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Originally published In Press as doi:10.1074/jbc.M706399200 on October 31, 2007

J. Biol. Chem., Vol. 282, Issue 52, 37515-37528, December 28, 2007
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The Interactions of Cell Division Protein FtsZ with Guanine Nucleotides*Formula

Sonia Huecas{ddagger}1, Claudia Schaffner-Barbero{ddagger}, Wanius García{ddagger}2, Hugo Yébenes{ddagger}3, Juan Manuel Palacios{ddagger}4, José Fernando Díaz{ddagger}, Margarita Menéndez§, and José Manuel Andreu{ddagger}5

From the {ddagger}Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain and §Instituto de Química-Fisica Rocasolano, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain

Received for publication, August 2, 2007 , and in revised form, October 16, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prokaryotic cell division protein FtsZ, an assembling GTPase, directs the formation of the septosome between daughter cells. FtsZ is an attractive target for the development of new antibiotics. Assembly dynamics of FtsZ is regulated by the binding, hydrolysis, and exchange of GTP. We have determined the energetics of nucleotide binding to model apoFtsZ from Methanococcus jannaschii and studied the kinetics of 2'/3'-O-(N-methylanthraniloyl) (mant)-nucleotide binding and dissociation from FtsZ polymers, employing calorimetric, fluorescence, and stopped-flow methods. FtsZ binds GTP and GDP with Kb values ranging from 20 to 300 µM-1 under various conditions. GTP·Mg2+ and GDP·Mg2+ bind with slightly reduced affinity. Bound GTP and the coordinated Mg2+ ion play a minor structural role in FtsZ monomers, but Mg2+-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with ~10-fold lower affinity than GTP. Mant-GTP displacement measured by fluorescence anisotropy provides a method to test the binding of any competing molecules to the FtsZ nucleotide site. Mant-GTP is very slowly hydrolyzed and remains exchangeable in FtsZ polymers, but it becomes kinetically stabilized, with a 30-fold slower k+ and ~500-fold slower k- than in monomers. The mant-GTP dissociation rate from FtsZ polymers is comparable with the GTP hydrolysis turnover and with the reported subunit turnover in Escherichia coli FtsZ polymers. Although FtsZ polymers can exchange nucleotide, unlike its eukaryotic structural homologue tubulin, GDP dissociation may be slow enough for polymer disassembly to take place first, resulting in FtsZ polymers cycling with GTP hydrolysis similarly to microtubules.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
FtsZ is a cytoskeletal protein essential to bacterial cytokinesis and a member of the tubulin family of GTPases, which also includes {alpha}β-tubulin (1), {gamma}-tubulin (2), bacterial tubulin BtubA/B (3, 4), and TubZ (5). FtsZ assembles by forming filaments that constitute the Z-ring at the cell division site in bacteria. The Z-ring, a dynamic structure maintained by assembly and disassembly of FtsZ, recruits the other elements of the division machinery following chromosome segregation (6-10). Bacterial cell growth and division are regulated by nutrient availability; a metabolic sensor has been recently identified in Bacillus subtilis, including an effector, the glucosyltransferase UgtP, which modulates FtsZ assembly (11). GTP binding, hydrolysis, and exchange constitute the regulatory mechanism responsible for dynamics of FtsZ and tubulin polymers. The nucleotide switches of these assembling GTPases appear to involve polymerization-driven structural changes (12), although FtsZ and tubulin form different end polymers. The GTPase activity of FtsZ is modified by the polymerization inhibitory protein MipZ (13) and, weakly, by EzrA (14).

The hydrolyzable nucleotide bound to tubulin becomes occluded in microtubule protofilaments (15). Microtubules hydrolyze all bound GTP to GDP except at their very ends and become metastable, giving rise to microtubule dynamic instability (16). In contrast, polymers of FtsZ from E. coli were reported to contain mostly GTP, and, under certain conditions, nucleotide exchange proceeds faster than hydrolysis (17). This suggested that the nucleotide binding site remains exchangeable in FtsZ polymers, which would therefore be devoid of dynamic instability. Polymers of Methanococcus jannaschii FtsZ were found to contain different proportions of GTP and GDP (depending on the hydrolysis rate) and to rapidly depolymerize upon either GTP consumption or GDP addition (18, 19). GDP binding destabilizes M. jannaschhi FtsZ polymers compared with polymers with GTP or without a bound nucleotide (20). In E. coli FtsZ polymers the main rate-limiting step in nucleotide turnover was found to be nucleotide hydrolysis, rapidly followed by phosphate release, whereas a second rate-limiting step could be nucleotide dissociation. However, whether nucleotide dissociation took place directly from the polymer or through depolymerization into subunits, followed by GDP release, was not determined (21).

An important problem yet to be solved for FtsZ assembly dynamics is whether, following GTP hydrolysis (i) GDP dissociates from subunits in the FtsZ polymer which directly reload with GTP, (ii) polymer subunits exchange with GTP-bound subunits in solution, or (iii) the FtsZ-GDP polymer fully disassembles and reassembles again from GTP-bound subunits. Consistent with an exchangeable nucleotide in FtsZ polymers, the nucleotide was observed to be largely accessible in the crystal structure of a protofilament-like dimer of M. jannaschii FtsZ (22). On the other hand, exchange of GFP-FtsZ fusions in bacterial Z-rings was found to proceed with a half-time of 8-9 s in vivo, by means of fluorescence recovery after photobleaching (23, 24). As observed in an in vitro fluorescence resonance energy transfer assay, subunit turnover in filaments of E. coli FtsZ took place with a half-time of 7 s with GTP, which was slowed down under conditions reducing the nucleotide hydrolysis rate (25). This rate of subunit turnover is comparable with the turnover rate of GTP hydrolysis (21) and with the rate of depolymerization in GDP excess, suggesting that GDP does not exchange into intact filaments (23). This favors the interpretation that the rapid assembly dynamics of FtsZ filaments may operate by a mechanism related to microtubule dynamic instability (25). In addition, subunit turnover and GTPase in FtsZ from Mycobacterium tuberculosis are both about 10 times slower than in E. coli FtsZ (26).

FtsZ and its nucleotide binding site are attractive targets for cell division inhibitors, which may lead to new classes of antibacterial compounds (27) to fight the continuous emergence of antibiotic resistance. Small molecules reported to modulate FtsZ assembly include 8-bromo-GTP (28) and other nucleotide analogues (29), 3-metoxybenzamide (30), viriditoxin (31), ruthenium red (32), zantrins (33), SRI-3072 (34), polyphenols (35), PC58538 and PC170942 (36), sanguinarine (37), certain taxanes (38), A189 (39), amikacin (40), totarol (41), and cinnamalehyde (42).

This study focused on fundamental processes of FtsZ-nucleotide interactions. We have determined the energetics of GTP and GDP binding to FtsZ and the kinetics of binding and dissociation in FtsZ monomers and polymers using fluorescent (mant)6-nucleotides. The results reveal functional differences with nucleotide binding to tubulin that will facilitate screening for compounds binding to the nucleotide site of FtsZ. They also indicate a slowed down nucleotide exchange in FtsZ polymers, which provides insight to their dynamics.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Nucleotides—GDP was obtained from Sigma, and GTP (lithium salt) was from Roche Applied Science or Sigma. mant-GTP and mant-GDP were from Jena Bioscience. [8-3H]GTP (6 Ci/mmol) and [{alpha}-32P]GTP (~400 Ci/mmol) were from Amersham Biosciences. Nucleotides were analyzed (after extraction with perchloric acid in the case of protein samples) (18) by HPLC with a Grace Vydac 3021c4.6 anion exchange column (0.46 x 25 cm) eluted with a linear gradient of 25 mM NaH2PO4/Na2HPO4, pH 2.8, to 125 mM NaH2PO4/Na2HPO4, pH 2.9. All other chemicals (analytical grade) used were from Merck or Sigma.

Preparation of Nucleotide-free FtsZ from M. jannaschii—FtsZ (without histidine tag) was overproduced in E. coli BL21(DE3)pLys and was purified as described (18, 19). Nucleotide-free FtsZ (apoFtsZ) was prepared as described (20) with minor modifications. FtsZ was incubated in 2.5 M guanidinium chloride (GdmCl) for 30 min at room temperature, followed by gel filtration in a 0.9 x 25-cm Sephadex G-25 column in 25 mM Pipes/KOH and 2.5 M GdmCl, pH 7.5, to separate the released nucleotide from protein (monitored spectrophotometrically at 254 and 280 nm). A second G-25 column in 25 mM Pipes/KOH, 50 mM KCl, and 1 mM EDTA, pH 7.5 (Pipes-KCl buffer) was used to eliminate GdmCl and equilibrate the protein in this experimental buffer. ApoFtsZ concentration was measured spectrophotometrically employing an extinction coefficient {epsilon}280 = 6990 M-1 cm-1 (calculated for 1 Trp, 1 Tyr). ApoFtsZ was frozen and stored in liquid nitrogen and was melted on ice before use.

