Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants

Abstract

Amyotrophic lateral sclerosis is a neurodegenerative syndrome associated with 114 mutations in the gene encoding the cytosolic homodimeric enzyme Cu/Zn superoxide dismutase (SOD). In this article, we report that amyotrophic lateral sclerosis-associated SOD mutations with distinctly different disease progression can be rationalized in terms of their folding patterns. The mutations are found to perturb the protein in multiple ways; they destabilize the precursor monomers (class 1), weaken the dimer interface (class 2), or both at the same time (class 1 + 2). A shared feature of the mutational perturbations is a shift of the folding equilibrium toward poorly structured SOD monomers. We observed a link, coupled to the altered folding patterns, between protein stability, net charge, and survival time for the patients carrying the mutations.

Fig. 1.

The structure of homodimeric SOD (PDB ID code 1SPD), showing the positions of the 15 ALS-associated mutations analyzed in this study.

Fig. 2.

The folding kinetic of SOD. (Upper) Chevron plots for pseudo-WT SOD (black) and representative mutants (red) exemplifying the class 1, 2, and 1 + 2 behavior associated with neurotoxic function in ALS. Units are given in s^–1. (•), (▪), (□), and (▵). The fits are from Schemes 1 (Eq. 1) and 2 (25, 51), and the dotted line shows specifically the urea dependence of log k_d for . The small offset in log k_f observed for all monomeric proteins is due to the dimer-splitting substitutions F50E/G51E (25). (Lower) Schematic folding free-energy profiles at 0 M urea showing the energetic perturbations induced by the mutants. The class 1 mutant D90A shows a selective destabilization of the monomer without affecting the dimer interface (Δlog k_d = 0). In contrast, the class 2 mutant L144F has no effect on the monomer stability but fails to dimerize. The class 1 + 2 mutants G41S and I104F affect both the monomer and interface stability. Chevron plots for the other mutants in Tables 1 and 2 are given in the supporting information.

Fig. 3.

Size-exclusion chromatograms showing the dimer and monomer composition of (black), (blue), (green), and (red). Dimers elute at 10.5 ml, and folded monomers elute at 12 ml. Consistent with the kinetic data, L144F displays an intermediate elution position indicating that this mutant describes a dynamic equilibrium between the monomeric and homodimeric state.

Fig. 4.

Schematic representation of the three-state folding reaction of SOD, including the different effects of the 15 ALS-associated mutations characterized in this study. D, unfolded protein; M, folded monomer; M₂, homodimer. Common for the class 1, 2, and 1 + 2 mutations is a shift of the folding equilibrium toward unfolded and partly structure monomers, indicating that the starting material for the toxic gain of function is to be found within this broad ensemble of poorly defined species.

Fig. 5.

Plots of protein stability changes (ΔΔG) vs. average survival time after diagnosis. ΔΔG for ALS-associated mutations that do not alter the net charge of SOD shows a high correlation with survival time (R = 0.91). Increasing the net charge of the protein causes a shift toward longer survival time, whereas decreasing the charge has the opposite effect. This behavior is expected for a disease mechanism associated with protein aggregation (43).