Zinc in folding and misfolding of SOD1: implications for ALS

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1). Nevertheless it is likely that sporadic and familiar forms of the disease share a common mechanism, where SOD1 plays an important role. Eukaryotic SOD1 is a homodimeric metalloenzyme, functioning as the primary cytoplasmic scavenger of superoxide radicals (O2-). Despite the toxicity of superoxide radicals, SOD1 knockout mice do not develop ALS. Whereas the expression of human SOD1 in mice, either in its wild-type or in several mutated forms, were shown to display ALS-like symptoms. It is believed, that by a yet mysterious mechanism, the SOD1 structure gains toxic properties triggering apoptosis of motor nerve cells. Several lines of evidence suggest that the cytotoxic property of SOD1 arises from immature monomeric apo-SOD1, but a more detailed structural description of the cytotoxic species is missing. The thesis work presented herein is focused on understanding how structural and dynamic properties of this particular protein molecule change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc ion controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands is kinetically beneficial and speeds up folding of the SOD1 core structure, however, it stabilizes the SOD1 monomer in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, which occurs after the main folding transition is passed, induces the folding of loop VII and stabilizes the native and functional fold of both active-site loops IV and VII and (iii) the tremendous stability gain due to Zn-site metallation and its associated folding events corresponds to a life-time of hundreds of years for the folded state, thus the cellular life-time of SOD1 is likely to be controlled by Zn2+ release, which, in turn, is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. In support of this, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect structural integrity of active-site loops in holo-SOD1. The local destabilization effect of these mutations unbalance also the natural conformational equilibrium of differently matured SOD1 species, i.e. it increases population of SOD1 species with disorganized functional loops. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: SOD1 may influence the cellular Zn2+-homeostasis, especially taking into account the relatively high intracellular concentration of SOD1 and the relatively low intracellular concentration of free Zn2+.