(b)). The ratio between the calculated DHvH

Figure 1. Reversibility of ecGMPK unfolding anddeconvolution of the excess heat absorption of GMPK athigh salt. (a) Two consecutive DSC scans at 302 mg/ml ofecGMPK (12.8 mM in terms of monomer) and a scan rate of1 deg. C/min in solutions of 50 mM Pipes (pH 7.4), 0.25 MNaCl. The continuous and dotted lines are experimentalcurves after subtraction of the instrumental baseline. Theconstruction of the chemical baseline is illustrated: thedotted straight segments are extrapolations of pre andpost-translational baselines, and the broken curve represents thesmooth transition between the extremes, weighted at eachtemperature according to the integrated area of the DSCcurve up to that temperature. Integration of the excess heatcapacity curves (the areas between the Cp curves and thechemical baseline) gives total calorimetric enthalpies(DHcal) of 107 kcal molK1 and 91 kcal molK1 for the first(continuous line) and second (dotted line) scans indicatingthat the unfolding is approximately 80% reversible afterheating to 70 8C. (b) Resolution of the excess heat capacitycurve for ecGMPK into two non-two-state transitions withzeroDCp. The continuous line represents the first DSC scanafter subtraction of the chemical baseline. Lines with filledand open squares represent the individual thermaltransitions obtained by non-linear least-squares fit of thedata to the non-two-statemodel. The calculated excess heatcapacity curve is shown in thin line. In (b), the inset showsthe enthalpy contributions of the two thermal transitions tothe total enthalpy of unfolding as a function of the totalmonomer concentration. Symbols are experimental points,broken lines are calculated considering a dimer–tetramerequilibrium with no free monomer and a dissociationconstant (Kd) of 10 mM. Thermodynamic parameters aregiven in Table 1.

1046 Oligomeric E. coli GMP Kinase

(the heat change per unfolding unit) and themeasured DHcal (the heat change per mole ofmonomer) values provided further information onthe denaturation process. The fact that the van’tHoff enthalpy is larger than the calorimetricenthalpy can only be accounted for by a highdegree of intermolecular cooperation, since in theabsence of such cooperation the van’t Hoff enthalpycannot exceed the calorimetric value. The DHvH/DHcal ratio of 2.2 and 3.8 for respectively the lowand high temperature transitions suggested thatecGMPK exists as a dimer of lower thermal stabilityand a tetramer of higher thermal stability, dis-sociation and unfolding being coupled reactions foreach species. It should be noted that the van’t Hoffenthalpies used here are the true van’t Hoffenthalpies determined by using all of the data todefine the temperature-dependence of the unfold-ing equilibrium, rather than the often-used effectivevan’t Hoff enthalpy, which depends too strongly onthe value of a single data point (i.e. Cp,max

14). Theoligomeric nature of ecGMPK was further con-firmed by the observation that the contribution ofeach unfolding transition to the total heat ofunfolding (DHcal of a transition in % of the totalcalorimetric enthalpy of denaturation DHcal,tot,DHcal,totZ107 kcal molK1 at 0.25 M NaCl) variedwith protein concentration with low enzymeconcentration favoring the low-temperature tran-sition (Figure 1(b), inset). These data could be cal-culated considering a simple dimer/tetramerequilibrium with a dissociation constant (Kd) of10 mM (total ecGMPK monomer concentrations inthe DSC runs were between 0.1 Kd and 1.3 Kd, seeMaterials and Methods).

Similar experiments were performed withoutadded salt (Figure 2). Complete denaturation ofthe protein was not reversible due to aggregation atthe unfolded state. However, Cp profiles withoutsalt were analyzed in terms of equilibrium thermo-dynamics as described.17,18 These studies showedwith various proteins that the effects of variations ofprotein concentration on denaturational parameterscan be accurately and consistently expressed interms of the van’t Hoff equation:

d ln K=dT ZDHvH=RT2 (1)

where K is the equilibrium constant for thedenaturation and DHvH is the (van’t Hoff) enthalpy,which determines the variation of K with absolutetemperature. Furthermore, when a protein under-goes a reversible denaturation followed by anirreversible step, the data for such a system can beanalyzed according to the van’t Hoff equation togive parameters close to those originally assigned tothe reversible system.17,18 At pH 7.4 in the absenceof NaCl the Cp profile of ecGMPK was clearlyasymmetric, being sharper on the high-temperatureside than on the low-temperature side (Figure 2(a)).A possible cause for this type of asymmetry is adecrease in the extent of oligomerization during thetransition.17 The excess heat absorption of ecGMPK

Table 1. Thermal unfolding parameters for ecGMPK

Tm (8C) DHcal (kcal molK1) DHvH (kcal molK1) DHvH/DHcal

No NaCl, no ATP 49.5G0.8 27.3G4.5 58.2G5.9 2.1G0.554.9G0.2 47.8G3.7 165.7G15.7 3.5G0.658.0G0.1 30.8G3.7 201.2G4.9 6.5G0.9

No NaCl, 133 mM ATP 52.2G0.4 38.9G3.1 76.8G4.5 2.0G0.358.0G0.1 67.5G2.6 246.8G8.7 3.7G0.3

250 mM NaCl, no ATP 54.1G0.3 36.8G3.4 79.2G4.5 2.2G0.359.3G0.1 70.3G2.8 265.5G5.5 3.8G0.2

Figure 2. Deconvolution of the excess heat absorptionof ecGMPK at low salt. (a) The experimental heat capacitycurve at 302 mg/ml of ecGMPK (12.8 mM in terms ofmonomer) at a scan rate of 1 deg. C/min in solutions of50 mM Pipes (pH 7.4) without added salt. The continuousline represents the experimental curve after subtraction ofthe instrumental baseline. The construction of thechemical baseline is illustrated as described for Figure 1.(b) Resolution of the excess heat capacity curve forecGMPK into three non-two-state transitions with zeroDCp. The continuous line represents the experimentalheat capacity curve after subtraction of the chemicalbaseline. Broken lines with filled and open squares andfilled triangles represent the individual thermal tran-sitions obtained by non-linear least-squares fit of the datato the non-two-state model. The calculated excess heatcapacity curve is shown in thin line. In (b), the inset showsthe relative enthalpy contribution of each thermaltransition to the total enthalpy of unfolding. Symbolsare experimental points and the broken lines are linearregression analyses of the data. Thermodynamic para-meters are given in Table 1.


