GTP hydrolysis of cell division protein FtsZ: evidence that the active site is formed by the association of monomers.

The essential prokaryotic cell division protein FtsZ is a tubulin homologue that forms a ring at the division site. FtsZ forms polymers in a GTP-dependent manner. Recent biochemical evidence has shown that FtsZ forms multimeric structures in vitro and in vivo and functions as a self-activating GTPase. Structural analysis of FtsZ points to an important role for the highly conserved tubulin-like loop 7 (T7-loop) in the self-activation of GTP hydrolysis. The T7-loop was postulated to form the active site together with the nucleotide-binding site on an adjacent FtsZ monomer. To characterize the role of the T7-loop of Escherichia coli FtsZ, we have mutagenized residues M206, N207, D209, D212, and R214. All the mutant proteins, except the R214 mutant, are severely affected in polymerization and GTP hydrolysis. Charged residues D209 and D212 cannot be substituted with a glutamate residue. All mutants interact with wild-type FtsZ in vitro, indicating that the T7-loop mutations do not abolish FtsZ self-association. Strikingly, in mixtures of wild-type and mutant proteins, most mutants are capable of inhibiting wild-type GTP hydrolysis. We conclude that the T7-loop is part of the active site for GTP hydrolysis, formed by the association of two FtsZ monomers.     

Structural Change in FtsZ Induced by Intermolecular Interactions between Bound GTP and the T7 Loop

FtsZ is a prokaryotic homolog of tubulin and is a key molecule in bacterial cell division. FtsZ with bound GTP polymerizes into tubulin-like protofilaments. Upon polymerization, the T7 loop of one subunit is inserted into the nucleotide-binding pocket of the second subunit, which results in GTP hydrolysis. Thus, the T7 loop is important for both polymerization and hydrolysis in the tubulin/FtsZ family. Although x-ray crystallography revealed both straight and curved conformations of tubulin, only a curved structure was known for FtsZ. Recently, however, FtsZ from Staphylococcus aureus has been shown to have a very different conformation from the canonical FtsZ structure. The present study was performed to investigate the structure of FtsZ from Staphylococcus aureus by mutagenesis experiments; the effects of amino acid changes in the T7 loop on the structure as well as on GTPase activity were studied. These analyses indicated that FtsZ changes its conformation suitable for polymerization and GTP hydrolysis by movement between N- and C-subdomains via intermolecular interactions between bound nucleotide and residues in the T7 loop.     

Polymerization of Ftsz, a bacterial homolog of tubulin. is assembly cooperative?

FtsZ is a bacterial homolog of tubulin that is essential for prokaryotic cytokinesis. In vitro, GTP induces FtsZ to assemble into straight, 5-nm-wide polymers. Here we show that the polymerization of these FtsZ filaments most closely resembles noncooperative (or "isodesmic") assembly; the polymers are single-stranded and assemble with no evidence of a nucleation phase and without a critical concentration. We have developed a model for the isodesmic polymerization that includes GTP hydrolysis in the scheme. The model can account for the lengths of the FtsZ polymers and their maximum steady state nucleotide hydrolysis rates. It predicts that unlike microtubules, FtsZ protofilaments consist of GTP-bound FtsZ subunits that hydrolyze their nucleotide only slowly and are connected by high affinity longitudinal bonds with a nanomolar K(D).     

Rate-limiting guanosine 5'-triphosphate hydrolysis during nucleotide turnover by FtsZ, a prokaryotic tubulin homologue involved in bacterial cell division

FtsZ is a prokaryotic tubulin homologue that polymerizes into a dynamic ring during cell division. GTP binding and hydrolysis provide the energy for FtsZ dynamics. However, the precise role of hydrolysis in polymer assembly and turnover is not understood, limiting our understanding of how FtsZ functions in the cell. Here we investigate GTP hydrolysis during the FtsZ polymerization cycle using several complementary approaches that avoid technical caveats of previous studies. We find that at steady state approximately 80% of FtsZ polymer subunits are bound to GTP. In addition, we use pre-steady-state, single turnover assays to directly measure the rate of hydrolysis. Hydrolysis was found to occur at approximately 8/min and to be a rate-limiting step in GTP turnover; phosphate release rapidly followed. These results clarify previously conflicting results in the literature and suggest that pure FtsZ polymers, unlike microtubules, may not be able to undergo dynamic instability or to store energy in the polymer for force production.     

