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

Nature volume 525, pages 486–490 (2015)Cite this article

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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.

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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.

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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.

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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.

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Author notes

  1. Magdalena I. Ivanova, Lin Jiang & Marc Messerschmidt

    Present address: †Present addresses: Department of Neurology and Program of Biophysics, University of Michigan School of Medicine, Ann Arbor, Michigan 48109, USA (M.I.I.); Department of Neurology, UCLA, Los Angeles, California 90095, USA (L.J.); National Science Foundation BioXFEL Science and Technology Center, Buffalo, New York 14203, USA (M.M.).,

  2. Jose A. Rodriguez, Magdalena I. Ivanova, Michael R. Sawaya, Duilio Cascio and Francis E. Reyes: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Biological Chemistry and Chemistry and Biochemistry, Howard Hughes Medical Institute, UCLA-DOE Institute, Box 951570, UCLA, Los Angeles, 90095-1570, California, USA

    Jose A. Rodriguez, Magdalena I. Ivanova, Michael R. Sawaya, Duilio Cascio, Smriti Sangwan, Elizabeth L. Guenther, Lisa M. Johnson, Meng Zhang, Lin Jiang, Mark A. Arbing & David S. Eisenberg

  2. Howard Hughes Medical Institute, Janelia Research Campus, 19700 Helix Drive, Ashburn, 20147, Virginia, USA

    Francis E. Reyes, Dan Shi, Brent L. Nannenga, Johan Hattne & Tamir Gonen

  3. Box 42, NPI-Semel Institute, 760 Westwood Plaza, UCLA, Los Angeles, 90024, California, USA

    Julian Whitelegge

  4. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Aaron S. Brewster & Nicholas K. Sauter

  5. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, 94025, California, USA

    Marc Messerschmidt & Sébastien Boutet

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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.

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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 Fo − Fc 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 2Fo − Fc density contoured at 1.2σ. The green and red mesh represent Fo − Fc 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
Full size table
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.
Full size table

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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

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