Nickel binding properties of Helicobacter pylori UreF an accessory protein in the nickel-based activation of urease

摘要

Helicobacter pylori UreF is involved in the insertion of Ni²⁺ in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive α-helical structure and a well-folded tertiary structure. HpUreF binds two Ni²⁺ ions per dimer, with micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopic analysis indicates that the Ni²⁺ ions reside in a five-coordinate pyramidal geometry comprised exclusively of N/O-donor ligands derived from the protein, including one or two histidine imidazoles and carboxylate ligands. Binding of Ni²⁺ does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interacts with HpUreD, altered the affinity of the protein for Ni²⁺. This result, complemented by the XAS analysis, indicates that the Ni²⁺ binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A-HpUreF, C231A-HpUreF and H229/C231-HpUreF are significantly less competent in this process, suggesting a role for a Ni²⁺ complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apo-to-holo enzyme activation.