Mechanism of Regulation of Prokaryotic Tubulin-like GTPase FtsZ by Membrane Protein EzrA

Abstract

At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed. At initiation of cell division, FtsZ, a tubulin-like GTPase, assembles into a so-called Z-ring structure at the site of division. The formation of Z ring is negatively regulated by EzrA, which ensures only one ring at the midcell per cell cycle. The mechanism leading to the negative regulation of Z-ring formation by EzrA has been analyzed. Our data reveal that the interaction between EzrA and FtsZ not only reduces the GTP-binding ability of FtsZ but also accelerates the rate of GTP hydrolysis, both of which are unfavorable for the polymerization of FtsZ. Moreover, the acceleration in rate of GTP hydrolysis by EzrA is attributed to stabilization of the transition state for GTP hydrolysis and reduction in the affinity of GDP for FtsZ. Clearly, EzrA is able to modify the GTP hydrolysis cycle of FtsZ. On the basis of these results, a model for how EzrA acts to negatively regulate Z-ring formation is proposed. Cell division in bacteria is mediated by the Z ring, of which FtsZ is the key component (1Bi E.F. Lutkenhaus J. Nature. 1991; 354: 161-164Crossref PubMed Scopus (1126) Google Scholar). At initiation of cell division, FtsZ polymerizes at the midcell into a ring structure that is required for subsequent recruitment of other cell division proteins and assembly of the cell division machinery (2Chen J.C. Weiss D.S. Ghigo J.M. Beckwith J. J. Bacteriol. 1999; 181: 521-530Crossref PubMed Google Scholar, 3Daniel R.A. Harry E.J. Katis V.L. Wake R.G. Errington J. Mol. Microbiol. 1998; 29: 593-604Crossref PubMed Scopus (88) Google Scholar, 4Lutkenhaus J. Addinall S.G. Annu. Rev. Biochem. 1997; 66: 93-116Crossref PubMed Scopus (406) Google Scholar, 5Rothfield L. Justice S. Garcia-Lara J. Annu. Rev. Genet. 1999; 33: 423-448Crossref PubMed Scopus (235) Google Scholar, 6Weiss D.S. Chen J.C. Ghigo J.M. Boyd D. Beckwith J. J. Bacteriol. 1999; 181: 508-520Crossref PubMed Google Scholar). FtsZ is a prokaryotic tubulin homologue that possesses GTPase activity. Although its amino acid sequence includes only a short segment with a high degree of sequence homology to tubulins, there is a remarkable similarity between the higher order structures of FtsZ and tubulin (7Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1771) Google Scholar, 8Nogales E. Whittaker M. Milligan R.A. Downing K.H. Cell. 1999; 96: 79-88Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar, 9Lowe J. Li H. Downing K.H. Nogales E. J. Mol. Biol. 2001; 313: 1045-1057Crossref PubMed Scopus (971) Google Scholar, 10Lowe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (705) Google Scholar). Polymerization of FtsZ along the inner circumference of the cytoplasmic membrane is GTP-dependent (4Lutkenhaus J. Addinall S.G. Annu. Rev. Biochem. 1997; 66: 93-116Crossref PubMed Scopus (406) Google Scholar) and is regulated by the GTP hydrolysis cycle of FtsZ (11Romerg L. Levin P.A. Annu. Rev. Microbiol. 2003; 57: 125-154Crossref PubMed Scopus (222) Google Scholar, 12Romberg L. Mitchison T.J. Biochemistry. 2004; 43: 282-288Crossref PubMed Scopus (83) Google Scholar). Mutational analysis of Escherichia coli FtsZ has identified three catalytic amino acid residues in the GTPase-activating T7 loop of FtsZ, which are located far from the active site and are important for the GTPase but not nucleotide-binding activity of FtsZ (13Lu C. Stricker J. Erickson H.P. BMC Microbiol. 2001; 1: 7Crossref PubMed Scopus (79) Google Scholar, 14Scheffers D.J. de Wit J.G. den Blaauwen T. Driessen A.J. Biochemistry. 2002; 41: 521-529Crossref PubMed Scopus (122) Google Scholar, 15Wang X. Huang J. Mukherjee A. Cao C. Luthkenhaus J. J. Bacteriol. 