Differential Scanning Calorimetry (DSC)—Measurements were performed using a VP-DSC microcalorimeter (Microcal, Inc.). Samples were degassed at room temperature prior to calorimetric experiments. Calorimetric cells (operative volume ~0.5 ml) were kept under an extra constant pressure of 2 atm to prevent degassing during the scan. Standard VP-Viewer and Origin-DSC software (MicroCal) were used for data acquisition and analysis. Excess heat capacity (Cp) was obtained after subtraction of the buffer-buffer base line, and the denaturation enthalpy ({Delta}HD) was determined from the area under the absorption peak. Measurements were performed at a scan rate of 30 °C/h in Pipes-KCl buffer using 12 µM FtsZ. GXP and Mg2+ concentrations were 100 µM and 10 mM, respectively.

Isothermal Titration Calorimetry (ITC)—Calorimetric titrations of FtsZ with GXP, GXP-Mg, and Mg2+ were performed at 25 °C using a MCS titration calorimeter (MicroCal). Measurements were carried out in Pipes-KCl buffer, supplemented with 10 mM Mg2+ in both protein and nucleotide solutions for titration experiments with GXP·Mg2+ (EDTA was omitted for titration with Mg2+). Samples were dialyzed against buffer before measurements. Ligand solutions (~150 µM GXP or 50 mM Mg2+) were prepared in the dialysis buffer. FtsZ (10-25 µM) solution was loaded into the calorimeter cell and titrated, typically, by adding 1x 1 µl, plus 16-22 injections (10-12 µl), of a concentrated solution of the ligand. Heats of titrant dilution were determined in separate runs and subtracted, when required, to obtain the heat of binding. Binding isotherms were analyzed by nonlinear regression analysis to a single set of sites model, using software supplied by the manufacturer, to calculate the number of binding sites (n), the binding constant (Kb), and the enthalpy of binding ({Delta}H).

Stoichiometry of Binding of Nucleotides and ApoFtsZ Polymerization—The stoichiometry of binding of GTP, GDP, mant-GTP, and mant-GDP to soluble apoFtsZ was measured using a centrifugation assay. ApoFtsZ (6 or 8 µM) was incubated at 25 °C for 30 min with nucleotides at different known concentrations (3-15 µM) in a final volume of 0.6 ml of Pipes-KCl buffer. Samples were then centrifuged for 2.5 h at 100,000 rpm and 25 °C in a TLA-120.2 rotor employing a Beckman Optima TLX ultracentrifuge. After centrifugation, the top 0.3 ml were carefully withdrawn, and the concentration of free nucleotide was determined spectrophotometrically, employing the extinction coefficients {epsilon}254 = 23,300 M-1 cm-1 for mant-nucleotides and {epsilon}254 = 13,620 M-1 cm-1 for GTP and GDP. The top half contained only free nucleotide and essentially no protein, as checked by control measurements, and the bottom half contained all of the protein, in chemical equilibrium with free nucleotide. The nucleotide bound to FtsZ was calculated as the difference of the known total concentration of nucleotide minus the free concentration in the top part of the solution.

To measure the stoichiometry of binding of mant-GTP to apoFtsZ polymers, 20 µM apoFtsZ was polymerized at 55 °C in Pipes-KCl buffer with 10 mM MgCl2. After 10 min, 20 µM mant-GTP was added. After 1, 10, and 20 min, different aliquots of 0.1 ml were taken and centrifuged for 6 min at 80,000 rpm in a prewarmed TLA-100 rotor employing a Beckman Optima TLX ultracentrifuge (18). After centrifugation, the supernatant was carefully withdrawn and the pellet was resuspended in buffer. FtsZ concentration was measured in the supernatant and in the pellet with the Bio-Rad protein assay kit (43) in multiwell plates, employing spectrophotometrically calibrated FtsZ standards and a Titertek Multiskan MC plate reader with a 595-nm filter. Mant-GTP bound to FtsZ was fluorometrically determined in the resuspended pellet employing a Shimadzu RF-540 spectrofluorometer (excitation wavelength 357 nm, emission wavelength 445 nm, 5-nm excitation and emission slits) using standards of FtsZ-bound mant-GTP.

Affinity of Binding of [3H]GTP to ApoFtsZ—Binding of [8-3H]GTP to apoFtsZ was measured by protein depletion (44) as follows. Varying [8-3H]GTP concentrations were added to aliquots of apoFtsZ (500 nM) in Pipes-KCl buffer (0.2 ml). Mixtures were centrifuged for 1.5 h at 100,000 rpm and 25 °C in a Beckman TLA-100 rotor. The total [8-3H]GTP concentration was determined in the bottom half, and the free concentration was determined in the protein-depleted top half of tubes, after dilution in 1.5 ml of Beckman ReadySafe solution, employing a Wallac Trilux 1450 Microbeta liquid scintillation counter (PerkinElmer Life Sciences). In each assay, controls with [8-3H]GTP alone were included, and concentrations were corrected for the small amount of nucleotide sedimented in the absence of protein. When we measured the binding of [8-3H]GTP to an excess of apoFtsZ, 1.8% of inactive ligand was found in the stock solution. This percentage was subtracted from the concentration of free [8-3H]GTP calculated in each assay.

Affinity of Binding of Mant-nucleotides to ApoFtsZ—Binding of mant-nucleotides to FtsZ was measured by the increase in fluorescence intensity and anisotropy of the probe. It was first confirmed that more than 95% of mant-GXP co-sedimented with an excess of apoFtsZ upon high speed centrifugation. Fluorescence of free and FtsZ-bound mant-GXP was measured with a Fluorolog 3-221 instrument (Jobin Yvon-Spex, Longiumeau, France) employing an excitation wavelength of 357 nm and an emission wavelength of 445 nm, with 3- and 5-nm bandwidths, respectively, and 2 x 10-mm cells. Anisotropy was measured in T-format with 5-nm excitation bandwidth and 10-nm emission bandwidths. Protein-fluorescent ligand interaction was determined as reported (45), with modifications. Fixed concentrations of mant-GXP (10-500 nM) were first titrated with different apoFtsZ concentrations (0-6 µM) in Pipes-KCl buffer, with or without 10 mM Mg2+, to obtain the anisotropy increment, {Delta}rmax, corresponding to all of the mant-GTP bound. To do this, the increase in anisotropy was plotted against apoFtsZ concentration and iteratively least-squares fitted with an isotherm of binding to one site. The estimated values of {Delta}rmax were used to approximate the free apoFtsZ concentrations, and these new values were employed again, until an unchanging {Delta}rmax value was obtained. The convergent data were used to calculate the binding constant of apoFtsZ to mant-GXP. Titration of apoFtsZ (500 nM) with different mant-GTP concentrations was also measured, and the data were model-fitted (employing the {Delta}rmax value) to yield the number of binding sites and the equilibrium binding constant of mant-GTP to apoFtsZ.

Affinity of Ligands Competing with Mant-GTP—Competition assays were performed by measuring, through the decrease in fluorescence anisotropy, the displacement of mant-GTP from FtsZ. Different concentrations of competing ligand were mixed with apoFtsZ (500 nM) and mant-GTP (500 nM) in Pipes-KCl, 10 mM MgCl2 buffer (final volume of 0.4 ml), and the anisotropy was measured at 25 °C. The fraction of the reference ligand mant-GTP bound was plotted against the competing ligand concentration, and data were fitted assuming that the two ligands bind to the same site. The resulting system of equations (45) was numerically solved with the program Equigra version 5.0 (46) or with a MATLAB script (available upon request),7 which provided the best fitted value of the equilibrium binding constant of the competing ligand.

The relative affinity of FtsZ for GDP and GTP was directly determined by incubating apoFtsZ with solutions of different ratios of GTP/GDP for 1 h at 25 °C in 50 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, pH 7.5 (Tris-KCl buffer). Excess nucleotide was removed by a chromatography in a fast desalting column HR 10/10 (Amersham Biosciences) equilibrated in the same buffer with 10 µM nucleotide at the same GDP/GTP ratio. Eluted protein was precipitated with perchloric acid, and nucleotide content was measured by HPLC.