could be precisely predicted using the non-two-state denaturation model. Analysis into threetransitions gave significantly better fits to theexperimental data than those obtained with onlytwo transitions (c2 was reduced 25-fold between fitswith two and three transitions; Figure 2(b); Table 1).The DHvH/DHcal ratio of 2.1, 3.5, and 6.5 for the low,medium and high-temperature transitions, respect-ively, suggested that ecGMPK exists in solution asdimeric, tetrameric, and hexameric species ofincreasing thermal stabilities, dissociation andunfolding being coupled reactions for each species.The oligomeric nature of ecGMPK was furtherconfirmed by the observation that the contributionof each unfolding transition (DHcal %) to the totalunfolding energy (DHcal,totZ105.9 kcal molK1) var-ied with protein concentration. Increasing ecGMPKmonomer concentration from 2 mM to 18 mMdecreased the contribution of the low-temperaturetransition and increased the contributions of themedium and high-temperature transitions(Figure 2(b), inset).In order to validate our analysis of a non-reversible

system using equilibrium thermodynamics, DHvH

was also evaluated by an independent method.12,17 Itcan be shown for the process in which an oligomericprotein Pn undergoes dissociation:

Pn4nD (2)

that if the degree of oligomerization of Pn decreasesduring aDSCupscan, the value ofTm should increasewith protein concentration. For the case representedin equation (2), a van’tHoff plot of ln (P)0 versus 1/Tm,(P)0 being the total protein concentration, will have aslope:12,18,19

S ZKDHvH=ðnK1ÞR (3)

Values of DHvH for each transition as derived fromthe van’tHoff plots in Figure 3werewithin 15–25%ofthe corresponding values of DHvH in Table 1, exceptfor the high-temperature transitions at low and highsalt concentration where they were within 30–50%.This acceptable agreement gives further support tothe DHvH /DHcal ratio values calculated by deconvo-lution of the excess heat capacity curves (Table 1) andto our application of equilibrium thermodynamics tothe irreversible unfolding of oligomeric ecGMPK.The effect of ATP, added in tenfold excess, on the

oligomeric equilibrium of ecGMPK in the absenceof NaCl was also analyzed. The Cp profile closelyresembled that of ecGMPK at 0.25 M NaCl (Cp

Figure 3. van’t Hoff plots of the logarithm of the totalconcentration of ecGMPK versus 1000/Tm. van’t Hoffplots are shown for the independent transitions 1 and 2 at(a) 0.25 M NaCl and (b) 1, 2, and 3, without added NaClobserved by resolution of the DSC curves. Tm is thetemperature at which unfolding is half completed (TmZT1/2), (GMPK)0 is the total protein molar concentration.

Figure 4. Chemical cross-linking of ecGMPK analyzedby SDS-PAGE. EcGMPK at 360 mM (lanes 3–5) wasincubated with (lanes 3 and 4) or without (lane 5) EGSat 20 mM. Lane 4 is with 250 mM NaCl. The predictednumber of cross-linked monomers is indicated at theright. Lanes 1 and 6 show molecular mass markers andlane 2 EGS-treated monomeric M. tuberculosis GMPK(22 kDa) at 280 mM.


curve not shown). Thermodynamic parameters ofthe observed transitions using a non-two-statemodel for deconvolution are given in Table 1. TheDHvH/DHcal ratios of 2.0 and 3.7 for, respectively,the low and high-temperature transitionssuggested that ATP binding stabilized thedimer and tetramer species. Indeed, Tm valuesfor the low and high-temperature species wereincreased by 2.7 deg. C and 3.1 deg. C, respect-ively, compared in the same experiment in theabsence of ATP. DHcal values for unfolding ofthe ATP-bound dimer and tetramer werebetween those observed with the free proteinwithout NaCl and at 0.25 M NaCl. Finally, it isnoteworthy that fitting the calorimetric data witha sequential model, which is more appropriatein the case of strongly interacting structuraldomains, leads to virtually identical results (notshown). Such a situation is often observed whenthe difference in Tm of two transitions is sufficientlylarge, as is the case here (Table 120,21).

The quaternary structure of ecGMPK in solutionat higher enzyme concentrations was investigatedby dynamic light-scattering (DLS) and cross-link-ing. DLS of ecGMPK at 360 mM, a concentration inthe range used for crystallization, indicated that theenzyme is mostly monodisperse, with a hydrodyn-amic radius that was independent from the saltconcentration andwas compatiblewith anhexamericstructure (data not shown). Chemical cross-linkingwith ethylene glycol bis(succinimidyl succinate)(EGS) at the same enzyme concentration caused theappearance of five adducts of molecular masscompatible with two, three, four, five and six cross-linked monomers, as expected for a hexamericstructure (Figure 4). Addition of 250 mM NaCl inthe assay did not significantly modify the nature oramount of cross-linked species.

Overall fold and quaternary structure of ecGMPKin three crystal forms

The crystal structures of ecGMPK in complexwith GDP (ecGMPK$GDP), GMP and SO2K

4(ecGMPK$GMP) and in the nucleotide-free form(apo-ecGMPK) were solved by the molecularreplacement method at 2.4 A, 2.9 A and 3.2 Aresolution, respectively (crystallization conditions,data collection and refinement statistics summar-ized in Table 2). Despite the crystals belonging tothree different space groups (P63, R32 and P41212)with, respectively, two, one and six molecules in theasymmetric unit, ecGMPK assembles as an hexamerwith dihedral D3 symmetry in all crystal forms


(Figure 5). The hexamer corresponds to the asym-metric unit for apo-ecGMPK, or can be reconstitutedby crystal symmetries for ecGMPK$GMP andecGMPK$GDP. The ecGMPK subunit has, however,the characteristic fold of monomeric GMPKs fromyeast and mouse4–6 and from M. tuberculosis,9 con-sisting of a CORE, LID, and NMP-binding domains.These domains have different positions in apo-ecGMPK, ecGMPK$GMP and ecGMPK$ GDP, indi-cating that substrate-induced domain closure cantake place within the hexamer (see below).

