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FtsZ is a bacterial protein that plays a crucial role in cytokinesis by forming the Z-ring. This ring acts as a scaffold to recruit other division proteins and guide the synthesis of septal peptidoglycan, which leads to cell constriction. In its native state, the FtsZ protein from Escherichia coli (EcFtsZ) is a multi-oligomer comprising dimers, trimers, tetramers, and hexamers in a dynamic self-association equilibrium depending on its concentration. This study employed classical methods of analytical biochemistry that included native polyacrylamide gel electrophoresis, size-exclusion chromatography, sedimentation through sucrose gradients, and chemical cross-linking with formaldehyde to characterize the EcFtsZ. The dimers, trimers, and tetramers are the most prevalent oligomers of the EcFtsZ protein; however, the trimer has been understudied compared to the dimer. In this study, we characterized uncross-linked trimers by exclusion chromatography and crosslinked trimers by sedimentation. The results of size-exclusion chromatography demonstrated that the uncross-linked trimer of EcFtsZ has a mass of 128.8 kDa and a frictional ratio f/f o of 1.96, which coincides with the theoretical frictional ratio of 1.80 for a linear trimer. The EcFtsZ protein treated with formaldehyde resulted in a polypeptide band of 128 kDa recognized by anti-FtsZ antibodies and a frictional ratio S max /S 20,w equal to 1.95, which agrees with the theoretical calculation of the frictional ratio of a lateral trimer. The protein-protein interaction prediction program (PEPPI) identified a contact site between subunits in the C-terminal linker region of the EcFtsZ protein, which has the potential to interfere with the recognition of the C-terminal linker by the ClpX(P) protease. [Display omitted] • Two types of trimers (linear and lateral) are present in the FtsZ protein from Escherichia coli (EcFtsZ). • Uncross-linked trimers with a mass of 128.8 kDa and a frictional ratio f/f o of 1.96. Corresponding to linear trimers. • Cross-linked trimers with a mass of 128 kDa and a frictional ratio S max /S 20,w of 1.95. Corresponding to lateral trimers. • Size-exclusion chromatography and sedimentation through sucrose gradients were used for characterization. • The PEPPI program predicted protein-protein interaction in the C-terminal linker region of the EcFtsZ protein. [ABSTRACT FROM AUTHOR]

Abstract The dominance of multi drug resistance in the clinically significant bacteria led to urgency in the development of new antibiotics with novel mechanism of action. Among the biochemical targets explored for selective toxicity, molecular mechanisms involving cell division remained focal point for novel antimicrobial drug discovery. For this purpose we have performed in-silico studies of FtsZ protein and obtained benzimidazolium compounds as potential hits. These molecules obtained in the dock results were synthesized via reacting benzimidazoles with appropriate benzyl halides. The structures of the synthesized compounds were confirmed by their 1H NMR, 13C NMR, IR and mass spectral data. These were evaluated for anti-microbial activity. Among the tested compounds B14, B15 and B20 have shown highest activity (MIC 5 μg/mL) against Staphylococcus aureus , Macrococcus caseolyticus, Escherichia coli and Pseudomonas aeruginosa.. Microscopic examination of drug-treated cultures of Staphylococcus aureus and Pseudomonas aeruginosa showed rod-shaped filamentous growth of the dividing cells, which is a characteristic feature of FtsZ inhibition. Graphical abstract Image 1 Highlights • Novel FtsZ enzyme inhibitors with benzimidazole nucleus were designed and then docking analysis was carried out. • Hit molecules were synthesized and structures were confirmed by IR, NMR and Mass spectra. • They are evaluated for antimicrobial activity and anti fungal activity. • Cell divison inhibitory assay was performed as a proof of concept that it acts on FtsZ protein. [ABSTRACT FROM AUTHOR]

The dimerization rate of the bacterial cell division protein FtsZ is strongly affected by the intracellular crowding. Yet the complexity of the intracellular environment makes it difficult to investigate via all-atom molecular dynamics or other detailed theoretical methods. We study the crowding effect on FtsZ dimerization which is the first step of an oligomerization process that results in more elaborate supramolecular structures. In particular, we consider the effect of intracellular crowding on the reaction rates, and their dependence on the different concentrations of crowding agents. We achieved this goal by using Brownian dynamics (BD) simulation techniques and a modified post-processing approach in which we decompose the rate constant in crowded media as a product of the rate constant in the dilute solution times a factor that incorporates the crowding effect. The latter factor accounts for the diffusion reduction and crowder induced energy. In addition we include the crowding effects on water viscosity in the BD simulations of crowded media. We finally show that biomolecular crowding has a considerable effect on the FtsZ dimerization by increasing the dimerization rate constant from 2.6 × 10 7 M −1 s −1 in the absence of crowders to 1.0 × 10 8 M −1 s −1 at crowding level of 0.30. [ABSTRACT FROM AUTHOR]

Abstract: ZipA is an element of the bacterial division ring complex that provides an anchor to the membrane to FtsZ, a GTPase ancestor of tubulin. In vitro reconstitution and characterization of these interactions is challenged by the difficulty to integrate a physiological membrane environment. Here a single copy of the full-length ZipA protein from Escherichia coli incorporated into phospholipid bilayer nanodiscs (Nd-ZipA) has been visualized using negative-staining electron microscopy (EM). The EM images reveal the presence of discs, mostly organized in two distinct populations of 11 and 13nm in diameter. The globular FtsZ-binding C-terminal domain of ZipA (ZBD) was not visible in 3D reconstructions of Nd-ZipA or 2D averages, suggesting that this domain is separated from the membrane by the large flexible domain connecting the N-terminal trans-membrane region to the ZBD. We tested if Nd-ZipA were appropriate models for the in vitro reconstitution of ZipA–FtsZ interactions. First we observed that the ZBD region of ZipA was accessible for the interaction with other proteins in the context of the nanodisc, as revealed by its recognition by specific antibodies. In addition, Nd-ZipA attached to carbon coated EM grids, but not empty nanodiscs, were able to capture FtsZ filaments without inducing significant filament bundling, consistent with a model in which FtsZ filaments are loosely attached to the cell-membrane. These observations are compatible with the plastic nature of the ZipA–FtsZ complexes formed at the membrane, evidenced in the moderate binding affinity of Nd-ZipA to FtsZ oligomers and polymers recently measured. [Copyright &y& Elsevier]

Abstract: Factors contributing to the stability of bacterial cell division protein FtsZ remain unknown. In order to identify FtsZ-stabilizing factor(s), we exploited FtsH protease-based in vitro FtsZ degradation assay system. Whole cell lysate from an ftsH-null strain of Escherichia coli inhibited degradation of FtsZ by FtsH in vitro. However, activated charcoal-treated lysate did not inhibit degradation. The loss of ability of the activated charcoal-treated lysate to inhibit degradation of FtsZ was restored when it was replenished with GTP, but not when replenished with other NTPs or dNTPs. The lysate did not protect either FtsZ deletion mutants, which do not bind GTP, or FtsH substrates, σ32 and cI-108 proteins, against FtsH. GDP and GTPγS also stabilized FtsZ against FtsH. Neither GTP nor GDP inhibited proteolytic activity of FtsH per se. These observations demonstrate that binding of GTP/GDP ligands is responsible for the proteolytic stability of FtsZ against FtsH. [Copyright &y& Elsevier]