Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Structure of the toxic core of α-synuclein from invisible crystals

Abstract

The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: NACore (residues 68–78) is the fibril-forming core of the NAC domain of full-length α-synuclein.
Figure 2: Diffraction from NACore nanocrystals is similar to that from full length α-synuclein fibrils.
Figure 3: Structure of the amyloid core of α-synuclein.
Figure 4: NACore aggregates faster than SubNACore and is more cytotoxic to cultured cells.

Similar content being viewed by others

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 4RIK (SubNACore), 4RIL (NACore) and 4ZNN (PreNAC). The maps for PreNAC and NACore have been deposited in the EMDB with accession codes EMD-3001 and EMD-3028, respectively.

References

  1. Spillantini, M. G. et al. α-Synuclein in Lewy bodies. Nature 388, 839–840 (1997)

    Article  ADS  CAS  Google Scholar 

  2. Goedert, M., Spillantini, M. G., Del Tredici, K. & Braak, H. 100 years of Lewy pathology. Nature Rev. Neurol. 9, 13–24 (2013)

    Article  CAS  Google Scholar 

  3. Polymeropoulos, M. H. et al. Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047 (1997)

    Article  CAS  Google Scholar 

  4. Krüger, R. et al. Ala30Pro mutation in the gene encoding α-synuclein in Parkinson’s disease. Nature Genet. 18, 106–108 (1998)

    Article  Google Scholar 

  5. Zarranz, J. J. et al. The new mutation, E46K, of α-synuclein causes Parkinson and Lewy body dementia. Ann. Neurol. 55, 164–173 (2004)

    Article  CAS  Google Scholar 

  6. Ibáñez, P. et al. Causal relation between α-synuclein gene duplication and familial Parkinson’s disease. Lancet 364, 1169–1171 (2004)

    Article  Google Scholar 

  7. Singleton, A. B. et al. α-Synuclein locus triplication causes Parkinson’s disease. Science 302, 841 (2003)

    Article  CAS  Google Scholar 

  8. Uéda, K. et al. Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease. Proc. Natl Acad. Sci. USA 90, 11282–11286 (1993)

    Article  ADS  Google Scholar 

  9. Biere, A. L. et al. Parkinson’s disease-associated α-synuclein is more fibrillogenic than β- and γ-synuclein and cannot cross-seed its homologs. J. Biol. Chem. 275, 34574–34579 (2000)

    Article  CAS  Google Scholar 

  10. Giasson, B. I., Murray, I. V. J., Trojanowski, J. Q. & Lee, V. M.-Y. A hydrophobic stretch of 12 amino acid residues in the middle of α-synuclein is essential for filament assembly. J. Biol. Chem. 276, 2380–2386 (2001)

    Article  CAS  Google Scholar 

  11. Du, H.-N. et al. A peptide motif consisting of glycine, alanine, and valine is required for the fibrillization and cytotoxicity of human α-synuclein. Biochemistry 42, 8870–8878 (2003)

    Article  CAS  Google Scholar 

  12. Periquet, M., Fulga, T., Myllykangas, L., Schlossmacher, M. G. & Feany, M. B. Aggregated α-synuclein mediates dopaminergic neurotoxicity in vivo. J. Neurosci. 27, 3338–3346 (2007)

    Article  CAS  Google Scholar 

  13. Han, H., Weinreb, P. H. & Lansbury, P. T. The core Alzheimer’s peptide NAC forms amyloid fibrils, which seed and are seeded by β-amyloid: is NAC a common trigger or target in neurodegenerative disease? Chem. Biol. 2, 163–169 (1995)

    Article  CAS  Google Scholar 

  14. El-Agnaf, O. M. et al. Aggregates from mutant and wild-type α-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β-sheet and amyloid-like filaments. FEBS Lett. 440, 71–75 (1998)

    Article  CAS  Google Scholar 

  15. Crowther, R. A., Daniel, S. E. & Goedert, M. Characterisation of isolated α-synuclein filaments from substantia nigra of Parkinson’s disease brain. Neurosci. Lett. 292, 128–130 (2000)

