Extreme diversity of 12 cations in folding ALS-linked hSOD1 unveils novel hSOD1-dependent mechanisms for Fe(2+)/Cu(2+)-induced cytotoxicity

153-Residue copper-zinc superoxide dismutase 1 (hSOD1) is the first gene whose mutation was linked to FALS. To date, > 180 ALS-causing mutations have been identified within hSOD1, yet the underlying mechanism still remains mysterious. Mature hSOD1 is exceptionally stable constrained by a disulfide bridge to adopt a Greek-key β-barrel fold that accommodates copper/zinc cofactors. Conversely, nascent hSOD1 is unfolded and susceptible to aggregation and amyloid formation, requiring Zn(2+) to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations that disrupt Zn(2+)-binding correlate with their ability to form toxic aggregates. Therefore, to decode the role of cations in hSOD1 folding provides not only mechanistic insights, but may bear therapeutic implications for hSOD1-linked ALS. Here by NMR, we visualized the effect of 12 cations: 8 essential for humans (Na(+), K(+), Ca(2+), Zn(2+), Mg(2+), Mn(2+), Cu(2+), Fe(2+)), 3 mimicking zinc (Ni(2+), Cd(2+), Co(2+)), and environmentally abundant Al(3+). Surprisingly, most cations, including Zn(2+)-mimics, showed negligible binding or induction for folding of nascent hSOD1. Cu(2+) exhibited extensive binding to the unfolded state but led to severe aggregation. Unexpectedly, for the first time Fe(2+) was deciphered to have Zn(2+)-like folding-inducing capacity. Zn(2+) was unable to induce folding of H80S/D83S-hSOD1, while Fe(2+) could. In contrast, Zn(2+) could trigger folding of G93A-hSOD1, but Fe(2+) failed. Notably, pre-existing Fe(2+) disrupted the Zn(2+)-induced folding of G93A-hSOD1. Comparing with the ATP-induced folded state, our findings delineate that hSOD1 maturation requires: (1) intrinsic folding capacity encoded by the sequence; (2) specific Zn(2+)-coordination; (3) disulfide formation and Cu-load catalyzed by hCCS. This study unveils a previously-unknown interplay of cations in governing the initial folding of hSOD1, emphasizing the pivotal role of Zn(2+) in hSOD1-related ALS and implying new hSOD1-dependent mechanisms for Cu(2+)/Fe(2+)-induced cytotoxicity, likely relevant to aging and other diseases.