Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of
            <i>Helicobacter pylori</i>

Wolfram, Lutz; Bauerfeind, Peter

doi:10.1128/jb.184.5.1438-1443.2002

Cited by 33 publications

(39 citation statements)

References 37 publications

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“…As in the case of HoxN, a hydrophobic Ile residue is not tolerated at this position. Strong reduction of transport activity has also been observed for a similar variant of NixA of H. pylori (32). The H34N exchange had a slight inhibitory effect on Co 2ϩ uptake but caused significant inhibition of Ni 2ϩ uptake.…”

Section: Fig 7 Relative Amounts Of Hoxn and Nhlf Variants In Mem-mentioning

confidence: 63%

“…The roles of other conserved features, for instance, the conserved signature in TMD III, the essential hydrophilic loop connecting TMDs IV and V, and the pairs of acidic residues in TMDs V and VI (reviewed in reference 9) for NiCoT activity need closer investigation. A very recent report on H. pylori NixA mutants assigned an essential role in nickel transport to the FX 2 GH sequence conserved in TMD III of NiCoTs (32).…”

Section: Fig 7 Relative Amounts Of Hoxn and Nhlf Variants In Mem-mentioning

confidence: 99%

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Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II

Degen

Eitinger

2002

J Bacteriol

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HoxN, a high-affinity, nickel-specific permease of Ralstonia eutropha H16, and NhlF, a nickel/cobalt permease of Rhodococcus rhodochrous J1, are structurally related members of the nickel/cobalt transporter (NiCoT) family. These transporters have an eight-helix structure and are characterized by highly conserved segments with polar or charged amino acid residues in transmembrane domains (TMDs) II, III, V, and VI. . NhlF activity dropped in response to the converse mutation. Our data predict that TMDs I and II in NiCoTs spatially interact to form a critical part of the selectivity filter. As seen for the V64F variant of HoxN, modification of this site can increase the velocity of transport and concomitantly reduce the specificity.In the functional phylogenetic transporter classification system developed by Saier (26,27), the family with the code TC 2.A.52 is described as the nickel/cobalt transporter (NiCoT) family. The family comprises structurally related membrane proteins in gram-negative and gram-positive bacteria, in archaea, and in fungi. An eight-helix structure, conserved signatures containing charged residues in transmembrane domains (TMDs), and a large hydrophilic loop connecting TMDs IV and V are common features of the members of this family (reviewed in references 5, 8, and 9). High-affinity Ni 2ϩ uptake to provide the metal ion for incorporation into Ni-containing metalloenzymes (see reference 14 for a review) is the physiological role of the NiCoTs of Ralstonia eutropha (HoxN), Helicobacter pylori (NixA), and the fission yeast Schizosaccharomyces pombe. Despite the family name, HoxN acts as a selective Ni 2ϩ permease and does not transport Co 2ϩ (4). NixA activity in the human pathogen H. pylori is essential to virulence, since nickel-containing urease is a central pathogenicity determinant in this species. Site-directed mutagenesis has been employed to localize residues and motifs in HoxN (10) and NixA (12) that cannot be altered without a dramatic or complete loss of activity. These studies uncovered the relevance of a strongly conserved segment (GLR/KHAV/ FDADHI/LAAI) in TMD II that can be considered a signature sequence for NiCoTs. NhlF, the NiCoT of the gram-positive Rhodococcus rhodochrous J1, provides Co 2ϩ ion for incorporation into Co 2ϩ -containing nitrile hydratases (17), industrial catalysts that contain a non-corrin cobalt metallocenter (reviewed in reference 16). In contrast to HoxN, NhlF is literally a NiCoT and transports Ni 2ϩ and Co 2ϩ with high affinity (4). Experimental analysis of NiCoT activity is difficult. These permeases transport their substrates with very high affinity but extremely low capacity. For reproducible measurement of HoxN activity, hoxN has been expressed in Escherichia coli and metal uptake has been analyzed during growth in complex medium (33). At a 63 Ni 2ϩ concentration of 500 nM in the growth medium, the resulting cellular 63 Ni content was in the range of 25 to 50 pmol per mg of protein, corresponding to approximately 2,500 to 5,000 Ni 2ϩ ions per cell. T...

