Nonnative structure in a peptide model of the unfolded state of superoxide dismutase 1 (SOD1): Implications for ALS-linked aggregation

Abstract

Dozens of mutations throughout the sequence of the gene encoding superoxide dismutase 1 (SOD1) have been linked to toxic protein aggregation in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). A parsimonious explanation for numerous genotypes resulting in a common phenotype would be mutation-induced perturbation of the folding freeenergy surface that increases the populations of high-energy states prone to aggregation. The absence of intermediates in the folding of monomeric SOD1 suggests that the unfolded ensemble is a potential source of aggregation. To test this hypothesis, here we dissected SOD1 into a set of peptides end-labeled with FRET probes to model the local behavior of the corresponding sequences in the unfolded ensemble. Using timeresolved FRET, we observed that the peptide corresponding to the loop VII-β8 sequence at the SOD1 C-terminus was uniquely sensitive to denaturant. Utilizing a two-dimensional form of maximum entropy modeling, we demonstrate that the sensitivity to denaturant is the surprising result of a two-state-like transition from a compact to an expanded state. Variations of the peptide sequence revealed that the compact state involves a nonnative interaction between the disordered Nterminus and the hydrophobic C-terminus of the peptide. This nonnative intramolecular structure could serve as a precursor for intermolecular association and result in aggregation associated with ALS. We propose that this precursor would provide a common molecular target for therapeutic intervention in the dozens of ALS-linked SOD1 mutations.

Probing Intermolecular Interactions of Amyloidogenic Fragments of SOD1 by Site-Specific Tryptophan and Its Noncanonical Derivative

“…The misfolding and aggregation of superoxide dismutase 1 (SOD1) are associated with the fetal neurodegenerative disease amyotrophic lateral sclerosis (ALS). − Recent X-ray crystallographic studies have captured a corkscrew-like oligomeric species formed by the amyloidogenic segment residues 28–38 of SOD1 (with the sequence KVKVWGSIKGL). , This segment is the core fragment of the SOD1 protein and has been reported to play a critical role in SOD1-mediated toxicity . The physiological relevance of the oligomeric species formed by SOD1 28–38 calls for the dynamic studies to follow its evolution, which will provide important insights into the aggregation mechanism of the full-length protein. , Particularly, it is unknown whether the corkscrew-like oligomer is an on-pathway intermediate or an off-pathway by-product.…”

Trifluoroethanol Partially Unfolds G93A SOD1 Leading to Protein Aggregation: A Study by Native Mass Spectrometry and FPOP Protein Footprinting

“…Given that the results indicate that the monomer is the likely precursor that triggers the downstream oligomerization cascade, ,, understanding the G93A SOD1 structural changes from native dimer to monomeric species may shed light upon the molecular basis of the aggregation mechanism. In the following study, we focus on the elucidation of the unfolding events and localization of the regions associated with the unfolding of G93A SOD1 induced by TFE.…”

“…Furukawa et al identified a protease-resistant core structure within various fALS-inducing mutant SOD1 aggregates and found three regions (amino acids 1–30, 90–120, and 135–153) that are likely to form a scaffold core for the aggregates. Matthews and co-workers recently reported that the C-terminus of SOD1 (loop VII-β8 regions) is involved in a non-native interaction with a disordered N-terminus, a non-native intramolecular structure that may provide a nucleation site for ALS aggregations. In addition, Kumar and co-workers, using molecular dynamics simulations, showed that SOD1 undergoes partial unfolding of β-strands 4, 5, and 6 before TFE-induced aggregation.…”

Friction-Limited Folding of Disulfide-Reduced Monomeric SOD1

“…The C-terminus of SOD1 would have ample opportunity to collapse on itself and introduce chain friction for both reactions. Although the marginal stability of the Loop VII-b8 peptide to denaturant appears to challenge this explanation for friction-limited unfolding, the presence of the TSE in the full-length protein and high concentrations of viscogen make comparisons ambiguous and highlight the limitations of peptide models for unfolded proteins (25).…”

“…Loop IV binds the zinc ion, whereas Loop VII, the electrostatic loop, serves to support Loop IV and guides the anionic substrate to the redox-active copper ion. A recent peptide dissection analysis of SOD1 from our lab demonstrated that the 35-residue C-terminal peptide, containing Loop VII and b8, folds on itself to bring the polar and charged N-terminus and the nonpolar C-terminus into a nonnative juxtaposition (25). This interaction cannot exist in the disulfide-containing form of SOD1, in which C146 in b8 is covalently linked to C57.…”