Nickel binding to NikA: An additional binding site reconciles spectroscopy, calorimetry and crystallography

Graduate School of Integrated Science, Yokohama City University, Tsurumi, Suehiro 1-7-29, Yokohama 230-0045, Japan.
Acta Crystallographica Section D Biological Crystallography (Impact Factor: 2.67). 03/2007; 63(Pt 2):221-9. DOI: 10.1107/S0907444906048712
Source: PubMed


Intracellular nickel is required by Escherichia coli as a cofactor for a number of enzymes and is necessary for anaerobic respiration. However, high concentrations of nickel are toxic, so both import and export systems have evolved to control the cellular level of the metal. The nik operon in E. coli encodes a nickel-uptake system that includes the periplasmic nickel-binding protein NikA. The crystal structures of wild-type NikA both bound to nickel and in the apo form have been solved previously. The liganded structure appeared to show an unusual interaction between the nickel and the protein in which no direct bonds are formed. The highly unusual nickel coordination suggested by the crystal structure contrasted strongly with earlier X-ray spectroscopic studies. The known nickel-binding site has been probed by extensive mutagenesis and isothermal titration calorimetry and it has been found that even large numbers of disruptive mutations appear to have little effect on the nickel affinity. The crystal structure of a binding-site mutant with nickel bound has been solved and it is found that nickel is bound to two histidine residues at a position distant from the previously characterized binding site. This novel site immediately resolves the conflict between the crystal structures and other biophysical analyses. The physiological relevance of the two binding sites is discussed.

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    • "The current consensus is that NikA binds different forms of nickel at two sites, one site that binds free nickel which is distinct from the usual binding cleft (Addy et al., 2007), and a second site within the cleft that binds chelated nickel (Addy et al., 2007; Cherrier et al., 2005). However, neither of the measured binding affinities (K d ) of ~11 mM for free nickel and ~30 mM for nickel–EDTA is consistent with high-affinity transport, although NikA binds iron– EDTA with very high affinity (Addy et al., 2007; Cherrier et al., 2005). More recent evidence suggests that compounds similar to butane-1,2,4-tricarboxylate (BTC) are possible nickel metallophores that provide a higher binding affinity and represent physiological ligands (Cherrier et al., 2008). "
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    ABSTRACT: Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.
    Preview · Article · Mar 2012 · Microbiology
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    • "This result also applied to the structure determined by Heddle and collaborators. Indeed, in 2007, these authors revised their NikA structure and agreed with our previous interpretation [16]. Therefore, all these studies suggested the existence of a EDTA-like natural chelator involved in nickel binding. "
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    ABSTRACT: Escherichia coli require nickel for the synthesis of [NiFe] hydrogenases under anaerobic growth conditions. Nickel import depends on the specific ABC-transporter NikABCDE encoded by the nik operon, which deletion causes the complete abolition of hydrogenase activity. We have previously postulated that the periplasmic binding protein NikA binds a natural metallophore containing three carboxylate functions that coordinate a Ni(II) ion, the fourth ligand being His416, the only direct metal-protein contact, completing a square-planar coordination for the metal. The crystal structure of the H416I mutant showed no electron density corresponding to a metal-chelator complex. In vivo experiments indicate that the mutation causes a significant decrease in nickel uptake and hydrogenase activity. These results confirm the essential role of His416 in nickel transport by NikA.
    Full-text · Article · Feb 2011 · FEBS letters
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    • "Cherrier et al. suggested that NikA binds Ni chelated by a small organic molecule, such as butane-1,2,4-tricarboxylate (BTC), and that some residues, including Tyr402, Arg137, Arg97 and His416, form a binding site that is involved in the BTC-Ni-NikA interaction [78]. On the other hand, Addy and coworkers showed that Ni may bind E. coli NikA without chelators and is bound to two histidine residues (His56 and His442, although not conserved in other NikA proteins) at a position distant from the previously characterized binding site [79]. Here, the presence of the majority of these residues was used to help predict NikA proteins. "
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    ABSTRACT: Nickel (Ni) and cobalt (Co) are trace elements required for a variety of biological processes. Ni is directly coordinated by proteins, whereas Co is mainly used as a component of vitamin B12. Although a number of Ni and Co-dependent enzymes have been characterized, systematic evolutionary analyses of utilization of these metals are limited. We carried out comparative genomic analyses to examine occurrence and evolutionary dynamics of the use of Ni and Co at the level of (i) transport systems, and (ii) metalloproteomes. Our data show that both metals are widely used in bacteria and archaea. Cbi/NikMNQO is the most common prokaryotic Ni/Co transporter, while Ni-dependent urease and Ni-Fe hydrogenase, and B12-dependent methionine synthase (MetH), ribonucleotide reductase and methylmalonyl-CoA mutase are the most widespread metalloproteins for Ni and Co, respectively. Occurrence of other metalloenzymes showed a mosaic distribution and a new B12-dependent protein family was predicted. Deltaproteobacteria and Methanosarcina generally have larger Ni- and Co-dependent proteomes. On the other hand, utilization of these two metals is limited in eukaryotes, and very few of these organisms utilize both of them. The Ni-utilizing eukaryotes are mostly fungi (except saccharomycotina) and plants, whereas most B12-utilizing organisms are animals. The NiCoT transporter family is the most widespread eukaryotic Ni transporter, and eukaryotic urease and MetH are the most common Ni- and B12-dependent enzymes, respectively. Finally, investigation of environmental and other conditions and identity of organisms that show dependence on Ni or Co revealed that host-associated organisms (particularly obligate intracellular parasites and endosymbionts) have a tendency for loss of Ni/Co utilization. Our data provide information on the evolutionary dynamics of Ni and Co utilization and highlight widespread use of these metals in the three domains of life, yet only a limited number of user proteins.
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