Familial Amyotrophic Lateral Sclerosis-associated Mutations Decrease the Thermal Stability of Distinctly Metallated Species of Human Copper/Zinc Superoxide Dismutase

Abstract

We report the thermal stability of wild type (WT) and 14 different variants of human copper/zinc superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system. We correlated the metal ion contents of SOD1 variants with the occurrence of distinct melting transitions. Altered thermal stability upon reduction of copper with dithionite identified transitions resulting from the unfolding of copper-containing SOD1 species. We demonstrated that copper or zinc binding to a subset of "WT-like" FALS mutants (A4V, L38V, G41S, G72S, D76Y, D90A, G93A, and E133⌬) conferred a similar degree of incremental stabilization as did metal ion binding to WT SOD1. However, these mutants were all destabilized by ϳ1-6°C compared with the corresponding WT SOD1 species. Most of the "metal binding region" FALS mutants (H46R, G85R, D124V, D125H, and S134N) exhibited transitions that probably resulted from unfolding of metal-free species at ϳ4 -12°C below the observed melting of the least stable WT species. We conclude that decreased conformational stability shared by all of these mutant SOD1s may contribute to SOD1 toxicity in FALS.Copper/zinc superoxide dismutase (SOD1) 1 catalyzes the disproportionation of two molecules of superoxide anion (O 2 . )into O 2 and H 2 O 2 (1, 2) in all eukaryotic cells. Many specific, highly conserved structural interactions confer upon SOD1 a remarkable thermal stability (3-6) and resistance to chemical denaturation (7-9).Each subunit of homodimeric SOD1 is built upon a flattened ␤-barrel motif with additional loop regions that contribute to metal ion binding and formation of the active site (10). One catalytic copper ion and one buried zinc ion per subunit are bound at the active site on the external surface of the ␤-barrel. Occupancy of the metal ion binding sites confers greater thermal stabilization to the bovine SOD1 apoenzyme (3, 4). The copper and zinc ions are linked directly via the imidazolate side chain of the shared His-63 residue 2 and indirectly via extended interactions between their respective ligands. SOD1 dimerization is stabilized by optimized hydrophobic interactions at the contact interface between complementary patches on each subunit (10 -12). A conserved intrasubunit disulfide bond involving Cys-57 also stabilizes the enzyme by anchoring a loop that forms part of the dimer interface to the ␤-barrel at Cys-146.A subset of SOD1 mutations in familial amyotrophic lateral sclerosis (FALS) have been proposed to destabilize the ␤-barrel or disrupt dimerization of SOD1 monomers (13,14). A crystal structure obtained for the G37R SOD1 mutant shows minimal perturbation of the averaged backbone conformation but exhibits unusually high atomic displacement parameters, suggestive of increased molecular flexibility in some regions of the molecule (15). Consistent with this, some mutant SOD1s exhibit acceler...

Familial Amyotrophic Lateral Sclerosis Mutants of Copper/Zinc Superoxide Dismutase Are Susceptible to Disulfide Reduction

“…SDS (second lane of each set). This result suggested increased SDS binding to these mutants, possibly as a consequence of unfolding in this gel system, which was consistent with the established importance of metal binding to SOD1 for both chemical and thermal stability (19,21,22).…”

“…Our data suggest that cellular disulfide reducing influences at physiological temperature and pH are sufficient to convert relatively well folded WT-like SOD1 mutants (18) or less stable metal-binding region mutants (19) into more severely destabilized species. These non-native mutant forms might resemble the subset of highly unstable SOD1 C-terminal truncation mutants (15,64), which also lack the disulfide bond consequent to deletion of Cys-146.…”

“…We hypothesized that increased stability afforded by metal ion binding to SOD1 (19,21,22) could contribute to protease resistance despite the presence of a destabilizing mutation. To test this possibility, preparations of D76Y and E133⌬ that differed only with respect to metal ion occupancy (18) were exposed to these proteases under reducing conditions (Fig.…”

“…The other six "metal-binding region" mutants (H46R, H48Q, G85R, D124V, D125H, and S134N) were clearly distinguished from WT SOD1 according to decreased metal ion contents, altered visible absorption spectra, or decreased specific activities. In a further analysis by differential scanning calorimetry, we found that metal-binding region mutants generally exhibited a larger fraction of species that unfolded at low T m values compared with those of the WT-like mutants and normal SOD1 (19). Although purified SOD1 mutants can lose in vitro metal ion binding specificity following partial denaturation at non-physiological pH (20), the retention of many native properties in the "as-isolated" WT-like mutants suggests that other influences may destabilize these SOD1 mutants in vivo.…”

Different Immunoreactivity against Monoclonal Antibodies between Wild-type and Mutant Copper/Zinc Superoxide Dismutase Linked to Amyotrophic Lateral Sclerosis

“…9). Several papers (41,46) have also reported that H46R is less stable than other FALS mutants in vitro. Most interestingly, the immunoreactivities of H46R against the mAbs in Western blot and ELISA were similar to that of WT or C111S (Figs.…”