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.     

Activation of the Escherichia coli cell division protein FtsZ by a low-affinity interaction with monovalent cations

We have investigated the activation of FtsZ by monovalent cations. FtsZ polymerization was dependent on the concentrations of protein and monovalent salts, and was accompanied by the uptake of a single ion per monomer added. The affinity and the specificity for the cation were low. Potassium, ammonium, rubidium or sodium activated FtsZ to different extents. Electron microscopy showed that polymers formed with either rubidium, or potassium, were very similar, as were their nucleotide turnover rates. The GTPase activity was lower with rubidium than with potassium, indicating that nucleotide exchange is independent of nucleotide hydrolysis. Control of polymerization by binding of a low affinity cation might govern the dynamic behavior of the FtsZ polymers.     

Glutamate-induced assembly of bacterial cell division protein FtsZ

The polymerization of FtsZ is a finely regulated process that plays an essential role in the bacterial cell division process. However, only a few modulators of FtsZ polymerization are known. We identified monosodium glutamate as a potent inducer of FtsZ polymerization. In the presence of GTP, glutamate enhanced the rate and extent of polymerization of FtsZ in a concentration-dependent manner; approximately 90% of the protein was sedimented as polymer in the presence of 1 m glutamate. Electron micrographs of glutamate-induced polymers showed large filamentous structures with extensive bundling. Furthermore, glutamate strongly stabilized the polymers against dilution-induced disassembly, and it decreased the GTPase activity of FtsZ. Calcium induced FtsZ polymerization and bundling of FtsZ polymers; interestingly, although 1 m glutamate produced a larger light-scattering signal than produced by 10 mm calcium, the amount of polymer sedimented in the presence of 1 m glutamate and 10 mm calcium was similar. Thus, the increased light scattering in the presence of glutamate must be due to its ability to induce more extensive bundling of FtsZ polymers than calcium. The data suggest that calcium and glutamate might induce FtsZ polymerization by different mechanisms.     

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.     

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.     

The polymerization mechanism of the bacterial cell division protein FtsZ

Translin and Trax are components of an RNA binding complex initially detected in extracts of brain and testes. Although other tissues appear to contain much lower or negligible levels of the Translin/Trax gel-shift complex, we found, unexpectedly, that several of these peripheral tissues express Translin and Trax proteins at levels comparable to those present in brain. In this study, we demonstrate that the paradoxically low levels of the Translin/Trax complex in kidney and other peripheral tissues are due to masking of these sites by endogenous RNA. Thus, these findings indicate that the Translin/Trax complex is involved in RNA processing in a broader range of tissues than previously recognized.     

EzrA prevents aberrant cell division by modulating assembly of the cytoskeletal protein FtsZ

In response to a cell cycle signal, the cytoskeletal protein FtsZ assembles into a ring structure that establishes the location of the division site and serves as a framework for assembly of the division machinery. A battery of factors control FtsZ assembly to ensure that the ring forms in the correct position and at the precise time. EzrA, a negative regulator of FtsZ ring formation, is important for ensuring that the ring forms only once per cell cycle and that cytokinesis is restricted to mid-cell. EzrA is distributed throughout the plasma membrane and localizes to the ring in an FtsZ-dependent manner, suggesting that it interacts directly with FtsZ to modulate assembly. We have performed a series of experiments examining the interaction between EzrA and FtsZ. As little as twofold overexpression of EzrA blocks FtsZ ring formation in a sensitized genetic background, consistent with its predicted function. A purified EzrA fusion protein interacts directly with FtsZ to block assembly in vitro. Although EzrA is able to inhibit FtsZ assembly, it is unable to disassemble preformed polymers. These data support a model in which EzrA interacts directly with FtsZ at the plasma membrane to prevent polymerization and aberrant FtsZ ring formation.     