The ecGMPK hexamer can be described as threedimers related by 3-fold symmetry, thus definingtwo different interfaces along the 2-fold and 3-foldaxis (Figure 5(a) and (b)). The symmetrical dimericinterface buries an extensive 3200 A2 surface area,and involves the LID, CORE and C-terminal regions(Figure 5(c)). It features both hydrophobic patchesand polar and charged interactions, including eighthydrogen bonds and two salt-bridges. The largestcontribution is provided by the C terminus (helicesa7, a8 and a9), which is longer by ten to 21 residuesin ecGMPK than in other eukaryotic or bacterialGMPKs (Figure 5(e)). Compared to other knownGMPK structures, the C terminus differs in thelength and position of helices a7 and a8, andfeatures a supplementary helix (a9). Remarkably,the interface is identical in our three crystalstructures, indicating that it is insensitive tosubstrate-induced domain motions.

The trimeric interface is smaller (w1100 A2 surfacearea), with the CORE domain, the hinges of theNMP-binding domain and the C terminus from onesubunit facing the CORE and LID domains from a

Table 2. Crystallization buffer, data collection and refinemen

ecGMPK$GDP ecGM

Crystallizationconditions

4% (w/v) PEG 3350, 0.04 M potassiumthiocyanate

1.4 M ammlithium chlor

A. Data collectionX-ray source ESRF, BM30A EUnit cell (A) aZ68.2, cZ151.2 aZ1Space group P63Molecules/A.U. 2Resolution limit (A) 20–2.35Measured reflections 198,342Unique reflections 16,498Completeness (%) 99.3 (97.7)I/sI 28.8 (16.4)Rsym

a 7.1 (14.4)B. RefinementReflections(working/free)

14839/833

Rcrystb (%) 18.5 (19.1) 2

Rfreeb (%) 25.7 (31.7) 2

No. residues/watermolecules

410/185

rmsd bond lengths (A) 0.015rmsd bond angles(degree)

1.48

PEG, polyethylene glycol; A.U., asymmetric unit; rmsd, root-mean-sshells (2.41 to 2.35 A for ecGMPK$GDP, 2.97 to 2.9 A for ecGMP

a RsymZSh Si jIhiKhIhij=Sh Si Ihi, were Ihi is the ith observation of tb RcrystZSjjFojKjFcjj=jFoj. Rfree was calculated with a fraction (5%

3-fold related subunit (Figure 5(d)). As for thedimeric interface, hydrophobic and polar inter-actions contribute to the trimeric interface, includ-ing four to six hydrogen bonds and two to threesalt-bridges. Part of the interface is insensitive todomain motions, while contacts that involve thehinge regions are modulated by the positions of theNMP-binding and LID domains. This is notablythe case for the contact between the hinge of theNMP-binding domain (residues 88–97) and the LID(residues 126–140), whose buried surface variesfrom 250 A2 to 440 A2 in the three ecGMPK crystalstructures depending on their degree of closure.Interestingly, the hinges of the NMP-bindingdomain have B-factors that are in the same rangeas those of the rest of the protein, in contrast to thehigh B-factors that were reported for yGMPK$GMP,5 which may reflect their contribution to thehexameric interfaces in ecGMPK.

Intrinsic and nucleotide-induced domainmotions within the ecGMPK hexamer

This ensemble of structures provides six inde-pendent copies of the ecGMPK subunit in theapoform, two in the GDP-bound form and one inthe GMP-bound form, thus allowing for theanalysis of both spontaneous and nucleotide-induced motions (Figure 6). In apo-ecGMPK, theNMP-binding domain, although it is well defined inthe electron density, adopts a variety of orientations,suggesting that this domain is mobile in the absenceof substrates. Such an intrinsic flexibilitywas reportedbefore for apo-yGMPK5 and other apo-NMPK

t statistics

PK$GMP$SO2K4 apo-ecGMPK

onium sulfate, 0.2 Mide, 0.1 M Na acetate(pH 5.5)

16% (w/v) PEG 10000, 0.08 M NaHepes (pH 7.5), 0.1 M Na cacodylate

(pH 6.5)

SRF, ID29 ESRF, ID14-206.7, cZ155.2 aZ108.2, cZ273.9

R32 P412121 6

19–2.9 34–3.282,483 292,2917689 27,681

99 (99.1) 99.7 (99.7)23.6 (5.8) 26.9 (11.6)7.4 (43.9) 7.1 (22.5)

6986/368 24899/1397

0.3 (24.5) 26.5 (32.2)4.2 (27.3) 28.3 (32.0)206/12 1,175/0

0.010 0.0131.43 1.10

quare deviation. Values in parentheses are for highest resolutionK$GMP and 3.28 to 3.2 A for apo-ecGMPK).he reflection h, while hIhi is the mean intensity of reflection h.

) of randomly selected reflections excluded from refinement.