1997; 179: 5551-5559Crossref PubMed Scopus (179) Google Scholar). On the basis of the structural data of FtsZ, it was assumed that the three catalytic amino acid residues in the T7 loop of one FtsZ subunit of the protofilament can be brought into the vicinity of the catalytic site of the second FtsZ by making head-to-tail contact (16Leung A.K. Leucile White E. Ross L.J. Reynolds R.C. DeVito J.A. Borhani D.W. J. Mol. Biol. 2004; 342: 953-970Crossref PubMed Scopus (96) Google Scholar). It is the FtsZ in head-to-tail contact that possesses the GTPase activity (14Scheffers D.J. de Wit J.G. den Blaauwen T. Driessen A.J. Biochemistry. 2002; 41: 521-529Crossref PubMed Scopus (122) Google Scholar). However, the main form of nucleotide in FtsZ polymers was found to be GDP-Pi (17Scheffers D.J. Driessen A.J. Mol. Microbiol. 2002; 43: 1517-1521Crossref PubMed Scopus (33) Google Scholar) or GTP (12Romberg L. Mitchison T.J. Biochemistry. 2004; 43: 282-288Crossref PubMed Scopus (83) Google Scholar), depending on the treatment of FtsZ polymers. In addition, excess GDP was found to promote disassembly of the preformed FtsZ polymers (18Scheffers D.J. den Blaauwen T. Driessen A.J. Mol. Microbiol. 2000; 35: 1211-1219Crossref PubMed Scopus (46) Google Scholar). EzrA is a transmembrane protein involved in spatial regulation of Z-ring formation in Bacillus subtilis (19Levin P.A. Kurster I.G. Grossman A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9642-9647Crossref PubMed Scopus (168) Google Scholar). It is uniformly distributed throughout the plasma membrane in both nondividing and dividing cells and concentrates at the cytokinetic ring in an FtsZ-dependent manner during cell division (19Levin P.A. Kurster I.G. Grossman A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9642-9647Crossref PubMed Scopus (168) Google Scholar). It has been also reported that EzrA plays both positive and negative roles in cell division (19Levin P.A. Kurster I.G. Grossman A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9642-9647Crossref PubMed Scopus (168) Google Scholar, 20Chung K.M. Hsu H.H. Govindan S. Chang B.Y. J. Bacteriol. 2004; 186: 5926-5932Crossref PubMed Scopus (13) Google Scholar). Evidence for the positive role of EzrA came from the observation that low level expression of EzrA increases both cell length and percentage of filamentous cells of B. subtilis during growth (20Chung K.M. Hsu H.H. Govindan S. Chang B.Y. J. Bacteriol. 2004; 186: 5926-5932Crossref PubMed Scopus (13) Google Scholar). The negative role of EzrA was noted by the fact that null mutation of EzrA lowers the critical concentration of FtsZ required for Z-ring assembly and leads to the formation of polar Z rings and septa (19Levin P.A. Kurster I.G. Grossman A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9642-9647Crossref PubMed Scopus (168) Google Scholar). In addition, the null mutation of EzrA is able to suppress the instability of FtsZ polymer associated with minCD overexpression (21Levin P.A. Schwartz R.L. Grossman A.D. J. Bacteriol. 2001; 183: 449-5452Crossref Google Scholar). However, how EzrA acts to fulfill both the positive and negative roles remains unclear. Recently, a purified thioredoxin-EzrA fusion protein was found to interact with FtsZ and to block FtsZ assembly but not disassembly in vitro. However, it did not significantly alter the GTPase activity of FtsZ (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google Scholar). This implies that EzrA binds neither to the nucleotide-binding site nor to the catalytic amino acids in the GTPase-activating T7 loop of FtsZ to drastically block the interaction between the FtsZ monomers and thus to eliminate the GTPase activity of FtsZ. Rather, EzrA