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 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.
Highlights
FtsZ is a cytoskeletal protein essential to bacterial cytokinesis and a member of the tubulin family of GTPases, which includes ␣-tubulin [1], ␥-tubulin [2], bacterial tubulin BtubA/B [3, 4], and TubZ [5]
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 be devoid of dynamic instability
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 disas
Summary
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 [␣-32P]GTP (ϳ400 Ci/mmol) were from Amersham Biosciences. ApoFtsZ-W319Y-His was prepared as apoFtsZ above, and its concentration was measured with an extinction coefficient ⑀280 ϭ 2980 MϪ1 cmϪ1 (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 Ͻ7 at room temperature). Copolymers of FtsZ-W319Y-His and FtsZ-His, 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-His single Trp was measured fluorometrically by excitation at 295 nm, employing FtsZ-His standards. Exchange of [␣-32P]GTP into FtsZ-W319Y-His or FtsZ-His, in Mes-KCl buffer with 6 mM MgCl2 and 1 mM GTP at 55 °C, was measured employing a nitrocellulose filtration assay [17]
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