UreG, a chaperone in the urease assembly process, is an intrinsically unstructured GTPase that specifically binds Zn2+

Bacillus pasteurii UreG, a chaperone involved in the urease active site assembly, was over-expressed in E. coli BL21(DE3) and purified to homogeneity. The identity of the recombinant protein was confirmed by SDS-PAGE, protein sequencing and mass spectrometry. Combination of size exclusion chromatography, multi-angle and dynamic laser light scattering established that BpUreG is present in solution as a dimer. Analysis of circular dichroism spectra indicated that the protein contains large portions of helices (15%) and strands (29%), while NMR spectroscopy indicated the presence of conformational fluxionality of the protein backbone in solution. BpUreG catalyzes the hydrolysis of GTP with a kcat = 0.04 min-1, confirming a role of this class of proteins in coupling energy requirements and nickel incorporation into the urease active site. BpUreG binds 2 Zn2+ ions per dimer with a KD = 42±3 ?M, and has ten-fold lower affinity for Ni2+. A structural model for BpUreG was calculated using threading algorithms. The protein, in the fully folded state, features the typical structural architecture of GTPases, with an open ?-barrel surrounded by ?-helices and a P-loop at the N-terminus. The protein dynamic behavior observed in solution is critically discussed relatively to the structural model, using algorithms for disorder predictions. The results suggest that UreG proteins belong to the class of intrinsically unstructured proteins (IUP) that need the interaction with co-factors or other protein partners to perform their function. It is also proposed that metal ions such as Zn2+ could have important structural roles in the urease activation process.