Kinetics of Binding and Dissociation of Mant-nucleotides to ApoFtsZ—Kinetic measurements were made with a Bio-Logic SFM-400 T-format stopped-flow device equipped with a fluorescence detection system. A wavelength of 368 nm in the excitation pathway and a filter with a cut-off of 450 nm in the emission pathway was employed. When measuring light scattering at the same time, a 350-nm band pass filter was included in the second emission pathway. 5-10 separate curves were averaged for each condition, and the curves so obtained were fitted to a single-, double- or triple-exponential equation of the form y(t) = at + b + {Sigma}Aie-kit (where the slope (a) and offset (b) correspond to the linear drift after the reaction). The best fitting rate constants (ki) and amplitudes (Ai) were determined with the Bio-Kine software (Bio-Logic) or with a nonlinear least squares fitting program based on the Marquardt algorithm (47).


Figure 1
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  		FIGURE 1.
A, DSC traces of ApoFtsZ (12.5 µM) with MgCl2 (10 mM), with GDP (100 µM), and with GDP (100 µM) plus MgCl2 (10 mM) in Pipes-KCl buffer. B and C, calorimetric titration (ITC) of FtsZ binding to GDP and GTP·Mg2+, respectively, at 25 °C. Each peak (upper panels) represents the heat (integrated area) resulting from ligand injection into FtsZ solution (see "Experimental Procedures"). Each point in the bottom panels is the heat evolved per mol of injected ligand in the corresponding peak in the upper panel, after subtraction of ligand dilution heat. Solid lines are the best fits to experimental data (see Table 1 for binding parameters).

 
Histidine-tagged FtsZ Polymers—FtsZ-His6 and mutant FtsZ-W319Y-His6 were overproduced in E. coli and affinity-purified as described (19). ApoFtsZ-W319Y-His6 was prepared as apoFtsZ above, and its concentration was measured with an extinction coefficient {epsilon}280 = 2980 M-1 cm-1 (2 Tyr). It was diluted at 55 °C into Pipes-KCl buffer, pH 6.5, supplemented with 6 mM MgCl2 and nucleotides (the His-tagged protein has a tendency to precipitate at pH <7 at room temperature). FtsZ polymers were negatively stained and observed under a Jeol 1230 electron microscope.

Copolymers of FtsZ-W319Y-His6 and FtsZ-His6, formed in 50 mM Mes, 50 mM KCl, 1 mM EDTA, pH 6.5 (Mes-KCl buffer) with 6 mM MgCl2 and 0.1 mM GTP at 55 °C, were pelleted by centrifugation at 60,000 rpm for 6 min at 55 °C in a prewarmed TLA-100 rotor. They were resuspended in 1% SDS, and the concentration of the FtsZ-His6 single Trp was measured fluorometrically by excitation at 295 nm, employing FtsZ-His6 standards. Concentration of FtsZ-W319Y-His6 polymers was measured with the Bio-Rad assay (43) with FtsZ-W319Y-His6 standards. Exchange of [{alpha}-32P]GTP into FtsZ-W319Y-His6 or FtsZ-His6, in Mes-KCl buffer with 6 mM MgCl2 and 1 mM GTP at 55 °C, was measured employing a nitrocellulose filtration assay (17).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Nucleotide on FtsZ Secondary Structure and Thermal Stability—Prior to studying the interactions of FtsZ with nucleotides, effects of the bound nucleotide on FtsZ stability were evaluated. The circular dichroism spectrum of stable nucleotide-free FtsZ from M. jannaschii (20) was not significantly different from that of FtsZ. The reversible unfolding profiles with GdmCl were also very similar in the absence and presence of 50 µM GTP plus 1 mM MgCl2, with a [GdmCl]1/2 value of 3.1 M (supplemental Fig. 1); this is compatible with the release of the nucleotide at lower GdmCl concentration (48).

Nucleotide binding would be expected to stabilize the protein against denaturation. This was examined by differential scanning calorimetry, which was done with GDP, in order to avoid FtsZ polymerization and GTP hydrolysis at high temperatures. Irreversible thermograms (Fig. 1A) showed that this thermophilic apoFtsZ (Tm = 90.16 ± 0.03 °C, {Delta}HD = 190 ± 20 kcal/mol) is further stabilized by GDP (100 µM), which increased the temperature of the transition by 10 °C (Tm = 100.72 ± 0.09 °C, {Delta}HD = 220 ± 10 kcal/mol). Magnesium (10 mM MgCl2) does not significantly stabilize apoFtsZ (Tm = 90.25 ± 0.02 °C, {Delta}HD 230 ± 10 kcal/mol) but apparently induces a destabilization of FtsZ-GDP (FtsZ-GDP-Mg2+ Tm = 96.2 ± 0.7 °C, {Delta}HD = 190 ± 10 kcal/mol). The contribution of GDP dissociation to the denaturation enthalpy, {Delta}HD, could not be estimated from these experiments, due to errors of the large denaturation enthalpy values.

Binding Equilibrium of Guanine Nucleotides to FtsZ—The stoichiometry of nucleotide binding to apoFtsZ was checked first. Different known concentrations of GTP, GDP, mant-GTP, or mant-GDP were added to FtsZ, and the solutions were centrifuged at high speed. The free nucleotide in the protein-depleted top half of tubes was measured, and the bound nucleotide was calculated by difference from the total. The stoichiometry values were as follows: 0.94 ± 0.03 GDP or GTP, 0.94 ± 0.06 mant-GDP, 0.83 ± 0.08 mant-GTP (i.e. essentially one nucleotide per FtsZ).

The energetics of the interaction of apoFtsZ (10-25 µM) with GDP and GTP were systematically examined by ITC. Nucleotide binding is moderately exothermic (Fig. 1, B and C, and Table 1) and the average stoichiometry of GXP binding from ITC experiments was 0.81 ± 0.06. Binding affinity increased in the presence of the nucleotide {gamma}-phosphate (6-fold without Mg2+, 1.5-fold with Mg2+) but decreased (2.5-10-fold) when an excess of Mg2+ is added to provide nucleotide·Mg2+ complexes. Mg2+ alone binds with very low affinity (Table 1).


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  		TABLE 1
Energetics of the interactions of FtsZ with nucleotides determined by ITC at 25 °C ITC experiments were made with 0 or 10 mM MgCl2 in both protein and GXP solutions (the titration with MgCl2 was done without EDTA). The stoichiometry of the low affinity Mg2+ binding cannot be measured from these experiments, and therefore, the enthalpy change is an estimate per mole of protein, not per mole of site. Titration of FtsZ-GTP (no EDTA) with equimolar Mg2+ gave insignificant heat.

 
The ITC binding measurements were made under conditions in which FtsZ does not polymerize but self-associates into dimers and trimers, in a magnesium- and nucleotide-insensitive manner; however, FtsZ is predictably monomeric at submicromolar concentrations (18). Therefore, the binding affinity of [8-3H]GTP to 500 nM apoFtsZ was also measured by protein depletion, counting the top and bottom half of each solution. The best fitting equilibrium association constant was Kb = 700 ± 100 µM-1, which decreased in excess Mg2+ to Kb = 90 ± 20 µM-1 (Fig. 2A). These values are 2-3-fold larger than association constants estimated by ITC (Table 1). This may be due to a slightly weaker binding of the nucleotide to FtsZ oligomers and the disparity of the methods. The average of the best fitting stoichiometries was 0.9 ± 0.1 [8-3H]GTP per FtsZ.

In order to conveniently measure the binding of nucleotides to FtsZ monomers with fluorescent methods, we employed the analogs mant-GTP and mant-GDP that contain a methyl-anthraniloyl group attached to the ribose moiety and have been widely employed to study nucleotide binding by proteins (49-51) and were found to bind specifically to FtsZ. The addition of apoFtsZ produced both a 3.5-fold increase of the mant-nucleotide fluorescence intensity and a shift of the emission maximum from 449 to 440 nm. Magnesium in the millimolar concentration range quenched the fluorescence of FtsZ-bound mant-GXP but not that of free mant-GXP. This impeded intensity measurement of the equilibrium binding of the fluorescent nucleotides to FtsZ in Mg2+ containing buffers. However, a protein concentration-dependent increment of anisotropy, {Delta}r, over that of the free fluorophore (0.04) was also observed, with a maximum value, {Delta}rmax, practically insensitive to Mg2+. Titration of mant-GTP with apoFtsZ in 10 mM MgCl2 allowed determination of best fitted values of {Delta}rmax = 0.24 ± 0.01 and Kb = 4.2 ± 0.4 µM-1 (Fig. 2B); titration of apoFtsZ with mant-GTP, employing the {Delta}rmax value, gave a coincident Kb value (4 ± 1 µM-1) and a stoichiometry of 1.12 ± 0.06 mant-GTP bound per FtsZ (Fig. 2C). Affinities of binding of mant-GDP and mant-GTP to FtsZ were systematically measured under several conditions (Table 2 and supplemental Fig. 2). Apparent affinities of mant derivatives are 3-16-fold lower than those of natural nucleotides. Triphosphate/diphosphate affinity ratios are small, and a similar weakening effect of Mg2+ is observed. Smaller Mg2+ concentrations (50 nM to 100 µM in buffer without EDTA) did not increase the apparent affinity of mant-GTP. On the other hand, the minor differences found between values measured at 25 or 55 °C can be explained by the small binding enthalpies of GTP and GDP measured by ITC.


