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

Nature Structural & Molecular Biology volume 27, pages 249–259 (2020)Cite this article

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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.

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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).

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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.

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Author notes

  1. These authors contributed equally: Ciaran P. A. Doherty, Sabine M. Ulamec.

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, United Kingdom

    Ciaran P. A. Doherty, Sabine M. Ulamec, Roberto Maya-Martinez, Sarah C. Good, Jemma Makepeace, G. Nasir Khan, Patricija van Oosten-Hawle, Sheena E. Radford & David J. Brockwell

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Contributions

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.

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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.

a−h, 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. b−g, 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 c−f. 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 b−g 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.

a−c, 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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