Defining essential charged residues in fibril formation of a lysosomal derived N-terminal α-synuclein truncation

Abstract

N- and C-terminal α-synuclein (α-syn) truncations are prevalent in Parkinson's disease. Effects of the N- and C-terminal residues on α-syn aggregation and fibril propagation are distinct, where the N-terminus dictates fibril structure. Here, the majority of α-syn truncations are assigned by intact mass spectrometry to lysosomal activity. To delineate essential charged residues in fibril formation, we selected an N-terminal truncation (66-140) that is generated solely from soluble α-syn by asparagine endopeptidase. Ala-substitutions at K80 and E83 impact aggregation kinetics, revealing their vital roles in defining fibril polymorphism. K80, E83, and K97 are identified to be critical for fibril elongation. Based on solid-state NMR, mutational and Raman studies, and molecular dynamics simulations, a E83-K97 salt bridge is proposed. Finally, participation of C-terminal Lys residues in the full-length α-syn fibril assembly process is also shown, highlighting that individual residues can be targeted for therapeutic intervention.

Fig. 1. Lysosomal degradation of α-syn.

a Schematic representation of the primary amino acid sequences of 1–140 (top) and 66–140 (bottom), coloring basic (blue) and acidic (red) residues. Charged residues that participate in salt bridges within the shared α-syn fibril core (residues 36–99) are indicated^{,,,,,,,–,–,–,,}. b Schematic representation of the primary amino acid sequence of α-syn with cleavage sites generated for either soluble (cyan) or fibrillar (black) α-syn. Residues 36–99 (light gray) show cryoEM fibril core while residues 61–95 denote the NAC region (dark gray). c α-Syn peptide fragments derived from lysosomal degradation of soluble (cyan) and fibrillar (black) α-syn. Brain lysosomal extracts were obtained from a 10-month-old male mouse. Fragment masses and residue assignments are reported in Supplementary Table 1. Previously identified fragments from PD patients are denoted by asterisks. Effect of mouse age on specific lysosomal protease activities. Brain lysosomal extracts were obtained from two female mice aged 2 and 17 months. Fluorogenic substrates d Ac-RR-AMC for CtsB, e Ac-FR-AMC for CtsL, f MCA-GKPILEFRKL(Dnp)-D-R-NH₂ for CtsD, and g AENK-AMC for AEP were incubated with lysosomal extracts (10–40 µg total protein) at pH 5.0 with 5 mM DTT, 37 °C. Fluorescence was recorded as a function of time (30 and 60 min), and relative fluorescence units (RFU) are reported (n = 3 technical replicates). Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Fig. 2. Aggregation kinetics and structural characterization of 66–140 fibrils.

a Aggregation reactions of Ac1‒140 (purple) and 66–140 (black) monitored by ThT fluorescence in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking supplemented with a 2-mm glass bead. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 4). For 66–140, average t_lag and t_½ values were 5 and 12 h for 40 μM and 3 and 9 h for 80 μM, respectively. For Ac1–140, average t_lag and t_½ values were 8 and 13 h for 40 μM and 5 and 14.5 h for 80 μM, respectively. b Representative TEM and AFM images of 66‒140 taken post-aggregation. Scale bars as indicated. Full-sized images are shown in Supplementary Fig. 5. c Raman spectra of Ac1‒140 (purple) and 66‒140 (black) fibrils. Solid lines and shaded regions represent the mean and SD, respectively (n ≥ 15). Amide-I, amide-II, and amide-III regions are as indicated. The frequency location of a distinguishing peak (1012.5 cm⁻¹) in 66–140 is denoted by an asterisk. Inset shows an expanded view of the boxed spectral area. Spectra have been normalized to the Phe breathing mode (1003 cm⁻¹). Difference spectrum is shown in Supplementary Fig. 6. Source data are provided as a Source Data file.

Fig. 3. ssNMR of 66‒140 fibrils.

a 2D ¹³C-¹³C ssNMR spectra of 66‒140 fibrils, recorded under conditions where only immobilized, structurally ordered segments are visible. Residue assignments are based on 2D NCACX/NCOCX, and 3D NCACX/NCOCX/CANCOCX spectra (Supplementary Figs. 7–12). Contour levels increase by successive factors of 1.3. b Secondary chemical shifts Δδ¹³Cα (top) and Δδ¹³Cβ (bottom) obtained by subtracting random-coil shifts from the observed chemical shifts. Blue arrows indicate β-sheet secondary structure predicted by TALOS-N. c Structural model of the 66‒140 fibril generated by modifying model 8 in PDB ID: 8FPT using UCSF Chimera. NMR-assigned residues consist of 66 to 95, with blue arrows depicting the β-sheet secondary structure for residues 69 to 72, 74 to 79, and 87 to 92. Fibril axis as indicated. Source data are provided as a Source Data file.

Fig. 4. Aggregation kinetics and structural characterization of single-Ala mutants of 66–140.

a Schematic representation of the primary amino acid sequences of 66–140 showing sites of mutations in this study. Fibril core assigned by ssNMR is underlined. b Aggregation reactions monitored by ThT fluorescence of 40 (top) and 80 (bottom) µM of 66–140 (black), K80A (blue), E83A (magenta), K96A (purple), K97A (green), D98A (gold) and K102A (teal) in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking supplemented with a 2-mm glass bead. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 5). Complete aggregation kinetics to 160 h are shown in Supplementary Fig. 14. c Fibril morphologies of single-Ala mutants visualized by TEM (top) and AFM (bottom). TEM scale bars are 200 nm. Full-size images are shown in Supplementary Fig. 15. Source data are provided as a Source Data file.

