The Caulobacter crescentus CgtA Protein Displays Unusual Guanine Nucleotide Binding and Exchange Properties

Abstract

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 Mg 2+ . In the absence of Mg 2+ , 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 Mg 2+ was extremely rapid ( k d = 1.4 and 1.5 s −1 , respectively), 10 3 - to 10 5 -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 Mg 2+ . Finally, there was a low inherent GTPase activity with a single-turnover rate constant of 5.0 × 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.