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  		TABLE 2
Equilibrium association constants of mant-nucleotides to apoFtsZ and their dependence on MgCl2 concentration and temperature 10 nM mant-GXP was titrated with apoFtsZ in Pipes-KCl buffer with 0, 2, or 10 mM MgCl2. Free Mg2+ concentrations are ~4 nM, 1 mM, and 9 mM, respectively (our cation-free buffers typically contain ~1 µM residual Mg2+). Binding was measured from the increase in fluorescence anisotropy of mant-GXP. Shown in parentheses are equilibrium constants measured by fluorescence intensity.

 


Figure 2
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  		FIGURE 2.
A, isotherms of binding of [8-3H]GTP to apoFtsZ. ApoFtsZ (500 nM) in Pipes-KCl buffer, in the absence (solid triangles) or presence of 10 mM MgCl2 (solid circles) at 25 °C. Open symbols are the [8-3H]GTP bound in the presence of an excess of GTP (200 µM). B and C, isotherms of binding of apoFtsZ and mant-GTP, measured by fluorescence anisotropy of the ligand. B, titration of mant-GTP (50 nM) with apoFtsZ (fitted {Delta}rmax = 0.234 ± 0.005 in this experiment). C, titration of apoFtsZ (500 nM) with mant-GTP. Open symbols are nonspecific binding, measured with an added excess of GTP (250 µM). Lines in each case are the best fitted model isotherms (see "Experimental Procedures" for binding parameters).

 


Figure 3
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  		FIGURE 3.
Relative affinity of FtsZ for guanine nucleotides. A, displacement curves of mant-GTP (500 nM total) from FtsZ (500 nM apoFtsZ total) by GTP (solid circles), GDP (open circles), and GMP (squares) in Pipes-KCl buffer, 10 mM MgCl2 at 25 °C. Data were determined from the change in mant-GTP anisotropy with the competing ligands (see "Experimental Procedures"). Lines (solid, dashed, and dashed and dotted) correspond to the best fitted Kb to each data set (see "Experimental Procedures" and "Results"). B, relative affinity of FtsZ for GTP and GDP. ApoFtsZ (32 µM) was incubated with GTP/GDP mixtures (total GXP 200 µM) in 50 mM Tris-HCl, 50 mM KCl, 1 mM EDTA, pH 7.5 (0.5 ml) at 25 °C for 60 min, excess nucleotide was removed, and protein-bound nucleotides were analyzed. For two ligands binding to the same site, the slope of this plot is the ratio of their equilibrium binding constants. Empty triangles, no MgCl2; solid triangles, 10 mM MgCl2.

 
Finally, affinities of binding of natural guanine nucleotides to FtsZ were measured by competition with mant-GTP in the presence of Mg2+ (Fig. 3A). Kb values for GTP, GDP, and GMP were 330 ± 80, 110 ± 40, and 0.022 ± 0.007 µM-1, respectively. The Kb value of GTP determined by competition is 3.6-fold larger than the one determined directly by [8-3H]GTP co-sedimentation with FtsZ. The relative affinity of FtsZ for GTP and GDP was measured directly by incubating apoFtsZ with nucleotide mixtures of varying GDP/GTP ratios, separating and quantifying the protein-bound nucleotides. The affinity of GTP binding is slightly larger than GDP binding, 11 ± 1- and 3.2 ± 0.3-fold with 0 and 10 mM Mg2+, respectively (Fig. 3B).

Kinetics of Nucleotide Interactions with Unassembled FtsZ—The kinetics of mant-GXP binding and dissociation from FtsZ were studied by employing stopped-flow methods at 25 °C (in the presence and absence of magnesium) and at 55 °C (without magnesium to avoid polymer formation). To measure the association under pseudo-first-order conditions, mant-nucleotide was mixed with a large excess of apoFtsZ, and the increments in fluorescence intensity (Fig. 4A) and anisotropy (Fig. 4B) of mant were recorded. The reaction time courses were fitted by single exponentials. The rate constant values determined by intensity and anisotropy were identical within experimental error, although the noise was smaller for the intensity measurements (and it could be further reduced by removing the polarizers). The small increase in fluorescence intensity with 10 mM MgCl2 could also be monitored with the stopped-flow instrument. The observed rate constant values, kapp, depend linearly on the concentration of binding sites (apoFtsZ) (Fig. 4C), which is compatible with a one-step binding mechanism, for which the following relationship holds.

Formula(Eq. 1)
We could determine the association rate constant, k+, from the slope of the regression line, but not, with sufficient precision, the dissociation rate constant k-. The association rate constant is reduced by Mg2+ and increases weakly with temperature (Table 3).


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  		TABLE 3
Kinetics of binding and dissociation of mant-nucleotides from FtsZ Association and dissociation rate constants were determined from the fluorescence intensity change of mant-GXP in Pipes-KCl buffer; constants obtained from the anisotropy increment are indicated in parentheses. Predicted values of association and dissociation rate constants in magnesium at 55 °C, obtained by multiplying the values with magnesium at 25 °C by the ratio of values at 55 and 25 °C without magnesium, are indicated solely for the purpose of comparison with further measurements (Table 4).

 
The dissociation rate was determined in displacement experiments in which an excess of unlabeled GTP was used to displace mant-GXP from its complex with FtsZ. Time courses of mant-GXP dissociation monitored by the decrease in fluorescence intensity could be fitted to single exponentials (Fig. 5), giving the first-order dissociation rate constant values (Table 3). The dissociation rate was increased by Mg2+ and temperature. Calculation of equilibrium binding constants from the association and dissociation rate constants (Kb = k+/k-) gives values within a factor of 2 from the measured equilibrium values (Table 2). This supports the simple kinetic mechanism here proposed for binding of nucleotide to unassembled FtsZ. Under conditions for FtsZ polymerization (Mg2+, 55 °C), and depending on the protein concentration, both association and dissociation became multiphasic; their kinetics are analyzed below.

Kinetics of Nucleotide Binding and Dissociation from FtsZ Polymers—The interaction kinetics of mant-nucleotides with FtsZ polymers were compared with the interaction kinetics of unassembled protein. The experiments were facilitated by polymerization of nucleotide-free FtsZ (20), although a complete kinetic analysis is hampered by the system heterogeneity, consisting of unassembled FtsZ (monomers and oligomers) and FtsZ polymers.


Figure 4
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  		FIGURE 4.
Kinetics of binding of mant-GDP and mant-GTP to ApoFtsZ. A and B, a 1 µM solution of mant-GDP was mixed with 10 µM ApoFtsZ in the stopped-flow instrument at 25 °C (final concentrations of 500 nM mant-GDP and 5 µM ApoFtsZ). Binding was followed by fluorescence intensity (A) and by anisotropy (B). Empty symbols in A and B are controls of mant-GDP mixed with buffer. Solid lines, best fits of a single exponential to experimental data (k+ (intensity) = 101 ± 2 s-1, {Delta}Fmax = 2.00 ± 0.03; k+ (anisotropy) = 110 ± 10 s-1, {Delta}rmax = 0.07 ± 0.005). C, dependence on the concentration of apoFtsZ of the observed rate constants of binding of mant-GDP (circles) and mant-GTP (squares) with 10 mM MgCl2 (solid symbols) and without MgCl2 (empty symbols) at 25 °C.

