Characterization of interaction between the spatial regulator for bacterial division MinC and its target FtsZ in Bacillus subtilis

A divisão celular bacteriana é orquestrada por FtsZ, uma proteína homóloga à tubulina eucariótica que possui a capacidade de polimerizar e gerar uma estrutura chamada de anel Z. O local onde esta estrutura citoesquelética contrátil é formada determina o futuro sítio de divisão. O complexo MinCD é um dos principais reguladores da posição da divisão, favorecendo a montagem do anel Z precisamente na região medial da bactéria. MinCD age como um inibidor sítio específico da polimerização de FtsZ, atuando preferencialmente nos polos celulares. MinC é a proteína do complexo que atua diretamente sobre FtsZ e inibe sua polimerização. Essa tese elucida a interação entre FtsZ e MinC e sugere o mecanismo exercido por MinC em Bacillus subtilis. Foi triada uma biblioteca de mutantes randômicos de FtsZ para identificação de mutantes resistentes à ação de MinC. Dentre estes, as substituições K243R e D287V, quando caracterizados usando espalhamento de luz e espectroscopia de fluorescência impediram a interação com MinC. Como as mutações estavam localizados em torno das hélices H-9 e H-10 no domínio C-terminal de FtsZ, concluímos que esta região representa o sítio de interação com MinC desta proteína. Como complemento ao mapeamento do sitio de ligação de MinC em FtsZ, identificamos a região de MinC que interage com FtsZ. Para tanto, escolhemos resíduos de MinC para mutagênese e caracterização. A escolha priorizou os resíduos conservados entre espécies Gram-positivas, experimentos de RMN, carga e exposição ao solvente dos mesmos. Dentre os resíduos de MinC mutados que afetaram sua capacidade de inibir a polimerização de FtsZ in vitro foram: Y8 e K12 (β-1), K15 (alça-2), H55 (β-3) , H84 (β-4) e K149 (C-terminal). Sendo assim, podemos concluir que a face de interação para FtsZ em MinC de B. subtilis é a única folha β do domínio N-terminal desta proteína. Com base nos sítios mapeados das duas proteínas experimentalmente, criamos um modelo in silico do complexo MinC-FtsZ por docking molecular. De acordo com o modelo gerado, MinC interage com a porção lateral de polímeros de FtsZ. Isto sugere que MinC atue na inibição da formação de feixes de filamentos de FtsZ, impedindo assim a formação de anéis Z funcionais. Esse mecanismo de ação do sistema Min é diferente do proposto para E. coli, no qual MinC interage com a face de polimerização FtsZ-FtsZ e impede a formação de protofilamentos de FtsZ.Bacterial 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 di srupted 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 β only 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

Genetic and biochemical characterization of the MinC-FtsZ interaction in Bacillus subtilis.

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

FtsZ filament capping by MciZ, a developmental regulator of bacterial division.

International audienceCytoskeletal 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

Polymerization of FtsZ mutants in vitro is not affected by MinC.

<p>A. Representative light scattering trace of an experiment performed with wild-type FtsZ in the absence of inhibitor and in the presence of MinC or MinC19. Reactions contained 7 µM of FtsZ and 20 µM of MinC or MinC19 in Mes/NaOH 50 mM, MgCl₂ 10 mM, KCl 133 mM, DEAE-dextran 0.6 mg/mL, pH 6.5. B. Experiments similar to A were performed and the inhibition of the polymerization of each FtsZ mutant by MinC was quantified relative to the maximum light scattering signal in the absence of MinC. The original light scattering traces corresponding to this graph can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060690#pone.0060690.s004" target="_blank">Fig. S4</a>. Measurements were repeated at least three times for each FtsZ mutant with similar results.</p

Trp fluorescence measurements of MinC binding to mutant FtsZ.

<p>Wild-type or double-mutant (K243R,D287V) FtsZ <b>(</b>2 µM) were mixed with 1 µM MinC Y44W in buffer Tris/HCl 20 mM, KCl 100 mM, EDTA 5 mM, pH 7,5 and fluorescence emission at 320–360 nm was recorded. A. Representative trace of one experiment. B. Averaged quantitation of 3 experiments, with standard deviations.</p

FtsZ mutant measurements.

<p>FtsZ mutant measurements.</p

Cell division phenotype of FtsZ mutants.

<p>Strains containing an IPTG inducible allele of <i>minD</i> (<i>thrC</i>::P_spac-hy-<i>minD</i>, <i>erm</i>) plus either wild-type or Min-resistant <i>ftsZ</i> alleles were grown on LB-agar chambers with or without 1 mM IPTG for 2 hours and imaged by microscopy. Membranes were stained with FM 5–95. Arrowheads point to a typical minicell (yellow) and an abnormally sized minicell (red). Strain numbers: FtsZ wild-type (AB164), T111A (AB70), K243R (AB168), D287V (AB174), R376T (AB177). The scale bar corresponds to 5 µm.</p

GTPase activity and Cc of FtsZ mutants.

<p>GTPase activity and Cc of FtsZ mutants.</p

The binding site for MinC differs in <i>B. subtilis</i> and <i>E. coli</i> FtsZ.

<p>A. Comparison between mutations that promote MinC resistance in <i>B. subtilis</i> (red residues) and <i>E. coli</i> (blue residues) mapped onto the 2VAM FtsZ structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060690#pone.0060690-Oliva2" target="_blank">[75]</a>. <i>E. coli</i> residue N280 corresponds to <i>B. subtilis</i> D280; E276 corresponds to Q276; R271 corresponds to S271. Two other important landmarks are highlighted in the structure: catalytic residue D213, shown in yellow, defines the center of the FtsZ-FtsZ interface (the polymerization axis follows a vertical line through this residue); and the point where the CTP should emerge from the structure (residue F315) is shown in orange. B. Surface electrostatic potential of <i>B. subtilis</i> FtsZ (2VAM) obtained with the “protein contact potential” tool of PyMOL. Note that the MinC binding site corresponds to a highly negative region of the FtsZ molecule.</p