SOD1 mutations targeting surface hydrogen bonds promote amyotrophic lateral sclerosis without reducing apo-state stability

Abstract

In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

FIGURE 1.

The distribution of charged, polar, and hydrophobic amino acids in the SOD1 structure (PDB entry 1HL5) is shown as sticks. Positions with ALS-associated mutations are marked as spheres in the right monomer only. A, SOD1 dimer showing the positions of charged side chains, D (blue), E (light blue), K (red), and R (bright red), and histidines (cyan). B, structural positions of the polar (beige) side chains (S, T, C, N, and Q). C, structural positions of the hydrophobic (green) side chains (A, V, F, M, I, W, and L). Proline (white) and glycine (yellow). D, SOD1 amino acid sequence indicating the degree of side chain conservation across 17 eukaryotic species as visualized by Homologene (53). The degree of conservation is measured in bits (log₂(20) = 4.3 bits, where 20 is the number of possible amino acids in a peptide chain). For each sequence position there is a stack of amino acid occurrences. The height of each stack indicates the sequence conservation for that position (54). The height of each letter reflects the relative frequency for the amino acid in question and the letter at the top of each stack is the most frequent in that position. Multiple alignments were made with the T-coffee software using default parameters (55). The consensus sequence was made with the WebLogo software (54).

FIGURE 2.

Structural positions of the ALS-associated SOD1 mutations examined in this study. Each mutant is color coded with a corresponding color-coded close-up on the affected hydrogen bonds. A, front view of SOD1 dimer and side view of monomer (PDB entry 1HL5). B–F, close-ups of the local structural environments of Asp⁹⁰, Asn¹³⁹, Asp⁷⁶, Asn⁸⁶, and Asp¹⁰¹, respectively.

FIGURE 3.

Chevron plots of D76V, D76Y, N86D, N86K, and N86S. k_f and k_u are the rate constants for folding and refolding, respectively, and the dashed line shows the urea dependence of log k_d. apoSOD_mono^pWT (●), apoSOD_mono^mut (○), apoSOD_dimer^pWT (■) and apoSOD_dimer^mut (□). The units of [urea] and k are M and s⁻¹, respectively.

FIGURE 4.

Chevron plots of D90V, D90A (17), D101N, D101G, N139D, and N139K. apoSOD_mono^pWT (●), apoSOD_mono^mut (○), apoSOD_dimer^pWT (■) and apoSOD_dimer^mut (□). The units of [urea] and k are M and s⁻¹, respectively.

FIGURE 5.

Plots of patient survival time after first diagnosis versus the stability loss of the ALS-provoking SOD1 mutation (ΔΔG^norm). Data are from supplemental Table S1. The dotted lines are tentatively set at 5 years to denote the division between short and medium/long survival times. A, plot of the combined data set, showing the mutations that do not affect charges (black), the mutations that decrease the net repulsive charge (blue), and the mutations that increase the net repulsive charge (red). Solid circles denotes n > 5 and open circles n < 5 (supplemental Table S1). The red, blue, and black arrows show the average ΔΔG^norm of each mutant group. B, subset of mutations that do not affect charges, hydrogen bonds or directly affect metal binding, yields an apparent correlation of r = 0.78. Although the statistical significance of these patterns are not yet clear, it is interesting to note that they comply with a reductionist disease model based on the propensity of SOD1 aggregation.

FIGURE 6.

The statistical distribution of charge alterations among ALS-associated SOD1 mutations with survival times below and above 5 years after first diagnosis. A, fractions of the total number of different ALS mutations. B, absolute numbers of different ALS mutations. C, fractions of the total number of ALS cases involving SOD1 mutations.