 
FtsZ polymer stability was examined first. Polymerization measurements with 10 mM MgCl2 at 55 °C showed that apoFtsZ formed pelletable polymers above a critical protein concentration Cr = 7.0 ± 0.5 µM, FtsZ-mant-GTP with Cr = 2.2 ± 0.2 µM, very similar to FtsZ-GTP with Cr = 2.2 ± 0.1 µM (Fig. 6A). The rate constant of GDP-induced depolymerization of apoFtsZ, mant-GTP-FtsZ, and GTP-FtsZ polymers were 2.6 ± 0.1, ~0.055, and 0.42 ± 0.05 s-1, respectively. Measurements of depolymerization rates by dilution in buffer without GDP were carried out in parallel (Fig. 6B) and gave the observed values of 0.16 ± 0.02 s-1 (apoFtsZ), 0.03 ± 0.01 s-1 (mant-GTP-FtsZ), and 1.2 ± 0.1 s-1 (GTP-FtsZ). FtsZ-mant-GTP polymers were observed under the electron microscope, and their morphology was found to be similar to that of GTP polymers. However, no polymers were detected with mant-GDP and up to 20 µM FtsZ by pelleting or electron microscopy. These observations resemble previous ones with GTP and GDP (18, 20). ApoFtsZ polymers bind mant-GTP in a molar ratio of 1.0 ± 0.1, determined by sedimentation of polymers at different times after adding mant-GTP and measuring protein and mant-GTP concentrations in the pellet and in the supernatant. These polymers were stable for at least 30 min after adding equimolar mant-GTP, suggesting that FtsZ very slowly hydrolyzes mant-GTP, in contrast with GTP, which is required in a larger excess to maintain the polymers. The hydrolysis rate of mant-GTP (20 µM) to mant-GDP by polymers of FtsZ (12 µM) was 4 x 10-5 s-1, 10 mM MgCl2 at 55 °C (determined by HPLC), whereas the hydrolysis rate of GTP is 0.1 s-1 under related conditions (19).


Figure 5
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  		FIGURE 5.
Kinetics of dissociation of mant-GTP and mant-GDP from unassembled FtsZ. The mant-nucleotide-FtsZ complex was formed by adding 2 µM ApoFtsZ to 1 µM mant-GXP, with or without 10 mM MgCl2. At time 0, this solution was mixed 1:1 with 400 µM GTP. The reaction was followed by the change in fluorescence of the mant-GXP (gray traces). Data were fitted to single exponentials (black lines; see rate constant values in Table 4). Trace 1, mant-GTP, 25 °C; trace 2, mant-GDP, 25 °C; trace 3, mant-GDP with Mg2+, 25 °C; trace 4, mant-GTP with Mg2+, 25 °C; trace 5, mant-GTP, 55 °C; trace 6, mant-GDP, 25 °C; trace 7, mant-GTP alone. Note that each pair of mant-GTP/mant-GDP measurements is arbitrarily displaced on the y axis to facilitate comparison.

 
Once the FtsZ polymer stability was determined, we proceeded to measure the mant-nucleotide association. Binding of mant-GTP to unassembled and polymerized apoFtsZ was first compared during the same experiment (with 10 mM MgCl2 at 55 °C) by loading, in the thermostated syringe of the stopped-flow instrument, either unassembled apoFtsZ (3 µM, under the 7.0 µM Cr of polymer formation) or partially polymerized apoFtsZ (15 µM) and then mixing it with the ligand to the same final concentration (1.5 µM). Nucleotide binding to the FtsZ polymer-containing solution was markedly slower than to unassembled FtsZ (note that the fast initial rise was smaller). Interestingly, both were complete within a few seconds and were clearly faster than the dilution-induced depolymerization measured in the same experiment (Fig. 7A); this is a model-free observation. The time courses in these experiments were best fitted by a sum of three exponentials. The slowest of them was independent of protein concentration and had a constant rate value of 0.42 ± 0.05 s-1. This phase was attributed to an uncharacterized rearrangement of the system, and its value was constrained in further analysis. The apparent rate constants of the fastest and second fastest phases in this experiment (Fig. 7A) were as follows: unassembled, ~70 and 4.6 s-1; polymerized, ~40 and 4.3 s-1. Their relative amplitudes were 10:1 in the unassembled sample and 0.4:1 in the polymerized sample. This suggests parallel reactions with two types of binding sites, fast (unassembled) and slow (polymerized FtsZ), present in different proportions in each sample. To estimate the bimolecular rate constants of binding of mant-nucleotides to unassembled and polymerized FtsZ, apoFtsZ solutions were mixed at different final concentrations in excess over the nucleotide, time courses were fitted as above, and observed rates were plotted against total protein concentration (Fig. 7, B and C). The results (Table 4) indicate that unassembled apoFtsZ binds mant-GTP with a rate constant k1 {approx} 30 µM-1 s-1. This fast rate is twice the rough value predicted from measurements under related conditions (Table 3). The equivalent fast component, which is observed with decreased amplitude in polymerized apoFtsZ solutions (Table 4), can be attributed to the fraction of unassembled protein. It may then be proposed that the second rate constant, k2 {approx} 1 µM-1 s-1, whose amplitude increases upon FtsZ polymerization (Table 4), reflects the binding of mant-GTP to apoFtsZ polymers. This process is more than 1 order of magnitude slower than the binding to unassembled apoFtsZ (the fact that this slow component can also be detected in a small proportion at FtsZ concentrations below the Cr measured by sedimentation might be explained by formation of polymer nucleation species which fail to pellet). Apparent rates of binding of mant-GDP and mant-GTP to polymerized apoFtsZ were similar (Fig. 7C). The inherent limitations of this phase analysis should be kept in mind, including the possibility that we are approximating a continuum of reaction rates from diverse unassembled and polymeric FtsZ species with a simple sum of a few exponentials. The dissociation rate of mant-GTP from unassembled FtsZ under polymerization conditions, ~40 s-1, estimated from the y axis intercepts in Fig. 7, B and C, is compatible with rough predicted values (Table 3); the dissociation rate from polymers could not be determined by this method.


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  		TABLE 4
Association kinetics of mant-GTP to apo-FtsZ under polymerization solution conditions Values in parenthesis are the average relative amplitude of each phase. Association values from apoFtsZ above Cr have been corrected by the fractions of unassembled and polymeric FtsZ in solution. Available mant-GDP data are roughly similar to the mant-GTP data (Fig. 7, B and C).

 


Figure 6
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  		FIGURE 6.
A, nucleated polymerization of apoFtsZ with mant-GTP (20 µM; solid circles) or GTP (1 mM; solid triangles) or without any nucleotide (empty circles) in Pipes-KCl buffer, 10 mM MgCl2, at 55 °C. The polymers formed were measured by sedimentation. B, kinetics of depolymerization monitored by light scattering in the stopped-flow under the same solution conditions as in A. ApoFtsZ polymers (15 µM) were diluted to 12 µM in 2 mM GDP (trace 1) or to 1.5 µM in buffer (trace 2). FtsZ-Mant-GTP polymers (15 µM) were diluted to 1.5 µM in buffer (trace 3) (for dissociation in GDP excess, see dashed trace 2 in Fig. 8). FtsZ-GTP polymers (15 µM) were diluted to 12 µM in 2 mM GDP (trace 4) or to 1.5 µM in buffer (trace 5).

 


Figure 7
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  		FIGURE 7.
A, kinetics of binding of mant-GTP to unassembled (trace 1) and to polymerized (trace 2) apoFtsZ. Polymers were formed from 15 µM apoFtsZ with 10 mM MgCl2 at 55 °C (8 µM apoFtsZ in polymers) and mixed in the stopped flow at final concentrations of 1.5 µM FtsZ (0.8 µM polymers) and 400 nM mant-GTP (trace 2). Light scattering was recorded during the experiment with apoFtsZ polymers to monitor their stability (trace 3). The light scattering signal changed from 2.22 to 2.18 during the ligand binding measurement and to 2.10 later when depolymerization was complete. Unassembled apoFtsZ (3 µM, under the same conditions) was mixed to 1.5µM final concentration (trace 1). Data were fitted by a sum of three exponentials, k1app, k2app, and k3app:72 s-1 (69%), 4.6 s-1 (7%), and 0.42 s-1 (24%) (trace 1); 43 s-1 (16%), 4.3 s-1 (32%), and 0.42 s-1 (52%) (trace 2). Residuals of the fit are shown in the small panel above. B, dependence of the observed rate constants k1app and k2app on the concentration of unassembled apoFtsZ (solid circles, 400 nM mant-GTP; empty circles, mant-GDP shown for comparison). C, dependence of the observed rate constants k1app and k2app on the final total concentration of apoFtsZ under polymerization conditions (400 nM mant-GXP); a very slow third rate constant, k3app = 0.42 s-1, was independent of protein concentration and is not plotted.

