A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function

Abstract

Aggregation of human α-synuclein (αSyn) is linked to Parkinson’s disease (PD) pathology. The central region of the αSyn sequence contains the non-amyloid β-component (NAC) crucial for aggregation. However, how NAC flanking regions modulate αSyn aggregation remains unclear. Using bioinformatics, mutation and NMR, we identify a 7-residue sequence, named P1 (residues 36–42), that controls αSyn aggregation. Deletion or substitution of this ‘master controller’ prevents aggregation at pH 7.5 in vitro. At lower pH, P1 synergises with a sequence containing the preNAC region (P2, residues 45–57) to prevent aggregation. Deleting P1 (ΔP1) or both P1 and P2 (ΔΔ) also prevents age-dependent αSyn aggregation and toxicity in C. elegans models and prevents αSyn-mediated vesicle fusion by altering the conformational properties of the protein when lipid bound. The results highlight the importance of a master-controller sequence motif that controls both αSyn aggregation and function—a region that could be targeted to prevent aggregation in disease.

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Fig. 1: Aggregation and solubility profiles of αSyn.
Fig. 2: The kinetics of aggregation of WT αSyn and P1 and P2 deletion variants.
Fig. 3: ThT fluorescence assays of disulfide-locked αSyn dimers.
Fig. 4: Intramolecular PRE experiments for WT αSyn.
Fig. 5: Intramolecular PRE experiments for ΔΔ αSyn.
Fig. 6: Deletion of P1 or both P1 and P2 in C. elegans expressing αSyn::YFP suppresses aggregation and proteotoxicity.
Fig. 7: Lipid-induced aggregation kinetics of WT αSyn and its variants.
Fig. 8: NMR experiments detailing the molecular basis of liposome binding of WT αSyn ΔΔ and P1P2-GS.

Data availability

Chemical shift assignments can be accessed using BMRB accession numbers 27900 (WT-αSyn), 27901 (ΔΔ αSyn) and 28045 (P1P2-GS αSyn). Source data for Fig. 1c−e, Fig. 2a−d, Fig. 3a−c, Fig. 4a−g, Fig. 5a−g. Fig 6b,d, Fig. 7a−f and Fig. 8d and Extended Fig. 1a−h, Extended Fig. 2a, Extended Fig. 3b−g, Extended Fig. 5a,b, Extended Fig. 6b,c and Extended Fig. 7a−d are available with the paper online. Other datasets generated during and/or analyzed during the current study are available in the University of Leeds data repository (https://doi.org/10.5518/707).

References

  1. 1.

    Iwai, A. et al. The precursor protein of non-Aβ component of Alzheimer’s-disease amyloid is a presynaptic protein of the central nervous system. Neuron 14, 467–475 (1995).

  2. 2.

    Dettmer, U., Selkoe, D. & Bartels, T. New insights into cellular α-synuclein homeostasis in health and disease. Curr. Opin. Neurobiol. 36, 15–22 (2016).

  3. 3.

    Iadanza, M. G., Jackson, M. P., Hewitt, E. W., Ranson, N. A. & Radford, S. E. A new era for understanding amyloid structures and disease. Nat. Rev. Mol. Cell Biol. 19, 755–773 (2018).

  4. 4.

    Tysnes, O.-B. & Storstein, A. Epidemiology of Parkinson’s disease. J. Neural Transm. 124, 901–905 (2017).

  5. 5.

    Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. & Lansbury, P. T. NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry 35, 13709–13715 (1996).

  6. 6.

    Theillet, F.-X. et al. Structural disorder of monomeric α-synuclein persists in mammalian cells. Nature 530, 45–50 (2016).

  7. 7.

    Fusco, G. et al. Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers. Science 358, 1440–1443 (2017).

  8. 8.

    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).

  9. 9.

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

  10. 10.

    Bartels, T. et al. The N-terminus of the intrinsically disordered protein α-synuclein triggers membrane binding and helix folding. Biophys. J. 99, 2116–2124 (2010).

  11. 11.

