The Role of zinc in the disulphide stress-regulated anti-sigma factor RsrA from Streptomyces coelicolor

The regulation of disulphide stress in actinomycetes such as Streptomyces coelicolor is known to involve the zinc-containing anti-sigma factor RsrA that binds and inactivates the redox-regulated sigma factor sigmaR. However, it is not known how RsrA senses disulphide stress nor what role the metal ion plays. Using in vitro assays, we show that while zinc is not required for sigmaR binding it is required for functional anti-sigma factor activity, and that it plays a critical role in modulating the reactivity of RsrA cysteine thiol groups towards oxidation. Apo-RsrA is easily oxidised and, while the Zn-bound form is relatively resistant, the metal ion is readily expelled when the protein is treated with strong oxidants such as diamide. We also show, using a combination of proteolysis and mass spectrometry, that the first critical disulphide to form in RsrA involves Cys11 and one of either Cys41 or Cys44, all previously implicated in metal binding. Circular dichroism spectroscopy was used to follow structural changes during oxidation of RsrA, which indicated that concomitant with formation of this critical disulphide bond is a major restructuring of the protein where its alpha-helical content increases. Our data demonstrate that RsrA can only bind sigmaR in the reduced state and that this state is stabilised by zinc. Redox stress induces disulphide bond formation amongst zinc-ligating residues, expelling the metal ion and stabilising a structure incapable of binding the sigma factor.     

Determinants of redox sensitivity in RsrA, a zinc-containing anti-sigma factor for regulating thiol oxidative stress response

Various environmental oxidative stresses are sensed by redox-sensitive regulators through cysteine thiol oxidation or modification. A few zinc-containing anti-sigma (ZAS) factors in actinomycetes have been reported to respond sensitively to thiol oxidation, among which RsrA from Streptomyces coelicolor is best characterized. It forms disulfide bonds upon oxidation and releases bound SigR to activate thiol oxidative stress response genes. Even though numerous ZAS proteins exist in bacteria, features that confer redox sensitivity to a subset of these have been uncharacterized. In this study, we identified seven additional redox-sensitive ZAS factors from actinomycetes. Comparison with redox-insensitive ZAS revealed characteristic sequence patterns. Domain swapping demonstrated the significance of the region K(33)FEHH(37)FEEC(41)SPC(44)LEK(47) that encompass the conserved HX(3)CX(2)C (HCC) motif. Mutational effect of each residue on diamide responsive induction of SigR target genes in vivo demonstrated that several residues, especially those that flank two cysteines (E39, E40, L45, E46), contribute to redox sensitivity. These residues are well conserved among redox-sensitive ZAS factors, and hence are proposed as redox-determinants in sensitive ZAS. H37A, C41A, C44A and F38A mutations, in contrast, compromised SigR-binding activity significantly, apparently affecting structural integrity of RsrA. The residue pattern around HCC motif could therefore serve as an indicator to predict redox-sensitive ZAS factors from sequence information.     

The zinc-dependent redox switch domain of the chaperone Hsp33 has a novel fold

The Escherichia coli chaperone Hsp33 contains a C-terminal zinc-binding domain that modulates activity by a so-called "redox switch". The oxidized form in the absence of zinc is active, while the reduced form in the presence of zinc is inactive. X-ray crystal structures of Hsp33 invariably omit details of the C-terminal domain, which is truncated in protein constructs that are capable of forming crystals. We report the solution structure of a recombinant 61-residue protein containing the zinc-binding domain (residues 227-287) of Hsp33, in the presence of stoichiometric amounts of Zn2+. The zinc-bound protein is well folded, and forms a novel structure unlike other published zinc-binding domains. The structure consists of two helices at right-angles to each other, a two-stranded B-hairpin and a third helix at the C terminus. The zinc site comprises the side-chains of the conserved cysteine residues 232, 234, 262 and 265, and connects a short sequence before the first helix with the tight turn in the middle of the B-hairpin. The structure of the C-terminal zinc-binding domain suggests a mechanism for the operation of the redox switch: loss of the bound zinc ion disrupts the folded structure, allowing the ligand cysteine residues to be oxidized, probably to disulfide bonds. The observation that the C-terminal domain is poorly structured in the active oxidized form suggests that the loss of zinc and unfolding of the domain precedes the oxidation of the thiolate groups of the cysteine residues, since the formation of disulfides between distant parts of the domain sequence would presumably promote the formation of stable three-dimensional structure in the oxidized form.Hsp33 provides an example of a redox signaling system that utilizes protein folding and unfolding together with chemical modification for transduction of external stimuli, in this case oxidative stress, to activate the machinery of the cell that is designed to deal with that stress.     

Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase

The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-proline-rich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zinc-binding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase.     

Escherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zinc-binding domains

Escherichia coli DNA topoisomerase I catalyzes interconversions of different DNA topological isomers by the breakage and rejoining of DNA phosphodiester bonds. It has a crucial role in maintaining an optimal DNA superhelicity in E. coli. It is a single polypeptide of 864 amino acids. Analysis of the amino acid sequence reveals three tandem repeat units each containing two pairs of cysteines suggesting that each unit may form a zinc-binding domain. We have determined that each enzyme molecule contains three to four zinc atoms using inductively coupled plasma-atomic emission analysis. Modification of the cysteine residues and removal of the zinc from the enzyme result in loss of activity. Zinc ions are needed for full recovery of enzyme activity when the cysteine modification is reversed. Comparison with the zinc-binding domains of the sequence-specific DNA-binding proteins shows significant differences.     

The Presence of Zinc-Binding Proteins in Brain

Abstract: Zinc is one of the most abundant divalent metal ions in the brain, its concentration being greater than those of copper and manganese. Since free zinc ion is a potent inhibitor of sulfhydryl enzymes, we postulated that zinc in the brain most probably exists bound to macromolecules. As zinc-binding proteins in brain have not been characterized, we attempted to discover the occurrence and properties of these proteins. By using Sephadex G-75 column chromatography calibrated with proteins of known molecular weights, and by other techniques, we detected separate zinc-binding proteins, with apparent estimated molecular weights ranging from 15,000 to 210,000. Unlike the hepatic or renal zinc thioneins, the zinc-binding proteins in brain are not inducible following administration of zinc. Our interpretation of the results is that the major portion of the existing zinc in the brain is bound, and does not exist in free form.     

The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver.

delta-Aminolevulinic acid dehydratase (5-aminolevulinic acid hydro-lyase (adding 5-aminolevulinic acid and cyclizing), EC 4.2.1.24 purified from bovine liver in the presence of both SH-reducing reagent and zinc during the purification contained one zinc atom and eight SH groups/subunit. This preparation showed the full enzymatic activity even in the absence of thiol activator. It was found that two cysteine residues, one zinc atom and two histidine residues were involved in the active site. The enzyme was fullly active as long as two SH groups in the active site remained in the reduced form even in the absence of zinc. However, the enzymatic activity was completely lost, with a concomitant loss of bound zinc, upon oxidation of the SH groups to a disulfide bond, modification of SH groups with chemical reagents, or mercaptide formation by heavy metals. Thus, it is apparent that the activity depends on the essential SH groups. The zinc is not absolutely essential for the activity but may be required to prevent the essential SH groups from autooxidation by coordination. Binding experiments indicated that there was one binding site of zinc/subunit. Photooxidation of histidine residues diminished both enzymatic activity and bound zinc, suggesting that the histidine residues not only constituted the active site but also served as a possible ligand to zinc.     

Human papillomavirus type 18 E7 protein requires intact Cys-X-X-Cys motifs for zinc binding, dimerization, and transformation but not for Rb binding.

