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Structures of fibrils formed by α-synuclein hereditary disease mutant H50Q reveal new polymorphs

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Abstract

Deposits of amyloid fibrils of α-synuclein are the histological hallmarks of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, with hereditary mutations in α-synuclein linked to the first two of these conditions. Seeing the changes to the structures of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of α-synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two previously unobserved polymorphs of α-synuclein: narrow and wide fibrils, formed from either one or two protofilaments, respectively. These structures recapitulate conserved features of the wild-type fold but reveal new structural elements, including a previously unobserved hydrogen-bond network and surprising new protofilament arrangements. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells and greater cytotoxicity that we observe for H50Q compared to wild-type α-synuclein.

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Fig. 1: Comparison of wild-type and H50Q polymorphs.
Fig. 2: Cryo-EM structures of H50Q polymorphs.
Fig. 3: Comparison of protofilaments A and B.
Fig. 4: Solvation energy maps and biochemical characterization of H50Q and wild-type α-syn.

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Data availability

All structural data have been deposited into the Protein Database (PDB) and the Electron Microscopy Data Bank (EMDB) with the following accession codes: H50Q narrow fibril (PDB 6PEO, EMD-20328) and H50Q wide fibril (PDB 6PES, EMD-20331). All other data are available from the authors upon reasonable request.

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Acknowledgements

We thank H. Zhou for use of Electron Imaging Center for Nanomachines (EICN) resources and P. Ge for assistance in cryo-EM data collection. We acknowledge the use of instruments at the EICN supported by NIH (grant nos. 1S10RR23057 and 1S10OD018111), NSF (grant no. DBI-1338135) and CNSI at UCLA. The authors acknowledge grant nos. NIH AG 060149, NIH AG 054022, NIH AG061847 and DOE DE-FC02-02ER63421 for support. D.R.B. was supported by the National Science Foundation Graduate Research Fellowship Program.

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Author notes

  1. These authors contributed equally: David R. Boyer, Binsen Li.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry and Biological Chemistry, UCLA-DOE Institute, Molecular Biology Institute and Howard Hughes Medical Institute, UCLA, Los Angeles, CA, USA

    David R. Boyer, Michael R. Sawaya & David S. Eisenberg

  2. Department of Neurology, David Geffen School of Medicine, Molecular Biology Institute, UCLA, Los Angeles, CA, USA

    Binsen Li, Chuanqi Sun, Weijia Fan & Lin Jiang

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Contributions

D.R.B. and B.L. designed experiments and performed data analysis. B.L. and C.S. expressed and purified the α-syn protein. B.L. grew fibrils of α-syn and performed biochemical experiments. D.R.B. and B.L prepared cryo-EM samples and performed cryo-EM data collection. B.L. and W.F. selected filaments from cryo-EM images. D.R.B. performed cryo-EM data processing and built the atomic models. M.R.S. wrote the software for and D.R.B. carried out solvation energy calculations. All authors analyzed the results and D.R.B wrote the manuscript with input from all authors. L.J. and D.S.E. supervised and guided the project.

Corresponding authors

Correspondence to Lin Jiang or David S. Eisenberg.

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Competing interests

D.S.E. is an advisor and equity shareholder in ADRx, Inc.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Fourier Shell Analysis.

a) Helical reconstructions of Narrow and Wide Fibrils with minimum and maximum widths labeled. b) Gold-standard half map FSC curves for Narrow (top, left) and Wide (top, right) Fibrils. Map-model FSC curve for Narrow and Wide Fibrils (bottom).

Extended Data Fig. 2 Cryo-EM images and processing.

a) Cryo-EM micrographs and 2D class averages of Narrow (left) and Wide (right) Fibrils. Scale bar = 50 nm. b) 1024 and 288 pixel box size class averages of the Narrow Fibril used to determine crossover distance. 288 pixel box map projections match 2D class averages. c) 686 pixel box size class averages used to determine crossover distance. 686 pixel box map projections match 2D class averages. d) Wide Fibril class averages with a 320 pixel box demonstrate a lack of two-fold symmetry across the fibril axis.