    Salveson, P. J., Spencer, R. K. & Nowick, J. S. X-ray crystallographic structure of oligomers formed by a toxic β-hairpin derived from α-synuclein: trimers and higher-order oligomers. J. Am. Chem. Soc. 138, 4458–4467 (2016).

  12. 12.

    Giasson, B. I., Murray, I. V., Trojanowski, J. Q. & Lee, V. M. 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).

  13. 13.

    Li, B. et al. Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel. Nat. Commun. 9, 3609 (2018).

  14. 14.

    Guerrero-Ferreira, R. et al. Cryo-EM structure of alpha-synuclein fibrils. Elife 7, e36402 (2018).

  15. 15.

    Tuttle, M. D. et al. Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein. Nat. Struct. Mol. Biol. 23, 409–415 (2016).

  16. 16.

    Allison, J. R., Varnai, P., Dobson, C. M. & Vendruscolo, M. Determination of the free energy landscape of α-synuclein using spin label nuclear magnetic resonance measurements. J. Am. Chem. Soc. 131, 18314–18326 (2009).

  17. 17.

    Bertoncini, C. W. et al. Release of long-range tertiary interactions potentiates aggregation of natively unstructured α-synuclein. Proc. Natl Acad. Sci. USA 102, 1430–1435 (2005).

  18. 18.

    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).

  19. 19.

    Phillips, A. S. et al. Conformational dynamics of α-synuclein: insights from mass spectrometry. Analyst 140, 3070–3081 (2015).

  20. 20.

    Uversky, V. N., Li, J. & Fink, A. L. Evidence for a partially folded intermediate in α-synuclein fibril formation. J. Biol. Chem. 276, 10737–10744 (2001).

  21. 21.

    Wu, K. P., Weinstock, D. S., Narayanan, C., Levy, R. M. & Baum, J. Structural reorganization of α-synuclein at low pH observed by NMR and REMD simulations. J. Mol. Biol. 391, 784–796 (2009).

  22. 22.

    Hoyer, W., Cherny, D., Subramaniam, V. & Jovin, T. M. Impact of the acidic C-terminal region comprising amino acids 109−140 on α-synuclein aggregation in vitro. Biochemistry 43, 16233–16242 (2004).

  23. 23.

    Stephens, A. D., Zacharopoulou, M. & Kaminski Schierle, G. S. The cellular environment affects monomeric α-synuclein structure. Trends Biochem. Sci. 44, 453–466 (2018).

  24. 24.

    Das, M., Mei, X., Jayaraman, S., Atkinson, D. & Gursky, O. Amyloidogenic mutations in human apolipoprotein A-I are not necessarily destabilizing–a common mechanism of apolipoprotein A-I misfolding in familial amyloidosis and atherosclerosis. FEBS J. 281, 2525–2542 (2014).

  25. 25.

    Hoop, C. L. et al. Polyglutamine amyloid core boundaries and flanking domain dynamics in huntingtin fragment fibrils determined by solid-state nuclear magnetic resonance. Biochemistry 53, 6653–6666 (2014).

  26. 26.

    Bugg, C. W., Isas, J. M., Fischer, T., Patterson, P. H. & Langen, R. Structural features and domain organization of huntingtin fibrils. J. Biol. Chem. 287, 31739–31746 (2012).

  27. 27.

    Colvin, M. T. et al. Atomic resolution structure of monomorphic Aβ42 amyloid fibrils. J. Am. Chem. Soc. 138, 9663–9674 (2016).

  28. 28.

    Lucato, C. M., Lupton, C. J., Halls, M. L. & Ellisdon, A. M. Amyloidogenicity at a distance: how distal protein regions modulate aggregation in disease. J. Mol. Biol. 429, 1289–1304 (2017).

  29. 29.

    Crowther, R. A., Jakes, R., Spillantini, M. G. & Goedert, M. Synthetic filaments assembled from C-terminally truncated α-synuclein. FEBS Lett. 436, 309–312 (1998).

  30. 30.

    Kessler, J. C., Rochet, J.-C. & Lansbury, P. T. The N-terminal repeat domain of α-synuclein inhibits β-sheet and amyloid fibril formation. Biochemistry 42, 672–678 (2003).

