Peter T. Chivers

The CXXC motif: imperatives for the formation of native disulfide bonds in the cell

The rapid formation of native disulfide bonds in cellular proteins is necessary for the efficient use of cellular resources. This process is catalyzed in vitro by protein disulfide isomerase (PDI), with the PDI1 gene being essential for the viability of Saccharomyces cerevisiae. PDI is a member of the thioredoxin (Trx) family of proteins, which have the active‐site motif CXXC. PDI contains two Trx domains as well as two domains unrelated to the Trx family. We find that the gene encoding Escherichia coli Trx is unable to complement PDI1 null mutants of S.cerevisiae. Yet, Trx can replace PDI if it is mutated to have a CXXC motif with a disulfide bond of high reduction potential and a thiol group of low pKa. Thus, an enzymic thiolate is both necessary and sufficient for the formation of native disulfide bonds in the cell.

Crystal structure of the nickel-responsive transcription factor NikR.

NikR is a metal-responsive transcription factor that controls nickel uptake in Escherichia coli by regulating expression of a nickel-specific ATP-binding cassette (ABC) transporter. We have determined the first two structures of NikR: the full-length apo repressor at a resolution of 2.3 Å and the nickel-bound C-terminal regulatory domain at a resolution of 1.4 Å. NikR is the only known metal-responsive member of the ribbon-helix-helix family of transcription factors, and its structure has a quaternary arrangement consisting of two dimeric DNA-binding domains separated by a tetrameric regulatory domain that binds nickel. The position of the C-terminal regulatory domain enforces a large spacing between the contacts that each NikR DNA-binding domain can make with the nik operator. The regulatory domain of NikR contains four nickel-binding sites at the tetramer interface, each exhibiting a novel square-planar coordination by three histidines and one cysteine side chain.

Nickel homeostasis in Escherichia coli – the rcnR‐rcnA efflux pathway and its linkage to NikR function

The nickel physiology of Escherichia coli is dominated by its Ni‐Fe hydrogenase isozymes, which are expressed under anaerobic growth conditions. Hydrogenase activity in E. coli requires the NikABCDE nickel transporter, which is transcriptionally repressed by NikR in the presence of excess nickel. Recently, a nickel and cobalt‐efflux protein, RcnA, was identified in E. coli. This study examines the effect of RcnA on nickel homeostasis in E. coli. Under nickel‐limiting conditions, deletion of rcnA increased NikR activity in vivo. Nickel and cobalt‐dependent regulation of rcnA expression required the newly identified transcriptional repressor RcnR (formerly YohL). Deletion of rcnR results in constitutive rcnA expression and a corresponding decrease in NikR activity. Purified RcnR binds directly to the rcnA promoter DNA fragment and this interaction is inhibited by nickel and cobalt. Nickel accumulation is affected differently among deletion strains with impaired nickel homeostasis. Surprisingly, in low nickel growth conditions rcnA expression is required for nickel import via NikABCDE. The data support a model with two distinct pools of nickel ions in E. coli. NikR bridges these two pools by controlling the levels of the hydrogenase‐associated pool based on the nickel levels in the second pool.

Ni(II) and Co(II) sensing by Escherichia coli RcnR

Escherichia coli RcnR and Mycobacterium tuberculosis CsoR are the founding members of a recently identified, large family of bacterial metal-responsive DNA-binding proteins. RcnR controls the expression of the metal efflux protein RcnA only in response to Ni(II) and Co(II) ions. Here, the interaction of Ni(II) and Co(II) with wild-type and mutant RcnR proteins is examined to understand how these metals function as allosteric effectors. Both metals bind to RcnR with nanomolar affinity and stabilize the protein to denaturation. X-ray absorption and electron paramagnetic resonance spectroscopies reveal six-coordinate high-spin sites for each metal that contains a thiolate ligand. Experimental data support a tripartite N-terminal coordination motif (NH2-Xaa-NH-His) that is common for both metals. However, the Ni(II)- and Co(II)-RcnR complexes are shown to differ in the remaining coordination environment. Each metal coordinates a conserved Cys ligand but with distinct M-S distances. Co(II)-thiolate coordination has not been observed previously in Ni(II)-/Co(II)-responsive metalloregulators. The ability of RcnR to recruit ligands from the N-terminal region of the protein distinguishes it from CsoR, which uses a lower coordination geometry to bind Cu(I). These studies facilitate comparisons between Ni(II)-RcnR and NikR, the other Ni(II)-responsive transcriptional regulator in E. coli, to provide a better understanding how different nickel levels are sensed in E. coli. The characterization of the Ni(II)- and Co(II)-binding sites in RcnR, in combination with bioinformatics analysis of all RcnR/CsoR family members, identified a four amino acid fingerprint that likely defines ligand-binding specificity, leading to an emerging picture of the similarities and differences between different classes of RcnR/CsoR proteins.

