Cofactor-dependent structural transition of SOD1 modulates its ability to undergo liquid-liquid phase separation.

Abstract

Impetus leading to the toxic gain of function of Cu/Zn superoxide dismutase (SOD1) whose aggregation forms the hallmark of amyotrophic lateral sclerosis (ALS) is still a controversial area. The formation of membraneless organelles such as stress granules (SGs) is believed to occur through the process of liquid-liquid phase separation (LLPS) and involves numerous proteins containing intrinsically disordered regions. Whether SOD1, which is also associated with SGs and whose aggregation is associated with Amyotrophic lateral sclerosis (ALS), can independently undergo LLPS, is not known. Using a combination of experiments and computational methods, we report here that local structural disorder in two loop regions of SOD1 induced by the absence of metal cofactor - Zn, triggers its LLPS. The liquid condensates give rise to aggregates which eventually form toxic amyloids in a longer timescale. The addition of exogenous Zn to immature, metal-free SOD1 and the disease-relate ALS mutant - I113T, stabilized the loops and restored the folded structure, thereby inhibiting LLPS and subsequent aggregation. On the contrary, the Zn-induced inhibition of phase separation and aggregation was found to be partial in the case of another severe ALS-associated mutant - G85R, which exhibits reduced Zn-binding. Moreover, a less-severe ALS mutant - G37R with perturbed Cu binding does not undergo LLPS. In this work, we want to highlight the modulation of SOD1 LLPS propensity in a Zn-dependent manner due to underlying conformational transitions between folded and partially disordered states. Our work establishes a link between SOD1 LLPS and aggregation, which is relevant to ALS pathogenesis.