  31. 31.

    Izawa, Y. et al. Role of C-terminal negative charges and tyrosine residues in fibril formation of α-synuclein. Brain Behav. 2, 595–605 (2012).

  32. 32.

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

  33. 33.

    Li, Y. et al. Amyloid fibril structure of α-synuclein determined by cryo-electron microscopy. Cell Res. 28, 897–903 (2018).

  34. 34.

    van Ham, T. J. et al. C. elegans model identifies genetic modifiers of α-synuclein inclusion formation during aging. PLoS Genet. 4, e1000027 (2008).

  35. 35.

    Fusco, G. et al. Structural basis of synaptic vesicle assembly promoted by α-synuclein. Nat. Commun. 7, 12563 (2016).

  36. 36.

    Lautenschläger, J. et al. C-terminal calcium binding of α-synuclein modulates synaptic vesicle interaction. Nat. Commun. 9, 712 (2018).

  37. 37.

    Tartaglia, G. G. & Vendruscolo, M. The Zyggregator method for predicting protein aggregation propensities. Chem. Soc. Rev. 37, 1395–1401 (2008).

  38. 38.

    Sormanni, P., Aprile, F. A. & Vendruscolo, M. The CamSol method of rational design of protein mutants with enhanced solubility. J. Mol. Biol. 427, 478–490 (2015).

  39. 39.

    Thompson, M. J. et al. The 3D profile method for identifying fibril-forming segments of proteins. Proc. Natl Acad. Sci. USA 103, 4074–4078 (2006).

  40. 40.

    Terada, M. et al. The effect of truncation on prion-like properties of α-synuclein. J. Biol. Chem. 293, 13910–13920 (2018).

  41. 41.

    Mirecka, E. A. et al. Sequestration of a β-hairpin for control of α-synuclein aggregation. Angew. Chem. Int. Ed. Engl. 53, 4227–4230 (2014).

  42. 42.

    Shaykhalishahi, H. et al. Contact between the β1 and β2 segments of α-synuclein that inhibits amyloid formation. Angew. Chem. Int. Ed. Engl. 54, 8837–8840 (2015).

  43. 43.

    Agerschou, E. D. et al. An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils. Elife 8, e46112 (2019).

  44. 44.

    Cho, M.-K. et al. Structural characterization of α-synuclein in an aggregation prone state. Protein Sci. 18, 1840–1846 (2009).

  45. 45.

    Buell, A. K. et al. Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation. Proc. Natl Acad. Sci. USA 111, 7671–7676 (2014).

  46. 46.

    Hoyer, W. et al. Dependence of α-synuclein aggregate morphology on solution conditions. J. Mol. Biol. 322, 383–393 (2002).

  47. 47.

    Wördehoff, M. M. et al. Opposed effects of dityrosine formation in soluble and aggregated α-synuclein on fibril growth. J. Mol. Biol. 429, 3018–3030 (2017).

  48. 48.

    Wu, K.-P. & Baum, J. Detection of transient interchain interactions in the intrinsically disordered protein α-synuclein by NMR paramagnetic relaxation enhancement. J. Am. Chem. Soc. 132, 5546–5547 (2010).

  49. 49.

    Dedmon, M. M., Lindorff-Larsen, K., Christodoulou, J., Vendruscolo, M. & Dobson, C. M. Mapping long-range interactions in α-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J. Am. Chem. Soc. 127, 476–477 (2005).

  50. 50.

    Wu, K.-P., Kim, S., Fela, D. A. & Baum, J. Characterization of conformational and dynamic properties of natively unfolded human and mouse α-synuclein ensembles by NMR: implication for aggregation. J. Mol. Biol. 378, 1104–1115 (2008).

  51. 51.

    Bertoncini, C. W., Fernandez, C. O., Griesinger, C., Jovin, T. M. & Zweckstetter, M. Familial mutants of α-synuclein with increased neurotoxicity have a destabilized conformation. J. Biol. Chem. 280, 30649–30652 (2005).

  52. 52.

