The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties

Abstract

The Caulobacter crescentus CgtA protein is a member of the Obg-GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA specifically bound GTP and GDP but not GMP or ATP. CgtA bound GTP and GDP with moderate affinity at 30°C and displayed equilibrium binding constants of 1.2 and 0.5 μM, respectively, in the presence of Mg2+. In the absence of Mg2+, the affinity of CgtA for GTP and GDP was reduced 59- and 6-fold, respectively. N- Methyl-3'-O-anthranoyl (mant)-guanine nucleotide analogs were used to quantify GDP and GTP exchange. Spontaneous dissociation of both GDP and GTP in the presence of 5 to 12 mM Mg2+ was extremely rapid (k(d) = 1.4 and 1.5 s-1, respectively), 103- to 105-fold faster than that of the well- characterized eukaryotic Ras-like GTP-binding proteins. The dissociation rate constant of GDP increased sevenfold in the absence of Mg2+. Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 x 10-4 S-1 corresponding to a half-life of hydrolysis of 23 min. These data clearly demonstrate that the guanine nucleotide binding and exchange properties of CgtA are different from those of the well- characterized Ras-like GTP-binding proteins. Furthermore, these data are consistent with a model whereby the nucleotide occupancy of CgtA is controlled by the intracellular levels of guanine nucleotides.