Figure 5 (legend next page)

Figure 5 (a)–(d) (legend next page)


Figure 5. The hexameric structure of ecGMPK. (a) and (b) The ecGMPK$GMP hexamer shown along (a) a 2-fold and(b) the 3-fold axis. The 2-fold related subunits are shown in blue/cyan, dark green/light green and red/pink. GMPnucleotides are shown in yellow ball-and-stick. (c) Close-up view of the dimeric interface. Orientation is as in Figure 1(a).The CORE domain is shown in grey, the NMP-binding domain in blue, the LID domain in green, hinges in yellow andthe C-terminal extension in red. GDP is shown in red ball-and-stick. One monomer is contoured with its van der Waalssurface. (d) Close-up view of the trimeric interface. Orientation and colour-coding are as in Figure 1(b). (e) Sequencealignment of bacterial and eukaryotic GMPKs. From top to bottom, GMPK sequences are from: E. coli, Salmonellatyphimurium (95% identity with E. coli), Yersinia pestis (87% identity), Photorhabdus luminescens (80% identity),Vibrio cholerae (66% identity), Bacillus subtilis (42% identity), Mycobacterium tuberculosis (29% identity), Saccharomycescerevisiae (44% identity), Mus musculus (38% identity) and Homo sapiens (37% identity). Invariant residues are shownwitha black background and residues similar in at least 50% of the sequences with a grey background. On the top of thesequences are indicated residues involved in dimer interface (cyan boxes) and trimer interface (brown and purple boxesfor each monomer of the interface), the ecGMPK secondary structures, and the ecGMPK domains. The CORE regionincludes all regions outside the NMP-binding and LID domains. Hinges between domains are in yellow. Below thesequences are indicated residues that interact with the GDP (#) and residues that bind the non-substrate GDP (*). Notethe conserved tyrosine (in red) that occludes the ATP binding site in all conformations of the protein.



structures22 and was recently proposed as a majorcontributor to the catalytic efficiency of AMPKsbased on an NMR relaxation analysis.23 In apo-ecGMPK, the NMP-binding domain moves as arigid body with an amplitude of up to 88, which issimilar to the amplitude observed in apo-yGMPK.5

However, the NMP-binding domain is significantlyless open in all subunits compared to apo-yGMPK,as can be estimated by the average angular

Figure 6. Nucleotide-induced conformational changesin the ecGMPK hexamer. (a) Domain motion within thehexamer. Superimposition of apo (red) and GMP-bound(yellow) NMP-binding and LID domains onto a GDP-bound monomer (blue) within the GDP-bound hexamer(in light blue). (b) NMP-binding and LID domain closure.Superimposition of the apo (red) and GMP-bound(yellow) ecGMPK monomers. GMP and the sulfate ionin ecGMPK$GMP are shown in ball-and-stick and van derWaals surface. Residues involved in subunit interfaces aremarked by small spheres.

difference of 338 relative to mGMPK$ADP$GMPtaken as reference for the fully closed conformation(compared to 568 for apo-yGMPK). Thus, althoughintrinsic mobility of the apo-enzyme is retainedwithin the hexamer, the extent of domain openingmay be restricted compared to monomeric GMPKenzymes due to the oligomeric contacts with thehinges of the NMP-binding domain.

Upon binding of GDP, the NMP-binding domaincloses by 23–318 (depending on the reference apo-ecGMPK subunit chosen) whereas the LID domainretains a similar conformation (Figure 6(a)). Theresulting structurehasmuch lessflexibility comparedto apo-ecGMPK, as shownby the good superpositionbetween the two ecGMPK$GDP monomers in theasymmetricunit. Bothdomainsof ecGMPK$GDPare,however, more open than in the ecGMPK$GMPstructure, where the NMP-binding domain hasclosed by a further 48, while the LID domain closesbyw238 and features an additional rearrangement ofhelices a5 and a6 (Figure 6(a) and (b)). Part of thesedifferences between the GMP and GDP-boundstructures may be due to the binding of a sulfate ionin the P-loop (11APSGAGKS18) in ecGMPK$GMP,whichmimics the b-phosphate group ofADP orATP.An SO2K

4 is frequently bound to the P-loop in NMPKstructures but does not lead, in general, to the closureof the LID domain, although this was observed in themtTMPK$TMP structure.24 Here, the LID domainbinds to the SO2K

4 throughArg138LID and forms a salt-bridge with the NMP-binding domain (Asp141LID-Arg45NMP), yielding a conformation and interactionsthat are similar to those observed in the closedconformation of the mGMPK$GMP$ ADP complex(Figure 6(b)).6 Thus, the SO2K

4 in the ecGMPK$GMPstructuremaymimic ADP in this complex, yielding adead-end complex in which the active site holds oneproduct (mimicked by the sulfate ion) and onereactant (GMP). Altogether, our ensemble of struc-tures shows that the ecGMPK hexamer can accom-modate both intrinsic and substrate-inducedLIDandNMP-binding domain motions, and that the closedconformation can be reached within the hexamer.

Interactions of ecGMPK with substrate andproduct guanine nucleotides

The ecGMPK$GDP structure is the first exampleof a GMPK with the product of the phosphoryltransfer reaction bound at the active site.EcGMPK$GDP is in an intermediate conformationbetween the apo and GMP-bound structures, withthe LID open as in the apo-enzyme and the NMP-binding domain slightly less closed than in theecGMPK$GMP$SO2K

4 structure (Figure 6(a)). Theinteractions of GMP and of the GMPmoiety of GDPwith the NMP-binding domain are nevertheless thesame and are also similar to those in structures ofeukaryotic GMPK4,6 (Figure 7(a)). The b-phosphategroup of GDP is loosely coordinated to the side-chains of P-loop residues Ser13, Ser18 and Lys17(distances ranging from 3.4 A to 3.8 A), and byArg45 in the NMP-binding domain, which also

Figure 7. The ecGMPK catalytic site. (a) Schematic diagram of the interactions of GDP with ecGMPK. Broken lines inorange, pink and black correspond, respectively, to the interactions found in monomer A, monomer B or bothmonomers. Labels for the NMP-binding region are in blue, for the CORE region in black. (b) Close-up view of theputative ATP-binding site at the trimeric interface (subunit carrying the ATP-binding site in blue, neighboring subunit ingreen). The location of ADP in the mGMPK$GMP$ADP structure (ADPm) is overlayed in transparency in grey ball-and-stick. The non-substrate GDP molecule is shown in red. Note that both Tyr31 from the neighboring subunit and the non-substrate GDP molecule would conflict with the binding of the ATP substrate. (c) Schematic diagram of the interactionsof the guanosinemoiety of the non-substrate GDP. Labels for 3-fold related subunits are in green and blue. (d) Amodel ofcatalytic interactions of conserved arginine residues. Overlay of GDP (red) from the ecGMPK$GDP structure onto GMPin the closed conformation of ecGMPK$GMP$SO2K

