Disorder in CENP-ACse4 tail-chaperone interaction facilitates binding with Ame1/Okp1 at the kinetochore.

The centromere is epigenetically marked by a histone H3 variant-CENP-A. The budding yeast CENP-A called Cse4, consists of an unusually long N-terminus that is known to be involved in kinetochore assembly. Its disordered chaperone, Scm3 is responsible for the centromeric deposition of Cse4 as well as in the maintenance of a segregation-competent kinetochore. In this study, we show that the Cse4 N-terminus is intrinsically disordered and interacts with Scm3 at multiple sites, and the complex does not gain any substantial structure. Additionally, the complex forms a synergistic association with an essential inner kinetochore component (Ctf19-Mcm21-Okp1-Ame1), and a model has been suggested to this effect. Thus, our study provides mechanistic insights into the Cse4 N-terminus-chaperone interaction and also illustrates how intrinsically disordered proteins mediate assembly of complex multiprotein networks, in general.

Backbone and side-chain resonance assignments of centromeric protein Scm3 from Saccharomyces cerevisiae.

The centromeric chromatin plays an essential role in regulating the attachment of microtubules and controlling the segregation of sister chromatids during mitosis. In budding yeast, the evolutionary conserved histone variant, Cse4 is a vital component of the multiprotein kinetochore complex and is recruited to the centromere through its chaperone, Suppressor of chromosome mis-segregation (Scm3). Scm3 is an inner kinetochore protein crucial for the formation of a functional inner kinetochore. Scm3 has been known to play an active role in the assembly of the centromeric nucleosome and its deletion has been found to have deleterious effects on the cells leading to chromosome segregation defects. However, structural details of monomeric full length Scm3 have remained elusive so far. Here, we report the backbone and side-chain resonance assignments of centromeric protein, Scm3. 1H, 13C and 15N chemical shifts of Scm3 have been obtained by various 2D and 3D heteronuclear NMR experiments at pH 7.4 and 283 K.

Triphala inhibits alpha-synuclein fibrillization and their interaction study by NMR provides insights into the self-association of the protein.

The process of assembly and accumulation of the intrinsically disordered protein (IDP), alpha-synuclein (αSyn) into amyloid fibrils is a pathogenic process leading to several neurodegenerative disorders such as Parkinson's disease, multiple system atrophy and others. Although several molecules are known to inhibit αSyn fibrillization, the mechanism of inhibition is just beginning to emerge. Here, we report the inhibition of fibrillization of αSyn by Triphala, a herbal preparation in the traditional Indian medical system of Ayurveda. Triphala was found to be a rich source of polyphenols which are known to act as amyloid inhibitors. ThT fluorescence and TEM studies showed that Triphala inhibited the fibrillization of αSyn. However, it was observed that Triphala does not disaggregate preformed αSyn fibrils. Further, native-PAGE showed that Triphala reduces the propensity of αSyn to oligomerize during the lag phase of fibrillization. Our NMR results showed that certain stretches of residues in the N-terminal and NAC regions of αSyn play an anchor role in the self-association process of the protein, thereby providing mechanistic insights into the early events during αSyn fibrillization.

A Carbocyclic Curcumin Inhibits Proliferation of Gram-Positive Bacteria by Targeting FtsZ.

Inhibition of FtsZ assembly has been found to stall bacterial cell division. Here, we report the identification of a potent carbocyclic curcumin analogue (2d) that inhibits Bacillus subtilis 168 cell proliferation by targeting the assembly of FtsZ. 2d also showed potent inhibitory activity (minimum inhibitory concentrations of 2-4 mg/L) against several clinically important species of Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. In addition, 2d displayed a significantly reduced inhibitory effect on human cervical cancer cells in comparison to its effect on bacterial cells. Using live cell imaging of GFP-FtsZ by confocal microscopy, 2d was found to rapidly perturb the cytokinetic FtsZ rings in Bacillus subtilis cells. The immunofluorescence imaging of FtsZ also showed that 2d destroyed the Z-ring in bacteria within 5 min. Prolonged treatment with 2d produced filamentous bacteria, but 2d had no detectable effect either on the nucleoids or on the membrane potential of bacteria. 2d inhibited FtsZ assembly in vitro, whereas it had minimal effects on tubulin assembly. Interestingly, 2d strongly enhanced the GTPase activity of FtsZ and reduced the GTPase activity of tubulin. Furthermore, 2d bound to purified FtsZ with a dissociation constant of 4.0 ± 1.1 µM, and the binding of 2d altered the secondary structures of FtsZ. The results together suggested that the non-natural curcumin analogue 2d possesses powerful antibacterial activity against important pathogenic bacteria, and the evidence indicates that 2d inhibits bacterial proliferation by targeting FtsZ.

