Characterization of the copper-thiolate cluster in yeast metallothionein and two truncated mutants

Cu-metallothionein was purified from Saccharomyces cerevisiae harboring plasmids containing mutated CUP1 metallothionein genes resulting in deletions at the carboxy-terminal end of the polypeptide. The truncated polypeptides are recovered as polypeptides of 35 and 48 residues in length. The Cu-S cluster in the wild-type metallothionein and the two truncates were characterized. The truncated proteins, designated T35 and T48, contain 4 and 2 fewer cysteinyl residues, respectively, compared to the 12 cysteines in wild-type metallothionein; yet the mutant molecules bind Cu(I) ions in a stoichiometry comparable to the wild-type protein, i.e. 7-8 mol eq. The Cu(I) ions bound to T48 are as tenaciously bound as those bound to the wild-type molecule. The electronic transitions in the ultraviolet are similar for Cu-T48 and the wild-type protein. Both mutants and wild-type Cu-protein exhibit luminescence. The corrected emission maxima occurs at 609 nm with a corrected excitation peak near 277 nm. The luminescence quantum yield and lifetime of fluorescence decay of Cu-T48 and wild-type Cu-metallothionein are similar. The absolute quantum yield of the wild-type Cu-protein luminescence is 0.0058 and has a 440-ns lifetime. The similar fluorescence rate constant in the two molecules suggests they possess a similar chromophore. The Cu-T35 protein is more labile than Cu-T48 or the wild-type protein in the association of Cu(I) ions and the air sensitivity of the electronic transitions and luminescence. Although T48 lacks 2 of the 12 cysteines in the wild-type protein, we are unable to detect any differences in the properties of the native metal clusters in the two molecules; T35 lacking 4 cysteinyl residues forms a Cu(I) cluster with properties significantly different from the wild-type molecule. Properties of the Cu-thiolate cluster were also studied in Cu(I)-reconstituted samples. The cluster in wild-type metallothionein forms in all-or-nothing fashion. This conclusion is based on copper binding stoichiometry and luminescence studies. The relative quantum yield of samples with intermediate Cu(I) levels was constant, consistent with all-or-none cluster formation.     

Structural and functional studies of the amino terminus of yeast metallothionein

Purified yeast copper-metallothionein lacks 8 amino-terminal residues that are predicted from the DNA sequence of its gene. The removed sequence is unusual for metallothionein in its high content of hydrophobic and aromatic residues and its similarity to mitochondrial leader sequences. To study the significance of this amino-terminal cleavage, several mutations were introduced into the metallothionein coding gene, CUP1. One mutant, which deletes amino acid residues 2-8, had a minor effect on the ability of the molecule to confer copper resistance to yeast but did not affect CUP1 gene regulation. A second mutation, which changes two amino acids adjacent to the cleavage site, blocked removal of the extension peptide but had no effect on copper detoxification or gene regulation. Immunofluorescence studies showed that both the wild-type and these two mutant proteins are predominantly cytoplasmic with no evidence for mitochondrial localization. The cleavage site mutation allowed isolation and structural characterization of a full length metallothionein polypeptide. The copper content and luminescent properties of this molecule were identical to those of the truncated wild-type protein indicating a homologous cluster structure. Moreover, the amino-terminal peptide was selectively removed by various endopeptidases and an exopeptidase suggesting that it does not participate in the tertiary fold. These results argue that the amino-terminal peptide is not required for either the structural integrity or biological function of yeast metallothionein.     

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.     

Mutational analysis of the mitochondrial copper metallochaperone Cox17

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochrome c oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys --> Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys --> Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.     

Spectroscopic studies on Neurospora copper metallothionein

The spectral properties of Neurospora copper metallothionein were investigated and compared with those of the Cu(I)-2-mercaptoethanesulfonic acid complex. In both cases, the absorption spectra are rather similar, showing a characteristic shoulder at approximately 250 nm. However, marked differences were observed in their emissive properties. Thus, only metallothionein emits detectable luminescence in solution, but both the copper protein and the Cu(I) complex are luminescent at 77 K. The circular dichroism spectrum of Neurospora copper metallothionein shows several Cotton extrema attributable to asymmetry in metal coordination. The influence of HgCl2 and p-(chloromercuri)benzoate on the spectral properties of metallothionein was also investigated. The two mercurials exerted a pronounced effect on the electronic absorption, chiroptical, and emissive properties of the protein. Spectroscopic titrations followed by gel filtration experiments indicate that two mercurials can be bound per metallothionein molecule without loss of copper. This binding is responsible for the disappearance of the emissive properties of metallothionein and for the distinct changes in its electronic absorption and circular dichroism spectra. From these data, it is suggested that the Cu(I) ions are coordinated to the cysteinyl residues in the form of a single metal cluster.     

