title: Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG.
abstract: The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) 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 Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), 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 Zn(2+), and not for Ni(2+), 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 Zn(2+), 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 Zn(2+) in the Ni(2+)-dependent urease system is envisaged.
authors.affiliation: Laboratory of Bioinorganic Chemistry, Department of Agro-Environmental Science and Technology, University of Bologna, Bologna, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Bellucci
authors.forename: Barbara
authors.initials: B
authors.lastname: Zambelli
authors.forename: Francesco
authors.initials: F
authors.lastname: Musiani
authors.forename: Paola
authors.initials: P
authors.lastname: Turano
authors.forename: Stefano
authors.initials: S
authors.lastname: Ciurli
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002151:Calorimetry; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D016480:Helicobacter pylori / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D028044:Phosphate-Binding Proteins; D011485:Protein Binding; D016415:Sequence Alignment; D040681:Structural Homology, Protein; D015032:Zinc / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D050505:Mutant Proteins; D028044:Phosphate-Binding Proteins; C090404:UreE protein, Bacteria; C082270:ureG protein, Bacteria; D009532:Nickel; D015032:Zinc