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

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. A key unanswered question is how FtsZ achieves its kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we report the characterization of FtsZ from the cell wall-less bacteria,Spiroplasma melliferum(SmFtsZ). SmFtsZ is a slower GTPase and has higher critical concentration (CC) for polymerization compared toEscherichia coliFtsZ (EcFtsZ). Analysis of the crystal structures of FtsZ structures reveal that the interaction of gamma phosphate of the nucleotide with the T3 loop leads to a peptide flip at Gly71. We propose that the flipped peptide conformation results in a key interaction that facilitates preferential binding of the N-terminal domain (NTD) of a GTP-bound FtsZ monomer to the C-terminal domain (CTD) exposed end of FtsZ filament. In FtsZs, a conformational switch from R- to T-state favors polymerization. We identified a residue, Phe224, located at the interdomain cleft of SmFtsZ, which is crucial for R- to T-state transition. The mutation F224M in SmFtsZ cleft resulted in higher GTPase activity and lower CC, whereas the corresponding M225F in EcFtsZ resulted in cell division defects inE. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. This step, in addition to the GTP-dependence of the T3 loop conformation, slows down the addition of monomers to the NTD-exposed end of filament in comparison to CTD end, thus explaining kinetic polarity.