    Sung, Y.-H. & Eliezer, D. Residual structure, backbone dynamics, and interactions within the synuclein family. J. Mol. Biol. 372, 689–707 (2007).

  53. 53.

    Esteban-Martín, S., Silvestre-Ryan, J., Bertoncini, C. W. & Salvatella, X. Identification of fibril-like tertiary contacts in soluble monomeric α-synuclein. Biophys. J. 105, 1192–1198 (2013).

  54. 54.

    Janowska, M. K., Wu, K.-P. & Baum, J. Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein. Sci. Reports 5, 15164–15164 (2015).

  55. 55.

    Ben-Zvi, A., Miller, E. A. & Morimoto, R. I. Collapse of proteostasis represents an early molecular event in Caenorhabditis elegans aging. Proc. Natl Acad. Sci. USA 106, 14914–14919 (2009).

  56. 56.

    Labbadia, J. & Morimoto, R. I. Repression of the heat shock response is a programmed event at the onset of reproduction. Mol. Cell 59, 639–650 (2015).

  57. 57.

    Diao, J. et al. Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2. Elife 2, e00592 (2013).

  58. 58.

    Bodner, C. R., Dobson, C. M. & Bax, A. Multiple tight phospholipid-binding modes of α-synuclein revealed by solution NMR spectroscopy. J. Mol. Biol. 390, 775–790 (2009).

  59. 59.

    Fusco, G. et al. Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour. Nat. Commun. 5, 3827 (2014).

  60. 60.

    Jao, C. C., Hegde, B. G., Chen, J., Haworth, I. S. & Langen, R. Structure of membrane-bound α-synuclein from site-directed spin labeling and computational refinement. Proc. Natl Acad. Sci. USA 105, 19666–19671 (2008).

  61. 61.

    Galvagnion, C. et al. Lipid vesicles trigger α-synuclein aggregation by stimulating primary nucleation. Nat. Chem. Biol. 11, 229–234 (2015).

  62. 62.

    Fonseca-Ornelas, L. et al. Small molecule-mediated stabilization of vesicle-associated helical α-synuclein inhibits pathogenic misfolding and aggregation. Nat. Commun. 5, 5857 (2014).

  63. 63.

    Jackson, M. P. & Hewitt, E. W. Cellular proteostasis: degradation of misfolded proteins by lysosomes. Essays Biochem. 60, 173–180 (2016).

  64. 64.

    Brännström, K. et al. The N-terminal region of amyloid β controls the aggregation rate and fibril stability at low pH through a gain of function mechanism. J. Am. Chem. Soc. 136, 10956–10964 (2014).

  65. 65.

    Chen, D. et al. Tau local structure shields an amyloid-forming motif and controls aggregation propensity. Nat. Commun. 10, 2493 (2019).

  66. 66.

    Esposito, G. et al. Removal of the N-terminal hexapeptide from human β2-microglobulin facilitates protein aggregation and fibril formation. Protein Sci. 9, 831–845 (2000).

  67. 67.

    Goedert, M. Alpha-synuclein and neurodegenerative diseases. Nat. Rev. Neurosci. 2, 492–501 (2001).

  68. 68.

    Mehra, S., Sahay, S. & Maji, S. K. α-synuclein misfolding and aggregation: Implications in Parkinson’s disease pathogenesis. Biochim. Biophys. Acta Proteins Proteom. 1867, 890–908 (2019).

  69. 69.

    Cabin, D. E. et al. Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking α-synuclein. J. Neurosci. 22, 8797–8807 (2002).

  70. 70.

    Martin, E. M. et al. Conformational flexibility within the nascent polypeptide-associated complex enables its interactions with structurally diverse client proteins. J. Biol. Chem. 293, 8554–8568 (2018).

  71. 71.

    Masuda, M. et al. Cysteine misincorporation in bacterially expressed human α‐synuclein. FEBS Lett. 580, 1775–1779 (2006).

  72. 72.

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

  73. 73.

    Skinner, S. P. et al. CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis. J. Biomol. NMR 66, 111–124 (2016).

  74. 74.