 
Following the analysis of binding kinetics, the dissociation of mant-nucleotide from unassembled FtsZ, oligomers, and FtsZ polymers were compared. To do this, mant-GXP-FtsZ was mixed with a large excess of GDP or GTP in the stopped flow, with a minimal (10%) dilution of protein to avoid depolymerization. Dissociation of mant-GTP and mant-GDP from unassembled FtsZ (0.55 µM initial concentration, well below the 2.2 µM Cr for polymerization) took place with rate constants of ~20 and ~50 s-1, respectively (Table 5 and supplemental Fig. 3), which are compatible with predicted values (Table 3). Dissociation of mant-GDP from oligomeric FtsZ (10 µM FtsZ-mant-GDP initial concentration) included a principal component with a rate constant of 0.20 s-1 (Table 5 and supplemental Fig. 3) possibly due to the FtsZ oligomers. Dissociation of mant-GTP was found to be markedly slower in polymerized FtsZ solutions (10 µM FtsZ-mant-GTP initial concentration, well above the 2.2 µM polymerization Cr). In an excess of GTP, dissociation consisted of two phases (0.018 ± 0.001 s-1, 85% amplitude; 0.20 ± 0.01 s-1, 15% amplitude). It was followed by partial depolymerization at a rate of 0.012 ± 0.001 s-1 (Fig. 8, trace 1, and Table 5) and by new GTP-induced polymerization at longer times (not shown). These results indicate that mant-GTP-FtsZ polymers depolymerize upon substitution of the fluorescent derivative by GTP, suggesting that GTP-FtsZ and mant-GTP-FtsZ do not freely co-polymerize into exactly the same polymer, possibly due to some structural perturbation induced by the fluorescent group, which also inhibits the nucleotide hydrolysis. With an excess of GDP, mant-GTP dissociation proceeded at a rate of ~0.057 s-1 (which could not be fitted by a sum of exponentials) and was closely followed by depolymerization at ~0.055 s-1 monitored by light scattering (Fig. 8, trace 2, and Table 5). Since dilution-induced depolymerization takes place in a similar time scale (see above), an experimental comparison of dissociation from unassembled and polymerized FtsZ at the same final low protein concentration (as in the case of the association) could not be made.


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  		TABLE 5
Observed dissociation rate constants (s–1) of mant-GXP from FtsZ under polymerization solution conditions ApoFtsZ with added mant-GTP or mant-GDP (1.1 nucleotide/1 FtsZ) was allowed to polymerize at 55 °C with 10 mM MgCl2 and mixed in the stopped flow with an excess (1 mM) of GTP or GDP as indicated. The reaction time courses were recorded during 400 s. NO, a second phase was not observed.

 
The 0.02-0.06 s-1 mant-GTP dissociation rate, which may be attributed to FtsZ polymers, is several hundred-fold slower than dissociation from unassembled FtsZ under the same solution conditions. These results could be interpreted as either a lower intrinsic dissociation rate of mant-GTP from FtsZ polymers or as the result of a rate-limiting exchange of FtsZ monomers in these polymers, followed by fast nucleotide dissociation from the unassembled subunits. It should be kept in mind that this, necessarily simplified, analysis only partially resolves the dissociation rates of the nucleotide from the multiple FtsZ species present.

Accessibility of the Nucleotide Binding Site in Stable Sheet of FtsZ-W319Y-His6—In order to probe the accessibility of the nucleotide binding site in FtsZ polymers without the complications due to subunit exchange, it was desirable to use stabilized FtsZ polymers. Under standard conditions, histidine-tagged FtsZ-His6 polymerizes, hydrolyzes GTP, and depolymerizes similarly to FtsZ, but large stable sheets are formed by the nonhydrolyzing point mutant FtsZ-W319Y-His6 (19), which were employed as stable model FtsZ polymers. X-ray structures of FtsZ-W319Y and FtsZ-His6 are superimposable (22). The FtsZ-W319Y-His6 sheets are made up of double protofilaments with the same 4-nm tubulin-like subunit spacing as in wild-type FtsZ filaments. They hardly disassemble with an excess of GDP or in the cold (19). Polymerized apoFtsZ-W319Y-His6 readily binds mant-GTP, with a marked increase in fluorescence intensity of the ligand; the addition of an excess of GTP reduced fluorescence to the level of free mant-GTP (Fig. 9A). Both mant-GTP association and dissociation were essentially complete (>90%) within the dead time of measurement (~20 s, therefore proceeding at an apparent rate of >0.1 s-1). This implies, for the reactant concentrations employed (12.5 µM mant-GTP and ~6 µM polymerized FtsZ-W319Y-His6, determined by sedimentation) a bimolecular association constant of >0.05 µM-1 s-1 for the slowest FtsZ species (52) and a dissociation constant of >0.1 s-1. These rate constant are compatible with the corresponding values for wild-type FtsZ polymers (Table 5). The association time course of 0.4 µM mant-GTP to 9 µM apoFtsZ-W319Y-His6 (~4 µM polymers) (Fig. 9B) was biphasic, with apparent rate constants of 8.7 ± 0.2 s-1 (48%) and 0.290 ± 0.003 s-1 (52%), which may be assigned to FtsZ-W319Y-His6 monomer and polymer, respectively. The dissociation time course could not be measured due to destruction of the FtsZ-W319Y-His6-mant-GTP polymers in the stopped flow.


Figure 8
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  		FIGURE 8.
Kinetics of dissociation of mant-GTP and mant-GDP from FtsZ under polymerization conditions. Dissociation of mant-GTP from 10 µM FtsZ polymers diluted to 9 µM in 1 mM GTP (line 1) or 1 mM GDP (line 2) or without nucleotide (line 3). Light scattering was also recorded during the experiment to follow depolymerization (corresponding dashed lines). Residuals are of a double exponential fit to fluorescence intensity (line 1) and a single exponential fit to light scattering (line 2) (see rate values under "Results"; a multiple exponential did not improve fit 2).

 
In order to estimate the rate of subunit exchange in the polymers of FtsZ-W319Y-His6, a small proportion (5%) of tracer FtsZ-His6 was added to the solution of preformed polymers. FtsZ-His6 (in a 1-10% proportion) had been observed to freely copolymerize with FtsZ-W319Y-His6 (from which it can be distinguished by fluorescence of the single tryptophan Trp319) without significantly perturbing polymer formation. The exchange process was very slow (Fig. 9C), with an observed rate constant of 0.0004 s-1, which clearly cannot account for the much faster observed binding of the mant-nucleotide (apparent rate of >0.1 s-1). Finally, mant-GTP binding results were complemented by independent measurements of the exchange of [{alpha}-32P]GTP into unassembled and polymerized FtsZ-W319Y-His6 and FtsZ-His6, which exchanged one GTP in less than 20 s in all cases (Fig. 9D). It can therefore be concluded that the nucleotide binding site is accessible in FtsZ-W319Y-His6 stable model polymers.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Energetics of Nucleotide Binding to FtsZ and Functional Consequences—Guanine nucleotide binding and dissociation are central to the dynamics of FtsZ and tubulin polymers, which are in turn essential for their respective cellular functions. Thermophilic apoFtsZ from M. jannaschii was employed in this work as a conveniently stable model protein for the study of the interactions of FtsZ with nucleotides. Similar experiments with nucleotide-free mesophilic FtsZ from E. coli were precluded by its instability. GDP stabilizes FtsZ against thermal denaturation. The destabilizing effect of Mg2+ on FtsZ-GDP may be explained by a reduction in the binding affinity of GDP (see below); alternately, the cation may be increasing the rate of irreversible thermal denaturation of the protein and therefore decreasing the apparent Tm. GTP binding imperceptibly modifies the average secondary structure of the protein, in agreement with the similar polymerization properties (20) and crystal structures of the nucleotide-free and GTP-liganded forms of this FtsZ (22). These results support the notion that the bound nucleotide has little structural role in M. jannaschii FtsZ monomers and polymers, but it is employed to trigger disassembly upon hydrolysis (20).