may regulate FtsZ assembly through modulating the functional conformation of FtsZ. To verify the idea, we analyzed the effects of EzrA on the GTP hydrolysis cycle of FtsZ. On the basis of our data, a model for how EzrA acts to negatively regulate Z-ring formation during B. subtilis cell division is proposed. Construction of Plasmids for Expression of EzrA and FtsZ—The DNA fragment encoding the cytoplasmic domain (amino acid residues 27–562) of EzrA was amplified from the chromosome DNA of B. subtilis by polymerase chain reaction and cloned into pET21d (Novagen). The resultant plasmid, pKM4, expressed a 59-kDa EzrA with a His6 tag fused at its C terminus. The sequences of the forward and reverse primers used for ezrA synthesis and cloning were 5′-AGACTACCATGGCCGAAATCGACCGGCTG-3′ and 5′-CGTTACCTCGAGAGCGGATATGTCAGCTTTG-3′, respectively. The FtsZ having a C-terminal His6 tag was expressed using the pET21d-derived plasmid, pKM2. The sequences of the forward and reverse primers used for the synthesis and cloning of ftsZ were 5′-AGACTACCATGGCTTTGGAGTTCGAAACAAACATAG-3′ and 5′-CGTTACCTCGAGGCCGCGTTTATTACGGTTTC-3′, respectively. Purification of EzrA and FtsZ—E. coli BL21/DE3 harboring pKM2 or pKM4 plasmid was inoculated into the 2xYT medium supplemented with 100 μg/ml ampicillin and grown at 37 °C. Protein expression was induced at A550 of 0.8 by 0.4 mm isopropyl 1-thio-β-d-galactopyranoside. The cells were harvested 4 h later, pelleted at 8,000 rpm and 4 °C for 10 min, and resuspended with 20 ml of ice-cold buffer (25 mm Tris-HCl, pH 7.0, 100 mm KCl, 5% glycerol, and 1 mm phenylmethylsulfonyl fluoride). Both His-tagged EzrA and FtsZ were purified using the same method. Briefly, the cells containing the target proteins were lysed with a French press at 16,000 p.s.i. The lysate was cleared by centrifugation at 12,000 rpm, 4 °C for 20 min. The target proteins in the supernatant were absorbed with TALON™ IMAC resin (Clontech) and washed extensively with the buffer (25 mm Tris-HCl, pH 8.0, 100 mm KCl, and 5% glycerol) supplemented with 10 mm imidazole. The target proteins were then eluted with the same buffer supplemented with 150 mm imidazole. Sample fractions containing the target proteins were pooled, applied onto Superdex 200HR column (Amersham Biosciences) preequilibrated with HEPES buffer (20 mm HEPES, pH 7.0, 100 mm KCl, and 5% glycerol). The purified EzrA and FtsZ were concentrated by Centricon 10 (Millipore), estimated to be at least higher than 95% in homogeneity, and stored in buffer containing 50 mm Tris-HCl, pH 8.0, 100 mm KCl, 1 mm phenylmethylsulfonyl fluoride, and 50% glycerol at -20 °C. The proteins were dialyzed against a specific buffer before being used to assay the effect of EzrA on a specific property of FtsZ. Cross-linking Assay—The protein mixture containing 5 μm each of FtsZ and EzrA in HEPES buffer (20 mm HEPES, pH 7.0, 100 mm KCl, and 5% glycerol) was cross-linked with a thiolcleavable primary amine-reactive homobifunctional cross-linker, 3,3-dithiobis(sulfosuccinimidylpropionate) (Pierce) at room temperature for 30 min. The concentration of the cross-linker was 250 μm. The cross-linking reaction was quenched with 20 mm Tris-HCl (pH 7.4) by incubation at room temperature for 15 min. Finally, the sample was subjected to SDS-PAGE and immunoblot analysis with anti-FtsZ and anti-EzrA. Effect of EzrA on Binding of used to FtsZ—The analysis was on of the of a GTP its to FtsZ. To the effect of EzrA on of the GTP to FtsZ, of in buffer (25 mm Tris-HCl, pH 7.0, 100 mm KCl, and 10 mm was into a and of the GTP was at room temperature with an 250 FtsZ or with an concentration of EzrA for 30 was into the of the GTP for each treatment was The for FtsZ and was by the of the GTP to with 