Human papillomavirus type 18 (HPV-18) E7 proteins bind zinc through Cys-X-X-Cys repeats located at the C terminus of the protein. In order to examine the role of these cysteine motifs in E7 function, we expressed the HPV-18 E7 protein in bacteria and found that purified E7 forms a dimer through interactions with zinc. Mutants with single mutations within the Cys-X-X-Cys motifs bound a reduced level of zinc in a zinc blot assay, while a double mutant lost all zinc-binding activity. When expressed in vivo, none of the mutants cooperated with an activated ras oncogene to transform primary rat embryo fibroblasts, but all mutants retained nearly wild-type Rb-binding activity. The results indicate that the cysteine motifs play an important role in transformation by HPV-18 E7 but do not contribute to Rb binding.     

Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase.

Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His. This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging. In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition. We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity. Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate. The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme. The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry. The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.     

Significance of zinc in a regulatory protein, CCM1, which regulates the carbon-concentrating mechanism in Chlamydomonas reinhardtii

In conditions with the poor availability of inorganic carbon(CO₂ and HCO₃ ^–: Ci) for photosynthesis, aquatic photosyntheticorganisms induce active Ci uptake systems that allow accumulationof Ci within the cell, the so-called carbon-concentrating mechanism(CCM). In a unicellular green alga, Chlamydomonas reinhardtii,a regulatory factor CCM1 is indispensable for the regulationof the CCM by sensing CO₂ availability. CCM1 has two putativezinc-binding domains with several conserved cysteine and histidineresidues in its N-terminal region. To determine whether thedomains actually bind zinc atoms, the N-terminal parts of CCM1were expressed as glutathione S-transferase fusion proteinsand subjected to atomic absorption spectrometry. It was foundthat 1 mol of zinc is bound to 1 mol of amino acid regions 1–71and 72–101 of CCM1, respectively. In the case of the site-directedmutant proteins, H54Y, C77V and C80V, the zinc-binding abilitywas lost. Physiological analyses of the transgenic Chlamydomonascells harboring a mutated Ccm1 gene revealed that amino acidresidues such as C36, C41, H54, C77, C80, H90 and C93 were indispensablefor induction of the CCM in response to Ci-limiting stress conditions.Size exclusion chromatography followed by immunoblot analysesindicated that CCM1 is present as a protein complex of approximately290–580 kDa independent of Ci availability.     

Mechanistic studies on Pyrobaculum calidifontis porphobilinogen synthase (5-aminolevulinic acid dehydratase)

Porphobilinogen synthase (PBG synthase) gene from Pyrobaculum calidifontis was cloned and expressed in E. coli. The recombinant enzyme was purified as an octamer and was found by mass spectrometry to have a subunit M_r of 37676.59 (calculated, 37676.3). The enzyme showed high thermal stability and retained almost all of its activity after incubation at 70 °C for 16 h in the presence of β-mercaptoethanol (β-ME) and zinc chloride. However, in the absence of the latter the enzyme was inactivated after 16 h although it regained full activity in the presence of β-ME and zinc chloride. The protein contained 4 mol of tightly bound zinc per octamer. Further, 4 mol of low affinity zinc could be incorporated following incubation with exogenous zinc salts. The enzyme was inactivated by incubation with levulinic acid followed by treatment with sodium borohydride. Tryptic digest of the modified enzyme and mass spectrometric analysis showed that Lys²⁵⁷ was the site of modification, which has previously been shown to be the site for the binding of 5-aminolevulinic acid giving rise to the propionate-half of porphobilinogen. P. calidifontis PBG synthase was inactivated by 5-chlorolevulinic acid and the residue modified was shown to be the central cysteine (Cys¹²⁷) of the zinc-binding cysteine-triad, comprising Cys^{125, 127, 135}. The present results in conjunction with earlier findings on zinc containing PBG synthases, are discussed which advocate that the catalytic role of zinc in the activation of the 5-aminolevulinic acid molecule forming the acetate-half of PBG is possible.     

