UreG is a GTPase required for assembly of the nickel-containing active site of urease. Herein, a Strep-tagged Klebsiella aerogenes UreG (UreGStr) and selected site-directed variants of UreGStr 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 UreGStr 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. UreGStr binds one nickel or zinc ion per monomer (Kd = ~5 μM for each metal ion) at a binding site that includes Cys72, as shown by a 12-fold increased Kd for nickel ions using C72A UreGStr 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 UreGStr 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.

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. SAXS analyses of urease, (UreABC-UreD) 3 , and (UreABC-UreDF) 3 confirm that UreD and UreF bind near UreB at the periphery of the (UreAC) 3 structure. This study supports an activation model in which a domain-shifted UreB conformation in (UreABC-UreDF) 3 allows CO 2 and nickel ions to gain access to the nascent active site. KeywordsUrease; Activation; Flexibility; Small-angle X-ray scattering Urease is a nickel-containing enzyme that hydrolyzes urea [1,2]. Crystallographic analyses of ureases from bacterial and plant sources [3][4][5][6][7] reveal a basic trimeric structure with three active sites, each composed of two nickel ions coordinated by a carboxylated Lys, four His and an Asp. Genetic and biochemical studies carried out with plants, fungi, and bacteria [reviewed in [8][9][10]] have shown that additional genes encoding accessory proteins are required for proper assembly of the urease metallocenter, with the possible exception of that from Bacillus *Corresponding author. Address: Department of Microbiology & Molecular Genetics, 2215 Biomedical Physical Sciences, Michigan State University, East Lansing, Michigan 48824-4320, . E-mail: hausinge@msu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [17,18]. Urease activity is generated by incubating these complexes with high concentrations of bicarbonate (to supply the CO 2 needed for Lys carboxylation) and nickel ions, but the required levels of these additives (100 mM and 100 µM, respectively) are not physiologically relevant and only a portion of the proteins are activated [19,20]. In contrast, fully active urease is generated with only 100 µM bicarbonate and 20 µM nickel ions using (UreABC-UreDFG) 3 plus UreE (M r 17,558) and GTP [21]. UreE functions as a nickel-binding protein [22,23] that delivers the metal ion to (UreABC-UreDFG) 3 as GTP is hydrolyzed [24]. Although UreE is often referred to as...