Escherichia coli FtsZ polymers contain mostly GTP and have a high nucleotide turnover

The cell division protein FtsZ is a GTPase structurally related to tubulin and, like tubulin, it assembles in vitro into filaments, sheets and other structures. To study the roles that GTP binding and hydrolysis play in the dynamics of FtsZ polymerization, the nucleotide contents of FtsZ were measured under different polymerizing conditions using a nitrocellulose filter-binding assay, whereas polymerization of the protein was followed in parallel by light scattering. Unpolymerized FtsZ bound 1 mol of GTP mol(-1) protein monomer. At pH 7.5 and in the presence of Mg(2+) and K(+), there was a strong GTPase activity; most of the bound nucleotide was GTP during the first few minutes but, later, the amount of GTP decreased in parallel with depolymerization, whereas the total nucleotide contents remained invariant. These results show that the long FtsZ polymers formed in solution contain mostly GTP. Incorporation of nucleotides into the protein was very fast either when the label was introduced at the onset of the reaction or subsequently during polymerization. Molecular modelling of an FtsZ dimer showed the presence of a cleft between the two subunits maintaining the nucleotide binding site open to the medium. These results show that the FtsZ polymers are highly dynamic structures that quickly exchange the bound nucleotide, and this exchange can occur in all the subunits.     

The interactions of cell division protein FtsZ with guanine nucleotides

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 K(b) values ranging from 20 to 300 microm(-1) under various conditions. GTP.Mg(2+) and GDP.Mg(2+) bind with slightly reduced affinity. Bound GTP and the coordinated Mg(2+) ion play a minor structural role in FtsZ monomers, but Mg(2+)-assisted GTP hydrolysis triggers polymer disassembly. Mant-GTP binds and dissociates quickly from FtsZ monomers, with approximately 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 approximately 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.     

Structural insights into FtsZ protofilament formation

The prokaryotic tubulin homolog FtsZ polymerizes into a ring structure essential for bacterial cell division. We have used refolded FtsZ to crystallize a tubulin-like protofilament. The N- and C-terminal domains of two consecutive subunits in the filament assemble to form the GTPase site, with the C-terminal domain providing water-polarizing residues. A domain-swapped structure of FtsZ and biochemical data on purified N- and C-terminal domains show that they are independent. This leads to a model of how FtsZ and tubulin polymerization evolved by fusing two domains. In polymerized tubulin, the nucleotide-binding pocket is occluded, which leads to nucleotide exchange being the rate-limiting step and to dynamic instability. In our FtsZ filament structure the nucleotide is exchangeable, explaining why, in this filament, nucleotide hydrolysis is the rate-limiting step during FtsZ polymerization. Furthermore, crystal structures of FtsZ in different nucleotide states reveal notably few differences.     

Non-hydrolysable GTP-gamma-S stabilizes the FtsZ polymer in a GDP-bound state

FtsZ, a tubulin homologue, forms a cytokinetic ring at the site of cell division in prokaryotes. The ring is thought to consist of polymers that assemble in a strictly GTP-dependent way. GTP, but not guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), has been shown to induce polymerization of FtsZ, whereas in vitro Ca2+ is known to inhibit the GTP hydrolysis activity of FtsZ. We have studied FtsZ dynamics at limiting GTP concentrations in the presence of 10 mM Ca2+. GTP and its non-hydrolysable analogue GTP-gamma-S bind FtsZ with similar affinity, whereas the non-hydrolysable analogue guanylyl-imidodiphosphate (GMP-PNP) is a poor substrate. Preformed FtsZ polymers can be stabilized by GTP-gamma-S and are destabilized by GDP. As more than 95% of the nucleotide associated with the FtsZ polymer is in the GDP form, it is concluded that GTP hydrolysis by itself does not trigger FtsZ polymer disassembly. Strikingly, GTP-gamma-S exchanges only a small portion of the FtsZ polymer-bound GDP. These data suggest that FtsZ polymers are stabilized by a small fraction of GTP-containing FtsZ subunits. These subunits may be located either throughout the polymer or at the polymer ends, forming a GTP cap similar to tubulin.     

Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ.

The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin. We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 microM. At 5 microM FtsZ, we have determined a K(m) of 82 microM GTP and a V(max) of 490 nmol of P(i) min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg(2+) and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 microM total protein concentration. Chemical cross-linking of FtsZ also shows that monomer, dimer, trimer, and tetramer species are present at higher (>2 microM) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization. We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg(2+) and Ca(2+) had significant effects on secondary structure. Taken together, our results support the hypothesis that Mg(2+)-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ. On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.     