    Fogh, R. et al. The CCPN project: an interim report on a data model for the NMR community. Nat. Struct. Mol. Biol. 9, 416–418 (2002).

  75. 75.

    Tang, C., Schwieters, C. D. & Clore, G. M. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449, 1078–1082 (2007).

  76. 76.

    Brenner, S. The genetics of Caenorhabditis elegans. Genetics 77, 71–94 (1974).

  77. 77.

    Nussbaum-Krammer, C. I., Neto, M. F., Brielmann, R. M., Pedersen, J. S. & Morimoto, R. I. Investigating the spreading and toxicity of prion-like proteins using the metazoan model organism C. elegans. J. Vis. Exp. 95, e52321 (2015).

  78. 78.

    Whitmore, L. & Wallace, B. A. DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 32, W668–W673 (2004).

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Acknowledgements

We thank members of our research groups for helpful discussions throughout this work. We also thank T. Karamanos for helpful advice about NMR PRE data analysis, E. Nollen (University of Groningen) for the kind gift of the plasmid encoding YFP-αSyn, L. Willis for help with SEC-MALS analysis, B. Schiffrin for his help with the Kd fitting and the MS facility for help with characterization of all purified proteins. S.E.R. acknowledges funding from the European Research Council under the European Union’s Seventh Framework Programme FP7.2007–2013/Grant agreement number 322408 and Wellcome Trust (204963). C.P.A.D. was supported by BBSRC (BB/K02101X/1) and by the ERC (322408); S.C.G. was supported by BBSRC (BB/M011151/1); R.M.-M. was supported by the Wellcome Trust (204963); and S.M.U. was supported by the Wellcome Trust (215062/Z/18/Z). P.v.O.-H. is also funded by an N3CR grant (NC/P001203/1). We thank the Wellcome Trust (094232) and University of Leeds for the purchase of the Chiroscan CD spectrometer, the electron microscopes and NMR instrumentation.

Author information

C.P.A.D. and S.M.U. prepared samples and designed and performed fluorescence, NMR, EM and other biochemical studies. J.M., S.C.G. and P.v.O.-H. performed the experiments with C. elegans. C.P.A.D., S.M.U. and G.N.K. performed CD experiments. R.M.-M. performed NMR assignments and assisted with NMR data analysis and interpretation. S.E.R. and D.J.B. developed the ideas and supervised the work. All authors contributed to the preparation of the manuscript.

Correspondence to Sheena E. Radford or David J. Brockwell.

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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 Aggregation kinetics of αSyn variants at different pH and salt conditions with TEM images of the aggregates (if any) formed at the end point.

ah, ThT fluorescence assays at pH 4.5 or pH 7.5 of WT αSyn (a,b), ΔP1 (c,d), ΔP2 (e,f) and ΔΔ (g,h) at high (200 mM NaCl) or low (20 mM NaCl) ionic strength. Negative-stain TEM images of representative samples of the aggregates formed at the end point (100 h) are shown alongside each plot using the same color scheme. The fibril yield under each condition, determined by SDS−PAGE after centrifugation (Methods), is shown in Supplementary Table 1. Data for graphs are available as Source data. Source data

Extended Data Fig. 2 Cross-seeding αSyn variants using seeds created from WT αSyn.

a, ThT fluorescence assays of αSyn variants (100 μM) WT (black), ΔP1 (red), ΔP2 (green) and ΔΔ (blue) seeded with 10 % (v/v) WT αSyn fibril seeds formed at pH 7.5. Seeding assays were performed at pH 7.5, low salt (20 mM added NaCl), 37 °C, quiescent. b, End point (42 h) TEM images of representative samples of fibrils from the seeding experiments using the same color scheme as in a. Scale bars, 200 nm. Data for graph in a are available as Source data. Source data

Extended Data Fig. 3 Aggregation kinetics of ΔC1 and P1P2-GS.

