Metal-Binding Properties of an Hpn-Like Histidine-Rich Protein

The Hpn-like protein (Hpnl), a histidine-and glutamine-rich protein, is critical for Helicobacter pylori colonization in human gastric muscosa. In this study, the thermodynamic properties of Ni-II, Cu-II, Co-II, and Zn-II toward Hpnl were studied by isothermal titration calorimetry (ITC). We found that Hpnl exhibits two independent binding sites for Ni-II as opposed to one site for Cu-II, Co-II, and Zn-II. Protease digestion and chemical denaturation analysis further revealed that Ni-II confers a higher stability upon Hpnl than other divalent metal ions. The potential Ni-II binding sites are localized in the His-rich domain of Hpnl as confirmed by mutagenesis in combination with modification of histidine residues of the protein. We also demonstrated that the single mutants (H29A and H31A) and tetrameric mutant (H29-32A) cut nearly half of the binding capacity of Hpnl towards nickel ions, whereas other histidine residues (His30, 32, 38, 39, 40, and 41) are nonessential for nickel coordination. Escherichia coli cells that harbored H29A, H31A, and H29-32A mutant genes exhibited less tolerance toward high concentrations of extracellular nickel ions than those with the wild-type gene. Our combined data indicated that the conserved histidine residues, His29 and His31 in the His-rich domain of Hpnl, are critical for nickel binding, and such a binding is important for Hpnl protein to fulfill its biological functions.