Structural and functional characterization of the NixA nickel II transporter Helicobacter pylori

Abstract

Synthesis of catalytically active urease is required for colonization by the human gastric pathogen Helicobacter pylori. NixA, the high affinity (KT = 11.3 nM) nickel transporter of H. pylori, imports Ni2+ across the membrane for insertion into the active site of the urease metalloenzyme. NixA fractionated with the cytoplasmic membrane and protein crosslinking indicated that NixA functions as a monomer. To determine the membrane topology of NixA, a series of 42 LacZ and PhoA reporter fusions were constructed, which are enzymatically active only when fused to cytoplasm- or periplasm-exposed determinants, respectively. Expression of reporter fusions in the bacterial membrane was confirmed by Western blotting with beta-galactosidase- and alkaline phosphatase-specific antisera. Analysis of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal domain including a membrane localization signal. All LacZ and PhoA fusions produced complementary activities and defined a topological model of NixA in which the amino- and carboxy-termini are located in the cytoplasm and the protein possesses eight transmembrane domains separated by four periplasmic and three cytoplasmic loops. Twelve conserved Asp, Glu, and His residues were identified by alignment of NixA with the homologous transporters HoxN, HupN, and UreH. Site-directed mutations in transmembrane domains (TMD) II and III of NixA abolished Ni2+ uptake and urease activity revealing two highly conserved, transport-dependent motifs: GX2HAXDADH and GX2FX2GHSSVV. Mutation of six additional conserved aspartates and glutamates reduced Ni2+ transport rates by ≥90% with correlating reductions in urease activities (r = 0.84). Four of these six additional Asp and Glu residues mutated were located in TMDs V and VI, with the two remaining transport-critical residues located near the cytoplasmic interface of TMD II and the periplasmic interface of TMD VII. 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. Immunoblotting with NixA-specific antibodies confirmed the expression of full length mutated proteins and confirmed their presence in the bacterial membrane.