a, Schematic of WT, ΔC1, ΔΔ and P1P2-GS αSyn variants including the amino acid sequence of the deleted C1 region and substituted P1-P2 region. bg, ThT assays at pH 4.5 or pH 7.5 of WT αSyn (b,c), ΔC1 (Δ14−20) (d,e), or P1P2-GS (f,g). Dark and light colors shows assays in high- (200 mM added NaCl) and low-salt (20 mM added NaCl) conditions, respectively. h,i, Negative-stain TEM images of representative samples at the endpoint (100 h) of the incubation of ΔC1 (h) or P1P2-GS (i), with same color scheme as in cf. Scale bars, 200 nm. The fibril yield under each condition, determined by SDS−PAGE after centrifugation (Methods) is shown in Supplementary Table 1. Data for graphs in bg are available as Source data. Source data

Extended Data Fig. 4 1H-15N HSQC NMR spectra showing intramolecular PRE NMR experiments on WT αSyn in 20 mM sodium acetate buffer, 20 mM NaCl, pH 4.5, 15 °C.

ac, Overlaid paramagnetic (green) and diamagnetic (orange) spectra for WT αSyn labeled at positions A18C (a), A90C (b) or A140C (c). Schematics are shown above each spectrum with the N-terminal (blue), NAC (pink) and C-terminal (red) regions highlighted. The location of the spin label is indicated by a yellow circle. Note that small chemical shift changes are observed upon reduction with ascorbic acid, which can be attributed to small changes in pH (Methods). As a consequence, 2 mM ascorbic acid was used throughout this study, resulting in incomplete reduction of the MTSL-labeled sample. This does not affect the pattern of PREs observed and results in an underestimate of the PRE effect (especially for residues in the NAC region such as K80, G84, S87, I88, A89, K96 and Q99).

Extended Data Fig. 5 Intramolecular PRE experiment for P1P2-GS αSyn.

a, Intramolecular PRE intensity ratios of amide protons (paramagnetic/diamagnetic) for P1P2-GS αSyn with the MTSL spin label at A90C at low ionic strengths (20 mM NaCl), 15 °C, pH 4.5. Blue, pink and red bars show intensity ratios for residues in the N-terminal, NAC and C-terminal regions, respectively. Dark blue bars highlight residues in the P1 and P2 regions that could be assigned and measured. The gray boxes mark the P1 and P2 regions. Black arrows show only a small PRE effect is observed in the P1-P2 region for P1P2-GS. Due to the repeating glycine and serine residues in the P1-P2 sequence, not all residues could be assigned (Methods). b, Comparison of a rolling window (over five residues for easier comparison) of the PRE effects for WT (blue), ΔΔ (red) and P1P2-GS (orange) αSyn. The black box is zoomed out in c to highlight residues in the P1-P2 region. The data for WT and ΔΔ are shown in Figs. 4d and 5d. Data for graphs in a,b are available as Source data. Source data

Extended Data Fig. 6 The role of P1 and P2 in intermolecular interactions.

a, Schematic of intermolecular PRE experiments. 14N and 15N αSyn are illustrated as cyan and dark blue chains, respectively. MTSL is shown as a yellow circle. b, HN-Γ2 rates for WT αSyn labeled with MTSL at position 40 (A40C) at pH 4.5 in low-salt (20 mM added NaCl) (black) or high-salt (200 mM added NaCl) (red) conditions, 15 °C. Bars depict residue-specific HN-Γ2 rates. c, HN-Γ2 rates at pH 4.5 under low-salt conditions (20 mM added NaCl) for WT (black) or ΔΔ (blue) αSyn, labeled at position 129 (S129C). Bars depict residue-specific HN-Γ2 rates. Data for graphs in b,c are available as Source data. Source data

Extended Data Fig. 7 CD binding assays of αSyn WT, ΔΔ and P1P2- GS to DMPS LUVs.