Mutation of Arg191 in FtsZ Impairs Cytokinetic Abscission of Bacillus subtilis Cells.

FtsZ monomers assemble to form a dynamic Z-ring at the midcell position in bacteria that coordinates bacterial cell division. Antibacterial agents plumbagin and SB-RA-2001 were found to bind to FtsZ and to inhibit Z-ring formation in bacteria. Docking analysis indicated similar binding regions for these two inhibitors on FtsZ, and residue R191 was involved in the binding interaction with both compounds. In this work, the importance of R191 in FtsZ assembly and in bacterial cell division was analyzed. R191A-FtsZ exhibited significantly poorer polymerization ability. Further, the mutant FtsZ could poison the assembly of wild-type FtsZ (WT-FtsZ). The expression of R191A-FtsZ in Bacillus subtilis strain PL2084 perturbed Z-ring formation and produced filamentous cells, indicating that the mutation hindered the division of these cells. The results suggested that the R191A mutation is a dominant negative mutation of FtsZ. Molecular dynamics simulations of R191A-FtsZ and WT-FtsZ revealed a kink in helices H5 and H7 in the active site of R191A-FtsZ compared to that of WT-FtsZ, which is required for FtsZ assembly. The findings suggested that R191 is an important residue for FtsZ assembly, which can be targeted for the design of FtsZ inhibitors.

ZapC promotes assembly and stability of FtsZ filaments by binding at a different site on FtsZ than ZipA.

ZapC, a component of the divisome in Escherichia coli, is known to co-localize with FtsZ at the mid-cell position. A deletion or an overexpression of ZapC has been found to induce elongation of bacterial cells implying a role of ZapC in the cell division. ZapC has also been shown to enhance the assembly of purified FtsZ. In this study, ZapC was found to prevent the dilution-induced disassembly of preformed FtsZ polymers and to decorate FtsZ protofilaments along the length. ZapC interacted with FtsZ with a dissociation constant of 30±7nM. Salt had no discernable effect on the binding of ZapC to FtsZ; however, bis-ANS inhibited the binding of ZapC to FtsZ suggesting that the interaction was predominantly hydrophobic in nature. Several of the positive regulators of FtsZ assembly including ZipA are shown to bind FtsZ at the C-terminal tail of FtsZ. Using a 12-residue C-terminal tail peptide (LDIPAFLRKQAD) of FtsZ and a C-terminal tail truncated FtsZ construct, we provided data suggesting that ZapC does not bind at the C-terminal tail of FtsZ. The results indicated that ZapC and ZipA, two functionally similar proteins of the divisome complex, regulate FtsZ assembly through different sites of action on FtsZ.

SB-RA-2001 inhibits bacterial proliferation by targeting FtsZ assembly.