250 FtsZ or 250 FtsZ with an concentration of EzrA for 30 min. The data of at of was to the of Effect of EzrA on Binding of or GDP for FtsZ—The protein sample containing FtsZ or each of FtsZ and EzrA was with in a of the GTP in each sample was and to respectively. of or GDP were into the to with for FtsZ The for each treatment was then by the to the percentage of Effect of EzrA on the GTPase of FtsZ—The GTPase activity of FtsZ was using the assay which of the of from to the is into and through the of to In order to the GTPase activity of FtsZ, 1 mm GTP was with the for 10 at °C to before with the protein which includes FtsZ, EzrA, or a mixture of FtsZ and EzrA in 50 mm HEPES, pH mm KCl, mm and mm The concentration of FtsZ or EzrA in each was 5 μm. The at of the sample was 10 at room temperature with a The rate of GTP hydrolysis was on the of To the effect of EzrA on the and of FtsZ, the GTPase activity of FtsZ or FtsZ with EzrA was at and μm The rate of GTP hydrolysis for each reaction thus was to the of to the and Effect of EzrA on FtsZ Polymerization by for analysis were to in a D. S. C. D. J. Bacteriol. 1998; PubMed Google Scholar). the purified FtsZ was cleared by at and 4 °C for 15 to or FtsZ or FtsZ with an concentration of EzrA was with polymerization buffer mm pH 10 mm and 30 mm at room temperature to a concentration of 5 μm for each 1 mm GTP or was to the polymerization of FtsZ. formation of FtsZ polymers was by to the assembly The of each assembly reaction was 100 The were at for 10 at 20 °C and resuspended in 100 of polymerization Both the supernatant and polymers were subjected to SDS-PAGE to the of FtsZ. The between EzrA and has been reported that the thioredoxin-EzrA fusion protein is able to interact with the interaction did not significantly alter the GTPase activity of FtsZ (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google Scholar). that the of the fusion protein may the of To the EzrA with a His6 tag fused at the C was and used to the effect of EzrA on FtsZ the the of interaction between EzrA and FtsZ was using a cross-linking method. in a protein with a for the cross-linked was the mixture of FtsZ and EzrA was with the homobifunctional cross-linker, and with anti-EzrA. To that protein was the specific protein was from the in with an immunoblot and then subjected to analysis using anti-FtsZ and or Both FtsZ and EzrA the protein was with the the fusion protein and with both anti-FtsZ and was that EzrA is able to interact with FtsZ. EzrA the Binding of the GTP to FtsZ—The ability of EzrA to interact with FtsZ the that EzrA may modify the GTP hydrolysis cycle of FtsZ through the in GTP-binding activity of FtsZ was analyzed. The GTP used to the functional mechanism of was for analysis to its and and A. D.P. 2003; PubMed Scopus Google Scholar). the of is significantly quenched in by interaction between and of the GTP the increases of the to the of was for the GTP was the was with EzrA, that EzrA is to the However, to FtsZ, of the GTP was and an 95% in was only was FtsZ was with concentration of EzrA these that EzrA is able to the affinity of the GTP for FtsZ. To idea, of the GTP to FtsZ with of EzrA was in of the GTP the of EzrA was was the of EzrA to FtsZ or and that EzrA and FtsZ are able to form a The ability of EzrA to of the GTP to FtsZ was by the fact that did not effect on of by FtsZ the effect of EzrA on of FtsZ was with the GTP to with EzrA we that EzrA plays its role not by on FtsZ polymers. analysis of the affinity of for FtsZ was also In the of the GTP were with FtsZ or FtsZ with an concentration of EzrA, FtsZ and EzrA may form a of the GTP was then and the data were to the of to the for FtsZ and The thus was or for FtsZ or FtsZ with EzrA, that EzrA