Visualization of single Escherichia coli FtsZ filament dynamics with atomic force microscopy

FtsZ, the prokaryotic homologue of tubulin, is an essential cell division protein. In the cell, it localizes at the center, forming a ring that constricts during division. In vitro, it binds and hydrolyzes GTP and polymerizes in a GTP-dependent manner. We have used atomic force microscopy to study the structure and dynamics of FtsZ polymer assembly on a mica surface under buffer solution. The polymers were highly dynamic and flexible, and they continuously rearranged over the surface. End-to-end joining of filaments and depolymerization from internal zones were observed, suggesting that fragmentation and reannealing may contribute significantly to the dynamics of FtsZ assembly. The shape evolution of the restructured polymers manifested a strong inherent tendency to curve. Polymers formed in the presence of non-hydrolyzable nucleotide analogues or in the presence of GDP and AlF(3) were structurally similar but showed a slower dynamic behavior. These results provide experimental evidence supporting the model of single-strand polymerization plus cyclization recently proposed to explain the hydrodynamic behavior of the polymers in solution.     

Modeling FtsZ ring formation in the bacterial cell-anisotropic aggregation via mutual interactions of polymer rods

The cytoskeletal protein FtsZ polymerizes to a ring structure (Z ring) at the inner cytoplasmic membrane that marks the future division site and scaffolds the division machinery in many bacterial species. FtsZ is known to polymerize in the presence of GTP into single-stranded protofilaments. In vivo, FtsZ polymers become associated with the cytoplasmic membrane via interaction with the membrane-binding proteins FtsA and ZipA. The FtsZ ring structure is highly dynamic and undergoes constantly polymerization and depolymerization processes and exchange with the cytoplasmic pool. In this theoretical study, we consider a scenario of Z ring self-organization via self-enhanced attachment of FtsZ polymers due to end-to-end interactions and lateral interactions of FtsZ polymers on the membrane. With the assumption of exclusively circumferential polymer orientations, we derive coarse-grained equations for the dynamics of the pool of cytoplasmic and membrane-bound FtsZ. To capture stochastic effects expected in the system due to low particle numbers, we simulate our computational model using a Gillespie-type algorithm. We obtain ring- and arc-shaped aggregations of FtsZ polymers on the membrane as a function of monomer numbers in the cell. In particular, our model predicts the number of FtsZ rings forming in the cell as a function of cell geometry and FtsZ concentration. We also calculate the time of FtsZ ring localization to the midplane in the presence of Min oscillations. Finally, we demonstrate that the assumptions and results of our model are confirmed by 3D reconstructions of fluorescently-labeled FtsZ structures in E. coli that we obtained.     

Deuterium oxide promotes assembly and bundling of FtsZ protofilaments

The assembly and bundling of FtsZ protofilaments play an important role during bacterial cell division. Deuterium oxide (D2O) is known to have strong stabilization effects on the assembly dynamics of several proteins including tubulin, a homologue of FtsZ. Here, we found that D2O enhanced the light-scattering intensity of the assembly reaction, increased sedimentable polymer mass, and induced bundling of FtsZ protofilaments. D2O also increased the stability of FtsZ polymers under challenged GTP conditions and suppressed dilution-induced disassembly of protofilaments. D2O enhances the assembly parameters of FtsZ and microtubules albeit differently. For example, D2O induced bundling of FtsZ protofilaments, whereas it did not induce bundling of microtubules in vitro. In addition, D2O strongly suppressed the GTP hydrolysis rate of microtubules, but it had no effect on the initial rate of GTP hydrolysis of the FtsZ assembly. D2O (80%) also increased the helical content of FtsZ by 25% compared to the helical content of FtsZ in aqueous buffer. D2O was shown to reduce the binding of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to tubulin. In contrast, we found that D2O strongly enhanced the binding of bis-ANS to FtsZ. The results indicated that D2O promotes assembly and bundling of FtsZ protofilaments by increasing hydrophobic interactions between the protofilaments. The results also suggest that the phosphate release rather than the on-site GTP hydrolysis is the rate-limiting step of the GTP turnover reaction.     