FtsZ has been recognized as a promising antimicrobial drug target because of its vital role in bacterial cell division. In this work, we found that a taxane SB-RA-2001 inhibited the proliferation of Bacillus subtilis 168 and Mycobacterium smegmatis cells with minimal inhibitory concentrations of 38 and 60 µM, respectively. Cell lengths of these microorganisms increased remarkably in the presence of SB-RA-2001, indicating that it inhibits bacterial cytokinesis. SB-RA-2001 perturbed the formation of the FtsZ ring in B. subtilis 168 cells and also affected the localization of the late cell division protein, DivIVA, at the midcell position. Flow cytometric analysis of the SB-RA-2001-treated cells indicated that the compound did not affect the duplication of DNA in B. subtilis 168 cells. Further, SB-RA-2001 treatment did not affect the localization of the chromosomal partitioning protein, Spo0J, along the two ends of the nucleoids and also had no discernible effect on the nucleoid segregation in B. subtilis 168 cells. The agent also did not appear to perturb the membrane potential of B. subtilis 168 cells. In vitro, SB-RA-2001 bound to FtsZ with modest affinity, promoted the assembly and bundling of FtsZ protofilaments, and reduced the GTPase activity of FtsZ. GTP did not inhibit the binding of SB-RA-2001 to FtsZ, suggesting that it does not bind to the GTP binding site on FtsZ. A computational analysis indicated that SB-RA-2001 binds to FtsZ in the cleft region between the C-terminal domain and helix H7, and the binding site of SB-RA-2001 on FtsZ resembled that of PC190723, a well-characterized inhibitor of FtsZ. The findings collectively suggested that SB-RA-2001 inhibits bacterial proliferation by targeting the assembly dynamics of FtsZ, and this can be exploited further to develop potent FtsZ-targeted antimicrobials.

Plumbagin inhibits cytokinesis in Bacillus subtilis by inhibiting FtsZ assembly--a mechanistic study of its antibacterial activity.

The assembly of FtsZ plays a central role in construction of the cytokinetic Z-ring that orchestrates bacterial cell division. A naturally occurring naphthoquinone, plumbagin, is known to exhibit antibacterial properties against several types of bacteria. In this study, plumbagin was found to perturb formation of the Z-ring in Bacillus subtilis 168 cells and to cause elongation of these cells without an apparent effect on nucleoid segregation, indicating that it may inhibit FtsZ assembly. Furthermore, it bound to purified B. subtilis FtsZ (BsFtsZ) with a dissociation constant of 20.7 ± 5.6 µM, and inhibited the assembly and GTPase activity of BsFtsZ in vitro. Interestingly, plumbagin did not inhibit either the assembly or GTPase activity of Escherichia coli FtsZ (EcFtsZ) in vitro. Using docking analysis, a putative plumbagin-binding site on BsFtsZ was identified, and the analysis indicated that hydrophobic interactions and hydrogen bonds predominate. Based on the in silico analysis, two variants of BsFtsZ, namely D199A and V307R, were constructed to explore the binding interaction of plumbagin and BsFtsZ. The effects of plumbagin on the assembly and GTPase activity of the variant BsFtsZ proteins in vitro indicated that the residues D199 and V307 may be involved in the binding of plumbagin to BsFtsZ. The results suggest that plumbagin inhibits bacterial proliferation by inhibiting the assembly of FtsZ, and provide insight into the binding site of plumbagin on BsFtsZ, which may help in the design of potent FtsZ-targeted antibacterial agents.

ZipA binds to FtsZ with high affinity and enhances the stability of FtsZ protofilaments.

A bacterial membrane protein ZipA that tethers FtsZ to the membrane is known to promote FtsZ assembly. In this study, the binding of ZipA to FtsZ was monitored using fluorescence spectroscopy. ZipA was found to bind to FtsZ with high affinities at three different (6.0, 6.8 and 8.0) pHs, albeit the binding affinity decreased with increasing pH. Further, thick bundles of FtsZ protofilaments were observed in the presence of ZipA under the pH conditions used in this study indicating that ZipA can promote FtsZ assembly and stabilize FtsZ polymers under unfavorable conditions. Bis-ANS, a hydrophobic probe, decreased the interaction of FtsZ and ZipA indicating that the interaction between FtsZ and ZipA is hydrophobic in nature. ZipA prevented the dilution induced disassembly of FtsZ polymers suggesting that it stabilizes FtsZ protofilaments. Fluorescein isothiocyanate-labeled ZipA was found to be uniformly distributed along the length of the FtsZ protofilaments indicating that ZipA stabilizes FtsZ protofilaments by cross-linking them.