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

Nature Structural & Molecular Biology volume 23, pages 409–415 (2016)Cite this article

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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.

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Figure 1: Effects of preformed α-syn fibril samples on neurons.
Figure 2: Long-range SSNMR structural restraints for an α-syn fibril.
Figure 3: Three-dimensional structure of an α-syn fibril.
Figure 4: Validation of α-syn fibril structure by microscopy and fiber diffraction.

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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

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

    Present address: 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.).,

Authors and 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

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  1. Marcus D Tuttle
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  2. Gemma Comellas
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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.

Corresponding author

Correspondence to Chad M Rienstra.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Mass spectrometry and solution NMR data for α-syn samples used in this study.

(a) Matrix-assisted laser-desorption and ionization time-of-flight (MALDI-TOF) mass spectrum of uniformly-13C,15N-enriched α-syn monomer (blue) and natural abundance monomer (red). The molecular mass of natural abundance α-syn monomer was calculated to be 14.46 kDa, in agreement with the experimental mass spectrum. The mass of 15.24 kDa for the U-13C,15N-labeled sample corresponded to a ~98% incorporation of 13C and 15N during protein expression. (b) 15N-1H HSQC spectrum of monomeric α-syn prior to fibrillization. The chemical shifts agreed well with the published assignments (Eliezer, D., Kutluay, E., Bussell, R. & Browne, G. Conformational properties of α-synuclein in its free and lipid-associated states. J. Mol. Biol. 307, 1061–1073 (2001).)

Supplementary Figure 2 SSNMR spectroscopy data for α-syn fibrils, exhibiting cross-peaks indicative of long-range restraints and intermolecular registry of the fibrils.

(a) 2D 13C-13C SSNMR {1H}-13C-{1H-1H}-13C spectrum (Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 9704–9705 (2002).) of dilute α-syn fibrils (sample D from Table 1) showing unambiguous, long-range intramolecular correlations. Data were acquired at 750 MHz 1H frequency, 12.5 kHz MAS and 400 μs 1H-1H mixing time, with 12 days signal averaging. Red labels correspond to unambiguous long-range distances, including those from A78 to V48, from A69 to G93, from F94 to I88, and from V82 to A89. These intramolecular long-range restraints were inconsistent with structural models that exhibit a domain swap. (b-e) 2D 15N,13C SSNMR transferred echo double resonance (TEDOR, Jaroniec, C.P., Filip, C. & Griffin, R.G. 3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly 13C, 15N-labeled solids. J. Am. Chem. Soc. 124, 10728–10742 (2002).) data showing intermolecular interactions. The data were collected for four samples: (b) 1,3-13C glycerol,15N-labeled fibrils (sample B), signal averaged for 28 hr; (c) 50:50 [1,3-13C]glycerol, natural abundance (n.a.) nitrogen: n.a. carbon, 15N labeled (sample E), signal averaged for 81 hr; (d) [2-13C]glycerol,15N labeled (sample C), signal averaged for 113 hr; and (e) 50:50 2-13C glycerol, n.a. nitrogen: n.a. carbon, 15N labeled (sample F), signal averaged for 124 hr. Data was collected with mixing times of (c, e) 14.4 ms at 500 MHz 1H frequency, 11.1 kHz MAS, and (b, d) 16.0 ms at 600 MHz 1H frequency, 10.0 kHz MAS. Cross-peaks present in both parts b and c, or d and e, demonstrate a parallel, in-register arrangement (Debelouchina, G.T., Platt, G.W., Bayro, M.J., Radford, S.E. & Griffin, R.G. Intermolecular alignment in β2-microglobulin amyloid fibrils. J. Am. Chem. Soc. 132, 17077–17079 (2010).) (f–k) Spectra of sample B collected at 750 MHz 1H frequency, 12.5 kHz MAS (f-i) 2D 13C-13C spectra using dipolar-assisted rotational resonance (DARR, Takegoshi, K., Nakamura, S. & Terao, T. 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637 (2001).) mixing times of (f) 50 ms, signal averaged for 10.9 hr, (g) 100 ms, signal averaged for 5.7 hr, (h) 200 ms, signal averaged for 12.0 hr, and (i) 300 ms, signal averaged for 12.7 hr. Several long-range correlations (red) were observed between the F94 aromatic ring resonances (CD and CE) and I88, A90 and A91. (j-k) 2D planes from a 3D 15N-13CO-13CX correlation spectrum signal averaged for 24 hr. Long-range correlations observed at this relatively short mixing time indicate internuclear distances <5 Å.