NikR Repressor: High-Affinity Nickel Binding to the C-Terminal Domain Regulates Binding to Operator DNA

E. coli NikR repressor binds operator DNA in a nickel-dependent fashion. The pM affinity of NikR for nickel is mediated by its C-terminal 86 residues. Nickel binding induced additional secondary structure, decreased the compactness, and increased the stability of NikR. Tetramer formation by the C-terminal domain and intact NikR did not require nickel. High-affinity nickel binding decreased the NikR concentration needed to half maximally protect operator DNA from undetectable levels to 30 nM. The intracellular concentration of NikR in E. coli is high enough that saturation of the high-affinity nickel sites should lead to substantial occupancy of the nik operator. Nickel binding to a set of low-affinity NikR sites resulted in an additional large increase in operator affinity and substantially increased the size of the NikR footprint on the operator.

Complex Transcriptional Control Links NikABCDE-Dependent Nickel Transport with Hydrogenase Expression in Escherichia coli

Escherichia coli requires nickel under anaerobic growth conditions for the synthesis of catalytically active NiFe hydrogenases. Transcription of the NikABCDE nickel transporter, which is required for NiFe hydrogenase synthesis, was previously shown to be upregulated by FNR (fumarate-nit rate regulator) in the absence of oxygen and repressed by the NikR repressor in the presence of high extracellular nickel levels. We present here a detailed analysis of nikABCDE transcriptional regulation and show that it closely correlates with hydrogenase expression levels. We identify a nitrate-dependent mechanism for nikABCDE repression that is linked to the NarLX two-component system. NikR is functional under all nickel conditions tested, but its activity is modulated by the total nickel concentration present as well as by one or more components of the hydrogenase assembly pathway. Unexpectedly, NikR function is independent of NikABCDE function, suggesting that NikABCDE is a hydrogenase-specific nickel transporter, consistent with its original identification as a hydrogenase (hyd) mutant. Further, the results suggest that the hydrogenase assembly pathway is sequestered within the cell. A second nickel import pathway in E. coli is implicated in NikR function.

Nickel coordination is regulated by the DNA-bound state of NikR

The uptake of nickel in Escherichia coli and other microorganisms is transcriptionally regulated by the NikR repressor or its homologs. Here we report the structure of the high-affinity nickel-binding site in NikR and show that it responds dramatically to DNA binding. X-ray absorption spectroscopy reveals that nickel in the holo-NikR protein is bound in a novel four-coordinate planar site consisting of two histidines, one additional O- or N-donor ligand and one S-donor ligand. Site-directed mutation of His87, His89, Cys95 or Glu97 in NikR to alanine eliminates high-affinity nickel binding and abolishes DNA binding but maintains stable protein folding. An unanticipated feature of the NikR structure is that the nickel coordination responds to DNA binding. A six-coordinate nickel site composed of O- or N-donor ligands, but lacking cysteine, forms when NikR binds to operator DNA. Because nickel binding and DNA binding are mediated by different domains within NikR, a communication link between the two domains is implicated, consistent with the finding that the nickel-binding site in a fragment corresponding to the C-terminal domain of NikR is structurally distinct from that found in holo-NikR.

