Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.T he discovery that bacteria have actin-, tubulin-, and intermediate filament-like proteins demonstrated that the cytoskeleton is an ancient invention, predating the divergence between prokaryotes and eukaryotes (1). The GTPase FtsZ (filamentation temperature-sensitive Z) was the first prokaryotic protein to be recognized as a cytoskeletal element (2, 3). FtsZ is a tubulin-like protein, which is widely conserved in bacteria and the main component of the bacterial cytokinesis machine, or "divisome." FtsZ self-assembles into single-stranded protofilaments and these associate further inside cells to form a superstructure known as the Z ring (4, 5). FtsZ alone can generate a constriction force to initiate division (6). The Z ring also provides a scaffold onto which several other components of the divisome-mostly cell wall synthesizing enzymes-are recruited and oriented so as to build the division septum, a cross-wall separating a progenitor cell into two isogenic daughter cells (7).FtsZ and tubulin share several essential properties: their assembly is cooperative, stimulated by GTP, and leads to GTP hydrolysis; they form dynamic polymers whose turnover is dependent on nucleotide hydrolysis (8); they use essentially the same bond for polymer formation (9); and recent evidence indicates that they undergo similar allosteric transitions upon polymerization (10, 11). Not surprisingly, however, the functional specialization of these proteins led to some significant differences between them, the most prominent being that FtsZ exists as single protofilaments, whereas tubulin always adopts a multifilament tubular structure. This difference in their higherorder structure implies that the reactions that lead to cooperativity and subunit turnover are likely different. It has also represented a significant technical challenge for the study of FtsZ. Because FtsZ filaments are smaller than the resolution of optical microscopy, so far it has been impossible to determine essential properties associated with its dynamic behavior.Similarly to actin filaments and microtubules, the assembly of FtsZ protofilaments into a Z ring is regulated by a number of proteins that bind directly...

Cell division in bacteria is regulated by proteins that interact with FtsZ and modulate its ability to polymerize into the Z ring structure. The best studied of these regulators is MinC, an inhibitor of FtsZ polymerization that plays a crucial role in the spatial control of Z ring formation. Recent work established that E. coli MinC interacts with two regions of FtsZ, the bottom face of the H10 helix and the extreme C-terminal peptide (CTP). Here we determined the binding site for MinC on Bacillus subtilis FtsZ. Selection of a library of FtsZ mutants for survival in the presence of Min overexpression resulted in the isolation of 13 Min-resistant mutants. Most of the substitutions that gave rise to Min resistance clustered around the H9 and H10 helices in the C-terminal domain of FtsZ. In addition, a mutation in the CTP of B. subtilis FtsZ also produced MinC resistance. Biochemical characterization of some of the mutant proteins showed that they exhibited normal polymerization properties but reduced interaction with MinC, as expected for binding site mutations. Thus, our study shows that the overall architecture of the MinC-FtsZ interaction is conserved in E. coli and B. subtilis. Nevertheless, there was a clear difference in the mutations that conferred Min resistance, with those in B. subtilis FtsZ pointing to the side of the molecule rather than to its polymerization interface. This observation suggests that the mechanism of Z ring inhibition by MinC differs in both species.

VALDIR BLASIOS JUNIOR Caracterização da interação entre o regulador espacial MinC e seu alvo FtsZ em Bacillus subtilisBacterial cell division is orchestrated by FtsZ, a protein homologous to eukaryotic tubulin that has the ability to polymerize and generate a cytoplasmic structure called the Z ring. The subcellular location where this cytoskeletal structure is formed determines the future division site. The MinCD complex is one of the main regulators of the position of cell division, driving the assembly of Z-ring precisely at the medial region of the cell. MinCD acts as a site-specific inhibitor of FtsZ polymerization, blocking Z ring formation at the cell poles. MinC is the protein of the complex that acts directly on FtsZ and inhibits its polymerization. This thesis elucidates the interaction between FtsZ and MinC and suggests the MinC mechanism in Bacillus subtilis. An ftsZ randomly mutagenized library was screened to identify mutants that are resistant to MinC action. Using right-angle light scattering and fluorescence spectroscopy we showed that substitutions K243R and D287V lost the interaction to MinC. These substituted residues clustered around the H-9 and H-10 helices in the C-terminal domain of FtsZ, thus, we conclude that this region is the binding site for MinC.In addition to mapping the MinC binding site on FtsZ, we also identified the FtsZ binding site in MinC. Based on residue conservation, NMR experiments and exposure to solvent, we chose residues of MinC for mutagenesis and characterization. The substituted residues that disrupted MinC ability to inhibit FtsZ polymerization in vitro were: Y8 and K12 (β-1), K15 (turn-2), H55 (β-3), H84 (β-4) and K149 (C-terminal). Thus, we conclude that the binding site of MinC for FtsZ is located on the only β sheet at the N-terminal domain of MinC from B. subtilis.Finally, based on the binding sites of the two proteins mapped experimentally, we created a model of the complex between MinC and FtsZ by molecular docking.According to the generated model, MinC interacts with the lateral portion of FtsZ polymers. This indicates that MinC should inhibit assembly of higher order FtsZ polymers, thereby preventing the formation of a functional Z-ring. This mechanism of Min is different from that proposed in E. coli, in which MinC interacts with FtsZ polymerization interface and inhibits FtsZ protofilament formation.Keywords: Cell division, FtsZ, MinC, Z-ring, cytoskeleton, protein-protein interaction. (Oliva, et al., 2004). Os contatos longitudinais do filamento são similares entre FtsZ e tubulina (Erickson et al., 1996). A semelhança estrutural e dos polímeros de FtsZ, quando comparado à tubulina eucariótica, se devem ao fato dessas duas proteínas derivarem de um ancestral comum (Erickson et al., 1996) (Lowe e Amos, 1998). LISTA DE ABREVIATURAS E SIGLAS Reguladores negativosDentre os reguladores negativos da polimerização de FtsZ que foram identificados até o momento, SulA e MinC são os melhores estudados. Porém, alguns inibidores como Noc/SlmA e EzrA tem destaque...

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