Magnesium-induced linear self-association of the FtsZ bacterial cell division protein monomer. The primary steps for FtsZ assembly

The bacterial cell division protein FtsZ from Escherichia coli has been purified with a new calcium precipitation method. The protein contains one GDP and one Mg(2+) bound, it shows GTPase activity, and requires GTP and Mg(2+) to polymerize into long thin filaments at pH 6.5. FtsZ, with moderate ionic strength and low Mg(2+) concentrations, at pH 7.5, is a compact and globular monomer. Mg(2+) induces FtsZ self-association into oligomers, which has been studied by sedimentation equilibrium over a wide range of Mg(2+) and FtsZ concentrations. The oligomer formation mechanism is best described as an indefinite self-association, with binding of an additional Mg(2+) for each FtsZ monomer added to the growing oligomer, and a slight gradual decrease of the affinity of addition of a protomer with increasing oligomer size. The sedimentation velocity of FtsZ oligomer populations is compatible with a linear single-stranded arrangement of FtsZ monomers and a spacing of 4 nm. It is proposed that these FtsZ oligomers and the polymers formed under assembly conditions share a similar axial interaction between monomers (like in the case of tubulin, the eukaryotic homolog of FtsZ). Similar mechanisms may apply to FtsZ assembly in vivo, but additional factors, such as macromolecular crowding, nucleoid occlusion, or specific interactions with other cellular components active in septation have to be invoked to explain FtsZ assembly into a division ring.     

Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ

3-Methoxybenzamide is a weak inhibitor of the essential bacterial cell division protein FtsZ. Exploration of the structure–activity relationships of 3-methoxybenzamide analogues led to the identification of potent anti-staphylococcal compounds.     

Berberine targets assembly of Escherichia coli cell division protein FtsZ

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.     

Ruthenium red-induced bundling of bacterial cell division protein, FtsZ

The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.     

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.     

Epitope mapping of Escherichia coli cell division protein FtsZ with monoclonal antibodies.

A fusion between lacZ and ftsZ of Escherichia coli was constructed to obtain a beta-galactosidase-FtsZ fusion protein. This fusion protein was used to raise antibodies against cell division protein FtsZ. Six monoclonal antibodies were obtained, and they reacted with FtsZ from cytoplasm and membrane fractions. The epitopes in FtsZ were localized by studying the reactions of the monoclonal antibodies with fusion proteins truncated at the carboxy terminus and with fragments that were obtained by CNBr cleavage of purified FtsZ. Five different epitopes were defined. Epitopes I and III reacted with the same monoclonal antibody, without showing apparent amino acid homology. Epitope II was defined by monoclonal antibodies that cross-reacted with an unknown cytoplasmic 50-kDa protein not related to FtsZ. Epitopes IV and V were recognized by different monoclonal antibodies. All monoclonal antibodies reacted strongly under native conditions, so it is likely that the five epitopes are situated on the surface of native FtsZ. By using these data and computer analysis, a provisional model of FtsZ is proposed. The FtsZ protein is considered to be globular, with a hydrophobic pocket containing GTP-binding elements. Epitopes I and II are situated on each side of the hydrophobic pocket. Because the carboxy terminus contains epitope V, the carboxy terminus of FtsZ is likely oriented toward the protein's surface.     

FtsZ and the division of bacterial cell

In this review we have tried to describe proteins and supermolecular structures which take part in the division of bacterial cell. The principal cell division protein of the most of prokaryotes is FtsZ, a homologue of eukaryotic tubulin. FtsZ just as tubulin is capable to bind and hydrolyze GTP. The division of bacterial cell begins with forming of so called divisome. The basis of such divisome is a contractile ring (Z ring); the ring encircles the cell about midcell. Z ring consists of a bundle of laterally bound protofilaments, which have been formed as a result of FtsZ polymerization. Z ring is rigidly bounded to cytozolic side of inner membrane with participation of FtsA, ZipA, FtsW and many other cell division proteins of divisome. The ring directs the process of cytokinesis transmitting power of constriction to membrane. Primary structures of members of the family of prokaryotic FtsZs differ from eukaryotic tubulines significantly except the region, where the site of GTP binding is placed. There is high degree of homology between structures of these proteins in the region. FtsZ is one of the most conservative proteins in prokaryotes, but ftsZ genes have not been found in completely sequenced genomes of several species of microorganisms. There are 2 species of mycoplasmas (Ureaplasma parvum and Mycoplasma mobile), Prostecobacter dejongeii, 10 species of chlamydia and 5 species of archaea among them. So these organisms divide without FtsZ. There are many open reading frames which encode proteins with unknown functions in genomes of U. parvum and M. mobile. The comparison of primary structures of these hypothetical proteins with structures of cell division proteins did not allow researchers to find similar proteins among them. We suppose that the process of cell division of these organisms should recruit proteins with function similar to FtsZ and having homologous with FtsZ or other cell division proteins spatial structures.     

