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.

Metal deficiency increases aberrant hydrophobicity of mutant superoxide dismutases that cause amyotrophic lateral sclerosis.

The mechanisms by which mutant variants of Cu/Zn-superoxide dismutase (SOD1) cause familial amyotrophic lateral sclerosis are not clearly understood. Evidence to date suggests that altered conformations of amyotrophic lateral sclerosis mutant SOD1s trigger perturbations of cellular homeostasis that ultimately cause motor neuron degeneration. In this study we correlated the metal contents and disulfide bond status of purified wild-type (WT) and mutant SOD1 proteins to changes in electrophoretic mobility and surface hydrophobicity as detected by 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence. As-isolated WT and mutant SOD1s were copper-deficient and exhibited mobilities that correlated with their expected negative charge. However, upon disulfide reduction and demetallation at physiological pH, both WT and mutant SOD1s underwent a conformational change that produced a slower mobility indicative of partial unfolding. Furthermore, although ANS did not bind appreciably to the WT holoenzyme, incubation of metal-deficient WT or mutant SOD1s with ANS increased the ANS fluorescence and shifted its peak toward shorter wavelengths. This increased interaction with ANS was greater for the mutant SOD1s and could be reversed by the addition of metal ions, especially Cu(2+), even for SOD1 variants incapable of forming the disulfide bond. Overall, our findings support the notion that misfolding associated with metal deficiency may facilitate aberrant interactions of SOD1 with itself or with other cellular constituents and may thereby contribute to neuronal toxicity.

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase.

Familial amyotrophic lateral sclerosis (fALS) caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. Similar inclusions observed in fALS transgenic mice have a fibrillar appearance suggestive of amyloid structure. Metal-free apo-SOD1 is a relatively stable protein and has been shown to form amyloid fibers in vitro only when it has been subjected to severely destabilizing conditions, such as low pH or reduction of its disulfide bonds. Here, by contrast, we show that a small amount of disulfide-reduced apo-SOD1 can rapidly initiate fibrillation of this exceptionally stable and highly structured protein under mild, physiologically accessible conditions, thus providing an unusual demonstration of a specific, physiologically relevant form of a protein acting as an initiating agent for the fibrillation of another form of the same protein. We also show that, once initiated, elongation can proceed via recruitment of either apo- or partially metallated disulfide-intact SOD1 and that the presence of copper, but not zinc, ions inhibits fibrillation. Our findings provide a rare glimpse into the specific changes in a protein that can lead to nucleation and into the ability of amyloid nuclei to recruit diverse forms of the same protein into fibrils.

Binding of a single zinc ion to one subunit of copper-zinc superoxide dismutase apoprotein substantially influences the structure and stability of the entire homodimeric protein.

The thermodynamics of zinc binding to metal-free (apo) human and bovine copper-zinc superoxide dismutases (SOD1) were measured using isothermal titration calorimetry. The apparent thermodynamics of zinc binding to the apoproteins were favorable (Ka > 108 M-1), with an observed stoichiometry of one zinc per homodimer. The change in heat capacity for the one-zinc binding event was large and negative (approximately -650 cal mol-1 K-1), suggestive of significant structural changes to the protein upon zinc binding. We further characterized the one-zinc derivative by circular dichroism and determined that this derivative had nearly the same secondary structure as the two-zinc derivative and that both are structurally distinct from the metal-free protein. In addition, we monitored the effect of zinc binding on hydrogen-deuterium exchange and accessibility of histidyl residues to modification by diethyl pyrocarbonate and observed that more than 50% protection was afforded by the binding of one zinc in both assays. Differential scanning calorimetry on the human SOD1 zinc derivatives also showed increased thermostability of the protein due to zinc binding. Further, the melting transitions observed for the one-zinc derivative closely resembled those of the two-zinc derivative. Finally, we observed that the quaternary structure of the protein is stabilized upon binding of one and two zinc ions in analytical ultracentrifugation experiments. Combined, these results suggest communication between the two monomers of SOD1 such that the binding of one zinc ion per homodimer has a more profound effect on the homodimeric protein structure than the binding of subsequent metal ions. The relevance of these findings to amyotrophic lateral sclerosis is discussed.

Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis.

The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H/D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6 degrees C to 56.2 degrees C, i.e., similar to or higher than that of the WT apoprotein (Tm = 52.5 degrees C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0 degrees C to 52.4 degrees C. The H/D exchange properties of the mutants were also wide-ranging: the MBR mutant apoproteins exhibited H/D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H/D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H/D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.