The metal binding properties of the zinc site of yeast copper-zinc superoxide dismutase: implications for amyotrophic lateral sclerosis.

We have investigated factors that influence the properties of the zinc binding site in yeast copper-zinc superoxide dismutase (CuZnSOD). The properties of yeast CuZnSOD are essentially invariant from pH 5 to pH 9. However, below this pH range there is a change in the nature of the zinc binding site which can be interpreted as either (1) a change in metal binding affinity from strong to weak, (2) the expulsion of the metal bound at this site, or (3) a transition from a normal distorted tetrahedral ligand orientation to a more symmetric arrangement of ligands. This change is strongly reminiscent of a similar pH-induced transition seen for the bovine protein and, based on the data presented herein, is proposed to be a property that is conserved among CuZnSODs. The transition demonstrated for the yeast protein is not only sensitive to the pH of the buffering solution but also to the occupancy and redox status of the adjacent copper binding site. Furthermore, we have investigated the effect of single site mutations on the pH- and redox-sensitivity of Co2+ binding at the zinc site. Each of the mutants H46R, H48Q, H63A, H63E, H80C, G85R, and D83H is capable of binding Co2+ to a zinc site with a distorted tetrahedral geometry similar to that of wild-type. However, they do so only if Cu+ is bound at the copper site or if the pH in raised to near physiological levels, indicating that the change at the zinc binding site seen in the wild-type is conserved in the mutants, albeit with an altered pKa. The mutants H71C and D83A did not bind Co2+ in a wild-type-like fashion under any of the conditions tested. This study reveals that the zinc binding site is exquisitely sensitive to changes in the protein environment. Since three of the mutant yeast proteins investigated here contain mutations analogous to those that cause ALS (amyotrophic lateral sclerosis) in humans, this finding implicates improper metal binding as a mechanism by which CuZnSOD mutants exert their toxic gain of function.

Copper-mediated repression of the activation domain in the yeast Mac1p transcription factor.

The expression of a number of genes encoding products involved in copper ion uptake in yeast is specifically inhibited by copper ions. We show here that copper metalloregulation occurs through Cu-dependent repression of the transactivation activity of Mac1p. A segment of the yeast transcription factor Mac1p was identified that activated transcription in vivo in a heterologous system using fusion polypeptides with the yeast Gal4 DNA-binding domain. The Gal4/Mac1p hybrid exhibits transactivation activity that is repressed in cells cultured in the presence of copper salts and derepressed in cells with reduced copper uptake. The repressive effect is specific for copper ions. The concentration dependency of the Cu-inactivation of Gal4/Mac1p is similar to that of Cu-inhibition of CTR1 expression, a known Cu-regulated gene in vivo. Copper inhibition of gene expression is not observed with a Gal4/Mac1p chimera containing the MAC1(up1) substitution within the transactivation domain. Cells harboring the MAC1(up1) allele fail to attenuate FRE1 and CTR1 expression in a Cu-dependent manner. Additional MAC1(up) alleles exist within the first of two cysteine-rich sequence motifs adjacent to the His --> Gln MAC1(up1) encoded substitution. Thus, Cu-regulation of Mac1p function arises from a novel Cu-specific repression of the transactivation domain function. Models for the mechanism of Cu-repression of Mac1p function will be discussed.

Presence of a copper(I)-thiolate regulatory domain in the copper-activated transcription factor Amt1.

The Amt1 transcription factor from Candida glabrata is activated by the formation of a tetracopper-thiolate cluster. Recombinant Amt1 (residues 1-110) is isolated as a Cu,ZnAmt1 complex. Previous mapping studies [Farrell et al. (1996) Biochemistry 35, 1571-1580] revealed that the Zn(II) site is enfolded by an independent, N-terminal domain consisting of residues 1-40. One prediction from the mapping study is that the tetracopper cluster is enfolded by residues 41-110. A truncated Amt1 peptide consisting of residues 37-110 was expressed and isolated as a CuAmt1 complex with 4 mol equiv of Cu(I) bound. The bound Cu(I) ions in the truncated Amt1 complex were spectroscopically similar to Cu(I) ions bound in the 110-mer Amt1 molecule in the energies and intensities of the ultraviolet S-->Cu charge transfer transitions and luminescence. Copper K-edge extended X-ray absorption fine structure spectroscopy (EXAFS) of the truncated CuAmt1 complex revealed the same 2.26 A mean Cu-S bond distance as in the Cu,ZnAmt1 complex. A diagnostic feature of the polycopper-thiolate cluster in Cu,-ZnAmtl1 is the short 2.7 A Cu-Cu distance determined by Cu K-edge EXAFS. The truncated CuAmt1 complex had the same short 2.7 A Cu-Cu distance. The truncated CuAmt1 complex bound DNA specifically and with high affinity consistent with residues 41-110 being an independent domain stabilized by the tetracopper cluster. Thus, Amt1 consists of three independent and contiguous domains, an N-terminal Zn module (residues 1-40), an adjacent Cu regulatory domain (residues 41-110), and a C-terminal transcriptional activation domain. Cu(I) activation of Amt1 appears to consist of conversion of the 70-residue Cu regulatory domain from an inactive conformer to a structure containing the tetracopper cluster.

Mutations in copper-zinc superoxide dismutase that cause amyotrophic lateral sclerosis alter the zinc binding site and the redox behavior of the protein.

A series of mutant human and yeast copper-zinc superoxide dismutases has been prepared, with mutations corresponding to those found in familial amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's disease). These proteins have been characterized with respect to their metal-binding characteristics and their redox reactivities. Replacement of Zn2+ ion in the zinc sites of several of these proteins with either Cu2+ or Co2+ gave metal-substituted derivatives with spectroscopic properties different from those of the analogous derivative of the wild-type proteins, indicating that the geometries of binding of these metal ions to the zinc site were affected by the mutations. Several of the ALS-associated mutant copper-zinc superoxide dismutases were also found to be reduced by ascorbate at significantly greater rate than the wild-type proteins. We conclude that similar alterations in the properties of the zinc binding site can be caused by mutations scattered throughout the protein structure. This finding may help to explain what is perhaps the most perplexing question in copper-zinc superoxide dismutase-associated familial ALS-i.e., how such a diverse set of mutations can result in the same gain of function that causes the disease.