Figure 9
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  		FIGURE 9.
Interactions of nonhydrolyzing mutant FtsZ-W319Y-His6 with nucleotides. A, mant-GTP binding to polymers of FtsZ-W319Y-His6. Solid line, fluorescence emission spectrum of 12.5 µM mant-GTP in a solution of 12.5 µM polymerized apoFtsZ-W319Y-His6; dashed line, mant-GTP alone; dotted line, mant-GTP + polymerized FtsZ-W319Y-His6 + 1 mM GTP; dashed and dotted line, polymerized FtsZ-W319Y-His6 alone. The spectrum of mant-GTP + GTP was identical to that of mant-GTP alone. Excitation was at 357 nm (1-nm bandwidth); emission maxima of bound mant-GTP was at 439 nm, free at 446 nm (2-nm bandwith). Inset, an electron micrograph of the FtsZ-W319Y-His6-mant-GTP polymers; the bar indicates 100 nm. B, association time course of 0.4 µM mant-GTP to 9 µM apoFtsZ-W319Y-His6 monitored with the stopped flow. Gray trace, data; dark line, biexponential best fit. C, time course of incorporation of 0.63 µM FtsZ-His6 into ~6 µM preformed polymers of the FtsZ-W319Y-His6 Trp-less mutant (12.5 µM total concentration), measured by polymer sedimentation. The solid line is a single exponential fit to data, giving a pseudo-first order rate constant of 4 ± 1 x 10-4 s-1 and an amplitude of 0.06 ± 0.01 (marked fit); maximum incorporation was 0.053 ± 0.003 (marked exp), determined by copolymerizing the FtsZ-wt-His6 together with FtsZ-W319Y-His6. D, time courses of exchange of [{alpha}-32P]GTP into 12.5 µM FtsZ-W319Y-His6 (solid circles) or FtsZ-His6 (empty circles) under no assembly (left, no MgCl2) or polymerization conditions (right, 6 mM MgCl2), measured with a filtration assay. Note that FtsZ-His6 polymers hydrolyze GTP, whereas FtsZ-W319Y-His6 ones do not.

 
ApoFtsZ binds guanine nucleotides with relatively high affinity. Kb values ranging from 20 to 300 µM-1 weakly increased with the presence of the nucleotide {gamma}-phosphate and decreased with a chelating Mg2+ ion (Table 1). An equivalent effect is observed with mant-GDP and mant-GTP (Table 2). MgCl2 moderately reduces the association rates and enhances the dissociation rates of mant-GDP and mant-GTP (Table 3). These results indicate that the Mg2+ ion bound to the nucleotide β- and {gamma}-phosphates and Gln75 observed in the crystal structure of FtsZ (22), suggested to assist the hydrolysis of the {gamma}-phosphate by FtsZ polymers, provides little additional stability to the FtsZ monomer-nucleotide complex. This may be explained by (i) Mg2+ binding to another low affinity site that has to be displaced by the nucleotide binding or that allosterically weakens the observed nucleotide binding affinity or (ii) the existence of a slightly unfavorable process that makes the binding of the nucleotide·Mg2+ complex less favorable than the binding of the nucleotide alone. The first explanation is consistent with the quenching of the fluorescence of FtsZ-bound mant-GXP (see "Results") induced by Mg2+ and with the low affinity binding of Mg2+ to tubulin (53). The second explanation would be compatible with a change in protonation upon Mg2+ binding or with the introduction of some strain by the binding of Mg2+ to the FtsZ-GTP complex.

FtsZ and tubulin form a distinct family of GTPases (1), but there are structural (22) and important functional differences between the FtsZ and tubulin nucleotide binding sites. Unlike FtsZ, nucleotide {gamma}-phosphate and Mg2+ binding are linked in {alpha}β-tubulin (54). The nucleotide {gamma}-phosphate and the coordinated Mg2+ ion bound at the functional GTP/GDP binding site of β-tubulin control microtubule stability, whereas the Mg2+ bound to the nonfunctional GTP site of {alpha}-tubulin controls the stability of the {alpha}β-dimer (55). In classical GTPases, GTP is bound in complex with Mg2+, which is coordinated to oxygens from the β- and {gamma}-phosphates. However, the functional roles of the {gamma}-phosphate and Mg2+ vary among different G-proteins. Thus, Ras and EF-Tu form tight GDP·Mg complexes, Mg2+ binding reduces the GDP off rate by 4 orders of magnitude, and GDP binds more tightly than GTP (56). As another example, Mg2+ is not required for GDP binding to eRF3 but strengthens GTP binding; no structural changes were observed for GTP·Mg2+ and GDP·Mg2+ binding to eRF3 (57). In Rho proteins, the Mg2+ cofactor does not affect the nucleotide binding affinity per se but rather acts as a kinetic stabilizer for bound nucleotides by slowing down both the off and on rates (58). The different properties of the FtsZ nucleotide binding site in comparison with tubulin and other GTPases suggest the possibility of fine tuning specific inhibitors for the FtsZ-GTP interaction.

Interactions of FtsZ Monomers with Fluorescent Mant-nucleotides, Kinetics of Binding, and Competitive Assay for Ligands of the FtsZ Nucleotide Site—Interactions of FtsZ monomers with GTP and GDP were probed by employing the fluorescence anisotropy change of their mant derivatives in dilute solutions. The kinetics of association of mant-nucleotides to unassembled FtsZ is compatible with a one-step reaction, with fast association rate constant values (10 < k+ < 40 µM-1 s-1) and dissociation rates (1 < k+ < 10 s-1), depending on solution conditions (Table 3). Rate constant values are weakly dependent on temperature, suggesting small activation energies for nucleotide association and dissociation from an easily accessible site.

The bound mant-GTP is specifically displaced by nonfluorescent nucleotides. Except for the possible offset in absolute Kb values determined by competition and ITC methods, the ratio Kb(GTP-Mg2+)/Kb(GDP-Mg2+) determined with the competition method is 3 ± 2, which is comparable with the 3.2 ± 0.3 ratio directly determined with GTP and GDP (Fig. 3B), with the ~1.5 ratio from ITC (Table 1), and with the 2.6 ± 0.6 ratio of the respective mant-derivatives (Table 2).

The mant-nucleotide displacement method outlined here is a homogeneous fluorescence assay that may, in principle, be conveniently employed to characterize the binding of any nucleotides or other substances, such as small molecule modulators of FtsZ assembly (see Introduction), to its nucleotide site, as well as to measure the effects of ligand modifications on binding affinity. This method may be eventually scaled up to screen for inhibitors binding to the FtsZ nucleotide site.


Figure 10
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  		FIGURE 10.
Interactions of FtsZ monomers and polymers with fluorescent and natural nucleotides. A, scheme of mant-GTP binding kinetics to FtsZ monomers and polymers. Exchangeable mant-GTP is kinetically stabilized in polymers of FtsZ from M. jannaschii. The nucleotide is represented by small black circles. Mant-GTP was not hydrolyzed during the experiments; therefore, all reactions represented in this scheme are chemical equilibria. Nucleotide binding and dissociation rates were determined from fluorescence measurements in this work. Polymer dissociation rates are preliminary estimates from light scattering measurements following dilution, both with a stopped-flow instrument. Polymer elongation rates (k+) were calculated from the critical concentration (Cr) and dissociation rate (k-) values (Cr = k-/k+) for an end-growing nucleated polymer in equilibrium with monomers (59). B, do FtsZ polymers cycle with GTP hydrolysis? In this scheme, GTP and GDP are represented by small filled and empty circles, respectively. Nucleotide exchange by FtsZ monomers in a GTP excess is expected to be relatively fast (similarly to mant-nucleotides; Table 3) and not rate-limiting for consecutive reactions. FtsZ-GTP polymers disassemble at a relatively fast rate (upon dilution or in GDP excess). Nucleotide exchange by FtsZ polymers in a GTP excess is limited by an as yet unknown rate of GDP dissociation from FtsZ polymers. Nucleotide exchange by the polymer has an effect opposite to that of hydrolysis. Depending on the ratio of GDP dissociation and GDP-polymer disassembly rates to the hydrolysis rate, FtsZ polymers may undergo cycles of assembly, GTP hydrolysis, disassembly, and nucleotide exchange as indicated by the curved arrow.

 
Exchangeable Nucleotide Is Kinetically Stabilized in FtsZ Polymers—How FtsZ polymers exchange the hydrolyzed nucleotide is a major unresolved issue, which impacts on the mechanism of their dynamics being possibly different or similar to microtubules (see Introduction). The results of this study indicate that the nucleotide remains exchangeable in polymers of FtsZ from M. jannaschii. The accessibility of the nucleotide binding site in FtsZ polymers was probed by measuring the kinetics of interactions of FtsZ with mant-GTP under polymerization conditions. The scheme in Fig. 10A summarizes relevant rate constants determined with unassembled FtsZ (at protein concentrations below Cr) and estimated for FtsZ polymers (above Cr). The apoFtsZ polymers are obviously devoid of any dynamics related to nucleotide hydrolysis, and FtsZ-mant-GTP polymers do not hydrolyze the nucleotide on the time scale of these experiments. Under polymerization conditions, binding kinetics becomes multiphasic due to the different FtsZ aggregation species found in solution. A nucleotide association phase with a rate constant 1 µM-1 s-1, whose amplitude increases upon polymerization (see "Results"), was ascribed to the binding of mant-GTP to FtsZ polymers, 30-fold slower than to unassembled FtsZ. This binding rate constant cannot come from the dissociation of apoFtsZ subunits or from nucleotide binding to the unassembled protein, since the apoFtsZ polymer dissociation rate is much lower than the observed mant-GTP binding rates (Fig. 7).

