Extreme diversity of 12 cations in folding ALS-linked hSOD1 unveils novel hSOD1-dependent mechanisms for Fe2+/Cu2+-induced cytotoxicity

Abstract

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 Zn2+ to initiate folding to a coexistence of folded and unfolded states. Recent studies demonstrate mutations that disrupt Zn2+-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+, Ca2+, Zn2+, Mg2+, Mn2+, Cu2+, Fe2+), 3 mimicking zinc (Ni2+, Cd2+, Co2+), and environmentally abundant Al3+. Surprisingly, most cations, including Zn2+-mimics, showed negligible binding or induction for folding of nascent hSOD1. Cu2+ exhibited extensive binding to the unfolded state but led to severe aggregation. Unexpectedly, for the first time Fe2+ was deciphered to have Zn2+-like folding-inducing capacity. Zn2+ was unable to induce folding of H80S/D83S-hSOD1, while Fe2+ could. In contrast, Zn2+ could trigger folding of G93A-hSOD1, but Fe2+ failed. Notably, pre-existing Fe2+ disrupted the Zn2+-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 Zn2+-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 Zn2+ in hSOD1-related ALS and implying new hSOD1-dependent mechanisms for Cu2+/Fe2+-induced cytotoxicity, likely relevant to aging and other diseases.