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) state transition. Our hypothesis was supported by structural, biochemical and in vivo experiments of the cleft mutant. Further, structure and sequence analyses of the interdomain cleft of FtsZs helped us obtain insights into the mechanism of binding of its inhibitor PC190723. We propose that the presence of occluding salt bridge interactions in the inter-domain cleft of FtsZ is responsible for the resistance of PC190723 binding to certain FtsZs. We finally propose a model for how the kinetic polarity of FtsZ filaments is opposite to that of microtubules, a polymer of its homolog tubulin and the model for the mechanism of the drug, PC190723, binding to FtsZ.