Fig. 5. Raman spectroscopy of single-Ala mutants and cross-seeding kinetics of 66–140 by single-Ala fibrils.

a Raman spectra of 66‒140 (black) and mutants (K80A (blue), E83A (magenta), K96A (purple), K97A (green), D98A (gold) and K102A (teal)) in the fingerprint (left), amide-III (middle) and amide-I (right) regions. Solid lines and shaded regions represent the mean and SD, respectively (n ≥ 15). Spectrum of 66‒140 is the same as shown in Fig. 2c. Full spectra are shown in Supplementary Fig. 16. b Difference spectra generated by subtracting the average spectrum of 66‒140 from that of the 66‒140 mutants. Shaded regions represent the propagated error. Dashed lines are guides to key spectral differences in the mutants. Color scheme is same as Fig. 5a. c Aggregation kinetics monitored by ThT fluorescence (10 µM) of soluble 66‒140 (30 µM) seeded with 0.3 µM 66–140 (black), K80A (blue), E83A (magenta), K96A (purple), K97A (green), D98A (gold) and K102A (teal) fibrils in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking in the absence of 2 mm glass beads. Unseeded 66‒140 is shown as a control. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 6). ppt refers to amount of insoluble material pelleted by ultracentrifugation. Source data are provided as a Source Data file.

Fig. 6. Cross-seeding kinetics, TEM, and Raman spectroscopy of single-Ala mutants seeded with 66‒140 fibrils.

a Aggregation kinetics monitored by ThT (10 µM) of soluble 66–140 (black), K80A (blue), E83A (magenta), K96A (purple), K97A (green), D98A (gold) and K102A (teal) (30 µM) seeded with 66–140 fibrils (0.3 µM) in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking in the absence of 2 mm glass beads. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 12). Precipitated (ppt) material calculated (%) after ultracentrifugation post-aggregation is shown for each mutant. b Representative TEM images taken post-seeding. Scale bar is 200 nm. Full images are shown in Supplementary Fig. 21. c Comparison of Raman fingerprint region of the cross-seeded (as colored in Fig. 6a) and unseeded Ala-mutant fibrils (black). Solid lines and shaded regions represent the mean and SD, respectively (n ≥ 15). Dashed lines indicate the frequency location of a distinguishing peak (1012.5 cm⁻¹) in 66–140. Spectra of the unseeded samples are the same as shown in Fig. 5a. Full spectra are shown in Supplementary Fig. 22. The bottom panel shows difference spectra generated by subtracting the average unseeded from the average seeded spectrum. The error has been propagated from the SD of the averaged spectra. Subscript f denotes fibrils. Source data are provided as a Source Data file.

Fig. 7. Aggregation and structural characterization of charge-switch mutants.

a Aggregation kinetics of 30 µM soluble 66–140 (black), K80E/E83K (blue), E83K/K96E (green), and E83K/K97E (gray) seeded with 66‒140 fibrils (1 mol%) in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking in the absence of 2 mm glass beads. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 6). b Representative TEM image taken post aggregation of E83K/K97E seeded with 66–140 fibrils. Full image is shown in Supplementary Fig. 23. c Comparison of Raman spectra of the fingerprint region of cross-seeded E83K/K97E with 66‒140 (gray) fibrils and 66‒140 (black) fibrils. Solid lines and shaded region represent the mean and SD, respectively (n ≥ 15). The bottom panel shows the difference spectrum generated by subtracting the spectrum of 66–140 from that of cross-seeded E83K/K97E. Dashed lines are guides to key spectral features in 66‒140. Spectrum of 66‒140 is the same as shown in Fig. 2c. Full spectrum of cross-seeded sample is shown in Supplementary Fig. 24. Subscript f denotes fibrils. Source data are provided as a Source Data file.

Fig. 8. Aggregation kinetics, fibril morphology characterization, and cross-seeding kinetics of single-Ala mutants of Ac1–140.

a Schematic representation of the primary amino acid sequences of Ac1–140 showing sites of mutations. CryoEM fibril core assigned to residues 36 to 99 (gray). Aggregation reactions monitored by ThT fluorescence (10 µM) of 50 µM Ac1–140 (b), AcK80A (c), AcK96A (d) and AcK97A (e) in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking supplemented with a 2-mm glass bead (n ≥ 4). f Fibril morphologies of single-Ala mutants visualized by TEM. Scale bars are 100 nm. Full-size images are shown in Supplementary Fig. 27. g Aggregation kinetics monitored by ThT fluorescence (10 µM) of soluble Ac1‒140 (50 µM) seeded with 2.5 µM Ac1–140 (black), K80A (blue), K96A (purple) and K97A (green) fibrils in pH 7.4 buffer (20 mM NaPi, 140 mM NaCl) at 37 °C with continuous linear shaking in the absence of 2 mm glass beads. Unseeded 66‒140 (black) is also shown as a control. Solid lines and shaded regions represent mean and SD, respectively (n ≥ 5). Source data are provided as a Source Data file.