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. Lys20 and Asp49 are likely to function in binding/hydrolysis of GTP and binding of Mg, respectively. UreG Str binds one nickel or zinc ion per monomer (K d ∼5 μM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased K d for nickel ions using C72A UreG Str and by a thiolate-to-nickel charge-transfer band that is absent in the mutant protein. Based on UreG homology to HypB, a GTPase needed for hydrogenase assembly, along with the mutation results, His74 is likely to be an additional metal ligand. In vivo pull-down assays revealed Asp80 as critical for stabilizing UreG Str interaction with the UreABC−UreDF complex. In vitro pull-down assays demonstrated UreG binding to UreE, with the interaction enhanced by nickel or zinc ions. The metallochaperone UreE is suggested to transfer its bound nickel to UreG in the UreABC−UreDFG complex, with the metal ion subsequently transferring to UreD and then into the nascent active site of urease in a GTP-dependent process.