Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel

Abstract

α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent β-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs.

Fig. 1

aSyn fibrils with distinct polymorphs have in vitro seeding and toxicity in cells. a, b Negative-stain EM (a) of full-length aSyn fibrils showing two distinct polymorphs—rod (non-twisted filaments, white arrow) and twister (twisted filaments, black arrow)—and a fibril width around 10 nm (b). c, d Direct visualization (c) and FRET-based quantification (d) of seeded intracellular aSyn aggregates. Fluorescent images obtained using the FITC channel (ex. 488 nm, em. 525 nm) showed aSyn aggregates as indicated by bright fluorescent puncta (white arrows in c). The diffuse background fluorescence came from endogenously expressed soluble, non-aggregated YFP-aSyn in the cells. Transduction of sonicated aSyn fibril seeds into the cells induced intracellular aSyn aggregation, which was also quantified using FRET analysis (d and Supplementary Fig. 3). e Cytotoxicity of aSyn fibrils evaluated by MTT-based cell viability assay of differentiated PC12, neuron-like cells. The aSyn fibrils used in the seeding experiment (c, d) have significant cytotoxicity (p < 0.0001) at 500 nM. Data are presented as mean ± standard error. Results are from multiple independent biological experiments with n = 3–5 per experiment. *p ≤ 0.01, ***p ≤ 0.0001 vs. control (buffer used to produce the aSyn fibrils). Scale bar: (a) 100 nm (c) 50 μm

Fig. 2

Cryo-EM structures and atomic models of the aSyn rod and twister polymorphs. a The cryo-EM structures of the rod (left) and twister (right) polymorphs of the full-length aSyn fibrils shown as density slices (top inlet), as semitransparent surfaces overlaid with their atomic models viewed from two different angles (lower panels). The rod (blue) and twister (red) polymorphs contain two protofilaments composed of stacked β-sheets and packed by an approximate 2₁ screw axis of symmetry. Shown on the left and right sides are the 3D model of the rod and twister fibril polymorphs, respectively, with their distinctively different helical pitches depicted. b Model validation. Representative regions of density maps of both polymorphs are superimposed with their models showing match of side chain with cryo-EM densities. Intra-protofilament hydrogen bonds are shown in black dashed lines, and inter-protofilament hydrogen bonds are shown in magenta dashed lines. See details in Supplementary Figures 7 and 8

Fig. 3

Distinct zipper interfaces between protofilament kernels in the two aSyn polymorphs. a, b Residue interactions of two asymmetric units in two opposing protofilaments elucidate packing between and within these two protofilaments in the rod (a) and twister (b) polymorphs (viewed down fibril axis). Residues are colored by hydrophobicity (yellow: hydrophobic; green: polar; red: negative charge; blue: positive charge). c, d An overlay of protofilaments of the rod (blue) and twister (red) polymorphs reveals a conserved kernel of a bent β-arch. e Diffraction patterns of the full-length aSyn fibrils agree with those of NACore and preNAC peptide fibrils. f, g The two protofilaments in the rod (f) and twister (g) polymorphs contact by different residues (space-filled) and have distinct fibril core of tightly packed steric zippers of preNAC (blue) and NACore (red), as previously observed in those peptide fibril structures. PD familial mutation residues are labeled with underlines. The cryo-EM density maps are shown as gray mesh surfaces. Intra-protofilament hydrogen bonds are shown in black dashed lines, and inter-protofilament hydrogen bonds are in magenta

Fig. 4

Morphogenesis of aSyn fibril polymorphs arising from inter-protofilament packing. a Primary sequence of preNAC (blue) and NACore (red) critical for the aggregation of aSyn 1–140 and six PD familial mutations (cyan) located near the preNAC region. b Protofilaments sharing a kernel structure of a bent β-arch assemble into the rod and twister fibril polymorphs by packing at preNAC and NACore zipper interfaces, respectively. The PD familial mutations (cyan) likely disfavor the rod structure over the twister structures, and alter the polymorphic composition of aSyn fibrils