Crystal structure of a truncated urease accessory protein UreF from Helicobacter pylori

Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.92). 10/2010; 78(13):2839-48. DOI: 10.1002/prot.22802
Source: PubMed

ABSTRACT Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG, which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C-terminal truncated UreF from H. pylori (residues 1-233), the first UreF structure to be determined, at 1.55 A resolution using SAD methods. UreF forms a dimer in vitro and adopts an all-helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.
    Journal of the American Society for Mass Spectrometry 06/2013; DOI:10.1007/s13361-013-0677-y · 3.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease.
    PLoS Biology 10/2013; 11(10):e1001678. DOI:10.1371/journal.pbio.1001678 · 11.77 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni(2+) 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 Ni(2+) ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni(2+) ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni(2+) 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 interact with H. pylori UreD, altered the affinity of the protein for Ni(2+). This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni(2+) 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 Ni(2+) 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 apoenzyme-to-holoenzyme activation.
    JBIC Journal of Biological Inorganic Chemistry 11/2013; DOI:10.1007/s00775-013-1068-3 · 3.16 Impact Factor

Full-text (2 Sources)

Available from
May 30, 2014