Folding of Cu/Zn superoxide dismutase suggests structural hotspots for gain of neurotoxic function in ALS: Parallels to precursors in amyloid disease

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to misfolding of the ubiquitous enzyme Cu͞Zn superoxide dismutase (SOD). In contrast to other protein-misfolding disorders with similar neuropathogenesis, ALS is not always associated with the in vivo deposition of protein aggregates. Thus, under the assumption that all protein-misfolding disorders share at primary level a similar disease mechanism, ALS constitutes an interesting disease model for identifying the yet-mysterious precursor states from which the cytotoxic pathway emerges. In this study, we have mapped out the conformational repertoire of the apoSOD monomer through analysis of its folding behavior. The results allow us to target the regions of the SOD structure that are most susceptible to unfolding locally under physiological conditions, leading to the exposure of structurally promiscuous interfaces that are normally hidden in the protein's interior. The structure of this putative ALS precursor is strikingly similar to those implicated in amyloid disease.neurodegenerative disease ͉ protein folding ͉ transition state A n increasing number of severe neurodegenerative disorders have been linked to misfolding and subsequent aggregation of proteins (1). In some cases the protein aggregates deposit in the form of highly ordered amyloid fibrils (2), whereas in other cases they appear more granular or even amorphous (3). Even so, the main cause of neurotoxic function seems not to be the macroscopically deposited protein itself but rather a mysterious precursor species in the aggregation pathway (1, 4-6). In this perspective, it is notable that there are also disease cases where in vivo depositions of the disease-associated proteins are hard to find and sometimes are not even observable. Characteristic examples are found among the sporadic cases of CreuzfeldtJacob disease and amyotropic lateral sclerosis (ALS) (7). It is further apparent that the ALS-associated enzyme Cu͞Zn superoxide dismutase (SOD) has relatively low propensities to aggregate in vitro: SOD can be produced and analyzed by standard protocols despite being destabilized to the extent that it is half unfolded in physiological buffer at 25°C (8). For comparison there are few nondisease proteins that would resist aggregation under these conditions. The question then arises, do the disease cases with and without in vivo deposition of protein aggregates share at the primary level the same toxicity mechanism? We have previously observed that ALS-provoking SOD mutations produce a common shift of the folding equilibrium toward unfolded and partly folded monomers, implicating that the noxious side reaction emerges from the early folding events. The observation is in full agreement with a series of reports pointing at the immature apoSOD monomer as precursor for cytotoxicity (8)(9)(10)(11)(12)(13)(14)(15). Accompanying the shift of the folding equilibrium, the ALS-associated SOD mutations indicate a quantitative relation between decreased protein stability, net charge, and diseas...

Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

“…We note that the detailed view of the excited state provided here differs from that inferred from previous -value analysis of the folding kinetics (42), possibly suggesting that the excited state is off the folding pathway; alternatively, the observed differences arguably could be explained by the limited structural resolution afforded by the -value approach. The chemical shifts of the perturbed residues in the excited state suggest that the protein is not simply undergoing local unfolding, which implies that the approach reported by Korzhnev et al (43) toward calculating the structure of the excited state cannot be applied in the present case, because one of the endpoint reference states remains unknown.…”

Oxidative Stress and Neurotoxicity

“…The most popular current theory linking the mutant SOD1 forms to neurological deficits seen in mouse models expressing them is that the different point mutations create a misfolding defect (269), leading to small amyloid-like aggregates that appear in late stages of the disease. Neurotoxicity is now being considered to arise from a toxic effect of the aggregated misfolded protein, similar to the neurotoxicity that arises in other amyloidoses (270). On the basis of studies showing that a number of fALS SOD1 mutants have increased affinity for copper (271), one must also consider the possibility that altered copper coordination may be associated with a misfolding tendency rather than with altered redox chemistry.…”

Global structural motions from the strain of a single hydrogen bond

“…As a clue to the functional role of the communication, the binding of a single Zn 2+ ion to one subunit of the dynamic apoSOD1 dimer is found to alter the structure and stability of the entire molecule (14). Based on the general view that globular proteins with compromised structural rigidity are at increased risk of misfolding (5,6), the dynamic apoSOD1 molecule also has drawn attention as a possible precursor for pathological aggregation in ALS (12,13,15). In this study, we show by a combination of NMR, X-ray crystallography, and protein engineering that the origin of these dynamic motions is the built-in strain of a conserved hydrogenbond linkage through the hydrophobic core.…”