Article | Published:

Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein

Nature Structural & Molecular Biology volume 23, pages 409415 (2016) | Download Citation

Abstract

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). We present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register β-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the structural similarity of early-onset-PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules, to aid in PD diagnosis and treatment.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Biological Magnetic Resonance Data Bank

Protein Data Bank

References

  1. 1.

    et al. α-synuclein in Lewy bodies. Nature 388, 839–840 (1997).

  2. 2.

    et al. Exogenous α-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc. Natl. Acad. Sci. USA 106, 20051–20056 (2009).

  3. 3.

    et al. Exogenous α-synuclein fibrils induce Lewy body pathology leading to synaptic dysfunction and neuron death. Neuron 72, 57–71 (2011).

  4. 4.

    et al. Inclusion formation and neuronal cell death through neuron-to-neuron transmission of α-synuclein. Proc. Natl. Acad. Sci. USA 106, 13010–13015 (2009).

  5. 5.

    et al. Pathological α-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice. Science 338, 949–953 (2012).

  6. 6.

    et al. α-Synuclein strains cause distinct synucleinopathies after local and systemic administration. Nature 522, 340–344 (2015).

  7. 7.

    et al. Molecular-level secondary structure, polymorphism, and dynamics of full-length α-synuclein fibrils studied by solid-state NMR. Proc. Natl. Acad. Sci. USA 102, 15871–15876 (2005).

  8. 8.

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

  9. 9.

    et al. Solid-state NMR sequential assignments of α-synuclein. Biomol. NMR Assign. 6, 51–55 (2012).

  10. 10.

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

  11. 11.

    et al. Amyloid fibrils of the HET-s(218-289) prion form a β solenoid with a triangular hydrophobic core. Science 319, 1523–1526 (2008).

  12. 12.

    et al. 3D structure of amyloid protofilaments of β2-microglobulin fragment probed by solid-state NMR. Proc. Natl. Acad. Sci. USA 103, 18119–18124 (2006).

  13. 13.

    , & Experimental constraints on quaternary structure in Alzheimer's β-amyloid fibrils. Biochemistry 45, 498–512 (2006).

  14. 14.

    et al. Molecular structure of β-amyloid fibrils in Alzheimer's disease brain tissue. Cell 154, 1257–1268 (2013).

  15. 15.

    et al. Atomic-resolution three-dimensional structure of amyloid β fibrils bearing the Osaka mutation. Angew. Chem. Int. Ed. Engl. 54, 331–335 (2015).

  16. 16.

    et al. Aβ(1–42) fibril structure illuminates self-recognition and replication of amyloid in Alzheimer's disease. Nat. Struct. Mol. Biol. 22, 499–505 (2015).

  17. 17.

    , , , & Preparation of α-synuclein fibrils for solid-state NMR: expression, purification, and incubation of wild-type and mutant forms. Protein Expr. Purif. 48, 112–117 (2006).

  18. 18.

    , , & Solid-state NMR spectroscopy reveals that water is nonessential to the core structure of α-synuclein fibrils. J. Phys. Chem. B 111, 13353–13356 (2007).

  19. 19.

    et al. Studies of lipopolysaccharide effects on the induction of α-synuclein pathology by exogenous fibrils in transgenic mice. Mol. Neurodegener. 10, 32 (2015).

  20. 20.

    & Protein structure determination by magic-angle spinning solid-state NMR, and insights into the formation, structure, and stability of amyloid fibrils. Annu. Rev. Biophys. 42, 515–536 (2013).

  21. 21.

    et al. Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420, 98–102 (2002).

  22. 22.

    , & 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637 (2001).

  23. 23.

    , & Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 9704–9705 (2002).

  24. 24.

    , , , & Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy. J. Chem. Phys. 131, 095101 (2009).

  25. 25.

    et al. Dipole tensor-based atomic-resolution structure determination of a nanocrystalline protein by solid-state NMR. Proc. Natl. Acad. Sci. USA 105, 4621–4626 (2008).

  26. 26.

    & Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227–241 (2013).

  27. 27.

    , & Using Xplor-NIH for NMR molecular structure determination. Prog. Nucl. Magn. Reson. Spectrosc. 48, 47–62 (2006).

  28. 28.

    & The Greek key motif: extraction, classification and analysis. Protein Eng. 6, 233–245 (1993).

  29. 29.

    , , & 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).

  30. 30.

    , & Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease. Nat. Med. 4, 1318–1320 (1998).

  31. 31.

    et al. A general model for amyloid fibril assembly based on morphological studies using atomic force microscopy. Biophys. J. 85, 1135–1144 (2003).

  32. 32.

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

  33. 33.

    et al. Architecture of Ure2p prion filaments: the N-terminal domains form a central core fiber. J. Biol. Chem. 278, 43717–43727 (2003).

  34. 34.

    & Fiber diffraction of the prion-forming domain HET-s(218-289) shows dehydration-induced deformation of a complex amyloid structure. Biochemistry 53, 2366–2370 (2014).

  35. 35.

    et al. A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR. Proc. Natl. Acad. Sci. USA 99, 16742–16747 (2002).

  36. 36.

    & A proposed atomic structure of the self-assembly of the non-amyloid-β-component of human α-synuclein as derived by computational tools. J. Phys. Chem. B 119, 10005–10015 (2015).

  37. 37.

    et al. Structure of the toxic core of α-synuclein from invisible crystals. Nature 525, 486–490 (2015).

  38. 38.

    & Fungal prion HET-s as a model for structural complexity and self-propagation in prions. Proc. Natl. Acad. Sci. USA 111, 5201–5206 (2014).

  39. 39.

    , , & Structure and dynamics of micelle-bound human α-synuclein. J. Biol. Chem. 280, 9595–9603 (2005).

  40. 40.

    & α-synuclein populates both elongated and broken helix states on small unilamellar vesicles. J. Biol. Chem. 286, 21450–21457 (2011).

  41. 41.

    , , , & Length preferences and periodicity in β-strands: antiparallel edge β-sheets are more likely to finish in non-hydrogen bonded rings. Protein Eng. 16, 957–961 (2003).

  42. 42.

    , & Characterisation of isolated α-synuclein filaments from substantia nigra of Parkinson's disease brain. Neurosci. Lett. 292, 128–130 (2000).

  43. 43.

    et al. Unlike twins: an NMR comparison of two α-synuclein polymorphs featuring different toxicity. PLoS One 9, e90659 (2014).

  44. 44.

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

  45. 45.

    et al. Ala30Pro mutation in the gene encoding α-synuclein in Parkinson's disease. Nat. Genet. 18, 106–108 (1998).

  46. 46.

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

  47. 47.

    et al. α-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease. Mov. Disord. 28, 811–813 (2013).

  48. 48.

    et al. G51D α-synuclein mutation causes a novel parkinsonian-pyramidal syndrome. Ann. Neurol. 73, 459–471 (2013).

  49. 49.

    et al. Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics. J. Biol. Chem. 287, 11526–11532 (2012).

  50. 50.

    et al. Site-specific perturbations of α-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K. PLoS One 8, e49750 (2013).

  51. 51.

    , , , & Structural intermediates during α-synuclein fibrillogenesis on phospholipid vesicles. J. Am. Chem. Soc. 134, 5090–5099 (2012).

  52. 52.

    et al. Structural comparison of mouse and human α-synuclein amyloid fibrils by solid-state NMR. J. Mol. Biol. 420, 99–111 (2012).

  53. 53.

    et al. Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta. eLife 2, e00857 (2013).

  54. 54.

    et al. Binding of the radioligand SIL23 to α-synuclein fibrils in Parkinson disease brain tissue establishes feasibility and screening approaches for developing a Parkinson disease imaging agent. PLoS One 8, e55031 (2013).

  55. 55.

    et al. Distinct α-synuclein strains differentially promote tau inclusions in neurons. Cell 154, 103–117 (2013).

