SOD1 exhibits allosteric frustration to facilitate metal binding affinity

Abstract

Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several unique computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally designed SOD1 variants. Allosteric interaction-free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together, reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.

Fig. 1.

Force (A, a), work (A), and effective modulus (A, b) as a function of extension. Tethers are placed at the C_α atom closest to the center of mass of the SOD1 monomer (H46), and the C_α atom of either residues G10 (red) or I17 (green), in separate pulling assays. Ribbon representations of the protein are also shown; the tethering residue is shown in licorice rendering (in red) and the center C_α as a red sphere. The initial equilibrated (at 0 Å, green ribbon) and final (at 5 Å, blue ribbon) structures are aligned to each other by minimizing RMSD. (B, a) Work profiles of Cu,Zn(SS) WT (black), E,E(SH) WT (blue), and E,E G127X SOD1 (red) vs. sequence index. Secondary structure schematic is shown underneath. (B) Cumulative distributions of the work values in B, a. E,E G127X is more stable than full-length E,E (SH) SOD1 (P = 9e-7). (B, b) Fraction of the 48 incidences that each variant had either the weakest, strongest, or middle work value—e.g., E,E(SH) SOD1 is weakest 80% of the time and is never the strongest variant. (C) Cumulative work distributions for Cu,Zn (SS) WT (black), Cu,Zn G127X (green), E,E G127X (red), and Cu,Zn (SH) WT (cyan). Cu,Zn G127X is destabilized with respect to full-length Cu,Zn (SS) WT (P = 6.2e-8). (C, a) Same analysis as B, b for the variants Cu,Zn(SS) WT, Cu,Zn G127X, and E,E G127X . (D) Cumulative distributions for serine mutant SOD1 variants demonstrate that C-terminal truncation stabilizes the apo form but destabilizes the holo form (Results).

Fig. 2.

(A) Cumulative distributions of work values for C-terminal–truncated SOD1 variants of variable sequence length show a nonmonotonic trend in mechanical stability. All variants are metal-depleted and have no disulfide bond. Sequences are given in the legend (Results). (A, a) Comparing the cumulative distributions in A, that of the mutant G127X is most commonly the strongest, full-length SOD1 is most commonly the weakest, and 1–140 is most often in the middle. (A, b) Change in work value averaged over residues, as a function truncation length, for the SOD1 variants in A. (A, c) Ribbon schematics of the various truncation mutants, colored blue to red from N to C terminus, labeled by C-terminal residue. (B) Simulated native-basin dynamical fluctuations (RMSF) in explicit simple point charge (SPC) solvent, for Cu,Zn(SS) (black) and E,E(SS) SOD1 monomer (red), along with the experimentally measured ratio of spectral density functions J(ω_H)/J(ω_N) of obligate monomeric E,E(SS) F50E/G51E/E133Q SOD1 (blue bars) (31). Correlation coefficient is r = 0.78. (C) Simulated RMSF for SOD1 variants E,E G127X (black), E,E(SS) (red), E,E(SH) (blue), and E,E(SH) with the ESL constrained to be natively structured (magenta). The presence of native stress is indicated by the increased disorder of the ZBL upon structuring the ESL (Results). (D) Snapshots of typical structures of E,E(SH) and G127X SOD1 from equilibrium simulations, color coded by the mean RMSF for each residue; RMSF increases from blue to red according to the scale bars shown.

Fig. 3.

(A, top cartoon) Frustrated contacts (in red) and unfrustrated contacts (in green) for E,E(SS) WT SOD1. (A, bottom cartoon) Same contacts as top cartoon for the average over 22 ALS E,E(SS) mutants (Results). (A) The mean number of frustrated contacts within a sphere of radius 5 Å centered on each C_α atom is found as a function of residue index. Ensemble averages are taken from 50 snapshots in an equilibrium simulation. This is done for both the Cu,Zn(SS) state and the E,E(SS) state, for both the WT sequence, and for 22 mutant sequences. The 22 mutant sequences are averaged to obtain the ensemble and mutant-averaged number of contacts as a function of residue index i, 〈n^HF(i)〉_MUT. Plotted is the difference between 〈n^HF(i)〉_MUT and the corresponding numbers for the WT sequence . A positive number would indicate an increase in frustration upon mutation. Holo state is shown in blue and has an average of +5 contacts; apo state is shown in red and has an average of –22 contacts. (B) Interaction-free energy between a residue’s side chain and the Cu ion, plotted as a function of the E,E(SS) ensemble-averaged number of highly frustrated contacts that residue has (r = –0.79, P = 8e-21). (C) Same as in B but for the Zn ion (r = –0.81, P = 2e-22).

Fig. 4.

(A) Change in potential energy ΔU(t) as a function of in silico time, before and after implementing the mutation G37R. (B) Distribution of the asymptotic potential energy change ΔU(∞) for 22 ALS mutants (Results). (C) Mean potential energy change averaged over mutants, for both the holo state and the apo state, along with the mean difference, WT minus mutants, in both Cu and Zn binding-free energy.

Fig. 5.

(A) Residues color-coded by interaction energy with the Cu ion (depicted as a cyan sphere). The extent of interaction is strongest in magnitude for red colored residues and decreases to blue. (B) Same as A for the Zn ion (depicted as a gray sphere). (C) Interaction energy with Cu correlates with the distance of the residue from the Cu ion; residues in close proximity more strongly interact. (D) Interaction energy with Zn does not correlate with distance of the residue to the Zn ion, indicating nonlocal allosteric effects.