    Article  CAS  Google Scholar 

  16. Der-Sarkissian, A., Jao, C. C., Chen, J. & Langen, R. Structural organization of α-synuclein fibrils studied by site-directed spin labeling. J. Biol. Chem. 278, 37530–37535 (2003)

    Article  CAS  Google Scholar 

  17. Chen, M., Margittai, M., Chen, J. & Langen, R. Investigation of α-synuclein fibril structure by site-directed spin labeling. J. Biol. Chem. 282, 24970–24979 (2007)

    Article  CAS  Google Scholar 

  18. Miake, H., Mizusawa, H., Iwatsubo, T. & Hasegawa, M. Biochemical characterization of the core structure of α-synuclein filaments. J. Biol. Chem. 277, 19213–19219 (2002)

    Article  CAS  Google Scholar 

  19. Conway, K. A., Harper, J. D. & Lansbury, P. T. Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease. Nature Med. 4, 1318–1320 (1998)

    Article  CAS  Google Scholar 

  20. Polymeropoulos, M. H. et al. Mutation in the α-synuclein gene identified in families with Parkinson’s disease. Science 276, 2045–2047 (1997)

    Article  CAS  Google Scholar 

  21. Nelson, R. et al. Structure of the cross-β spine of amyloid-like fibrils. Nature 435, 773–778 (2005)

    Article  ADS  CAS  Google Scholar 

  22. Sawaya, M. R. et al. Atomic structures of amyloid cross-β spines reveal varied steric zippers. Nature 447, 453–457 (2007)

    Article  ADS  CAS  Google Scholar 

  23. Bodles, A. M., Guthrie, D. J., Greer, B. & Irvine, G. B. Identification of the region of non-Aβ component (NAC) of Alzheimer’s disease amyloid responsible for its aggregation and toxicity. J. Neurochem. 78, 384–395 (2001)

    Article  CAS  Google Scholar 

  24. Nannenga, B. L. & Gonen, T. Protein structure determination by MicroED. Curr. Opin. Struct. Biol. 27, 24–31 (2014)

    Article  CAS  Google Scholar 

  25. Nannenga, B. L., Shi, D., Leslie, A. G. W. & Gonen, T. High-resolution structure determination by continuous-rotation data collection in MicroED. Nature Methods 11, 927–930 (2014)

    Article  CAS  Google Scholar 

  26. Shi, D., Nannenga, B. L., Iadanza, M. G. & Gonen, T. Three-dimensional electron crystallography of protein microcrystals. eLife 2, e01345 (2013)

    Article  Google Scholar 

  27. Nannenga, B. L., Shi, D., Hattne, J., Reyes, F. E. & Gonen, T. Structure of catalase determined by MicroED. eLife 3, e03600 (2014)

    Article  Google Scholar 

  28. Yonekura, K., Kato, K., Ogasawara, M., Tomita, M. & Toyoshima, C. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges. Proc. Natl Acad. Sci. USA 112, 3368–3373 (2015)

    Article  ADS  CAS  Google Scholar 

  29. Doyle, P. A. & Turner, P. S. Relativistic Hartree–Fock X-ray and electron scattering factors. Acta Crystallogr. A 24, 390–397 (1968)

    Article  ADS  CAS  Google Scholar 

  30. Sarafian, T. A. et al. Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein. PLoS ONE 8, e63557 (2013)

    Article  ADS  CAS  Google Scholar 

  31. Caughey, B. & Lansbury, P. T. Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders. Annu. Rev. Neurosci. 26, 267–298 (2003)

    Article  CAS  Google Scholar 

  32. Danzer, K. M., Schnack, C., Sutcliffe, A., Hengerer, B. & Gillardon, F. Functional protein kinase arrays reveal inhibition of p-21-activated kinase 4 by α-synuclein oligomers. J. Neurochem. 103, 2401–2407 (2007)

    Article  CAS  Google Scholar 

  33. Karpinar, D. P. et al. Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson’s disease models. EMBO J. 28, 3256–3268 (2009)

