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. This study focuses on the metal-binding properties of UreE from Helicobacter pylori (HpUreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. Isothermal titration calorimetry (ITC) showed that HpUreE binds one equivalent of Ni2+ (Kd = 0.15 muM) or Zn2+ (Kd = 0.49 muM) per dimer, 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 a novel binding site for Zn2+, actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidences of a UreE-UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.

M.Bellucci, B. Zambelli, F. Musiani, P. Turano, S. Ciurli (2009). Helicobacter pylori UreE, a urease accessory protein: specific Ni2+ and Zn2+ binding properties and interaction with its cognate UreG. BIOCHEMICAL JOURNAL, 422, 91-100 [10.1042/BJ20090434].

Helicobacter pylori UreE, a urease accessory protein: specific Ni2+ and Zn2+ binding properties and interaction with its cognate UreG

BELLUCCI, MATTEO;ZAMBELLI, BARBARA;MUSIANI, FRANCESCO;CIURLI, STEFANO LUCIANO
2009

Abstract

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. This study focuses on the metal-binding properties of UreE from Helicobacter pylori (HpUreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. Isothermal titration calorimetry (ITC) showed that HpUreE binds one equivalent of Ni2+ (Kd = 0.15 muM) or Zn2+ (Kd = 0.49 muM) per dimer, 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 a novel binding site for Zn2+, actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidences of a UreE-UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.
2009
M.Bellucci, B. Zambelli, F. Musiani, P. Turano, S. Ciurli (2009). Helicobacter pylori UreE, a urease accessory protein: specific Ni2+ and Zn2+ binding properties and interaction with its cognate UreG. BIOCHEMICAL JOURNAL, 422, 91-100 [10.1042/BJ20090434].
M.Bellucci; B. Zambelli; F. Musiani; P. Turano; S. Ciurli
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/76940
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