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

Article (PDF Available)inActa Crystallographica Section D Biological Crystallography 63(Pt 2):221-9 · March 2007with20 Reads
DOI: 10.1107/S0907444906048712 · Source: PubMed
Abstract
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.
Time evolutions of F
1
dihedral angle of Tyr127 in the MD simulations of unliganded
(upper panel) and liganded NikA (lower panel).
    • "6a and b, respectively). If the binding of EDTA to Ni 2+ ions is significantly tighter than nickel binding to Nd, then the nickel–EDTA complex binding constant can be measured (Addy et al., 2007). We found that EDTA binds nickel in the Nd solution extremely tightly and fitting of the data gave a dissociation constant of around 200 nM (Table 5). "
    [Show abstract] [Hide abstract] ABSTRACT: The crystal structure of the N-terminal domain of the RNA polymerase δ subunit (Nδ) from Bacillus subtilis solved at a resolution of 2.0 Å is compared with the NMR structure determined previously. The molecule crystallizes in the space group C222(1) with a dimer in the asymmetric unit. Importantly, the X-ray structure exhibits significant differences from the lowest energy NMR structure. In addition to the overall structure differences, structurally important β sheets found in the NMR structure are not present in the crystal structure. We systematically investigated the cause of the discrepancies between the NMR and X-ray structures of Nδ, addressing the pH dependence, presence of metal ions, and crystal packing forces. We convincingly showed that the crystal packing forces, together with the presence of Ni(2+) ions, are the main reason for such a difference. In summary, the study illustrates that the two structural approaches may give unequal results, which need to be interpreted with care to obtain reliable structural information in terms of biological relevance.
    Full-text · Article · Jun 2014
    • "This shows that none of the key residues known from the crystal structure of NikA to be involved in binding the Ni–metallophore (BTC) is conserved in Cj1584, including His416, which forms the only direct protein–metal bond to the Ni in the metallophore itself (Cavazza et al., 2011). His56 and His442, which bind nickel ions at a site remote from the metallophore binding cleft in NikA (Addy et al., 2007), are also not conserved (Fig. S1a). Given the obvious differences from NikA in both binding properties and lack of conservation of the key residues, we have designated Cj1584 as NikZ to distinguish it from NikA. "
    [Show abstract] [Hide abstract] 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.
    Article · Mar 2012
    • "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. "
    [Show abstract] [Hide abstract] 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
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