pubs.acs.org/Biochemistry Published on Web 09/10/2009 r 2009 American Chemical Society 9734 Biochemistry 2009, 48, 9734–9744 DOI: 10.1021/bi900556a The Stathmin-Derived I19L Peptide Interacts with FtsZ and Alters Its Bundling † Marie-Jeanne Cl  ement, ‡ Boi-trinh Kuoch, ‡ Tap Ha-Duong, § Vandana Joshi, ‡ Loic Hamon, ‡ Flavio Toma, ‡ Patrick A. Curmi,* ,‡ and Philippe Savarin* ,‡ ‡ Laboratoire Structure-Activit e des Biomol  ecules Normales et Pathologiques, INSERM/UEVE U829, Evry, 91025 France, and § Laboratoire Analyse et Mod elisation pour la Biologie et l’Environnement, UMR/UEVE 8587, Evry, 91025 France Received April 2, 2009; Revised Manuscript Received September 7, 2009 ABSTRACT: FtsZ is a prokaryotic tubulin-like protein. Despite a low degree of sequence identity with tubulin, it presents the same folding pattern and some similar functions, notably in cell division. Indeed, FtsZ and tubulin polymerize to form bundles and microtubules, respectively, which are essential for cell cytokinesis. We previously demonstrated that peptides derived from the N-terminal stathmin domain interact with tubulin and impede microtubule formation. We demonstrated here that I19L, the most efficient of these peptides, also alters FtsZ bundling assembly in vitro. STD-NMR and TRNOESY experiments revealed that I19L interacts with FtsZ and folds upon its binding but in a way different from what we observed with tubulin. These NMR data were used in molecular modeling calculations to propose models of the I19L-FtsZ complex. Interestingly, two models, consistent with NMR data, show an interaction of I19L near the T7 loop or near the GTP binding site of FtsZ, explaining the modifications of the bundling assembly observed with this peptide. The fine analysis of the structural differences of the complexes of I19L with FtsZ and tubulin should help for the rational development of new specific antibiotic agents. FtsZ is one of the major proteins implicated in cell division in bacteria (1). The FtsZ protein is implicated in the formation of the Z-ring, which determines the site of cytokinesis and appears to be a good candidate for discovering new antibiotics agents (2). FtsZ contains two domains arranged around a central helix; the first domain corresponds to a GTPase domain, and the other, the carboxy-terminal domain, is a four-stranded β sheet tilted by 90° against the β sheet of the GTPase domain (3). The Rβ-tubulin protein is the building block of microtubules in eukaryotes. Microtubules are major components of the eukar- yotic cytoskeleton and are implicated in various critical functions such as cell division, motility, and intracellular trafficking. Despite a low degree of sequence identity (<20%), the structures of FtsZ and of R- or β-tubulin are highly homologous (3, 4), and both proteins play an essential role in cell division. However, some remarkable differences exist between these two proteins. In contrast to tubulin, FtsZ is monomeric and does not assemble into cylindrical polymers (like microtubules) but rather forms filaments which also do not present a dynamic instability behavior as observed for microtubules (5). The GTP binding pocket of FtsZ is formed by helices H1-H6 and strands S1-S6. Loops T1-T6 are part of this pocket on one side of the molecule. Loop T7 is in the C-terminal domain and is involved in the longitudinal interactions between subunits and involved in the nucleotide hydrolysis process (6). In contrast to microtubules where the nucleotide-binding pocket is occluded, the nucleotide remains exchangeable in FtsZ protofilaments (7). Interestingly, some mutations in the T7 loop of FtsZ severely affect FtsZ polymerization and GTP hydrolysis (8), which underline its role in the regulation of FtsZ activity. Numerous studies have addressed the interaction between different kinds of drugs and tubulin or FtsZ polymers. Among the drugs used in clinical applications, paclitaxels (9) and peloru- side (10), epothilones (11) bind with higher affinity to polymer- ized tubulin than to soluble tubulin and stabilize microtubules. The second class concerns molecules that bind preferentially to tubulin and promote microtubule disassembly. Vinblastine, vincristine, the semisynthetic analogues vinflunine and vinorel- bine, and two antimitotic peptides phomopsin A and soblido- tin (12) bind to the vinca domain on tubulin. This domain is located adjacent to the exchangeable GTP-binding pocket on β-tubulin at the plus end (+) longitudinal interface (13). Finally, the colchicine binding site is located at the intradimer interface between R-tubulin and β-tubulin (14). In the case of FtsZ, drugs bind either to the GTP pocket (15) or to the T7 loop as cinnamaldehyde (16). Recently, PC190723 has been proposed to bind to a region analoguous to the paclitaxel binding site of tubulin (17). According to the great degree of structural similarity between FtsZ and tubulin, some ligands have been found to act on both proteins: bis-ANS (18) and sanguinarine (19, 20). Some compounds have a different impact on tubulin and FtsZ polymers. For example, calcium induces the formation of FtsZ bundles (21) but inhibits tubulin polymerization (22). Glutamate increases the GTPase activity in tubulin (23) but strongly suppresses this activity for FtsZ (24). Differences in the nucleotide binding site may also explain why some C8-substituted GTP analogues could inhibit FtsZ polymerization but have an opposite effect on tubulin polymerization (25). Interestingly, most of the † This work was supported by Institut National de la Sant e Et de la Recherche M edicale, Association pour la Recherche sur le Cancer (ARC). The R egion Ile de France, the Conseil G en eral de l’Essonne, Genopole, DSV/CEA, and the Association Franc -aise de lutte contre la Myopathie (AFM) are acknowledged for their contribution to the NMR equipment. *To whom correspondence should be addressed: Laboratoire Struc- ture-Activit e des Biomol  ecules Normales et Pathologiques, INSERM/ UEVE U829, Evry, 91025 France. Telephone: (33) 1 69 47 03 23. Fax: (33) 1 69 47 02 19. E-mail: philippe.savarin@univ-evry.fr or pcurmi@ univ-evry.fr.