Mutagenesis of Klebsiella aerogenes UreG to probe nickel binding and interactions with other urease-related proteins.

UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreG(Str)) and selected site-directed variants of UreG(Str) were constructed for studying the in vivo effects on urease activation in recombinant Escherichia coli cells, characterizing properties of the purified proteins, and analysis of in vivo and in vitro protein-protein interactions. Whereas the Strep tag had no effect on UreG's ability to activate urease, enzyme activity was essentially abolished in the K20A, D49A, C72A, H74A, D80A, and S111A UreG(Str) variants, with diminished activity also noted with E25A, C28A, and S115A proteins.

The structure of urease activation complexes examined by flexibility analysis, mutagenesis, and small-angle X-ray scattering.

Conformational changes of Klebsiella aerogenes urease apoprotein (UreABC)(3) induced upon binding of the UreD and UreF accessory proteins were examined by a combination of flexibility analysis, mutagenesis, and small-angle X-ray scattering (SAXS). ProFlex analysis of urease provided evidence that the major domain of UreB can move in a hinge-like motion to account for prior chemical cross-linking results. Rigidification of the UreB hinge region, accomplished through a G11P mutation, reduced the extent of urease activation, in part by decreasing the nickel content of the mutant enzyme, and by sequestering a portion of the urease apoprotein in a novel activation complex that includes all of the accessory proteins.