Apparent cooperative assembly of the bacterial cell division protein FtsZ demonstrated by isothermal titration calorimetry

The assembly dynamics of FtsZ, a prokaryotic homolog of tubulin, are important for their role in bacterial cytokinesis. Here we used isothermal titration calorimetry (ITC) to measure the heat of FtsZ self-association under various conditions. The measurements were designed to test whether FtsZ protofilaments are assembled by an isodesmic (linear aggregates in which each bond has an identical equilibrium constant) or a cooperative (aggregates only become stable after forming a oligomeric nucleus) assembly process. The isodesmic model can fit the assembly in GDP closely but cannot fit the assembly in GTP. FtsZ-GTP without Mg(2+) exhibits an apparent critical concentration, which is indicative of cooperative assembly, near 2.9 microm. With 2.5 mm Mg(2+) (which allows FtsZ to hydrolyze GTP) the critical concentration is reduced 10-fold to approximately 0.31 microm. Both with and without Mg(2+) there is no evidence for assembly below the critical concentration, but there is an abrupt transition to full assembly above. The ITC data are highly suggestive of a cooperative assembly, although this is difficult to reconcile with the 1-subunit-thick protofilaments observed by electron microscopy.     

Interaction between FtsZ and inhibitors of cell division.

The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.     

Acid-induced loss of functional properties of bacterial cell division protein FtsZ: evidence for an alternative conformation at acidic pH

Several types of bacteria live in highly acidic environments. Since the assembly of FtsZ is important for bacterial cytokinesis, the effects of pH on the assembly and structural properties of FtsZ were examined. FtsZ retained GTP binding ability but lost GTPase activity at pH 2.5. In the presence of GTP, FtsZ formed protofilaments at pH 7 while it formed aggregates instead of protofilaments at pH 2.5, indicating that GTP hydrolysis is important for the assembly of FtsZ into protofilaments. Further, the acid-inactivated state of FtsZ recovered its structural and functional properties upon refolding at pH 7, indicating that the cellular functions of FtsZ may be restored after removal of the external stress. In addition, the affinity of 1-anilinonaphthalene-8-sulfonic acid (ANS) binding to FtsZ was found to be higher at pH 2.5 than at pH 7. FtsZ-ANS complex had a higher quantum yield and lifetime at pH 2.5 than at pH 7. However, the secondary structures of FtsZ were similar at pH 7 and 2.5, indicating that FtsZ attained an alternatively folded state (A) at pH 2.5, which has some characteristics of a molten-globule-like state. The A state was more stable than the native state (N) against urea-induced unfolding. The transition from N to A state involves the formation of aggregates of FtsZ (I). The association of FtsZ monomers occurred in the narrow pH range (3.2-2.8) and it was found to be a fully reversible process. The results suggest that a productive intermediate (I) forms in the acid-induced unfolding pathway of FtsZ and that the unfolding pathway may be minimally described as N <==> I <==> A.     

Analysis of degradation of bacterial cell division protein FtsZ by the ATP-dependent zinc-metalloprotease FtsH in vitro

The identity of protease(s), which would degrade bacterial cell division protein FtsZ in vivo, remains unknown. However, we had earlier demonstrated that Escherichia coli metalloprotease FtsH degrades E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro. In this study, we examined FtsH protease-mediated degradation of FtsZ in vitro in detail using seven different deletion mutants of FtsZ as the substrates, which lack different extents of specific regions at the N- or C-terminus. FtsH protease assay in vitro on these mutants revealed that FtsH could degrade all the seven deletion mutants irrespective of the deletions or the extent of deletions at the N- or C-terminus. These observations indicated that neither the N-terminus nor the C-terminus was required for the degradation of FtsZ, like already known in the case of the FtsH substrate sigma(32) protein. The recombinant clones expressing full-length FtsZ protein and FtsZ deletion mutant proteins would be useful in investigating the possibility of FtsZ as a potential in vivo substrate for FtsH in ftsH-null cells carrying ftsH suppressor function and ectopically expressed FtsH protease.