Molecular Dynamics Simulation of the Escherichia coli NikR Protein: Equilibrium Conformational Fluctuations Reveal Interdomain Allosteric Communication Pathways

Escherichia coli NikR is a homotetrameric Ni(2+)- and DNA-binding protein that functions as a transcriptional repressor of the NikABCDE nickel permease. The protein is composed of two distinct domains. The N-terminal 50 amino acids of each chain forms part of the dimeric ribbon-helix-helix (RHH) domains, a well-studied DNA-binding fold. The 83-residue C-terminal nickel-binding domain forms an ACT (aspartokinase, chorismate mutase, and TyrA) fold and contains the tetrameric interface. In this study, we have utilized an equilibrium molecular dynamics simulation in order to explore the conformational dynamics of the NikR tetramer and determine important residue interactions within and between the RHH and ACT domains to gain insight into the effects of Ni(2+) on DNA-binding activity. The molecular simulation data were analyzed using two different correlation measures based on fluctuations in atomic position and noncovalent contacts together with a clustering algorithm to define groups of residues with similar correlation patterns for both types of correlation measure. Based on these analyses, we have defined a series of residue interrelationships that describe an allosteric communication pathway between the Ni(2+)- and DNA-binding sites, which are separated by 40 A. Several of the residues identified by our analyses have been previously shown experimentally to be important for NikR function. An additional subset of the identified residues structurally connects the experimentally implicated residues and may help coordinate the allosteric communication between the ACT and RHH domains.

The N-terminal Arm of the Helicobacter pylori Ni2+-dependent Transcription Factor NikR Is Required for Specific DNA Binding

The Ni2+-dependent transcription factor NikR is widespread among microbes. The two experimentally characterized NikR orthologs, from Helicobacter pylori and Escherichia coli, display vastly different regulatory capabilities in response to increased intracellular Ni2+. Here, we demonstrate that the nine-residue N-terminal arm present in H. pylori NikR plays a critical role in the expanded regulatory capabilities of this NikR family member. Specifically, the N-terminal arm is required to inhibit NikR binding to low affinity and nonspecific DNA sequences and is also linked to a cation requirement for NikR binding to the nixA promoter. Site-directed mutagenesis and arm-truncation variants of NikR indicate that two residues, Asp-7 and Asp-8, are linked to the cation requirement for binding. Pro-4 and Lys-6 are required for maximal DNA binding affinity of the full-length protein to both the nixA and ureA promoters. The N-terminal arm is highly variable among NikR family members, and these results suggest that it is an adaptable structural feature that can tune the regulatory capabilities of NikR to the nickel physiology of the microbe in which it is found.

Communication between the Zinc and Nickel Sites in Dimeric HypA: Metal Recognition and pH Sensing

Helicobacter pylori , a pathogen that colonizes the human stomach, requires the nickel-containing metalloenzymes urease and NiFe-hydrogenase to survive this low pH environment. The maturation of both enzymes depends on the metallochaperone, HypA. HypA contains two metal sites, an intrinsic zinc site and a low-affinity nickel binding site. X-ray absorption spectroscopy (XAS) shows that the structure of the intrinsic zinc site of HypA is dynamic and able to sense both nickel loading and pH changes. At pH 6.3, an internal pH that occurs during acid shock, the zinc site undergoes unprecedented ligand substitutions to convert from a Zn(Cys)(4) site to a Zn(His)(2)(Cys)(2) site. NMR spectroscopy shows that binding of Ni(II) to HypA results in paramagnetic broadening of resonances near the N-terminus. NOEs between the beta-CH(2) protons of Zn cysteinyl ligands are consistent with a strand-swapped HypA dimer. Addition of nickel causes resonances from the zinc binding motif and other regions to double, indicating more than one conformation can exist in solution. Although the structure of the high-spin, 5-6 coordinate Ni(II) site is relatively unaffected by pH, the nickel binding stoichiometry is decreased from one per monomer to one per dimer at pH = 6.3. Mutation of any cysteine residue in the zinc binding motif results in a zinc site structure similar to that found for holo-WT-HypA at low pH and is unperturbed by the addition of nickel. Mutation of the histidines that flank the CXXC motifs results in a zinc site structure that is similar to holo-WT-HypA at neutral pH (Zn(Cys)(4)) and is no longer responsive to nickel binding or pH changes. Using an in vitro urease activity assay, it is shown that the recombinant protein is sufficient for recovery of urease activity in cell lysate from a HypA deletion mutant, and that mutations in the zinc-binding motif result in a decrease in recovered urease activity. The results are interpreted in terms of a model wherein HypA controls the flow of nickel traffic in the cell in response to nickel availability and pH.