FtsZ Polymerization Assays: Simple Protocols and Considerations

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long filaments in the presence of GTP in vitro, and polymerization is regulated by several accessory proteins. FtsZ polymerization has been extensively studied in vitro using basic methods including light scattering, sedimentation, GTP hydrolysis assays and electron microscopy. Buffer conditions influence both the polymerization properties of FtsZ, and the ability of FtsZ to interact with regulatory proteins. Here, we describe protocols for FtsZ polymerization studies and validate conditions and controls using Escherichia coli and Bacillus subtilis FtsZ as model proteins. A low speed sedimentation assay is introduced that allows the study of the interaction of FtsZ with proteins that bundle or tubulate FtsZ polymers. An improved GTPase assay protocol is described that allows testing of GTP hydrolysis over time using various conditions in a 96-well plate setup, with standardized incubation times that abolish variation in color development in the phosphate detection reaction. The preparation of samples for light scattering studies and electron microscopy is described. Several buffers are used to establish suitable buffer pH and salt concentration for FtsZ polymerization studies. A high concentration of KCl is the best for most of the experiments. Our methods provide a starting point for the in vitro characterization of FtsZ, not only from E. coli and B. subtilis but from any other bacterium. As such, the methods can be used for studies of the interaction of FtsZ with regulatory proteins or the testing of antibacterial drugs which may affect FtsZ polymerization.     

FtsZ filament structures in different nucleotide states reveal the mechanism of assembly dynamics

Treadmilling protein filaments perform essential cellular functions by growing from one end while shrinking from the other, driven by nucleotide hydrolysis. Bacterial cell division relies on the primitive tubulin homolog FtsZ, a target for antibiotic discovery that assembles into single treadmilling filaments that hydrolyse GTP at an active site formed upon subunit association. We determined high-resolution filament structures of FtsZ from the pathogen Staphylococcus aureus in complex with different nucleotide analogs and cations, including mimetics of the ground and transition states of catalysis. Together with mutational and biochemical analyses, our structures reveal interactions made by the GTP γ-phosphate and Mg²⁺ at the subunit interface, a K⁺ ion stabilizing loop T7 for co-catalysis, new roles of key residues at the active site and a nearby crosstalk area, and rearrangements of a dynamic water shell bridging adjacent subunits upon GTP hydrolysis. We propose a mechanistic model that integrates nucleotide hydrolysis signaling with assembly-associated conformational changes and filament treadmilling. Equivalent assembly mechanisms may apply to more complex tubulin and actin cytomotive filaments that share analogous features with FtsZ.

Bacterial cell division critically relies on the tubulin homolog FtsZ, which assembles into filaments that treadmill, fuelled by GTP hydrolysis. This structural and biochemical study of FtsZ from Staphylocuccus aureus reveals the mechanism of GTP hydrolysis and its connection with filament dynamics.     

Mutations in the GTP-binding and synergy loop domains of Mycobacterium tuberculosis ftsZ compromise its function in vitro and in vivo

The Mycobacterium tuberculosis FtsZ (FtsZ(TB)), unlike other eubacterial FtsZ proteins, shows slow GTP-dependent polymerization and weak GTP hydrolysis activities [E.L. White, L.J. Ross, R.C. Reynolds, L.E. Seitz, G.D. Moore, D.W. Borhani, Slow polymerization of Mycobacterium tuberculosis FtsZ, J. Bacteriol. 182 (2000) 4028-4034]. In an attempt to understand the biological significance of these findings, we created mutations in the GTP-binding (FtsZ(G103S)) and GTP hydrolysis (FtsZ(D210G)) domains of FtsZ and characterized the activities of the mutant proteins in vitro and in vivo. We show that FtsZ(G103S) is defective for binding to GTP and polymerization activities, and exhibited reduced GTPase activity whereas FtsZ(D210G) protein is proficient in binding to GTP, showing reduced polymerization activity but did not show any measurable GTPase activity. Visualization of FtsZ-GFP structures in ftsZ merodiploid strains by fluorescent microscopy revealed that FtsZ(D210G) is proficient in associating with Z-ring structures whereas FtsZ(G103S) is not. Finally, we show that Mycobacterium smegmatis ftsZ mutant strains producing corresponding mutant FtsZ proteins are non-viable indicating that mutant FtsZ proteins cannot function as the sole source for FtsZ, a result distinctly different from that reported for Escherichia coli. Together, our results indicate that optimal GTPase and polymerization activities of FtsZ are required to sustain cell division in mycobacteria and that the same conserved mutations in different bacterial species have distinct phenotypes.     

