Characterization of spiroplasma ftsz insights into kinetic polarity and inhibitor binding

Abstract

FtsZ, a tubulin homolog, marks the site of division and forms ring-like structure, the Z-ring, in bacteria. The Z-ring gets tethered to the membrane via FtsA, an actin homolog. Unlike the dynamic instability seen in tubulin, FtsZ undergoes treadmilling to guide the peptidoglycan synthesis machinery for the formation of the septum. However, the role of FtsZ filament dynamics in absence of peptidoglycan synthesis is unclear. Study of FtsZ filaments from cell wall-less bacteria could provide insights into these mechanistic aspects. A conformational switch in FtsZ from low-affinity R state to high-affinity T state favours cooperativity and filament elongation from one end and depolymerization from the other. The difference in the rate of assembly and disassembly between two ends of the filaments decides kinetic polarity. However, the structural basis of the kinetic polarity of FtsZ polymerization is not fully understood. Since FtsZ is an essential protein in bacterial cell division, it can be the target for anti-microbial drugs. But the mechanism of the drug, PC190723, binding to FtsZ is not known. In my PhD research, I have performed comparative biochemical characterization of FtsZ from a cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ) and FtsZ from Escherichia coli, a cell-walled bacterium (EcFtsZ). We observed that features like GTP binding, hydrolysis and filament formation are conserved for FtsZ from the organism devoid of cell wall but SmFtsZ has lower GTPase activity and higher critical concentration of polymerization than that of EcFtsZ under similar in vitro conditions. We then determined the crystal structures of SmFtsZ, with GDP and GTP respectively, which captured the NTD in both R and T states and the CTD in an R state conformation, revealing an intermediate conformational state of FtsZ during R to T state transition. This led to the identification of a residue which could help in the cleft opening between N- terminal domain (NTD) and C-terminal domain (CTD) during the R (closed) to T (opened)