Self-activation of guanosine triphosphatase activity by oligomerization of the bacterial cell division protein FtsZ

The essential bacterial cell division protein FtsZ (filamentation temperature-sensitive protein Z) is a distant homologue to the eukaryotic cytoskeletal protein tubulin, We have examined the GTP hydrolytic activity of Escherichia coli FtsZ using a real-time fluorescence assay that monitors phosphate production. The GTPase activity shows a dramatic, nonlinear dependence on FtsZ concentration, with activity only observed at enzyme concentrations greater than 1 mu M. At 5 mu M FtsZ, we have determined a K-m of 82 mu M GTP and a V-max of 490 nmol of P-i min(-1) (mg of protein)(-1). Hydrolysis of GTP requires Mg2+ and other divalent cations substitute only poorly for this requirement. We have compared the concentration dependence of FtsZ GTPase activity with the oligomeric state by use of analytical ultracentrifugation and chemical cross-linking. Equilibrium analytical ultracentrifugation experiments show that FtsZ exists as 68% dimer and 13% trimer at 2 mu M total protein concentration. Chemical crosslinking of FtsZ also shows that monomer, dimer, trimer, and: tetramer species are present at higher (>2 mu M) FtsZ concentrations. However, as shown by analytical centrifugation, GDP-bound FtsZ is significantly shifted to the monomeric state, which suggests that GTP hydrolysis regulates polymer destabilization, We also monitored the effect of nucleotide and metal ion on the secondary structure of FtsZ; nucleotide yielded no evidence of structural changes in FtsZ, but both Mg2+ and Ca2+ had significant effects on secondary structure. Taken together, our results support the Hypothesis that Mg2+-dependent GTP hydrolysis by FtsZ requires oligomerization of FtsZ, On the basis of these results and structural comparisons with the alpha-beta tubulin dimer, GTP is likely hydrolyzed in a shared active site formed between two monomer subunits.