a, Far-UV CD spectra of 25 μM WT αSyn (blue) or ΔΔ (red) incubated in the absence or presence of liposomes (100:1 (M/M) DMPS:αSyn). b, Change of CD signal of WT αSyn (blue), ΔΔ (red) or P1P2-GS (orange) at 220 nm as a function of [DMPS]/[αSyn] ratio. Data were fitted (solid lines) to a single-step binding model, yielding the affinity (KD) and stoichiometry value (L, the number of DMPS molecules in the bilayer that are involved in binding to one molecule of αSyn). c, Far-UV CD spectra of 25 μM WT αSyn (blue) or P1P2-GS (orange) incubated in the absence or presence of 100 times molar excess of DMPS LUVs. d, Dynamic light scattering of DMPS liposomes showing they have a hydrodynamic radius (Rh) of on 81 nm. Data for graphs in a,c,d are available as Source data. Source data

Supplementary information

Source data

Source Data Fig. 1

Data for Fig. 1c−e; in silico data for α-synuclein WT (Zyggregator, CamSol and Rosetta Energy).

Source Data Fig. 2

Data for Fig. 2a−d; ThT raw data for WT, ΔP1, ΔP2 and ΔΔ at pH 4.5 and 7.5 at 200 mM NaCl.

Source Data Fig. 3

Data for Fig. 3a−c; ThT raw data for A140C (monomer/dimer), V40C (monomer/dimer), V52C (monomer/dimer) and WT.

Source Data Fig. 4

Data for Fig. 4b−g; intramolecular PRE intensities for paramagnetic and diamagnetic spectra and height intensity ratios, data for 20 mM NaCl and 200 mM NaCl, WT α-synuclein with spin label at positions A18C, A90C and A140C. ThT data in Source Data Extended Data Fig. 1.

Source Data Fig. 5

Data for Fig. 5b−g; intramolecular PRE intensities for paramagnetic and diamagnetic spectra and height intensity ratios, data for 20 mM NaCl and 200 mM NaCl, ΔΔ α-synuclein with spin label at positions A18C, A90C and A140C. ThT data in Source Data Extended Data Fig. 1.

Source Data Fig. 6

Data for Fig. 6b−d; FRAP and mobility raw data for experiments in C. elegans for WT, ΔP1 and ΔΔ at day 0, 3, 5, 7, 11 and 13.

Source Data Fig. 6

Uncropped western blot to show the expression level of synuclein in worms.

Source Data Fig. 7

Data for Fig. 7a−f; CD raw data for binding studies with liposomes for WT, ΔΔ and P1P2-GS.

Source Data Fig. 8

Data for Fig. 8d; ThT raw data for aggregation assays in the presence of DMPS liposomes for WT, ΔΔ and P1P2-GS.

Source Data Extended Data Fig. 1

Data for Extended Data Fig. 1a−h; ThT raw data for WT, ΔP1, ΔP2 and ΔΔ at pH 4.5 and 7.5 at 200 mM NaCl and 20 mM NaCl.

Source Data Extended Data Fig. 2

Data for Extended Data Fig. 2a; ThT raw data for WT, ΔP1, ΔP2 and ΔΔ seeded with WT α-synuclein seeds.

Source Data Extended Data Fig. 3

Data for Extended Data Fig. 3d−g; ThT raw data of ΔC1 and P1P2-GS at pH 4.5 and 7.5 at 200 mM NaCl and 20 mM NaCl.

Source Data Extended Data Fig. 5

Data for Extended Data Fig. 5a,b; NMR height intensities for paramagnetic and diamagnetic spectra of P1P2-GS including peak intensity ratios. Smoothed PRE ratios for WT, ΔΔ and P1P2-GS.

Source Data Extended Data Fig. 6

Data for Extended Data Fig. 6b,c; intermolecular PRE height intensities of paramagnetic and diamagnetic spectra of WT (spin label at position V40C and S129C) and ΔΔ (spin label at position S129C), including peak intensity ratios.

Source Data Extended Data Fig. 7

Data for Extended Data Fig. 7d; DLS data for hydrodynamic radius determination of DMPS liposomes. CD data in Source Data Fig 7.

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Doherty, C.P.A., Ulamec, S.M., Maya-Martinez, R. et al. A short motif in the N-terminal region of α-synuclein is critical for both aggregation and function. Nat Struct Mol Biol 27, 249–259 (2020). https://doi.org/10.1038/s41594-020-0384-x

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