Supplementary Figure 3 Full-length structure of the α-syn fibril, illustrating important features in expanded regions.

(a) View along the fibril axis showing the highly ordered core and the disordered tails. (b) Side view showing the β-sheet packing between each monomer as well as the disordered tails. (c-f) Core interactions illustrating NMR distance restraints with dotted lines. (g-l) Backbone traces for neighboring monomers drawn in blue, yellow, orange and red. (g, h) Side and top views of the salt bridge from E46 to K80 of the neighboring monomer. (i, j) Side and top views of the I88-A91-F94 pocket perpendicular to the fibril axis, exhibiting short intermolecular distances. (k, l) Side and top view of the Q79 side chain exhibiting a glutamine ladder, with intermolecular hydrogen bonds involving Nɛ2 and Oɛ1 moieties.

Supplementary Figure 4 SSNMR internal validation of structural restraints and dihedral angles for the α-syn fibril structure.

(a) The backbone trace is shown in grey, and black lines represent unambiguous distance restraints. Blue lines correspond to the shortest observed distance among the possible ambiguous assignments. The resulting structure is the lowest energy conformer consistent with all of the data. (b-c) Ramachandran probability maps of accepted dihedral angle regions (Lovell, S.C. et al. Structure validation by Cα geometry: φ,ψ and Cβ deviation. Proteins 50, 437–450 (2003).) and plotted in Chimera (Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).) for residues 41–100. The only residue not in the accepted range is E57, which is part of an unstructured loop and lacks NMR restraints in the simulated annealing calculations. All glycine residues are within the accepted Ramachandran space.

Supplementary Figure 5 Cross-validation of the SSNMR structure of α-syn fibrils with electron microscopy and X-ray fiber diffraction.

(a) Chemical shift comparison of the fibril sample for solid-state NMR before and after washing with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and sonication for electron microscopy. (b) Expansions of overlaid 2D 13C-13C spectra of the U-13C, 15N α-syn fibril as prepared for solid-state NMR (red) and fibrils washed with HEPES buffer as the samples for electron microscopy (blue). Spectra were acquired at 600 MHz 1H frequency, 13.3 kHz magic-angle spinning (MAS) and 50 ms dipolar-assisted rotational resonance (DARR, Takegoshi, K., Nakamura, S. & Terao, T. 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631–637 (2001).). (c) Equatorial intensity plot of the calculated and experimental fiber diffraction pattern of the α-syn fibril. The solid line shows the experimental fiber diffraction pattern (equatorial intensity projected from Figure 4c) compared to the simulated pattern (dotted line) from the atomic coordinates of the structure presented in Figure 3. The simulated pattern recapitulates the main features of the experimental pattern including the fine features near 0.12 Å−1. The correlation coefficient between the two patterns is 0.77, in good agreement with previous amyloid fibril fiber diffraction comparisons.

Supplementary Figure 6 Comparison of the Cα chemical-shift perturbations for three early-onset Parkinson's disease–mutant forms of α-syn, relative to the wild-type chemical shifts.

(a–c) Chemical shift perturbation plots of the absolute deviation of the Cα shifts for A30P, E46K, and A53T previously published (Lemkau, L.R. et al. Mutant protein A30P α-synuclein adopts wild-type fibril structure, despite slower fibrillation kinetics. J. Biol. Chem. 287, 11526-11532 (2012); Lemkau, L.R. 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).) relative to the wild-type chemical shifts used in this work.

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Tuttle, M., Comellas, G., Nieuwkoop, A. et al. Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. Nat Struct Mol Biol 23, 409–415 (2016). https://doi.org/10.1038/nsmb.3194

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