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

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1).ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have remained enigmatic, however, as is the case in other protein-misfolding diseases. Here, we target the critical conformational change that defines the earliest step toward aggregation. Using nuclear spin relaxation dispersion experiments, we identified a short-lived (0.4 ms) and weakly populated (0.7%) conformation of metal-depleted SOD1 that triggers aberrant oligomerization. This excited state emanates from the folded ground state and is suppressed by metal binding, but is present in both the disulfide-oxidized and disulfide-reduced forms of the protein. Our results pinpoint a perturbed region of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/ oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among different SOD1 variants that may explain why diverse mutations cause the same disease, and (ii) a structural basis that may aid in understanding how different mutations affect disease propensity and progression.amyotrophic lateral sclerosis ͉ nuclear magnetic resonance relaxation ͉ protein misfolding

Global structural motions from the strain of a single hydrogen bond

“…The results show that the population of the high-energy state decreases with increasing temperature, from p HS = 1.8 ± 0.1% at 278 K to p HS = 0.16 ± 0.02% at 298K ( Fig. 1 and Table 1), consistent with previous data for apoSOD1 pwt (11). Correspondingly, the exchange rate constant decreases from k ex = 5,800 s −1 to k ex = 2,000 s −1 ( Table 1).…”

“…1), in good agreement with previous CPMG measurements of apoSOD1 pwt (11) and H/D exchange analysis (16). Besides some loss of signal at positions that previously interfaced the dynamic loop-IV structure, the main effect of loop removal overall is lower CPMG amplitudes.…”

“…The dynamic motions of the apoSOD1 barrel are localized mainly to the β-strands facing the active site, and are indicated experimentally by diminished NMR NOE couplings, relaxation-dispersion measurements (11), and backbone hydrogen-exchange [hydrogen/deuterium (H/D)] kinetics (16). To enhance the structural resolution of these dynamic motions as much as possible, we focus here on a SOD1 variant with truncated loops IV and VII (apoSOD ΔIVΔVII ) (16).…”

“…As a consequence, the dimerization of SOD1 becomes tightly controlled by metal binding via the structure of loops IV and VII, and the conserved C57-C146 disulfide linkage (9): if the metals and disulfide bond are lost, the protein dissociates into free monomers. Metal loss also unleashes characteristic dynamic motions of the central barrel scaffolds (10)(11)(12)(13) that are most pronounced in the β-sheets facing the metal-binding sites. Together, these features form an intricate network of structural communication within the SOD1 molecule, spanning from the dynamic motions of the monomeric scaffold, via metal binding and the active-site loops, to the dimer interface.…”

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

“…These oligomers are thought to be responsible for the toxic gain of function, similar to what has been proposed for other neurodegenerative diseases [24][25][26][27] . Generation of soluble oligomers was found to occur through oxidation of the two free cysteines of SOD1 (C6 and C111) 28,29 , as well as through other possible mechanisms 30,31 , which give rise to various aggregation products 32,33 , including amyloid-like structures 34,35 .…”