Chemical synthesis and expression of a cassette adapted ubiquitin gene

A gene encoding the yeast ubiquitin was chemically synthesized and expressed in yeast under regulatory control of the copper metallothionein (CUP1) promoter. The gene was assembled in a one-step ligation reaction from eight oligonucleotide fragments ranging in length from 50 to 64 nucleotides. To facilitate mutagenesis and gene fusion studies, eight unique 6-base-cutting restriction enzyme sites were placed in the reading frame which did not alter the encoded protein sequence or force the utilization of rare codons. In a copper-resistant yeast strain (CUP1r), expression of the gene was induced by copper to approximately 5% of the total yeast proteins, as determined by Coomassie-stained polyacrylamide gels. The protein, purified from yeast, reacted with ubiquitin-specific antibodies and was found to be biologically active in supporting ubiquitin-dependent protein degradation in vitro.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Role of a disulfide bond in the thermal stability of the LamB protein trimer in Escherichia coli outer membrane

In order to understand the unusual heat resistance of LamB protein (the outer membrane component of the maltose transport system in Escherichia coli and its receptor for bacteriophage lambda), we investigated the role of its 2 cysteinyl residues. Our studies show that Cys22 and Cys38 form an intrasubunit disulfide bond which contributes to the heat stability of the LamB protein trimer. Physical evidence for the disulfide was obtained by using site-directed mutagenesis to convert Asn36 to Met, which allowed cyanogen bromide cleavage between the 2 cysteines. Upon reduction one of the N36M fragments migrated as two pieces, resolved by two-dimensional polyacrylamide gel electrophoresis. Other mutagenized LamB proteins, in which 1 or both Cys residues were converted to Ser, exhibited a sharp loss of thermal stability. In contrast to wild-type LamB protein trimer, which does not dissociate to monomers even after 60 min at 100 degrees C, only 10-15% of the mutant LamB proteins remain trimeric after boiling 10 min. The disulfide bond in LamB protein is not required for its transport function, since both mutagenized LamB protein and N-ethylmaleimide-labeled LamB protein exhibit normal uptake of sugars in proteoliposomes. Finally, the disulfide bond must not be between subunits of the LamB trimer since reversible dissociation of trimer is achieved by low pH or denaturants in the absence of reducing agent.     

X-ray absorption studies of yeast copper metallothionein

The local structures of the metal sites in copper metallothionein from Saccharomyces cerevisiae have been investigated by x-ray absorption spectroscopy at the copper and sulfur K edges. Analysis of the EXAFS (extended x-ray absorption fine structure) data indicates that each copper is trigonally coordinated to sulfur at a distance of 2.23 A. Cu-Cu interactions at 2.7 and 3.9 A have also been tentatively identified. Sulfur K edge data are compatible with cysteinyl thiolates bridging each of the eight Cu(I) ions. The data support a model for the copper cluster in yeast metallothionein consisting of a Cu8S12 core. EXAFS data on two specifically engineered carboxyl-terminal truncated mutants reveal that the copper coordination in the mutants is similar to that observed in the wild-type protein.     

Mutagenesis of murine granulocyte/macrophage-colony-stimulating factor reveals critical residues near the N terminus

A number of cDNAs encoding mutant forms of the murine haemopoietic growth factor, granulocyte/macrophage-colony-stimulating factor (GM-CSF), have been derived by in vitro mutagenesis and expressed in simian COS cells. Determination of the biological activity of the mutant factors revealed that residues within the regions 11-15, 24-37, 47-49 and 81-89 are required for generating a functional GM-CSF molecule. In particular, truncation of either of two strongly predicted alpha helices near the N terminus of the molecule severely depresses the activity of the factor.     

Sequence replacements in the central beta-turn of plastocyanin.

The role of beta-turns in dictating the structure of a beta-barrel protein is assessed by probing the tolerance of the central beta-turn of poplar plastocyanin to substitution by arbitrary sequences. Native plastocyanin binds copper and is colored bright blue. However, when the wild-type Pro47-Ser48-Gly49-Val50 turn sequence is replaced by arbitrary tetrapeptides, the vast majority (92/98 = 94%) of mutant proteins cannot fold into the native blue structure. Characterization of the colorless mutant proteins demonstrates that the majority of substitutions in this type II beta-turn disrupt the native structure severely. Gross structural changes are indicated by major differences in the CD spectra of the mutants relative to the wild-type protein, and by the much larger apparent size of mutant proteins in gel filtration experiments. These mutant proteins do not bind copper. Furthermore, Cys84 forms a disulfide bond readily in the colorless mutant proteins, indicating that it has moved away from the buried position it occupies in the native copper binding site and has become exposed. These results indicate that the central beta-turn in plastocyanin is not merely a default structure arising in response to the surrounding context; rather, sequence information in this turn plays an active role in dictating the location of a chain reversal in the beta-barrel structure. These findings are discussed in terms of their implications for the folding of natural proteins, as well as the design of de novo proteins.     

A comparison of the effects of penicillamine, trientine, and trithiomolybdate on [35S]-labeled metallothionein in vitro; implications for Wilson's disease therapy

The synthesis of radiolabeled metallothionein was induced in rats in vivo by the injection of CuSO4 and [35S]-cysteine. Treatment of "cold" rat liver cytosol "spiked" with purified [35S] metallothionein with Penicillamine and Trientine showed that even at relatively high concentrations (up to 50 mg/g liver, wet weight), these compounds had no effect on the copper peak or the position of the [35S] label in the cytosol eluate after Sephadex G-75 gel filtration. By contrast, incubation of the "spiked" liver cytosol with Trithiomolybdate, even at relatively low concentrations (0.5 mg/g liver, wet weight), resulted in a transfer of metallothionein copper to high molecular weight protein fractions; the position of the [35S] apoprotein was unaffected. This copper "stripping" effect on metallothionein supports clinical and other evidence that thiomolybdates have a genuine decoppering effect in vivo whereas Penicillamine and Trientine have another mode of action and indicates that thiomolybdates might provide a more rational alternate therapy for Wilson's disease patients.     

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.