Mechanistic insights into GTP-dependence and kinetic polarity of FtsZ filament assembly

Abstract

FtsZ, a tubulin homolog, forms the Z-ring at the division site in bacteria. FtsZ filaments guide peptidoglycan synthesis machinery to drive cell division, while their role in cell wall-less bacteria is unclear. We report the structure and biochemical properties of FtsZ from the cell wall-less bacterium Spiroplasma melliferum (SmFtsZ). Compared to Escherichia coli FtsZ (EcFtsZ), SmFtsZ possessed lower GTPase activity and higher critical concentration (CC) of polymerization. In FtsZs, a conformational switch from R- to T-state favours polymerization. In the crystal structures of SmFtsZ captured as domain-swapped dimers, conformation of the GTP-bound N-terminal domain (NTD) was in the T-state, while the relative orientation of the NTD and the C-terminal domain (CTD) matched the R-state. The occurrence of GTP-bound NTD in the T-state and CTD in the R-state captures a conformational state that facilitates preferential binding of the NTD of the monomeric FtsZ to the CTD-exposed end of the FtsZ filament. CTD of the nucleotide-bound monomer cannot bind to the NTD-exposed end of the filament unless relative rotation of the domains leads to cleft opening. Mutation of SmFtsZ-Phe224 in the cleft to methionine, the corresponding residue in EcFtsZ (Met226), resulted in higher GTPase activity and lower CC. Mutation of EcFtsZM226F resulted in cell division defects. These results suggest that the relative rotation of the two domains leading to cleft opening is a rate-limiting step of polymerization. Hence, addition of monomers to the CTD end of the filament is hypothesized to be faster than to the NTD end, thus explaining the kinetic polarity.

An amphipathic helix facilitates direct membrane binding of Mycoplasma FtsZ

“…SmftsZ gene was PCR amplified from the chromosomal DNA of Spiroplasma melliferum KC3 followed by restriction digestion and ligation at the Nde1 and BamH1 sites into a (His) 6 tag containing pHis17 vector as reported earlier (68). Restriction free cloning (69) was used for cloning of untagged SmFtsZ construct using specific primers.…”