4 (in yellow), based on the superposition of the NMP-binding domain.Candidate hydrogen bonds of the conserved arginine residues to the phosphate groups are shown in dotted lines. ADP,GMP and the invariant arginine residues from the mGMPK$GMP$ADP structure are superposed to show theequivalence of the sulfate ion with the b-phosphate group of ADP (in blue).


binds the a-b bridging and an a-phosphate non-bridging oxygen atoms. As observed in themGMPK$GMP$ADP structure,6 a potassium ioncoming from the crystallization solution is coordi-nated by the guanine base, and by Asp102CORE andSer38NMP, two conserved residues of the GMPbinding site (Figure 7(a)). Altogether, the intermedi-ate conformation and the somewhat less tightinteractions of GDP compared to GMP suggestthat the ecGMPK$GDP structure may mimic a late

conformational step of the reaction, involving there-opening of the domains, which may be necessaryto facilitate product release.

The ATP-binding site is located on the trimericinterface

The ATP-binding site is conserved in manyNMPKs and is located between the CORE andLID domains.8 In GMPKs, ATP-interacting residues


have been identified by the mGMPK$GMP$ADPstructure.6 They are identical in ecGMPK, whichshould thus bind ATP as the mammalian enzymedoes. Surprisingly, the ATP-binding site is notreadily accessible in any of our hexameric ecGMPKstructures, as the adenine-binding site is occludedby subunit interactions at the trimeric interface(Figure 7(b)). The most striking protrusion is due toTyr31CORE from a neighboring subunit, whose aro-matic ring mimics the adenine base in the ATP-binding pocket, stacking against Arg134LID andforming hydrogen bonds with Arg134LID andAsn169CORE in all structures. In mGMPK$GMP$ADP, the corresponding residues (Arg133 andAsn171) form stacking interactions and hydrogenbonds with the adenine base.6 This suggests thatnone of the ecGMPK structures is competent forbinding ATP. In the ecGMPK$GDP structure, theATP-binding site is further occupied by a guaninenucleotide whose phosphate groups are disorderedand was identified as GDP, since this nucleotidewas in excess in the crystallization solution(Figure 7(b) and (c)). Binding of this non-substrateGDP molecule at the trimeric interface is incompa-tible with the binding of ATP, as it would bothconflict with the ADP ribose (Figure 7(b)), and blockthe rearrangement of Arg138LID into the catalyticsite that accompanies the closure of the LIDdomain.

Discussion

The oligomeric structure of ecGMPK

We have characterized the oligomeric structure ofecGMPK in dilute solution by DSC and in thecrystal in the absence of substrates and in thepresence of GMP or GDP. Analysis of ecGMPKthermal stability by DSC revealed a complexdenaturation process. The Cp function at high(O0.1 M NaCl; Figure 1) and low (without NaCl;Figure 2) ionic strength could be deconvolutedusing a non-two-state model with two and threeunfolding transitions, respectively. Comparison ofthe van’t Hoff enthalpy and the calorimetricenthalpy for each transition (Table 1) suggestedthat the transitions resulted from the unfolding ofdifferent oligomers. The DHvH/DHcal ratio wasgreater than unity, showing cooperative disruptionof inter ecGMPK monomer interactions uponunfolding.12,25–29 The ratio values of w2 and w4at high salt, and of w2, w4, and w6 at low salt(Table 1) predicted that the unfolding transitionsresulted from the coupled dissociation and unfold-ing reactions of dimeric and tetrameric, and ofdimeric, tetrameric and hexameric species, respec-tively.12,30 The concentration dependence of eachspecies contribution to the calorimetric enthalpyshowed that these species are in equilibrium insolution (insets in Figures 1 and 2 and the van’t Hoffplots in Figure 3). A Kd value of 10 mM was derivedfrom the dissociation of the tetramer into dimers at

high salt, showing that the equilibrium shiftstowards the dimer upon decreasing ecGMPKconcentration to the micromolar range. The exist-ence of a dimeric and higher order oligomericspecies is consistent with our gel-filtrationchromatography experiments (data not shown)and with earlier experiments,10,11 which detectedeither a dimer (high ionic strength) or a highermolecular mass (low ionic strength) speciestentatively identified as a tetramer. Takentogether, the DSC data showed that ecGMPK isin equilibrium between various oligomericspecies in dilute solution, the concentration ofwhich depends on the ionic strength and theprotein concentration. No deconvoluted tran-sition could be attributed to the monomer species,suggesting that the dimer is the basic folding unit ofecGMPK and that it has a dissociation constantlower than 0.1 mM.

In the crystal, ecGMPK was found to formhexamers with dihedral D3 symmetry in threedifferent space groups, regardless of the ionicstrength of the crystallization conditions and ofthe presence and nature of the bound nucleotide atthe phosphate acceptor site (Figure 5). Remarkably,a hexamer can also be reconstituted by crystalsymmetries from the asymetric unit of apo-ecGMPK crystallized in a fourth crystal form,different from those in the present study (coordin-ates deposited in the Protein Data Bank while thiswork was in progress, space group P213, PDB entrycode 1S96). The existence of a hexamer in fourdifferent crystal forms rules out that the hexamericform is an artifact of the crystallization conditions.DLS and EGS cross-linking experiments at enzymeconcentrations in the range used for crystallizationwere consistent with such a hexameric quaternarystructure (Figure 4). The D3 symmetry allows us toidentify a dimer within the hexamer, which wepropose corresponds to the dimer characterized bygel filtration and DSC. The inability to detect theecGMPKmonomer using DSC is consistent with thefact that the dimer buries a very large interface thatinvolves the unique C-terminal extension inecGMPK. Furthermore, truncation of the 20C-terminal residues, so as to resemble monomericGMPKs, resulted in an insoluble recombinantenzyme (data not shown). Based on establishedrules of protein quaternary structure,31 a stabletetramer cannot derive from the loss of a dimerfrom the hexamer. Therefore, the tetramer predictedfrom the DSC analysis would correspond to adistinct species that cannot be identified from theavailable crystal structures. Since ecGMPK has acatalytic activity that is similar to that of monomericGMPKs and shows no cooperativity for the GMPsubstrate, it is likely that its active sites areessentially independent in the oligomers.Altogether, our structures extend the growingrepertoire of oligomeric NMPKs, which alreadyincludes dimeric TMP kinases,24,32 hexameric bac-terial UMP kinases,33 and AMP kinases, which, asGMPK, also exist as monomers in many species or