  56. 56.

    , & A method for efficient isotopic labeling of recombinant proteins. J. Biomol. NMR 20, 71–75 (2001).

  57. 57.

    & Dynamical mapping of E. coli thioredoxin via 13C NMR relaxation analysis. J. Am. Chem. Soc. 118, 9255–9264 (1996).

  58. 58.

    Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

  59. 59.

    , , , & Straightforward, effective calibration of SPINAL-64 decoupling results in the enhancement of sensitivity and resolution of biomolecular solid-state NMR. J. Magn. Reson. 209, 131–135 (2011).

  60. 60.

    & Chemical shift referencing in MAS solid state NMR. J. Magn. Reson. 162, 479–486 (2003).

  61. 61.

    et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

  62. 62.

    , & Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration. J. Am. Chem. Soc. 121, 2337–2338 (1999).

  63. 63.

    & An empirical backbone-backbone hydrogen-bonding potential in proteins and its applications to NMR structure refinement and validation. J. Am. Chem. Soc. 126, 7281–7292 (2004).

  64. 64.

    , & Smooth statistical torsion angle potential derived from a large conformational database via adaptive kernel density estimation improves the quality of NMR protein structures. Protein Sci. 21, 1824–1836 (2012).

  65. 65.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

  66. 66.

    et al. Role of α-synuclein carboxy-terminus on fibril formation in vitro. Biochemistry 42, 8530–8540 (2003).

  67. 67.

    & Scanning transmission electron microscopy of DNA-protein complexes. Methods Mol. Biol. 148, 589–601 (2001).

  68. 68.

    et al. Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked β-solenoid model of HET-s prion fibrils. J. Biol. Chem. 282, 5545–5550 (2007).

  69. 69.

    , , , & Enclosed chambers for humidity control and sample containment in fiber diffraction. J. Appl. Crystallogr. 41, 206–209 (2008).

  70. 70.

    , & Crystal structure refinements of magnesite, calcite, rhodochrosite, siderite, smithonite, and dolomite, with discussion of some aspects of the stereochemistry of calcite type carbonates. Z. Kristallogr. Cryst. Mater. 156, 233–243 (1981).

  71. 71.

    , , & Digital processing of fibre diffraction patterns. J. Appl. Crystallogr. 9, 81–94 (1976).

  72. 72.

    , , & WCEN: a computer program for initial processing of fiber diffraction patterns. J. Appl. Crystallogr. 39, 752–756 (2006).

  73. 73.

    & in International Tables for Crystallography 2nd edn., Vol. F (eds. Arnold, E., Himmel, D.M. & Rossman, M.G.) 444–450 (Wiley, 2012).

  74. 74.

    & The effect of disorientation on the intensity distribution of non-crystalline fibres. I. Theory. Acta Crystallogr. A 30, 635–638 (1974).

Download references

Acknowledgements

This study was supported by the US National Institutes of Health (NIH) (grants R01-GM073770 to C.M.R., P50-NS053488 to V.M.Y.L. and P01-AG002132 to G.S.) and used SSNMR instrumentation procured with the support of grant S10-RR025037 (to C.M.R.) from the NIH National Center for Research Resources (NCRR). M.D.T., A.J.N. and A.M.B. were supported as members of the NIH Molecular Biophysics Training Grant at the University of Illinois at Urbana-Champaign (T32-GM008276), and D.J.C. is supported by grant T32-AG000255. J.M.C. was supported by a US National Science Foundation Graduate Research Fellowship. C.D.S. is supported by the Intramural Research Program of the Center for Information Technology at NIH. The authors thank J. Wall and B. Lin (Brookhaven National Laboratory) for STEM MPL sample preparation and data collection. The Brookhaven National Laboratory STEM was an NIH-supported Resource Center (P41-EB2181), and additional support was provided by the US Department of Energy (DOE), Office of Biological and Environmental Research. TEM images were collected at the Frederick Seitz Materials Research Laboratory Central Facilities, University of Illinois, which are partially supported by the DOE under grants DE-FG02-07ER46453 and DE-FG02-07ER46471. The Voyager-DE STR MALDI TOF mass spectrometer was purchased in part with a grant from the NIH NCRR (S10-RR011966). The Stanford Synchrotron Radiation Lightsource (SSRL) is a national user facility operated by Stanford University on behalf of the DOE, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the DOE and the NIH. The authors thank M. Tang (College of Staten Island) for helpful discussions.