Dissociation of mant-GTP from FtsZ polymers proceeds at an observed rate of 0.06 s-1 in GDP excess (0.02 s-1 in GTP excess), which is 3 orders of magnitude slower than dissociation from unassembled FtsZ. The value of 0.21 s-1 estimated for mant-GTP dissociation from FtsZ polymers using the rate and equilibrium constants depicted in the reaction box of Fig. 10A is only 3.5-fold higher (not too bad, considering the difficulty of several of the kinetic measurements). The fact that the mant-GTP dissociation time course shortly precedes polymer disassembly (Fig. 8) would be compatible with direct dissociation of mant-GTP from the polymer, closely followed by disassembly of the GDP-bound polymer at the rates indicated (Fig. 10A), However, we do not think that monitoring the polymer concentration by scattering is accurate enough to warrant this interpretation. Given the similarity of the apparent ligand dissociation rate and the polymer disassembly rate, this result may also be interpreted as due to FtsZ depolymerization followed by fast mant-GTP dissociation from FtsZ monomers. According to this interpretation, the 0.06 s-1 value would be only an upper limit to the true rate constant of mant-GTP dissociation from the polymers. The slower dissociation rate in excess of GTP indicates the participation of polymer disassembly in this process. In either case, our results indicate that the nucleotide is kinetically stabilized in FtsZ polymers with respect to monomers. This agrees with an accessible nucleotide binding site located between two consecutive monomers along the FtsZ protofilament (22). Mant-GTP binding and FtsZ polymer elongation moderately favor each other, with a linkage free energy of only -1.1 ± 0.4 kcal mol-1, calculated from data in Fig. 10A.

In order to unequivocally prove whether FtsZ polymers can bind and dissociate nucleotide without subunit exchange, stabilized FtsZ polymers were needed. These have been provided by the mutant FtsZ-W319Y-His6, which forms an inactive GTPase sheet (further stabilized by the His tag (19)) and co-polymerizes with wild-type FtsZ-His6. Wild-type subunits slowly exchange into mutant polymers at a rate of 0.0004 s-1, whereas polymers bind and dissociate mant-GTP nucleotide at a much faster rate, >0.1 s-1, under the same conditions. This shows that exchange of the bound nucleotide without subunit exchange is possible in these model FtsZ polymers.

Implications for FtsZ Polymer Dynamics—The observation that mant-nucleotide exchange can take place without hydrolysis in polymers of M. jannaschii FtsZ gives insight into FtsZ polymer dynamics. These results might superficially seem to favor models in which FtsZ is devoid of any microtubule-like dynamics. However, the problem is quantitative; the kinetic pathway actually operative will depend on the effective reaction rates under given conditions. Once FtsZ polymers eventually hydrolyze mant-GTP and release Pi, mant-GDP would be expected to induce disassembly, but, since mant-GTP hydrolysis is much slower than the mant-nucleotide exchange, it does not influence polymer dynamics. This is not the case with the natural nucleotide GTP. Models for FtsZ assembly with GTP are schematized in Fig. 10B. M. jannaschii FtsZ polymers hydrolyze GTP with a turnover of 0.10 s-1 (19), which is similar to the value of 0.07 s-1 reported for E. coli FtsZ, at lower temperature (21). M. jannaschii FtsZ polymers disassemble rapidly, with half-times of 0.6 s (FtsZ-GTP polymers) to 25 s (mant-GTP-FtsZ polymers); these values comprise the 5 s half-time for E. coli FtsZ polymer disassembly (in GDP excess) and the 7 s half-time of subunit exchange, reported under quite different conditions (25). GDP dissociation from FtsZ polymers, which is difficult to measure, is rate-limiting to the exchange of GTP into polymers. If it is faster than the rate of hydrolysis, the steady-state polymer may contain mainly GTP and a minor fraction of GDP-bound subunits, which will have a given probability of fragmenting the polymer. As long as the nucleotide exchange in the polymer is significantly faster than hydrolysis and disassembly, subunit turnover is expected to be independent of the GTPase rate. On the contrary, if GDP dissociation is slower than GTP hydrolysis, GDP-bound subunits will accumulate, and the polymer will disassemble. Subunits will then rapidly exchange nucleotide with the solution and recycle into new polymers (indicated by the circular arrow in Fig. 10B). In this case, subunit turnover is expected to depend on the GTPase rate. A steady-state population of recycling FtsZ polymers will contain mainly GTP polymers and a small fraction of GDP-containing FtsZ polymers. An estimate for the dissociation rate of GDP-FtsZ polymers is provided by the observed GDP-induced dissociation rate constant (2.6 s-1) of apoFtsZ polymers, assuming that the binding of GDP is not rate-limiting.

The observation that subunit exchange is very slow in polymers of the GTPase-deficient mutant FtsZ-W319Y-His6 (Fig. 9) compared with the fast disassembly of FtsZ-His6 active GTPase (19) and the important findings that (i) the turnover of FtsZ-GFP subunits in the Z-rings of E. coli cells is reduced in mutant ftsZ84, which has a slow GTPase in vitro (23, 24), (ii) FtsZ subunit exchange in a fluorescence resonance energy transfer assay is strongly reduced by the slowly hydrolyzable nucleotide GMPCMP (25), and (iii) the correlation very recently found, between the slower subunit turnover, GTPase, and GDP-induced disassembly in Mycobacterium tuberculosis FtsZ (26) favors a polymer recycling model of FtsZ assembly (Fig. 10B). In conclusion, FtsZ polymers can be observed to exchange nucleotide, unlike microtubules, but GDP dissociation may be slow enough for FtsZ polymer disassembly to take place first, as in microtubules, resulting in FtsZ polymers cycling with GTP hydrolysis. Since FtsZ polymers are typically single flexible protofilaments a few hundred nanometers long (60), it is possible that their relatively rapid assembly and disassembly but proceeds between membrane attachment points and provides continuously updated positional information for the assembly and operation of the septosome.


   FOOTNOTES
 
* This work was supported in part by grants Ministerio de Educacion y Ciencia (MEC) BFU 2005-00505/BMC (to J. M. A.), MEC BFU 2006-10288 (to M. M.), and Comunidad Autónoma de Madrid S-BIO-0214-2006 (to J. M. A. and J. F. D.), a Consejo Superior de Investigaciones Científicas-I3P postdoctoral contract (to S. H.), a MEC-FPI predoctoral fellowship (to C. S.), and Travel Grant FAPESP CBME-98/14138-2 (to W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

2 Present address: Centro de Biotecnologia Molecular e Estrutural, Instituto de Física de São Carlos, USP, Brazil. Back

3 Present address: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain. Back

4 Present address: GlaxoSmithKline, Madrid, Spain. Back

1 To whom correspondence may be addressed. Tel.: 34-918373112 (ext. 4381); Fax: 34-915360432; E-mail: sonia{at}cib.csic.es. 5 To whom correspondence may be addressed. Tel.: 34-918373112 (ext. 4381); Fax: 34-915360432; E-mail: j.m.andreu{at}cib.csic.es.

6 The abbreviations used are: mant, 2'/3'-O-(N-methyl-anthraniloyl); mant-GTP, 2'/3'-O-(N-methyl-anthraniloyl)-guanosine-5'-triphosphate; mant-GDP, 2'/3'-O-(N-methyl-anthraniloyl)-guanosine-5'-diphosphate; HPLC, high pressure liquid chromatography; GdmCl, guanidinium chloride; Pipes, 1,4-piperazinediethanesulfonic acid; DSC, differential scanning calorimetry; ITC, isothermal titration calorimetry; Mes, 4-morpholineethanesulfonic acid. Back

7 S. Huecas, C. Schaffner-Barbero, J. F. Díaz, and J. M. Andreu, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Martín Alba for FtsZ purification and Dr. Jesús Mingorance for help with the [{alpha}-32P]GTP filtration assay.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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