Zn²⁺‐linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation

Abstract

The biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease involves the formation of a lysine-carbamate functional group concomitantly with the delivery of two Ni(2+) ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P-loop GTPases, often required to complete the biosynthesis of nickel-enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On-line size exclusion chromatography and light scattering indicated that apo-HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well-defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc-binding GTPase involved in the in vivo assembly of [Ni,Fe]-hydrogenase. A homology-based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn(2+) per monomer (K(d) = 0.33 +/- 0.03 microM), whereas it has 20-fold lower affinity for Ni(2+) (K(d) = 10 +/- 1 microM). Zinc ion binding (but not Ni(2+) binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn(2+)-binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site-directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism.

Antigenic Proteins of Helicobacter pylori of Potential Diagnostic Value

“…In the biosynthesis of the active metal-bound form of the nickel-dependent enzyme urease, a lysinecarbamate functional group is formed alongside with the delivery of two Ni (2+) ions into the precast active site of the apoenzyme. UreG functions as a chaperone in the urease active site assembly and is often required to complete the biosynthesis of nickel-enzymes (Zambelli et al, 2009). Pyrroline-5-carboxylate dehydrogenase plays a key role in the metabolic pathway, as it catalyzes the oxidation of proline to glutamate in the presence of NAD (Inagaki et al, 2006;Parsons et al, 2011).…”

Effects of Metal on the Biochemical Properties of Helicobacter pylori HypB, a Maturation Factor of [NiFe]-Hydrogenase and Urease

“…For example, metal-dependent dimerization was observed for CooC (28,29), the ATPase involved in carbon monoxide dehydrogenase biosynthesis, but ATP hydrolysis was not dramatically affected by the addition of nickel. Furthermore, the GTPase dedicated to urease maturation, UreG, binds nickel or zinc with micromolar affinities at a site that likely includes the CxH motif found at a similar location with respect to the GTPase motifs as C106/H107 of HpHypB (5,70,71). However, the relative affinities reported for nickel versus zinc, and whether the metals affect quaternary structure, vary depending on the UreG homolog.…”

Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein

“…Two other urease accessory proteins, UreG and UreE, are known to bind various metal ions. For example, UreG from Bacillus pasteurii binds 2 Zn 2ϩ or 4 Ni 2ϩ ions per dimer, and that from H. pylori binds 0.5 Zn 2ϩ or 2 Ni 2ϩ ions per monomer (34,35). Similarly, UreE proteins from K. aerogenes, B. pasteurii, and H. pylori are known to bind both Ni 2ϩ and Zn 2ϩ (1,2,33).…”