    Article  CAS  Google Scholar 

  34. Winner, B. et al. In vivo demonstration that α-synuclein oligomers are toxic. Proc. Natl Acad. Sci. USA 108, 4194–4199 (2011)

    Article  ADS  CAS  Google Scholar 

  35. Chen, S. W. et al. Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation. Proc. Natl Acad. Sci. USA 112, E1994–E2003 (2015)

    Article  CAS  Google Scholar 

  36. Bousset, L. et al. Structural and functional characterization of two α-synuclein strains. Nature Commun. 4, 2575 (2013)

    Article  ADS  Google Scholar 

  37. Auluck, P. K., Caraveo, G. & Lindquist, S. α-Synuclein: membrane interactions and toxicity in Parkinson’s disease. Annu. Rev. Cell Dev. Biol. 26, 211–233 (2010)

    Article  CAS  Google Scholar 

  38. Lee, J. C., Langen, R., Hummel, P. A., Gray, H. B. & Winkler, J. R. α-Synuclein structures from fluorescence energy-transfer kinetics: implications for the role of the protein in Parkinson’s disease. Proc. Natl Acad. Sci. USA 101, 16466–16471 (2004)

    Article  ADS  CAS  Google Scholar 

  39. Sievers, S. A. et al. Structure-based design of non-natural amino-acid inhibitors of amyloid fibril formation. Nature 475, 96–100 (2011)

    Article  CAS  Google Scholar 

  40. Comellas, G. et al. Structured regions of α-synuclein fibrils include the early-onset Parkinson’s disease mutation sites. J. Mol. Biol. 411, 881–895 (2011)

    Article  CAS  Google Scholar 

  41. Vilar, M. et al. The fold of α-synuclein fibrils. Proc. Natl Acad. Sci. USA 105, 8637–8642 (2008)

    Article  ADS  CAS  Google Scholar 

  42. Goldschmidt, L., Teng, P. K., Riek, R. & Eisenberg, D. Identifying the amylome, proteins capable of forming amyloid-like fibrils. Proc. Natl Acad. Sci. USA 107, 3487–3492 (2010)

    Article  ADS  CAS  Google Scholar 

  43. Otwinowski, Z. & Minor, W. in Methods in Enzymology (eds Carter, C. W. Jr & Sweet, R. M.) Vol. 276 307–326 (Academic Press, 1997)

    Google Scholar 

  44. Weierstall, U., Spence, J. C. H. & Doak, R. B. Injector for scattering measurements on fully solvated biospecies. Rev. Sci. Instrum. 83, 035108 (2012)

    Article  ADS  CAS  Google Scholar 

  45. Hattne, J. et al. Accurate macromolecular structures using minimal measurements from X-ray free-electron lasers. Nature Methods 11, 545–548 (2014)

    Article  CAS  Google Scholar 

  46. Sauter, N. K., Hattne, J., Grosse-Kunstleve, R. W. & Echols, N. New Python-based methods for data processing. Acta Crystallogr. D 69, 1274–1282 (2013)

    Article  CAS  Google Scholar 

  47. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    Article  CAS  Google Scholar 

  48. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  49. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    Article  CAS  Google Scholar 

  50. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    Article  CAS  Google Scholar 

  51. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    Article  CAS  Google Scholar 

  52. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  53. Delano, W. The PyMOL Molecular Graphics System (Schrödinger LLC). http://www.pymol.org

  54. Jakes, R., Spillantini, M. G. & Goedert, M. Identification of two distinct synucleins from human brain. FEBS Lett. 345, 27–32 (1994)

    Article  CAS  Google Scholar 

  55. Johnson, M., Coulton, A. T., Geeves, M. A. & Mulvihill, D. P. Targeted amino-terminal acetylation of recombinant proteins in E. coli. PLoS ONE 5, e15801 (2010)

    Article  ADS  CAS  Google Scholar 

  56. Whitelegge, J. P., Zhang, H., Aguilera, R., Taylor, R. M. & Cramer, W. A. Full subunit coverage liquid chromatography electrospray ionization mass spectrometry (LCMS+) of an oligomeric membrane protein: cytochrome b 6 f complex from spinach and the cyanobacterium Mastigocladus laminosus. Mol. Cell. Proteomics MCP 1, 816–827 (2002)