as trimers in certain archbacteria22 and bacteria34 orhexamers in certain plants.48

Implications for the catalytic mechanism

The crystal structures of apo, GMP-bound andGDP-bound ecGMPK feature different levels ofnucleotide-induced domain closure, ranging from afully open conformation in apo-ecGMPK, a par-tially closed NMP-binding domain in ecGMPK$GDP and essentially closed LID and NMP-bindingdomains in ecGMPK$GMP$SO2K

4 . Thus, the classi-cal domain motions of NMPKs can take place in thehexamer and the ecGMPK monomer can reach thecatalytically competent conformation at the activesite without restraints due to subunit interactions(Figure 6). However, the ATP-binding site remainspartially occluded by inter-subunit interactions inall our crystal structures, indicating that either localconformational changes or dissociation of thehexamer are required. The thermodynamic proper-ties of ecGMPK in the presence of ATP, which werebest modelled with two unfolding transitionsinstead of three without ATP, suggest that thesetransitions are related to conformational changesnecessary to achieve an ATP-binding site, likelythrough dissociation of the hexamer. The nature ofthese changes will now require further investi-gation.

Our ensemble of structures can, however, becombined to analyze the contribution of activesite residues to the phosphate transfer reaction,as they identify the closed conformation of the LIDand NMP-binding domains (ecGMPK$GMP$SO2K

4structure), the location of the b-phosphate groupof ADP (mimicked by SO2K

4 in theecGMPK$GMP$SO2K

4 structure) and for the firsttime the location of the transferred phosphategroup (located from the b-phosphate group ofGDP in the ecGMPK$GDP structure) (Figure 7(d)).Previous structural studies on GMPKs and otherNMPKs pointed to the role of conserved arginineresidues in the stabilization of the transition state(TS),6,8 which our structures allow us to refine. InecGMPK, Arg138LID (Arg137 in mGMPK) may thusbridge the ADP leaving group and the transferredphosphate at the TS, while Arg45NMP (Arg44 inmGMPK) is well positioned to bridge the enteringGMP group to the transferred phosphate group.Both these invariant arginine residues reach thecatalytic site upon closure of the LID and NMP-binding domains. A third conserved arginine base,Arg149LID (Arg148 in mGMPK), as it binds to theb-g bridging oxygen atom of GDP, may rather beinvolved in stabilizing the GMP substrate at theground state, as charges at this atom are expected todecrease at the TS, hence the contribution of thisresidue to the stabilization of the TS.

ecGMPK as a model for GMPKs fromenterobacteria

The structures of ecGMPK have revealed unique

features that distinguish it from its eukaryoticorthologs, including the mouse enzyme, whichcan be considered as a model for human GMPKdue to the high percentage of sequence identity. Theunique C-terminal extension is a critical determi-nant in forming the basic dimeric folding unit of thehexamer, and the tyrosine insertion in the COREdomain is a specific feature of the trimeric interface.These elements are thus likely to represent asignature of GMPKs with a dimeric and/orhexameric quaternary structure. Remarkably, mostenterobacterial GMPKs are highly homologous toecGMPK with respect to these signature regions(Figure 5(e)). This is also the case for the moredistantly related Vibrio cholerae GMPK. Thus, theoligomeric nature of ecGMPK in solution and itshexameric arrangement in the crystal is likely torepresent a model for the quaternary structure ofGMPKs from these bacteria, some of which arehuman pathogens such as Salmonella typhimurium,Yersinia pestis and V. cholerae. GMPKs are essentialbacterial enzymes,11,35 making them potentialtargets for the design of specific inhibitors. Non-competitive inhibitors that trap abortive confor-mations by binding at protein interfaces areemerging as an attractive strategy for drug dis-covery.36 In this regard, the auto-inhibited confor-mation of the ATP-binding site by inter-subunitinteractions at the trimeric interfaces may be worthstabilizing, as it would be specific to the bacterialenzyme compared to the mammalian enzyme. Thenon-substrate GDP-binding site observed in ourecGMPK$GDP structure, where it is in stericconflict with ADP binding and LID closure(Figure 7(b)), could represent a starting model fora drug targeted specifically to ecGMPK-like GMPKsfrom pathogenic bacteria.

Materials and Methods

Expression, purification and enzymatic assay ofecGMPK

The gmk gene encoding ecGMPK was amplified bypolymerase chain reaction (PCR) using chromosomalDNA from E. coli K12 strain as the template, and using thefollowing primers: 5 0-ecGMPK, 5 0-GGGGAATTCCATATGGCTCAAGGCAGG-3 0; and 3 0-ecGMPK, 5 0-CGGGATCCTCAGTCTGCCAACAATTGC-3 0. The PCR productwas inserted between the NdeI and BamHI restrictionsites of the vector pET 24a (Novagen). The resultingplasmid harbors the gmk gene under the control of ahybrid promoter/operator region, consisting of the T7promoter followed by a lac operator. The protein wasexpressed in the BLI5 strain, derived from E. coli BL21(DE3) (Novagen). Bacteria were grown at 37 8C in 2YTmedium supplemented with kanamycin (30 mg/ml) andchloramphenicol (30 mg/ml), until A600 nm reached 1.3.Overproduction was then triggered by 1 mM isopropyl-b-D-thiogalactosidase for 3 h, and bacteria were harvestedby centrifugation.Recombinant ecGMPK (about 25% of total soluble

proteins) was purified by a two-step chromatographyprocedure involving affinity chromatography on Blue