Energetics of the cooperative assembly of cell division protein FtsZ and the nucleotide hydrolysis switch

FtsZ is the first protein recruited to the bacterial division site, where it forms the cytokinetic Z ring. We have determined the functional energetics of FtsZ assembly, employing FtsZ from the thermophilic Archaea Methanococcus jannaschii bound to GTP, GMPCPP, GDP, or GMPCP, under different solution conditions. FtsZ oligomerizes in a magnesium-insensitive manner. FtsZ cooperatively assembles with magnesium and GTP or GMPCPP into large polymers, following a nucleated condensation polymerization mechanism, under nucleotide hydrolyzing and non-hydrolyzing conditions. The effect of temperature on the critical concentration indicates polymer elongation with an apparent heat capacity change of -800 +/- 100 cal mol-1 K-1 and positive enthalpy and entropy changes, compatible with axial hydrophobic contacts of each FtsZ in the polymer, and predicts optimal polymer stability near 75 degrees C. Assembly entails the binding of one medium affinity magnesium ion and the uptake of one proton per FtsZ. Interestingly, GDP- or GMPCP-liganded FtsZ cooperatively form helically curved polymers, with an elongation only 1-2 kcal mol-1 more unfavorable than the straight polymers formed with nucleotide triphosphate, suggesting a physiological requirement for FtsZ polymerization inhibitors. This GTP hydrolysis switch should provide the basic properties for FtsZ polymer disassembly and its functional dynamics.     

Bacterial Division Proteins FtsZ and ZipA Induce Vesicle Shrinkage and Cell Membrane Invagination

Permeable vesicles containing the proto-ring anchoring ZipA protein shrink when FtsZ, the main cell division protein, polymerizes in the presence of GTP. Shrinkage, resembling the constriction of the cytoplasmic membrane, occurs at ZipA densities higher than those found in the cell and is modulated by the dynamics of the FtsZ polymer. In vivo, an excess of ZipA generates multilayered membrane inclusions within the cytoplasm and causes the loss of the membrane function as a permeability barrier. Overproduction of ZipA at levels that block septation is accompanied by the displacement of FtsZ and two additional division proteins, FtsA and FtsN, from potential septation sites to clusters that colocalize with ZipA near the membrane. The results show that elementary constriction events mediated by defined elements involved in cell division can be evidenced both in bacteria and in vesicles.     

Analysis of FtsZ Assembly by Light Scattering and Determination of the Role of Divalent Metal Cations

FtsZ is an ancestral homologue of tubulin that polymerizes in a GTP-dependent manner. In this study, we used 90° angle light scattering to investigate FtsZ polymerization. The critical concentration for polymerization obtained by this method is similar to that obtained by centrifugation, confirming that the light scattering is proportional to polymer mass. Furthermore, the dynamics of FtsZ polymerization could be readily monitored by light scattering. Polymerization was very rapid, reaching steady state within 30 s. The length of the steady-state phase was proportional to the GTP concentration and was followed by a rapid decrease in light scattering. This decrease indicated net depolymerization that always occurred as the GTP in the reaction was consumed. FtsZ polymerization was observed over the pH range 6.5 to 7.9. Importantly, Mg²⁺ was not required for polymerization although it was required for the dynamic behavior of the polymers. It was reported that 7 to 25 mM Ca²⁺ mediated dynamic assembly of FtsZ (X.-C. Yu and W. Margolin, EMBO J. 16:5455–5463, 1997). However, we found that Ca²⁺ was not required for FtsZ assembly and that this concentration of Ca²⁺ reduced the dynamic behavior of FtsZ assembly.     

The mechanics of FtsZ fibers

Inhibition of the Fts family of proteins causes the growth of long filamentous cells, indicating that they play some role in cell division. FtsZ polymerizes into protofilaments and assembles into the Z-ring at the future site of the septum of cell division. We analyze the rigidity of GTP-bound FtsZ protofilaments by using cryoelectron microscopy to sample their bending fluctuations. We find that the FtsZ-GTP filament rigidity is κ=4.7±1.0×10(-27) Nm(2), with a corresponding thermal persistence length of l(p)=1.15±0.25μm, much higher than previous estimates. In conjunction with other model studies, our new higher estimate for FtsZ rigidity suggests that contraction of the Z-ring may generate sufficient force to facilitate cell division. The good agreement between the measured mode amplitudes and that predicted by equipartition of energy supports our use of a simple mechanical model for FtsZ fibers. The study also provides evidence that the fibers have no intrinsic global or local curvatures, such as might be caused by partial hydrolysis of the GTP.     

Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics

Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.