The oxidation of yeast alcohol dehydrogenase-1 by hydrogen peroxide in vitro

Yeast alcohol dehydrogenase (YADH) plays an important role in the conversion of alcohols to aldehydes or ketones. YADH-1 is a zinc-containing protein, and it accounts for the major part of ADH activity in growing baker's yeast. To gain insight into how oxidative modification of the enzyme affects its function, we exposed YADH-1 to hydrogen peroxide in vitro and assessed the oxidized protein by LC-MS/MS analysis of proteolytic cleavage products of the protein and by measurements of enzymatic activity, zinc release, and thiol/thiolate loss. The results illustrated that Cys43 and Cys153, which reside at the active site of the protein, could be selectively oxidized to cysteine sulfinic acid (Cys-SO2H) and cysteine sulfonic acid (Cys-SO3H). In addition, H2O2 induced the formation of three disulfide bonds: Cys43-Cys153 in the catalytic domain, Cys103-Cys111 in the noncatalytic zinc center, and Cys276-Cys277. Therefore, our results support the notion that the oxidation of cysteine residues in the zinc-binding domain of proteins can go beyond the formation of disulfide bond(s); the formation of Cys-SO2H and Cys-SO3H is also possible. Furthermore, most methionines could be oxidized to methionine sulfoxides. Quantitative measurement results revealed that, among all the cysteine residues, Cys43 was the most susceptible to H2O2 oxidation, and the major oxidation products of this cysteine were Cys-SO2H and Cys-SO3H. The oxidation of Cys43 might be responsible for the inactivation of the enzyme upon H2O2 treatment.     

Zinc-coordination of aspartic acid-76 in Sulfolobus ferredoxin is not required for thermal stability of the molecule

The highly thermostable 7Fe-ferredoxin from Sulfolobus sp. strain 7 has tightly bound zinc at the interface between the N-terminal extra domain and the C-terminal core. The zinc is tetrahedrally ligated by His-16, His-19, His-34, and Asp-76. Previous studies on truncated mutants have shown that the zinc and certain parts, i.e. not all, of the N-terminal extra stretch are responsible for the thermal stabilization of the molecule. To study the role of Asp-76, a series of mutants were constructed with Asp-76 replaced by Glu (D76E), Asn (D76N), or Ala (D76A). All the mutants, as well as wild type ferredoxin, bound 1 mol zinc/mol protein, and showed similar kinetics for 2-oxoacid:ferredoxin oxidoreductase. The stability of the protein was examined by thermal degradation of the clusters. In the absence of guanidium thiocyanate, the T(m), defined as the mid-point temperature of the thermal transition from the native to the denatured state, for every mutant was above 100 degrees C. The T(m) values in the presence of 1 M guanidium thiocyanate were determined to be 90.8, 90.2, 87.1, 84.4, and 72.9 degrees C for the natural, recombinant, D76N-, D76A-, and D76E-ferredoxins, respectively. These results indicate that the interaction between zinc and the carboxyl oxygen of Asp-76 has subtle effects on both the zinc-ligation and stability, although the native zinc center is liganded with high symmetry, suggesting that the three His residues are more important for zinc-binding.     

Redox switch of hsp33 has a novel zinc-binding motif

The chaperone activity of the heat shock protein Hsp33 is regulated by reversible disulfide bond formation. Oxidized Hsp33 is active, and reduced Hsp33 is inactive. We show that zinc binding is essential for the function of this redox switch. Our results reveal that Hps33 contains a new, high affinity (K(a) > 10(17) m(-)(1)), zinc-binding motif in the form Cys-X-Cys-X(27-32)-Cys-X-X-Cys. All four conserved cysteines within this motif act to coordinate a single zinc atom. Experiments where reduced wild type Hsp33 is reconstituted with cobalt or cadmium demonstrate that the metal-coordinating cysteines are present as highly reactive thiolate anions. This ionization may allow for the fast and successful activation of the chaperone function of Hsp33 upon incubation in oxidizing agents.