Extended Data Fig. 3 Speculative Atomic Models for Islands 1 and 2.

a) Schematic illustrating possible sequences occupying Islands 1 and 2. 8mers from residues 1–19 and 112–140 were considered as possibilities to occupy Island 1. Island 2 is considered to consist of residues 26–31 followed by a disordered linker formed by residues 32KTKE35. b) Illustration of possible regions from either Protofilament A (left) or Protofilament B (right) that could occupy Island 1. Note that residues from the N-terminus of Protofilament A could account for Island 1 in both the Narrow and Wide Fibril; however, only the Narrow Fibril model is shown here. Island 2 is thought to be formed by the N-terminus of Protofilament A in both Narrow and Wide Fibrils. c) Speculative models for Islands 1 and 2. Check marks indicate plausible models while X’s indicate implausible models. Island 1 models are from either the N-terminus of Protofilament A (blue panels) or the C-terminus of Protofilament B (green panels). Examples of sequences that were found to not be allowed to occupy Island 1 are shown with red dashed circles highlighting steric clashes or β-strand breaking proline residues.

Extended Data Fig. 4 Alternate conformations of K58 and T59 and potential solvent molecules in the α-syn β-arch cavity.

a) Wild-type and H50Q fibrils display alternate conformations of K58 and T59. We note that in order for the Wide Fibril to form, T59 needs to be facing away from the fibril core. Therefore the formation of the Wide Fibril is mutually exclusive with our wild-type rod polymorph. b) Environmental distances of putative water molecule for Protofilament A and B in Wide Fibril and b) Protofilament A in Narrow Fibril.

Extended Data Fig. 5 PreNAC homozipper Island 1 model and additional solvation energy maps.

a) Speculative model of preNAC residues 50QGVATVA56 occupying Island 1 in Protofilament A. b) Atomic solvation map and energetic calculations for Protofilament A with Island 1 as 50QGVATVA56 and Island 2 as 26VAEAAG31. c) Atomic energy solvation map for Wild-type rod polymorph (6cu7). Notice that K58 and T59 can have favorable stabilization energies whether they are facing the solvent or facing the cavity in the β-arch.

Extended Data Fig. 6 Comparison of α-syn protofilament interfaces.

a) Wide Fibril overview (left). 56AEKTKEQV63 homointerface with Wide Fibril electron density (middle). 56AEKTKEQV63 homointerface showing a 2.4 Å rise between mated strands from Protofilament A and Protofilament B and a distance of 7.8 Å between mated sheets of Protofilament A and B (right). b) Van der Waal’s surface, buried surface area, and shape complementarity of 58KTKE61 homointerface, preNAC interface, and NACore interface.

Extended Data Fig. 7 H50Q disrupts the wild-type rod polymorph preNAC protofilament interface.

a) Conformation of H50Q Protofilament A K45 and H50Q. b) Interaction of K45-H50-E57 in the wild-type rod polymorph protofilament interface. c) Hypothetical H50Q double protofilament using the preNAC of Protofilament A as a steric zipper interface. Notice that the H50Q mutation disfavors the protofilament interface due to steric clashes with E57. d) Hypothetical H50Q protofilament interface using preNAC of Protofilament B. Notice the steric clashes between H50Q and E57 at the hypothetical protofilament interface as well as clashes of other parts of the protofilament with Protofilament A.

Extended Data Fig. 8 H50Q fibrils disrupt PC12 cell membranes more than WT fibrils.

Differentiated PC12 cells were treated with sonicated WT and H50Q fibrils and cell permeability was measured via LDH activity in the media (see Methods). H50Q leads to significantly higher cell permeabilization at 1000 and 2000 nM than WT a-syn. Error bars represent standard deviation of four independent experiments. **** = p-value ≤ 0.0001. *** = p-value ≤ 0.001. ns = p-value > 0.05. P-values were calculated using an unpaired, two-tailed t-test with a 95% CI.

Extended Data Fig. 9 Structural alignment of different wild-type and mutant α-syn polymorphs.

a) Structural alignment of H50Q Protofilament A with all wild-type structures determined thus far. b) Structural alignment of residues 50–57 in wild-type and mutant α-syn polymorphs reveals the kernel region is largely conserved while tail regions, especially the N-terminus, adopt variable conformations.

Extended Data Fig. 10

Schematic illustrating possible secondary nucleation of Protofilament B by Narrow Fibrils.

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Boyer, D.R., Li, B., Sun, C. et al. Structures of fibrils formed by α-synuclein hereditary disease mutant H50Q reveal new polymorphs. Nat Struct Mol Biol 26, 1044–1052 (2019). https://doi.org/10.1038/s41594-019-0322-y

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