Seminar

Targeting bacterial cell division protein FtsZ with assembly inhibitors and fluorescent probes

José Manuel Andreu, PhD

Assembly machines from the tubulin-FtsZ superfamily of GTPases are essential for most cells to segregate DNA or to divide. Their different polymers can do work even without the assistance of motor proteins. FtsZ forms the Z-ring that coordinates the divisomal machinery in most bacteria. FtsZ is a structural homolog of tubulin, assembles into dynamic polar filaments similar to microtubule protofilaments, and hydrolyzes bound GTP at the association interface between consecutive subunits, which triggers disassembly. FtsZ is an attractive target for new antibiotics, needed to fight the widespread emergence of resistant bacterial pathogens. Assembly of purified FtsZ is selectively inhibited with several modified nucleotides, which gives insight for further inhibitors. We have replaced FtsZ´s GTP with effective synthetic inhibitors of bacterial cell division. We identified a series of small molecules that bind to FtsZ monomers with micromolar affinities, selectively impair FtsZ assembly, the localization of FtsZ-GFP to the division ring in Bacillus subtilis (Bs), and display antibacterial activity against Gram-positive pathogens including antibiotic-resistant Staphylococcus aureus (Sa). Large scale molecular dynamics (MD) simulations of Sa-FtsZ filaments identify a coordinated Mg2+ ion as the key element in closing the nucleotide site and stabilizing the GTP association interface, whereas with bound GDP the loss of the contacts with loop T7 from the next monomer leads to opened interfaces and curving filaments that are more prone to depolymerization. In addition to the GTP/GDP chemical signal for assembly/disassembly, the structure of FtsZ subunits switches between a low association affinity conformation in monomers and a high affinity conformation when assembled in filaments. The MD simulations recapitulate the FtsZ assembly switch suggested by structural comparisons, which includes an opening of the cleft between the C-terminal domain and H7 helix, coupled to a tight association interface between consecutive subunits, whereas isolated and minus-end subunits relax into the closed cleft conformation. The effective antibacterial PC190723 binds into the open cleft and allosterically stabilizes FtsZ filaments. We have designed fluorescent PC190723 analogs that bind to Bs- and Sa-FtsZ polymers rather than to monomers, with which we can monitor FtsZ assembly. These small probes permit direct imaging of the Z-ring in living cells and may be also useful to find new antibacterials targeting the FtsZ assembly switch.