Sepharose and gel-permeation chromatography asdescribed,37 except that the enzyme adsorbed on BlueSepharose was eluted with 50 mM Tris–HCl (pH 7.4)containing 1 mM ATP and 5 mM GMP. ecGMPK wasdetected in the eluted fractions by GMPK activity using acoupled spectrophotometric assay at 30 8C and 334 nm onan Eppendorf ECOM 6122 photometer as described.38

The reaction medium contained 50 mM Tris–HCl (pH7.4), 50 mM KCl, 2 mM MgCl2, 1 mM phosphoenol-pyruvate, 0.2 mM NADH, different concentrations ofATP and GMP, and two units of lactate dehydrogenase,pyruvate kinase, or nucleoside diphosphate kinase. Thereaction was started with GMPK. One unit of GMPKcorresponds to 1 mmol of product formed per minute. Ionmass spectra were recorded on an API365 triple-quadru-pole mass spectrometer (Applied Biosystems-MDS-Sciex)equipped with a nano-electrospray source (Protana).

Differential scanning calorimetry

DSC endotherms were measured with the VP-DSCfrom MicroCal Inc. ecGMPK stock solution was dialyzedovernight at 4 8C against 50 mM Pipes buffer (pH 7.4),with or without 0.25 M NaCl, and diluted in the samebuffer (concentrations ranging from 1.7 mM to 17 mM,estimated by the Bradford method). Samples weredegassed under vacuum for 10 min with gentle stirringprior to loading into the calorimetric cell (0.5 ml). Sampleswere held in situ under a constant external pressure of25 psi to avoid bubble formation and evaporation at hightemperatures, equilibrated for 30 min at 25 8C, heated at aconstant scan rate of 1 deg. C/min, and the data collectedwith a 16 s filter. Data analysis was made with theOrigin7e software39 provided by the manufacturer. Theexcess heat capacity function (Cp,ex) was obtained aftersubtraction of two baselines form the heat capacityfunction, the buffer-reference thermogram and thechemical baseline calculated after normalizing to concen-tration from the progress of the unfolding transition (seethe legend to Figure 1).

Thermal unfolding data analysis

Raw DSC data were converted to excess heat capacity(cal degK1 molK1) by dividing each datum point by thescan rate and the concentration of protein in the samplecell. Instrumental baselines were removed by subtractionof a scan of the buffer against which the protein wasdialyzed. The excess heat capacity data were analyzedusing the program Origin7e provided by MicroCale.The equilibrium constant for an unfolding reaction at anytemperature is given by:

KðTÞZ eKDG=RT (4)

where R is the gas constant, T is the temperature inKelvin, and the Gibbs free energy change for unfolding attemperature T is given by a modified Gibbs-Helmoltzequation:

DGðTÞZDHvH;mðTmKTÞ=TmÞKðTmKTÞDCp

CTDCp lnðTm=TÞ (5)

where Tm is the midpoint of the transition when KZ1.29

DHvH,m is the van’t Hoff enthalpy of unfolding at Tm andDCp is the change in heat capacity upon unfolding. DCp isassumed to be temperature-independent over the tem-perature range studied. a(T), the progress of theunfolding reaction at T, is given by:

aðTÞZKðTÞ=ð1CKðTÞÞ (6)

which can be expressed as a function of T,DHvH,m, Tm andDCp after substitution of equations (4) and (5). The excessheat capacity measured in a DSC experiment is given bythe temperature-dependence of the reaction progressmultiplied by the enthalpy change for the reaction at T:12

Cp;ex ZDHðTÞdaðTÞ=dðTÞ (7)

where DH is temperature-dependent:

DHðTÞZDHvH;m C ðTKTmÞDCp (8)

The baseline can be fit as the sum of two straight linesweighted by the progress of the reaction:12

Cbaseline Z ð1KaðTÞÞðACBTÞCaðTÞðCCDTÞ (9)

T is expressed relative to the midpoint temperature, Tm,so that C is DCp at Tm and:

Cbaseline Z ð1KaðTÞÞðACBTÞ

C aðTÞðDCp CDðTKTmÞÞ(10)

The DSC endotherm is then given by CobsZCbaselineCCp,ex, and the data were fit to this equation by theLevenberg/Marquard non-linear least-squares method.39

Examples of such fits are shown in Figures 1 and 2, andthe progress curve in the form of Cbaseline is shown inFigure 1.The enthalpy used above is the van’t Hoff enthalpy. It

satisfies the van’t Hoff relationship (equation (1)) anddetermines the width of the DSC endotherm. Integrationof equation (7) over the transition provides the calori-metric enthalpy, DHcal, which equals the van’t Hoffenthalpy only for a two-state transition. If the endothermresults from two or more two-state transitions, the van’tHoff and calorimetric enthalpies are not equal and theratio:

bZDHcal=DHvH (11)

is not equal to unity. Equation (8) must be modified togive:

Cp;ex ZbDHðTÞdaðTÞ=dT (12)

A problem arises here in the analysis, since theobserved DCp is the sum of the DCp values for eachoverlapping transition. The magnitudes for DCp for eachoverlapping transition are not defined by the data. Anyattempt to extract both a DHcal and a DHvH from the datarequires that either DCp be apportioned to two or moretransitions according to an assumed unfolding model, orthe DCp values are assumed to be zero. The latterassumption was used in the present study, where amodel for independent non-two-state transitions wasused to fit the excess heat capacity curves.Estimation of the equilibrium constant between

ecGMPK dimer and tetramer species at 0.25 M NaCl(inset in Figure 1) was done using a simple equilibriummodel, making the assumption that no other molecularspecies is present in solution:

2!P24P4

ðP2ÞZ ðK2C ð4C16KaPtÞ1=2Þ=8Ka, where Ka is the equili-

brium constant for tetramer formation from the dimer,and Pt, the total protein monomer concentration. AsDHcal,2/DHcal,tZP2/Pt, the equation can be written as:8MtKaDH2=DHtKð4C16MtKaÞ

1=2C2Z0. The equation issolved numerically by varying Ka.