Author information

Author notes

    • Marcus D Tuttle
    • , Andrew J Nieuwkoop
    • , Kathryn D Kloepper
    •  & William Wan

    Present addresses: Department of Chemistry, Yale University, New Haven, Connecticut, USA (M.D.T.), Leibniz-Institut Für Molekulare Pharmakologie, Berlin, Germany (A.J.N.), Department of Chemistry, Mercer University, Macon, Georgia, USA (K.D.K.) and Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany (W.W.).

Affiliations

  1. Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Marcus D Tuttle
    • , Andrew J Nieuwkoop
    • , Deborah A Berthold
    • , Kathryn D Kloepper
    • , Joseph M Courtney
    • , Jae K Kim
    •  & Chad M Rienstra
  2. Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Gemma Comellas
    • , Alexander M Barclay
    •  & Chad M Rienstra
  3. Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    • Dustin J Covell
    •  & Virginia M Y Lee
  4. Institute on Aging, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    • Dustin J Covell
    •  & Virginia M Y Lee
  5. Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

    • Dustin J Covell
    •  & Virginia M Y Lee
  6. Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA.

    • Amy Kendall
    • , William Wan
    •  & Gerald Stubbs
  7. Center for Structural Biology, Vanderbilt University, Nashville, Tennessee, USA.

    • Amy Kendall
    • , William Wan
    •  & Gerald Stubbs
  8. Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, Maryland, USA.

    • Charles D Schwieters
  9. Department of Biological and Experimental Psychology, School of Biological and Chemical Sciences, Queen Mary University of London, London, UK.

    • Julia M George
  10. Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

    • Chad M Rienstra

Authors

  1. Search for Marcus D Tuttle in:

  2. Search for Gemma Comellas in:

  3. Search for Andrew J Nieuwkoop in:

  4. Search for Dustin J Covell in:

  5. Search for Deborah A Berthold in:

  6. Search for Kathryn D Kloepper in:

  7. Search for Joseph M Courtney in:

  8. Search for Jae K Kim in:

  9. Search for Alexander M Barclay in:

  10. Search for Amy Kendall in:

  11. Search for William Wan in:

  12. Search for Gerald Stubbs in:

  13. Search for Charles D Schwieters in:

  14. Search for Virginia M Y Lee in:

  15. Search for Julia M George in:

  16. Search for Chad M Rienstra in:

Contributions

M.D.T. analyzed and collected SSNMR data, performed structure calculations, analyzed structural features and was the primary author on the manuscript. G.C. analyzed and collected SSNMR data, performed MPL data analysis and provided input on the manuscript. A.J.N. analyzed and collected SSNMR data, performed initial structure calculations and provided input on the manuscript. D.J.C. performed the immunofluorescence, biochemical and biophysical assays and helped prepare the manuscript. D.A.B. prepared isotopically labeled samples and provided input on the manuscript. K.D.K. aided in sample preparation and manuscript preparation. J.M.C. created scripts for data conversion, performed analysis of final structures and provided input on the manuscript. J.K.K. contributed to the sample preparation methods. A.M.B. contributed to the mass spectrometry and solution NMR data and analysis. A.K. and W.W. prepared fiber diffraction samples and collected and analyzed fiber diffraction data. G.S. analyzed fiber diffraction data and aided in manuscript preparation. C.D.S. supported the development of structure calculations in XPLOR-NIH. V.M.Y.L., J.M.G. and C.M.R. were the primary investigators, designed the experiments and aided in manuscript preparation, data collection and interpretation.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Chad M Rienstra.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–6 and Supplementary Table 1

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3194