Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS) - PubMed (original) (raw)

. 2017 Aug 15;114(33):8770-8775.

doi: 10.1073/pnas.1705091114. Epub 2017 Jul 31.

Anni Zhao 1, Katrina L Adams 2, Christina K Jayson 3, Michael R Sawaya 1, Elizabeth L Guenther 1, Albert C Pan 4, Jennifer Ngo 3, Destaye M Moore 2, Angela B Soriaga 1, Thanh D Do 5, Lukasz Goldschmidt 1, Rebecca Nelson 1, Michael T Bowers 5, Carla M Koehler 3, David E Shaw 4 6, Bennett G Novitch 2, David S Eisenberg 7

Affiliations

Atomic structure of a toxic, oligomeric segment of SOD1 linked to amyotrophic lateral sclerosis (ALS)

Smriti Sangwan et al. Proc Natl Acad Sci U S A. 2017.

Abstract

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.

Keywords: ALS; SOD1; oligomer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Structure of an 11-residue segment derived from SOD1 in its oligomeric state. (A) Crystal structure of the SOD1 segment with the sequence KVKVWGSIKGL at 2.0-Å resolution shows antiparallel, out-of-register β-strands forming a continuous left-handed helix. Sixteen strands form one complete turn of the helix, with a 25-Å outer diameter and 71-Å pitch. The hydrophobic interior lined with valine, isoleucine, and leucine side chains (shown in spheres) excludes water molecules, as shown in side and top views. (B) Model of a full-length SOD1 toxic oligomer. This model contains 16 protomers. Strands 2 and 3 of each protomer detach from the native fold to form the corkscrew spine (green interior, blue exterior) as observed in the crystal structure of residues 28–38. The remaining protein decorates the exterior of corkscrew, retaining much of the SOD1 native structure. The model is illustrated in the same two orientations as A. One monomer is colored brown for clarity. (C) Gly33 is essential for creating the concave inner surface of the corkscrew. The lack of a side chain on Gly33 permits the close approach of hydrophobic side chains of Val29 and Val31 located on β-strands bordering opposite sides of Gly33. (D) Unavoidable steric clash results from mutating Gly33 to tryptophan. (E) All-atom MD simulations of the corkscrew-forming segment suggest that introduction of the G33W substitution destabilizes the structure. Blue and red curves correspond to Cα rmsd from the corkscrew crystal structure in MD simulations of eight chains of the corkscrew segment (KVKVWGSIKGL) and G33W mutant segment (KVKVWWSIKGL), respectively. The structure of the corkscrew segment remained stable throughout the length of the simulation, whereas the G33W mutant deviated from the corkscrew structure. (F) SOD1 segment 28–38 preferentially assembled into a corkscrew-like structure in an MD simulation. MD simulations of weakly restrained monomers of SOD1 spontaneously assembled into a corkscrew-like structure. A snapshot of an assembled corkscrew-like structure from the MD simulations (green) is overlaid onto the crystal structure (blue). As a control, we found that monomers of the cylindrin-forming segment of αB-crystallin spontaneously assembled into a cylindrin structure using the same simulation protocol (additional simulation details are provided in SI Appendix, Fig. S5_B_).

Fig. 2.

Fig. 2.

Corkscrew-forming segment 28–38 is necessary and sufficient for cytotoxicity. (A) Cell viability of motor neurons measured by an MTT reduction assay shows that the corkscrew segment (KVKVWGSIKGL) is toxic to primary motor neurons in a dose-dependent manner. Results are shown as mean ± SD (n = 3). Symbols represent individual values of triplicates, and bars represent average values. Statistical significance was analyzed using two-tailed t tests with Welch’s correction. (B) Corkscrew-forming segment (28–38) harboring single-point substitutions at Gly33 (G33V and G33W) is nontoxic to motor neurons. All peptide segments were prepared identically, and motor neurons were treated with different final concentrations. The statistical significance of G33V and G33W mutants was compared with segment 28–38 by two-way ANOVA. (C) Hb9-GFP–labeled motor neurons treated with 8 μM aggregated full-length familial mutants (A4V and G93A) lose neurites, but the corresponding corkscrew-disrupting mutants (G93A/G33V, G93A/G33W, A4V/G33V, and A4V/G33W) are nontoxic and neurons look healthy. (Scale bars, 20 μm.) (D) Cell viability measured by an MTT reduction assay confirming that the familial mutants A4V and G93A are toxic and that substitution of Gly33 with valine or tryptophan renders the protein nontoxic. Results are shown as mean ± SD (n = 3). Symbols represent individual values of triplicates, and bars represent average values. Statistical significance was analyzed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3.