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Section: Fig 7 Relative Amounts Of Hoxn and Nhlf Variants In Mem-mentioning

confidence: 63%

Section: Fig 7 Relative Amounts Of Hoxn and Nhlf Variants In Mem-mentioning

confidence: 99%

Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II

Degen

Eitinger

2002

J Bacteriol

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“…Conserved Asp, Glu and His residues are located in the transmembrane domains and appear to be critical for nickel transport (Fulkerson et al 1998); in addition, two essential domains have been characterized within helix II (GXXHAXDADH) and helix III (GX2FXXGHSSVV) of NixA (Fulkerson and Mobley 2000). Furthermore, other amino acids, including some with low in vitro affinity to nickel, have been shown to be also important for the function of NixA (Wolfram and Bauerfeind 2002). NixA has a high affinity for nickel, with an estimated Kd of 11.3 nM (Mobley et al 1995), thus enabling H. pylori to efficiently scavenge the low concentrations of nickel found in the human body, which are estimated to be in the range of 2-11 nM (Sundermann 1993).…”

Section: Nixa the Nickel-specific Permeasementioning

confidence: 99%

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

2007

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Helicobacter pylori colonizes the human gastric mucosa and this can lead to chronic gastritis, peptic and duodenal ulcers, and even gastric cancers. The bacterium colonizes over one-half of the worlds population. Nickel plays a major role in the bacteriums colonization and persistence attributes as two nickel enzyme sinks obligately contain the metal. Urease accounts for up to 10% of the total cellular protein made and is required for initial colonization processes, and the hydrogen oxidizing hydrogenase provides the bacterium a high-energy substrate yielding low potential electrons for energy generation. A battery of accessory proteins are needed for maturation or activation of each of the apoen-zymes. These include Ni-chaperones and GTPas-es, some of which are unique to each Ni-enzyme and others that are individually required for maturation of both the Ni-enzymes. H. pylori's need for some conventional hydrogenase maturation proteins playing roles in urease maturation may have to do with the poor nickel-sequestering ability of the UreE urease maturation protein compared to other systems. H. pylori also possesses a NixA nickel specific permease, a nickel dependent regulator (NikR), a recently identified nickel efflux system (CznABC), and a histidinerich heat shock protein, HspA. Based on mutant analysis approaches all these proteins have roles in nickel homeostasis, in urease expression, and in host colonization. The His-rich putative nickel storage proteins Hpn and Hpn-like play roles in nickel detoxification and may influence the levels of Ni-activated urease that can be achieved.

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“…Nickel incorporation in Escherichia coli hydrogenases also requires a GTP-binding accessory protein, HypB (11). The protein sequences of a number of microbial nickel uptake systems have been described (12)(13)(14), but to date no structural information has been obtained. Nickel uptake by bacteria is of interest for bioremediation and water purification as well as its biological function and involvement in pathogenesis.…”

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confidence: 99%

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

Heddle

Scott

Unzai

et al. 2003

Journal of Biological Chemistry

114

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Bacteria have evolved a number of tightly controlled import and export systems to maintain intracellular levels of the essential but potentially toxic metal nickel. Nickel homeostasis systems include the dedicated nickel uptake system nik found in Escherichia coli, a member of the ABC family of transporters, that involves a periplasmic nickel-binding protein, NikA. This is the initial nickel receptor and mediator of the chemotactic response away from nickel. We have solved the crystal structure of NikA protein in the presence and absence of nickel, showing that it behaves as a "classical" periplasmic binding protein. In contrast to other binding proteins, however, the ligand remains accessible to the solvent and is not completely enclosed. No direct bonds are formed between the metal cation and the protein. The nickel binding site is apolar, quite unlike any previously characterized protein nickel binding site. Despite relatively weak binding, NikA is specific for nickel. Using isothermal titration calorimetry, the dissociation constant for nickel was found to be ϳ10 M and that for cobalt was approximately 20 times higher.

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Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of Helicobacter pylori

Cited by 33 publications

References 37 publications

Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II

Substrate Specificity of Nickel/Cobalt Permeases: Insights from Mutants Altered in Transmembrane Domains I and II

Nickel-binding and accessory proteins facilitating Ni-enzyme maturation in Helicobacter pylori

Crystal Structures of the Liganded and Unliganded Nickel-binding Protein NikA from Escherichia coli

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