Dynamic light-scattering analysis and chemicalcross-linking

ecGMPK at 360 mM (8.5 mg/ml) in 20 mM Na Hepes(pH 7.5) and different NaCl concentrations (without,125 mM, 250 mM or 500 mM) was spun for 30 min at100,000g and analyzed by DLS using the DynaPromachine (Protein Solutions). About 50 observationswere used to calculate the hydrodynamic radius (Rh)using the DynaPro software, and data were fitted with amonomodal analysis. Cross-linking experiments wereperformed without NaCL and at 250 mM NaCl usingethylene glycol bis(succinimidyl) succinate (EGS) at20 mM for 7 h at 25 8C. The reaction was stopped byaddition of 80 mM Tris (pH 8.5), and the products weredetected by silver staining of an SDS-polyacrylamide gelmade of two concentrations of acrylamide (8% and 15%,w/v).

Crystallization, X-ray data collection and structurerefinement

All crystallizations were done by the vapor-diffusionmethod at 20 8C. The complex of ecGMPK with GDP(ecGMPK$GDP) was formed by mixing 490 mM proteinwith 10 mMGDP, 10 mMADP, 10 mMMgCl2 and 20 mMNa Hepes (pH 7.5). The complex of ecGMPK with GMP(ecGMPK$GMP) was formed by mixing 750 mM proteinwith 10 mM GMP, 10 mM MgCl2 and 20 mM Na Hepes(pH 7.5). The nucleotide-free form of ecGMPK (apo-ecGMPK) was obtained with 490 mM protein in thepresence of 10 mM adenosine-5 0-[(b,g)-imido]tripho-sphate, a non-hydrolyzable ATP analog, and 10 mMMgCl2. All crystals appeared within three to ten days.For data collection at 100 K, ecGMPK$GDP, ecGMPK$GMP and apo-ecGMPK crystals were transferred in theirmother liquor supplemented with, respectively, 35% (w/v) of PEG 3350, xylitol and PEG 10,000, before cooling inliquid ethane.Each X-ray data set was collected from a single crystal

on beamlines BM30A (lZ0.9793 A), ID14-2 (lZ0.9330 A)or ID29 (lZ0.9756 A) at the European SynchrotronRadiation Facility and was indexed and scaled usingXDS and XSCALE.40 Initial phases for ecGMPK$GDPwere obtained bymolecular replacement with MOLREP41

using the yGMPK$GMP structure5 as a search model. Therefined ecGMPK$GDP structure was then used asmolecular replacement model to solve the ecGMPK$GMPand apo-ecGMPK structures. Refinement was carried outusing CNS42 and Refmac5,41 andmodel building with O43

or TURBO44 programs. Non-crystallographic symmetryrestraints were removed in the last steps of refinement forthe ecGMPK$GDP structure (two molecules in theasymmetric unit), and for the NMP-binding in the apo-ecGMPK structure (six molecules in the asymmetric unit)as this region displayed significant flexibility betweenmonomers. TLS parametrization defining one group perprotein region (CORE, LID and NMP-binding domains)was used at the end of the refinement in Refmac5 for theecGMPK$GMP and ecGMPK$GDP structures. Crystals ofecGMPK$GDP contain two molecules in the asymmetricunit, which are related by 2-fold non-crystallographicsymmetry and show minor differences in their structureswith an average root-mean-square deviation (rmsd) on allCa atoms of 0.4 A. The final model includes for eachmolecule residues 2 to 206, two GDP nucleotides and apotassium ion. Crystals of ecGMPK$GMP contain onemolecule per asymmetric unit. The model includes

residues 2 to 207, a GMP nucleotide and a sulfate ion.Crystals of apo-ecGMPK contain six molecules perasymmetric unit that are very similar, apart from residues37 to 84, which have rsmd between the differentmonomers of up to 2.8 A. The model comprises residues2 to 206 for chains A and C, 3 to 206 for chain D, 2 to 205for chains B and E, and 2 to 206 for chain F excludingresidues 35 to 86, which are disordered in this chain. Allstructural Figures were generated using MOLSCRIPT45

and Raster3D,46 or PyMol.47

Protein Data Bank accession numbers.

Coordinates and structure factors of apo-ecGMPK,ecGMPK$GMP and ecGMPK$GDP have been depositedwith the RCSB Protein Data Bank with accession numbers2ANC, 2ANB and 2AN9, respectively.

Acknowledgements

This work was supported by a grant from theFrench research ministry (ACI-BCMS) to J.C. P.C.was supported by a grant from the Direction desRelations Internationales, CNRS. We thank the staffat the European Synchrotron Radiation Facility(Grenoble, France) for making beam lines BM30A,ID14-2 and ID29 available to us, and AndrewThompson and Eric Girard (SOLEIL synchrotronfacility, St Aubin) for help in X-ray data collection atthe ESRF synchrotron facility. We thank FrancoisTernois (VMS, CNRS, Gif-sur-Yvette) for help withthe DLS machine. We are grateful to Octavian Barzu(Institut Pasteur) for his encouragements andmentorship. We thank Helene Munier-Lehmann(Institut Pasteur) for the gift of M. tuberculosisGMPK and Joel Janin (LEBS, CNRS, Gif-sur-Yvette) for critical reading of the manuscript.

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Edited by J. E. Ladbury

(Received 5 April 2005; received in revised form 11 July 2005; accepted 14 July 2005)Available online 1 August 2005

Calorimetric and Crystallographic Analysis of the Oligomeric Structure of Escherichia coli GMP...

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