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J. Biol. Chem., Vol. 282, Issue 52, 37515-37528, December 28, 2007

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The Interactions of Cell Division Protein FtsZ with Guanine Nucleotides^*

Sonia Huecas¹, Claudia Schaffner-Barbero, Wanius García², Hugo Yébenes³, Juan Manuel Palacios⁴, José Fernando Díaz, Margarita Menéndez, and José Manuel Andreu⁵

From the Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu, 9, 28040 Madrid, Spain and Instituto de Química-Fisica Rocasolano, Consejo Superior de Investigaciones Científicas, 28006 Madrid, Spain

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 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.

Received for publication, August 2, 2007 , and in revised form, October 16, 2007.

^* This work was supported in part by grants Ministerio de Educacion y Ciencia (MEC) BFU 2005-00505/BMC (to J. M. A.), MEC BFU 2006-10288 (to M. M.), and Comunidad Autónoma de Madrid S-BIO-0214-2006 (to J. M. A. and J. F. D.), a Consejo Superior de Investigaciones Científicas-I3P postdoctoral contract (to S. H.), a MEC-FPI predoctoral fellowship (to C. S.), and Travel Grant FAPESP CBME-98/14138-2 (to W. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

² Present address: Centro de Biotecnologia Molecular e Estrutural, Instituto de Física de São Carlos, USP, Brazil.

³ Present address: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

⁴ Present address: GlaxoSmithKline, Madrid, Spain.

¹ To whom correspondence may be addressed. Tel.: 34-918373112 (ext. 4381); Fax: 34-915360432; E-mail: sonia{at}cib.csic.es. ⁵ To whom correspondence may be addressed. Tel.: 34-918373112 (ext. 4381); Fax: 34-915360432; E-mail: j.m.andreu{at}cib.csic.es.

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