Abstract
Cell division protein FtsZ can form single-stranded filaments with a cooperative behavior by self-switching assembly. Subsequent condensation and bending of FtsZ filaments are important for the formation and constriction of the cytokinetic ring. PC190723 is an effective bactericidal cell division inhibitor that targets FtsZ in the pathogen Staphylococcus aureus and Bacillus subtilis and does not affect Escherichia coli cells, which apparently binds to a zone equivalent to the binding site of the antitumor drug taxol in tubulin (Haydon, D. J., Stokes, N. R., Ure, R., Galbraith, G., Bennett, J. M., Brown, D. R., Baker, P. J., Barynin, V. V., Rice, D. W., Sedelnikova, S. E., Heal, J. R., Sheridan, J. M., Aiwale, S. T., Chauhan, P. K., Srivastava, A., Taneja, A., Collins, I., Errington, J., and Czaplewski, L. G. (2008) Science 312, 1673-1675). We have found that the benzamide derivative PC190723 is an FtsZ polymer-stabilizing agent. PC190723 induced nucleated assembly of Bs-FtsZ into single-stranded coiled protofilaments and polymorphic condensates, including bundles, coils, and toroids, whose formation could be modulated with different solution conditions. Under conditions for reversible assembly of Bs-FtsZ, PC190723 binding reduced the GTPase activity and induced the formation of straight bundles and ribbons, which was also observed with Sa-FtsZ but not with nonsusceptible Ec-FtsZ. The fragment 2,6-difluoro-3-methoxybenzamide also induced Bs-FtsZ bundling. We propose that polymer stabilization by PC190723 suppresses in vivo FtsZ polymer dynamics and bacterial division. The biochemical action of PC190723 on FtsZ parallels that of the microtubule-stabilizing agent taxol on the eukaryotic structural homologue tubulin. Both taxol and PC190723 stabilize polymers against disassembly by preferential binding to each assembled protein. It is yet to be investigated whether both ligands target structurally related assembly switches.
Highlights
FtsZ is a relatively simple assembly machine capable of nucleated linear polymerization
Several GTP analogues substituted at C-8 selectively inhibit FtsZ polymerization but support tubulin assembly into microtubules [31], indicating differences in nucleotide binding by each protein that may be exploited to selectively inhibit bacterial FtsZ without poisoning eukaryotic tubulin
PC Induces Bundles of B. subtilis FtsZ Filaments—To characterize the effects of PC on FtsZ assembly, full-length, untagged FtsZ from B. subtilis was first expressed and purified, and its reversible assembly was induced by 10 mM MgCl2 and 2 mM GTP in Hepes50, 25 °C without other additives, which were chosen for convenience as reference conditions
Summary
FtsZ is a relatively simple assembly machine capable of nucleated linear polymerization. Cryoelectron microscopy of unsupported samples has shown that FtsZ from Escherichia coli can form semiflexible single-stranded filaments [16] Their cooperative behavior is explained by an unfavorable monomer isomerization (activation switch) between an inactive, assembly incompetent, and active conformation, which is coupled to assembly, creating a nucleation barrier [16,17,18,19]. Druggable cavities in FtsZ structures [22] are the apical nucleotide-binding site and a lateral channel between the N- and C-terminal domains. The latter overlaps the binding site of the microtubule-stabilizing antitumor drug taxol in eukaryotic tubulin. Its putative binding site, mapped with resistance mutations, corresponds to the taxol-binding site in tubulin [32]
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