is able to the of the GTP to FtsZ. EzrA the Binding of GTP for FtsZ—The reduction in of the GTP to FtsZ by EzrA that EzrA be also able to the affinity of GTP for FtsZ. To verify a assay was FtsZ or FtsZ with EzrA was with before of between GTP and the GTP for FtsZ was then in was for both FtsZ and FtsZ with EzrA, that GTP is able to the GTP of or not FtsZ was with However, the concentration of GTP required to 50% of the GTP from FtsZ with of EzrA was higher than that from FtsZ This that EzrA is able to the GTP-binding activity of FtsZ. also analyzed the effects of of EzrA to FtsZ on of in of GTP was the of EzrA and FtsZ that a of EzrA and FtsZ may be EzrA the Binding of the of GTP but the Binding of the to FtsZ—The of EzrA to modify the of GTP to FtsZ to that EzrA may also modify the of FtsZ to a transition state of GTP A.M. E. M.E. PubMed Scopus Google Scholar, M. J. Biol. Full Text PDF PubMed Google Scholar, J. Biol. Full Text PDF PubMed Google Scholar, A. A.M. L. Nature. PubMed Scopus Google Scholar) and also to the The was to by of or GDP to FtsZ or FtsZ with concentration of EzrA in a concentration of the of the GTP was for FtsZ with EzrA than for FtsZ The concentration of required to 50% of the GTP from FtsZ in the or of EzrA was or The of for FtsZ with EzrA than for FtsZ that EzrA is able to the transition state for GTP hydrolysis of FtsZ. the was the of EzrA to FtsZ or higher than it was that EzrA is able to with FtsZ in To EzrA can the affinity of GDP for FtsZ, a assay was also FtsZ or FtsZ with EzrA was with before of in was for both FtsZ and FtsZ with EzrA The of GDP for FtsZ with EzrA was higher than from FtsZ that EzrA is able to the affinity of GDP for FtsZ. the of GDP was the of EzrA to FtsZ was or which is also an of the formation of a EzrA and FtsZ EzrA the GTPase of FtsZ—The in affinity of FtsZ for by EzrA that EzrA is able to the GTPase activity of FtsZ by the for GTP To idea, we analyzed the effect of EzrA on the GTP hydrolysis rate of FtsZ using a that the from GTP Our data that GTP was by FtsZ at a rate of in the of EzrA However, the rate was to an concentration of EzrA was and Moreover, the acceleration of GTP hydrolysis was on the of EzrA to the acceleration was the or higher The and for GTP hydrolysis by FtsZ or FtsZ with concentration of EzrA were also in the for GTP hydrolysis by B. subtilis FtsZ is with that reported (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google it is than reported for E. coli FtsZ (12Romberg L. Mitchison T.J. Biochemistry. 2004; 43: 282-288Crossref PubMed Scopus (83) Google Scholar, T. den Blaauwen T. Biochemistry. PubMed Scopus Google Scholar, L. Nature. PubMed Scopus Google Scholar, D. Nature. PubMed Scopus Google Scholar, L. M. Erickson H.P. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Moreover, the is also with that reported for E. coli FtsZ T. den Blaauwen T. Biochemistry. PubMed Scopus Google Scholar). In the of EzrA, the for GTP hydrolysis by B. subtilis FtsZ that EzrA is able to the GTP hydrolysis rate of FtsZ. Moreover, with the data from the assay a in for GTPase activity was FtsZ was with EzrA that EzrA is able to the GTP-binding activity of and of FtsZ or FtsZ with EzrA The concentration of GTP is high mm in cells Microbiol. 2000; PubMed Scopus Google Scholar), which is higher than the in the in a EzrA to FtsZ verify the reduction in GTP-binding activity of FtsZ by EzrA is unfavorable for FtsZ a GTP was to the of GTP The effect of EzrA on FtsZ polymerization was then using analysis D. S. C. D. J. Bacteriol. 