Structure of Cu/Zn superoxide dismutase from the heavy-metal-tolerant yeast Cryptococcus liquefaciens strain N6

The deep-sea yeast Cryptococcus liquefaciens strain N6 shows high tolerance towards heavy metals, and can grow in the presence of high concentrations of copper ions. Enzymatic analysis indicated that copper ions induced the Cu/Zn superoxide dismutase activity of strain N6 (Cl-SOD1). In this study, the 1.2 Å resolution crystal structure of Cl-SOD1 has revealed several significant residue substitutions compared to the other Cu/Zn SODs. In the electrostatic loop, notably, His135 and Pro136 replace the well-conserved linear residues, while Thr133 substitutes a highly conserved glycine. The electrostatic loop has been shown to be involved in the copper uptake process, and these substitutions have caused an inward dragging of the turn region of the loop. As the introduction of proline and abolishment of glycine decrease loop flexibility, this structural reorganization may have helped stabilize the loop conformation, possibly resulting in more efficient copper uptake and a more stabilized copper-bound form.     

Molecular cloning, phylogenetic analysis and three-dimensional modeling of Cu,Zn superoxide dismutase (CnSOD1) from three varieties of Cryptococcus neoformans

Cryptococcus neoformans (Cn), causal agent of fungal meningoencephalitis, has three varieties with variable host predilection. To explore mechanisms for these pathogenic differences, we have characterized Cu,Zn SOD gene (CnSOD1). A Saccharomyces cerevisiae sod1Delta mutant was complemented with Cn var. grubii yeast expression library. The complementing clone had an ORF of 462 bp and the deduced 154 aa sequence showed 61% identity with S. cerevisiae SOD1 and 53-65% with other eukaryotic SOD1s. Cn var. grubii CnSOD1 cDNA was used to clone corresponding cDNAs from var. neoformans and var. gattii. ORFs from three varieties revealed 20-29% differences in deduced aa (s) with a significant 6% non-synonymous aa substitution between Cn var. grubii and Cn var. gattii. Cosmid library screening and PCR cloning were used to obtain genomic SOD1, which was split by five introns with identical placements and a typical 5' splice junction sequence, GTNNGY. These introns also showed a large nt variation among the three Cn varieties. Phylogenetic analyses revealed CnSOD1 to be in a group distinct from other eukaryotic SOD1s and with a significant divergence of the var. grubii from var. gattii. The CnSOD1 -deduced protein was modeled based on the crystal structure of S. cerevisiae SOD1, which showed an excellent fit. Most of the non-synonymous aa substitutions occurred on the outside of the molecule and these may contribute to differences in antigenicity among the three varieties. Notably, Cn var. neoformans and var. gattii Cu,Zn SOD had three substitutions of glycine (Gly26, Gly92 and Gly123 for Asn26, Ser92 and Ser123) that may contribute to the observed lower thermostability of this enzyme vis-a-vis Cn var. grubii. This is the first nucleotide and structural comparison of a protein-encoding gene from the three Cn varieties, which may provide a framework for future studies on the role of Cu,Zn SOD in Cn pathogenesis.     

A pivotal role of Zn-binding residues in the function of the copper chaperone for SOD1

A Cu chaperone for SOD1 (CCS) is required for the incorporation of copper ion into the protein. To investigate the roles of the conserved metal-binding residues in CCS, we introduced amino acid substitutions into human CCS and examined the function of the mutant CCS by transforming a mutant yeast strain, SY2950, which lacks the lys7 gene, a yeast orthologue of the mammalian CCS. Mutant CCS in which amino acid residues His147 and Asp167 were substituted by Ala exhibited a decreased ability to complement the growth of SY2950 under Lys-deficient conditions. This is because the mutations made the human CCS function in a less efficient manner, especially under metal-restricted conditions, leaving Cu,Zn-SOD in an apo-form. Since the His and Asp residues are both responsible for binding Zn which would serve to maintain the folded structure, the structural integrity supported by the coordinated Zn ion would be essential for CCS function.     

The Schizosaccharomyces pombe Pccs protein functions in both copper trafficking and metal detoxification pathways

Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain.     

Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat

Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells. Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells. During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats. The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase). The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids. The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III. The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD.     

