Conserved residues and motifs in the NixA protein of Helicobacter pylori are critical for the high affinity transport of nickel ions

NixA, the high affinity nickel transport protein of Helicobacter pylori, imports Ni2+ ions across the cytoplasmic membrane for insertion into the active site of the urease metalloenzyme, which is essential for colonization of the gastric mucosa, Twelve conserved aspartate (aspartates 47, 49, 55, 194, 231, and 234), glutamate (glutamates 106, 198, and 274), and histidine (histidines 44, 50, and 79) residues were identified by alignment of NixA with homologous transporters, Polymerase chain reaction-generated site-directed mutants of these residues mere expressed in E. coli along with the H. pylori urease gene cluster. Mutations in residues within the predicted periplasmic domains of NixA maintained near wild type levels of Ni2+ uptake and urease activity, as did control mutations of conserved positively charged residues (lysines 140 and 268; arginines 162 and 167). Mutations in highly conserved motifs in predicted helices II and III of NixA abolished Ni2+ uptake and urease activity, Mutations in helices V and VI and the cytoplasmic domains decreased Ni2+ transport rates by greater than or equal to 90%. Reduction in rates of Ni2+ transport correlated with reduction in urease activities (r = 0.77), Ni2+ transport was inhibited in the presence of Co2+, Cu2+ and Zn2+ indicating that these ions may also be bound or trans ported by NixA. We conclude that conserved Asp, Glu, and His residues in the transmembrane domains of NixA are critical for the transport of the divalent cations Ni2+, Co2+, Cu2+, and Zn2+ into the cytoplasm of H. pylori.