期刊
BIOCHEMICAL JOURNAL
卷 422, 期 -, 页码 91-100出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20090434
关键词
accessory protein UreE; accessory protein UreG; Helicobacter pylori urease assembly; nickel trafficking; protein-protein complex formation; zinc binding
资金
- MIUR-PRIN
- CIRB-UniBO
- Consorzio Interuniversitario di Risonanze Magnetiche di Metalloproteine Paramagnetiche
The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni2+ for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni2+ insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the Urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni2+ (K-d = 0.15 mu M) or Zn2+ (K-d = 0.49 mu M) per dinner, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn2+ and Ni2+, which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn2+, and not for Ni2+, in the stabilization of this complex was demonstrated using size-exclusion chromatography. light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of it novel binding site for Zn2+, actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent Urease system is envisaged.
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