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The interactions of cell division protein FtsZ with guanine nucleotides
|Authors:||Huecas, Sonia ; Schaffner-Barbero, Claudia ; García, Wanius; Yébenes, Hugo; Palacios, Juan Manuel; Díaz, José Fernando ; Menéndez, Margarita ; Andreu, José Manuel|
|Publisher:||American Society for Biochemistry and Molecular Biology|
|Citation:||Journal of Biological Chemistry 288(52): 7515-37528(2007)|
|Abstract:||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.
Previous SectionNext SectionFtsZ is a cytoskeletal protein essential to bacterial cytokinesis and a member of the tubulin family of GTPases, which also includes αβ-tubulin (1), γ-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
|Description:||14 páginas, 10 figuras, 5 tablas -- PAGS nros. 37515-37528|
|Publisher version (URL):||http://dx.doi.org/10.1074/jbc.M706399200|
|Appears in Collections:||(CIB) Artículos|
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|JBC_Andreu, J. M. et al 2007.pdf||1,58 MB||Adobe PDF|