1998; PubMed Google Scholar, J. 1997; PubMed Scopus Google Scholar, A. J. 1998; PubMed Scopus Google Scholar). This analysis on the formation of of FtsZ polymers in the of and the of high during in EzrA FtsZ concentration of EzrA and FtsZ was to the of and were was with GTP However, of the FtsZ EzrA was with that the in GTP-binding activity of FtsZ by EzrA is to the formation of FtsZ polymers in vitro. Moreover, of the effect of EzrA on FtsZ polymerization by was in the of is for the assay (18Scheffers D.J. den Blaauwen T. Driessen A.J. Mol. Microbiol. 2000; 35: 1211-1219Crossref PubMed Scopus (46) Google Scholar). to that reported (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google Scholar), of FtsZ polymerization was also not the mechanism for the negative regulation of FtsZ polymerization by Our reveal that EzrA is able to the affinity of GTP for FtsZ and the GTPase activity of FtsZ, both of which the assembly of FtsZ and data, with the ability of EzrA to interact with FtsZ (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google Scholar), that EzrA in the FtsZ GTP hydrolysis cycle to regulate FtsZ polymer also a model for the negative regulation of Z-ring formation by EzrA a negative the plasma EzrA Z-ring formation at other than the midcell by interaction with FtsZ The interaction reduces the GTP-binding ability of FtsZ, which Z-ring It also the GTPase activity of FtsZ by the transition state for GTP hydrolysis and reduces the activity of FtsZ both of which promote the disassembly of FtsZ. The mechanism for the of the GTPase activity of FtsZ by EzrA is to that reported for a GTPase for protein K.M. Nature. PubMed Scopus Google Scholar, Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Both EzrA and to the transition state for GTP hydrolysis of the GTP-binding The in the GTPase activity of FtsZ by EzrA in the is but and is from that reported for the thioredoxin-EzrA fusion which effect on the GTPase activity of FtsZ (22Haeusser D.P. Schwartz A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (98) Google Scholar). the fused has the activity of Moreover, these that of the polymer of FtsZ by EzrA is also In other of Z-ring formation in not a in the GTPase activity of a in GTPase activity may the ability to the for of FtsZ polymer has been also reported by (11Romerg L. Levin P.A. Annu. Rev. Microbiol. 2003; 57: 125-154Crossref PubMed Scopus (222) Google Scholar). On the basis of the effects of of EzrA to FtsZ on the of and GDP and on the GTP hydrolysis rate of FtsZ it was that EzrA and FtsZ may form a This a that the rings of FtsZ and EzrA at the initiation of cell division (19Levin P.A. Kurster I.G. Grossman A.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9642-9647Crossref PubMed Scopus (168) Google Scholar) may in at for FtsZ assembly been identified in E. coli and B. E. coli is the one that is This protein negatively Z-ring formation by the GTPase activity of FtsZ D. S. C. D. J. Bacteriol. 1998; PubMed Google Scholar, A. Cao Lutkenhaus J. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). The is attributed to to the GTPase-activating T7 loop to the nucleotide-binding of FtsZ E.J. J. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar), which in the of FtsZ and its GTPase activity (14Scheffers D.J. de Wit J.G. den Blaauwen T. Driessen A.J. Biochemistry. 2002; 41: 521-529Crossref PubMed Scopus (122) Google Scholar, J. Amos L.A. J. 1999; Scopus (179) Google Scholar). However, the mechanism the of Z-ring formation by EzrA is from that found for in of of the GTPase activity of FtsZ The in the GTPase activity of FtsZ by EzrA that EzrA not catalytic amino acid to the active site of FtsZ to the activity of FtsZ. The effects of EzrA on FtsZ the in GTP-binding but in GTPase activity of the that EzrA may to fulfill its role by the conformation of FtsZ. This can be by the fact that EzrA is able to the transition state for GTP hydrolysis and to the activity of FtsZ