Conformational Switch in the Alpha-Synuclein C-Terminal Domain Directs Its Fibril Polymorphs

Abstract

α-Synuclein (αSyn) inclusions are a pathological hallmark of several neurodegenerative disorders. While cryo-electron microscopy studies have revealed distinct fibril polymorphs across different synucleinopathies, the molecular switches controlling polymorphism remain unveiled. In this study, we found that fibril morphology is associated with the conformational state of monomeric αSyn. Through systematic evaluation of the ionic strength and temperature, we generated two distinct polymorphs: a twisted morphology at low ionic strength and temperature, and a rod-like morphology at higher ionic strength and temperature. Using solid-state NMR, we revealed that both polymorphs share a highly conserved core structure, with morphological differences arising probably from distinct structural arrangements at the protofilament interfaces. Furthermore, we found that a specific conformational change in the C-terminal domain of the monomeric αSyn serves as a molecular switch for the formation of polymorphs. Interestingly, this conformational change can also be triggered by calcium binding to the C-terminus, connecting environmental factors to specific fibril architectures. Our results reveal a conformational role for the C-terminal domain that influences αSyn fibril morphology, providing significant insights into the fibrogenesis of αSyn.

Keywords: NMR; alpha synuclein; amyloid fibrils; fibril polymorphism; protein folding; proteins.

Figure 1

αSyn can generate rod‐like and twisted polymorphs under different fibrillation reaction conditions. A) αSyn fibril formation kinetics monitored by ThT intensity at 37 °C, at the indicated concentrations of NaCl. B) Maximum ThT intensity values from panel A are plotted as a function of NaCl concentration. Also shown are the TEM images for selected fibrils, revealing twisted fibrils at low maximal ThT intensity (blue and green squares), and rod‐like fibrils at high maximal ThT intensity (yellow and red squares). White scale bar: 100 nm. C) Maximal ThT intensity values as a function of temperature and ionic strength of 0.15. TEM imaging confirms the formation of twisted fibrils at low maximal ThT intensity (blue and green squares), and rod‐like fibrils at high maximal ThT intensity (yellow and red squares). Black scale bar: 100 nm. D) Maximal ThT intensity values as a function of the NaCl concentration at different temperatures. Except for 37 °C, each condition was replicated five times. E) Phase diagram mapping fibril morphology as a function of NaCl concentration and temperature. Blue region indicates predominant twisted fibrils; green region corresponds to the inflection point where both rod and twisted fibrils coexist; red region shows predominant rod‐like fibrils.

Figure 2

Solid‐state NMR analysis of twisted and rod‐like fibrils. A) Twisted and rod‐like fibrils were generated at the NaCl concentration and temperature conditions indicated in the upper part of the panels. The morphological properties of the fibrils were confirmed by TEM visualization (scale bar: 100 nm) and PKR after 30 min of digestion (arrows: distinctive bands of each polymorph). B) 2D ¹³C‐¹³C DARR spectra of twisted and rod‐like fibrils produced under the specified conditions. Aliphatic‐carbonyl and aliphatic‐aliphatic ¹³C correlations are shown in the left and right panels, respectively, for each sample. Intra‐residue correlations spanning 1–3 bonds were observed with a short ¹³C‐¹³C DARR mixing time of 20 ms. No apparent differences among all spectra.

Figure 3

Solution NMR analysis of αSyn monomer conformational states. A) Schematic representation of four experimental routes examining the monomeric αSyn conformational changes, using ¹H‐¹⁵ N HSQC measurements under polymorph‐inducing conditions. The reference spectrum was taken at 10 °C without NaCl (black cross). R1: Temperature variation (10–55 °C) at 0 mM NaCl; R2: Temperature variation (10–55 °C) at 150 mM NaCl; R3: NaCl concentration variation (0–500 mM) at 30 °C; and R4: NaCl variation (0–500 mM) at 37 °C. White circles represent the tested conditions. B) Peak intensity ratio observed in the ¹H‐¹⁵ N HSQC spectra at selected points along routes R1 to R4, normalized to the reference spectrum (no NaCl at 10 °C). Peaks corresponding to the C‐terminal residues (residues 110–130) are highlighted across all routes. C) Quantification of the overall peak intensity for residues 110–130 through Gaussian curve fitting and area integration. D) Integrated peak intensities (colored circles) from panel C mapped onto the fibril morphology phase diagram.

Figure 4

Calcium‐induced formation of rod‐like fibrils under modest ionic strength conditions. A) Peak intensity ratio from the ¹H‐¹⁵ N HSQC spectra at varying Ca²⁺ concentrations but constant physiological ionic strength (0.15), normalized to the reference spectrum taken without NaCl or Ca²⁺ at 10 °C. C‐terminal residues (110–130) showed a characteristic local peak pattern seen in Figure 3B. B) Box‐and‐whisker plot showing maximal ThT intensities from fibrillation reactions, at constant ionic strength of 0.15 across different Ca²⁺ concentrations. TEM images demonstrate the morphology difference: low maximal ThT intensity conditions produced twisted fibrils (blue square), while high maximal ThT intensity conditions yielded rod‐like fibrils (red square). Black scale bar: 100 nm. C) Integrated peak intensities for residues 110–130 mapped onto the morphology phase diagram for calcium‐containing samples (green diamonds) at ionic strength of 0.15 or 0.20, and calcium‐free samples at different ionic strength (black open circles, same values as those shown in Figure 3D), at 37 °C. Calcium addition induces a conformational shift favoring rod‐like over twisted fibril formation.

Figure 5

Relationship between αSyn C‐terminus dynamics and fibril morphology. The conformational state of the C‐terminal domain determines the fibril polymorphism: high C‐terminus flexibility promotes twisted fibrils, while partial C‐terminus folding drives the assembly of rod‐like fibrils. Created with BioRender.com.