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+ (Kd =0.15 μM) or Zn2+ (Kd = 0.49 μM) 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 evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.
Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG / M.Bellucci;B.Zambelli;F.Musiani;P.Turano;S.Ciurli. - In: BIOCHEMICAL JOURNAL. - ISSN 1470-8728. - STAMPA. - 422:(2009), pp. 91-100. [10.1042/BJ20090434]
Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG
TURANO, PAOLA;
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. 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+ (Kd =0.15 μM) or Zn2+ (Kd = 0.49 μM) 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 evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn2+ in the Ni2+-dependent urease system is envisaged.I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.