Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization - PubMed (original) (raw)

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

Kaare Teilum et al. Proc Natl Acad Sci U S A. 2009.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Transient structural distortion of monomeric WT SOD1. (A) Structure of the native Cu/Zn-bound SOD1 dimer (35). Green and magenta ribbons represent the two molecules. The solvent-accessible surface is outlined in green, and the dimer interface is colored magenta. The Cu (blue) and Zn (green) ions are indicated. β-strands are numbered 1–8 and loops I–VII. (B) Representative 15N CPMG relaxation dispersions (_R_2eff vs. νCPMG) recorded at 2 static magnetic fields (open symbols, 50.6 MHz; filled symbols, 60.8 MHz) and 3 temperatures (blue, 18 °C; green, 25 °C; red, 32 °C). The solid lines represent the global fit to a 2-state process. (C) Residues with perturbed structure in the excited state of apo-SOD1 are shown in red. The tube width encodes ΔωFE = 354-1720 s−1. Black denotes prolines and residues lacking data due to spectral overlap; gray denotes residues not showing relaxation dispersion. Left-side view: as for the right-side molecule in (A); right-hand view: rotated by 180° around the vertical axis. (D) Residues showing quantifiable exchange in Zn-SOD1 are colored blue; in other respects, the layout follows (C). The global fit yielded _k_ex = 22 ± 1 s−1 and 〈ΔωAB〉 = (14 ± 9) × 10 s−1.

Fig. 2.

Transient oligomerization in the excited state of WT apo-SOD1. (A) PRE, Γ2, of transverse 1H magnetization in WT apo-SOD1 in the presence of the spin-labeled apo-SOD1 variant Q153C-MTSL. The measured Γ2 is plotted versus residue number (black bars), with estimated errors indicated. The blue line shows the Γ2 profile expected for transient formation of the native dimer (3%). (B) Residues in WT apo-SOD1 with significant Γ2 (P < 0.01) are shown in red. The width of the worm diagram indicates the magnitude of Γ2, which scales with _r_−6, where _r_ is the distance from the spin label in the nonnative dimer or oligomer. Black denotes prolines and residues lacking data due to spectral overlap; gray denotes residues that do not experience significant Γ2. (_C_) Calculated values of Γ2 > 2 s−1 expected for transient formation of the native dimer are shown in blue [corresponding to the blue line in (A)]. (D) Representative experimental data showing PRE decays for A60 and G61 in the major and minor conformations of WT apo-SOD1 that are due to cis-trans isomerization around the G61–P62 peptide bond. The relative cross-peak intensity measured in MTSL-oxidized and MTSL-reduced samples is plotted versus the relaxation delay. The fitted values are Γ2 = 25.9 ± 1.4 s−1 for A60 and 9.1 ± 0.5 s−1 for G61.

Fig. 3.

Structural variation between the excited states of WT and mutant variants of apo-SOD1. (A) Structural location of the mutated residues in the SOD1 variants investigated herein. Blue, A4; green, G85; red, D90; yellow, G93. (B–D) The locations of residues that are perturbed in the excited state of the mutants, but not in WT apo-SOD1. (B) A4V: residues 4–6 (located in β1), 12 and 14 (loop I), and 45 (β4). (C) G85R: residues 34 (β3), 91 (loop V), and 98–99 (β6). (D) D90A: residues 39 (loop III), 45 (β4), 91–92 (loop V), 116 and 120 (β7). (E) Differences in the chemical shifts of the backbone amide 15N nuclei between the exchanging folded and excited states, obtained from CPMG relaxation dispersions. The magnitude of the chemical shift change, ΔωFE, is plotted versus residue number. The dashed lines indicate the locations of the mutated residues. The color code is as in (A)–(D). (F) The ΔωFE values obtained for each of the mutants plotted versus ΔωFE for WT SOD1. The Pearson correlation coefficient, r, and P values are as follows: A4V versus WT, r = 0.80 and P < 0.0001; G85R versus WT, r = 0.41 and P = 0.039; D90A versus WT, r = 0.86 and P < 0.0001.

Fig. 4.

Schematic diagram of the initial molecular events triggering aberrant oligomerization of apo-SOD1. (I) Unfolded apo-SOD1. (II) Folded ground state of monomeric apo-SOD1. (III) Native dimeric Cu/Zn-bound SOD1. (IV) Excited state of monomeric apo-SOD1 with destabilized edge β-strands and increased hydrophobic surface area. The excited state defines the gateway to oligomerization implicated in ALS. (V) Nonnative dimer or oligomer. Arrows indicate equilibria; the relative size of the arrows qualitatively depicts the balance across each equilibrium, which depends on the specific mutation, among other things.

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Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization - PubMed (original) (raw)