    Article  CAS  Google Scholar 

  57. Arvai, A. Adxv - A Program to Display X-ray Diffraction Images (2015)

    Google Scholar 

  58. Rao, J. N., Jao, C. C., Hegde, B. G., Langen, R. & Ulmer, T. S. A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins. J. Am. Chem. Soc. 132, 8657–8668 (2010)

    Article  CAS  Google Scholar 

  59. Brunger, A. T. Version 1.2 of the crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    Article  CAS  Google Scholar 

  60. Fabiola, F., Bertram, R., Korostelev, A. & Chapman, M. S. An improved hydrogen bond potential: impact on medium resolution protein structures. Protein Sci. 11, 1415–1423 (2002)

    Article  CAS  Google Scholar 

  61. Read, R. J. Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallogr. A 42, 140–149 (1986)

    Article  Google Scholar 

Download references

Acknowledgements

We thank C. Liu for supplying PC12 cells; APS staff for beam line help solving SubNACore: M. Capel, K. Rajashankar, N. Sukumar, J. Schuermann, I. Kourinov and F. Murphy at NECAT beam lines 24-ID at APS funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403) and the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We thank the LCLS injection staff support: S. Botha, R. Shoeman and I. Schlichting. A.S.B. and N.K.S. were supported by NIH grants GM095887 and GM102520 and by the Director, Office of Science, Department of Energy (DOE) under contract DE-AC02-05CH11231 for data-processing methods. This work was supported by the US Department of Energy Office of Science, Office of Biological and Environmental Research program under award number DE-FC02-02ER63421. We also acknowledge the award MCB-0958111 from the National Science Foundation, award 1R01-AG029430 from the National Institutes of Health, award NIH-AG016570 from Alzheimer’s Disease Research (ADRC) at UCLA, and HHMI for support. J.A.R. was supported by the Giannini Foundation.

Author information

Authors and Affiliations

Authors

Contributions

M.I.I. characterized the α-synuclein segments and crystals. M.I.I. and S.S. conducted the toxicity assays. L.M.J. synthesized and purified NACore peptide. M.A.A. prepared the N-terminally acetylated α-synuclein. S.S. and M.Z. prepared wild-type α-synuclein. J.W. performed the mass spectrometry analyses of α-synuclein. M.I.I. and L.M.J. crystallized NACore. E.G. grew crystals of SubNACore. E.G., M.I.I. and M.R.S. collected and processed the data and solved the structure of SubNACore. L.J. and J.A.R. identified and crystallized PreNAC. J.A.R., D.S., B.L.N. and T.G. collected MicroED data on PreNAC and NACore nanocrystals. J.A.R., F.E.R., J.H., T.G., L.J., M.R.S., and D.C. processed the MicroED data and solved the structure of PreNAC and NACore. J.A.R., M.R.S., D.C., M.M. and S.B. collected XFEL diffraction from NACore nanocrystals. A.S.B. and N.K.S. processed the XFEL data. M.R.S and L.J. built the structure model of A53T α-synuclein protofibril. J.A.R., M.I.I., M.R.S., D.C., S.S. and E.G. prepared the figures. J.A.R., M.I.I., M.R.S., D.C., T.G. and D.S.E. wrote the paper, and all authors commented on the paper.

Corresponding authors

Correspondence to Tamir Gonen or David S. Eisenberg.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 A schematic representation of α-synuclein, highlighting the NAC region (residues 61–95) and within it the NACore sequence (residues 68–78).

A series of bars span regions of α-synuclein that are of interest to this work. Among the three synuclein paralogues (α, β and γ), the region whose sequence is unique to α-synuclein is shown as a blue bar (residues 72–83) that overlaps with a large portion of NACore. Segments investigated in ref. 23 are also shown. These span a variety of regions within NACore. Two of the segments we investigate here, SubNACore and NACore, are shown in this context. Only one of the segments studied ref. 23 is an exact match to our NACore sequence, and only this segment is both toxic and fibrillar. The sequences of α-synuclein, β-synuclein, and γ-synuclein are shown as a reference with conserved residues in bold and the NACore sequence in red.