Fig. 3.

Toxicity of full-length SOD1 derives from soluble oligomers. (A) Electron micrographs of a non–fibril-forming SOD1 mutant (I104P) and the corresponding double mutant (I104P/G33W) show some aggregates but no large fibrils. (B) Motor neurons treated with I104P lose neurites and have shrunken cell bodies (Left), but I104P/G33W-treated cells look healthy (Right). (Scale bars, 20 μm.) (C) Cell viability measured by an MTT reduction assay confirmed that I104P is toxic and I104P/G33W is nontoxic. Statistical significance was analyzed using a two-tailed t test with Welch’s correction. (D and E) Toxic properties of SOD1 mutants depend on the duration of aggregation. The A4V and G93A mutants aggregated for 12–16 h are toxic to motor neurons, whereas extended agitation for 72 h renders the proteins nontoxic. The corkscrew-disrupted proteins (A4V/G33W and G93A/G33W) are nontoxic irrespective of the duration of aggregation. Results are shown as mean ± SD (n = 3). Symbols represent individual values of triplicates, and bars represent average values. Statistical significance was analyzed by two-tailed t tests with Welch’s correction (*P < 0.05, **P < 0.01). (F) Representative electron micrographs of various preparations of the familial mutants A4V and G93A and the double mutants A4V/G33W and G93A/G33W. Some large aggregates can be seen at 2- to 16-h time points, but no fibrils can be seen, whereas all constructs show large fibril loads at 72 h. (G) Immunoblots of the familial mutants aggregated for different time points. Samples aggregated for 12–16 h are A11-positive, and both proteins lose A11 reactivity when aggregated for 72 h. SOD100 was used as a loading control.

Fig. 4.

Fig. 4.

Corkscrew-disrupting substitution of G33V alleviates axonopathies in a D. rerio (zebrafish) ALS model. (A) Micrographs of zebrafish embryos at 2 d postfertilization (dpf) show a reduction in axon lengths of A4V-expressing embryos, whereas corkscrew-disrupted A4V/G33V-expressing embryos have significantly longer axon lengths. The first axon is highlighted in pink for clarity, and axons measured for quantification are marked by an asterisk. (Scale bars, 100 μm.) (B) Quantification of axon lengths shows that A4V-expressing embryos have shorter axons than WT-expressing embryos. The corkscrew-disrupting substitution G33V alleviates the defect. Results are shown as mean ± SEM relative to WT for at least 72 embryos. Statistical significance was analyzed by one-way ANOVA. (C) Micrographs of zebrafish embryos at 2 dpf show a reduction in axon lengths of G93A-expressing embryos, whereas corkscrew-disrupted G93A/G33V-expressing embryos have significantly longer axons. The first axon is highlighted in pink for clarity, and axons measured for length are marked by an asterisk. (Scale bars, 100 μm.) (D) Quantifications of axon lengths show that G93A-expressing fish have shorter axons than WT-expressing fish. The corkscrew-disrupting substitution G33V alleviates the defect. Results are shown as mean ± SEM relative to WT for axons 12–16 of at least 73 embryos. Statistical significance was analyzed by one-way ANOVA. (E) Micrographs show A4V-expressing zebrafish have impaired mitochondria, which are clustered at the branch points (encircled) compared with WT. The mitochondrial network of A4V/G33V-expressing fish is similar to WT. (Scale bar, 100 μm.) (F and G) Quantitative analysis of the mitochondrial network shows A4V-expressing fish have a larger mitochondrial size [30.17 arbitrary units (a.u.)] and diffused clustering (fluorescence intensity of 32 a.u.) in the axons, indicative of defective fission, whereas A4V/G33V-expressing fish have healthy mitochondria (size of 10.57 a.u. and fluorescence intensity of 54 a.u.) similar to WT (size of 14.08 a.u. and fluorescence intensity of 58 a.u.). Symbols represent individual measurements made for each group. Statistical significance was analyzed by one-way ANOVA. (H and I) Quantitative analysis of the mitochondria network shows G93A-expressing fish have larger, more diffusely clustered mitochondria (size of 14.68 a.u. and fluorescence intensity of 39 a.u.), indicative of defective fission. The mitochondrial network of G93A/G33V-expressing fish (size of 11.68 a.u. and fluorescence intensity of 59 a.u.) is similar to WT (size of 10.78 a.u. and fluorescence intensity of 78 a.u.). Statistical significance was analyzed by one-way ANOVA. Symbols represent individual measurements for each group (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

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