Role of glutathione in detoxification of metal(loid)s by Saccharomyces cerevisiae

Cellular glutathione (GSH) was implicated in tolerance to potentially toxic metal(loid)s using two strains of Saccharomyces cerevisiae, a wild-type (sigma 1278b) and a GSH-deficient mutant strain (gshA-2). Both yeast strains exhibited no significant difference in tolerance to tellurite, zinc, cobalt, copper, manganese, nickel and chromate. There was no marked influence of glutathione on the accumulation of Te, Co, Cu, and Mn, although the absence of cellular glutathione significantly increased the cellular content of Zn and Ni, but greatly decreased Cr content without significant alteration of tolerance. These results indicated the independence of cellular glutathione activity from tolerance to Te, Zn, Co, Cu, Mn, Ni, and Cr. However, involvement of glutathione in Zn, Ni and Cr uptake is possible. The glutathione-deficient strain displayed a high sensitivity to selenite and cadmium in comparison to the wild-type strain of S. cerevisiae. The minimum inhibitory concentrations of Se and Cd for the glutathione-deficient strain were 980 +/- 13 and 32 +/- 4 microM, respectively, whereas the wild strain tolerated up to 4080 +/- 198 microM Se and 148 +/- 5 microM Cd. A relationship between tolerance and reduced cellular content of both Se and Cd was also shown: the mutant strain accumulated approximately three-fold more Se and two-fold more Cd than that accumulated by the wild-type strain. This suggests an influence of GSH on cellular uptake of Se and Cd, and also directly confirms the protective action of such a cellular thiol compound against Se and Cd toxicity.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Yeast metallothionein. Sequence and metal-binding properties

The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein.     

Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS

The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood. Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor. Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme. While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer. This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy. Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein. Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase.     

Insights into the Role of the Unusual Disulfide Bond in Copper-Zinc Superoxide Dismutase

The functional and structural significance of the intrasubunit disulfide bond in copper-zinc superoxide dismutase (SOD1) was studied by characterizing mutant forms of human SOD1 (hSOD) and yeast SOD1 lacking the disulfide bond. We determined x-ray crystal structures of metal-bound and metal-deficient hC57S SOD1. C57S hSOD1 isolated from yeast contained four zinc ions per protein dimer and was structurally very similar to wild type. The addition of copper to this four-zinc protein gave properly reconstituted 2Cu,2Zn C57S hSOD, and its spectroscopic properties indicated that the coordination geometry of the copper was remarkably similar to that of holo wild type hSOD1. In contrast, the addition of copper and zinc ions to apo C57S human SOD1 failed to give proper reconstitution. Using pulse radiolysis, we determined SOD activities of yeast and human SOD1s lacking disulfide bonds and found that they were enzymatically active at ∼10% of the wild type rate. These results are contrary to earlier reports that the intrasubunit disulfide bonds in SOD1 are essential for SOD activity. Kinetic studies revealed further that the yeast mutant SOD1 had less ionic attraction for superoxide, possibly explaining the lower rates. Saccharomyces cerevisiae cells lacking the sod1 gene do not grow aerobically in the absence of lysine, but expression of C57S SOD1 increased growth to 30–50% of the growth of cells expressing wild type SOD1, supporting that C57S SOD1 retained a significant amount of activity.     

乳酸克鲁维酵母Cu/Zn-SOD基因的克隆及在酿酒酵母中的表达研究

根据Genbank中乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因序列设计引物,通过PCR扩增得到Cu/Zn-SOD基因。在PGK1启动子驱动下,将该基因与荧光报告基因GFP融合,分别构建重组质粒YEplac195-PSGA和YCplac33-PSGA,并转化酿酒酵母(Saccharomyces cerevisiae)W303α菌株。通过菌落PCR和荧光显微观察证实乳酸克鲁维酵母(Kluyveromyces lactis)的Cu/Zn-SOD基因在W303α中成功表达。将获得的阳性转化子在添加20mmol/L百草枯的发酵培养基中进行发酵,SOD的比活力和总活力分别是不添加百草枯培养基中发酵菌体的6.7倍和4.7倍。通过热激胁迫处理进一步探讨Cu/Zn-SOD对宿主sod1Δ酿酒酵母菌株EG118耐受力的影响,结果显示抗热击能力的顺序为EG118(YEplac195-PSGA)EG118(YCplac33-PSGA)EG118。以上结果为发酵工业中防止菌体老化和增强菌体的发酵能力提供一定的理论指导,也为后续的Cu/Zn-SOD体外分子定向进化改造奠定必要的基础。