Extended Data Figure 2 NACore difference density maps calculated after successful molecular replacement using the SubNACore search model clearly revealed the positions of the missing residues (positive FoFc density at N and C termini corresponding to G68 and A78) and one water molecule near a threonine side chain (red circle); a second water was located during the refinement process.

The blue mesh represents 2FoFc density contoured at 1.2σ. The green and red mesh represent FoFc densities contoured at 3.0 and −3.0σ, respectively. All maps were σA-weighted61.

Extended Data Figure 3 The crystal structure of NACore reveals pairs of sheets as in the spines of amyloid fibrils.

a, NACore’s two types of sheet–sheet interfaces: a larger interface (orange, 268 Å2 of buried accessible surface area per chain) we call interface A, and a weaker interface (blue, 167 Å2) we call interface B. The crystal is viewed along the hydrogen-bonding direction (crystal ‘b’ dimension). The red lines outline the unit cell. b, The van der Waals packing between sheets. The sheets are related by a 21 screw axis denoted in black. The only gaps left by the interface are filled with water molecules which hydrogen-bond to the threonine residues (partially showing aqua spheres). The shape complementarity of both interfaces is 0.7. The viewing direction is the same as in a. c, Orthogonal view of the fibrillar assembly. The protofibril axis, coinciding with the 21 screw axis designated by the arrow, runs vertically between the pairs of sheets.

Extended Data Figure 4 Comparison of the crystal packing for NACore and SubNACore.

a, The face-to-face interactions are virtually the same for the pairs of NACore segments (orange chains) in its crystal structure and the SubNACore segments (white chains) in its structure (interfaces A and B shown in gold and blue, respectively). The table below shows the pairwise r.m.s.d. values comparing the nine residues shared in common between the structures. RMSD_res is an all-atom comparison between residue pairs, while RMSD_ca compares only Cα pairs. b, PreNAC (blue) is compared with NACore (orange). Five residues from each strand are shown in darker colour and the r.m.s.d. values between their Cα pairs are compared in the table below. The PreNAC–NACore interaction mimics the weaker interface B in the NACore structure.

Extended Data Figure 5 Intense reflections common among the NACore and the two polymorphs of full length α-synuclein suggest common structural features.

Common structural features are illustrated here on the crystal packing diagrams of NACore. The (0,0,2) planes approximate the separation between sheets in interface A (orange). The (0,2,0), (−1,1,1), and (1,1,1) reflections are intense because the corresponding Bragg planes recapitulate the staggering of strands from opposing sheets. The red lines correspond to the unit cell boundaries and all planes are shown in black. The location of the unit cell origin is indicated by ‘O’. The unit cell dimensions a, b, and c are labelled. Bragg spacings (spacings between planes), indicated by ‘d’, are indicated in angstroms.

Extended Data Figure 6 Mass spectrometry analysis of recombinantly expressed, full-length α-synuclein, with and without N-terminal acetylation.

The mass profile of wild-type full length α-synuclein (left) is compared to that of an N-terminally acetylated form of the protein (right). The mass shift for the N-terminally acetylated form is appropriately shifted with respect to the native form of the protein (14464.0 Da for α-synuclein and 14506.0 Da for acetylated α-synuclein), within a margin of error of 4 Da.

Extended Data Table 1 Statistics of data collection and atomic refinement for NACore, its fragment SubNACore, and PreNAC
Extended Data Table 2 Comparison of reflections observed in powder diffraction of fibrils of full-length α-synuclein, N-acetyl α-synuclein, and a synthetic pattern calculated from our α-synuclein model, to aligned nanocrystals of NACore.

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rodriguez, J., Ivanova, M., Sawaya, M. et al. Structure of the toxic core of α-synuclein from invisible crystals. Nature 525, 486–490 (2015). https://doi.org/10.1038/nature15368

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature15368

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing