The fold of alpha-synuclein fibrils

Abstract

The aggregation of proteins into amyloid fibrils is associated with several neurodegenerative diseases. In Parkinson's disease it is believed that the aggregation of alpha-synuclein (alpha-syn) from monomers by intermediates into amyloid fibrils is the toxic disease-causative mechanism. Here, we studied the structure of alpha-syn in its amyloid state by using various biophysical approaches. Quenched hydrogen/deuterium exchange NMR spectroscopy identified five beta-strands within the fibril core comprising residues 35-96 and solid-state NMR data from amyloid fibrils comprising the fibril core residues 30-110 confirmed the presence of beta-sheet secondary structure. The data suggest that beta1-strand interacts with beta2, beta2 with beta3, beta3 with beta4, and beta4 with beta5. High-resolution cryoelectron microscopy revealed the protofilament boundaries of approximately 2 x 3.5 nm. Based on the combination of these data and published structural studies, a fold of alpha-syn in the fibrils is proposed and discussed.

Fig. 1.

A qualitative interpretation of quenched H/D exchange of α-syn and α-syn (30–110) fibrils based on NMR spectra. Fast [¹⁵N,¹H]-HMQC-spectra of solubilized ¹⁵N-labeled α-syn in d₆-DMSO containing 0.1% d₁-TFA, corresponding to fully protonated (A) and 1 h exchanged amyloid fibrils of α-syn (B). Many cross-peaks are lost on H/D exchange for 1 h. In C and D fast [¹⁵N,¹H]-HMQC-spectra of ¹⁵N-labeled α-syn (30–110) solubilized in d₆-DMSO containing 0.1% d₁-TFA, corresponding to fully protonated, and 1 h exchanged amyloid fibrils of α-syn (30–110), respectively. The amino acid assignment of the cross-peaks of the spectrum shown in C are shown in Fig. S2.

Fig. 2.

Detailed H/D exchange analysis and side-chain solvent accessibility of α-syn fibrils. (A) H/D exchange curves for several amide moieties as indicated. The peak volumes versus logarithm of the exchange time are shown. Data points are shown as spheres. Smooth red lines represent a superposition of two monoexponential fits of the raw data. For most residues a biphasic exchange is observed including a slow decay (≈1–10 h) and an extremely slow decay (>1,000 h). (B) Plot of the H/D exchange rates of the fast- and slow-exchanging population against the amino acid sequence. (C) Plot of the fraction of each amide moiety that exhibit extremely slow H/D exchange at rates <0.001 h⁻¹. This representation enables us to highlight the solvent protection of amides of the population defined as extremely slow-exchanging by taking into consideration that this conformation may comprise fast-exchanging amides in loops and turns. Only if the fraction is >0.15 is it considered that the amide undergoes a very slow exchange in the sample, because an accuracy of ≈10% is obtained by fitting the data with a superposition of two monoexponential decays. The amide moieties for which H/D exchange data were obtained are labeled with a single-letter amino acid code. (D) Side-chain solvent accessibility of Cys in fibrils of α-syn variants. The fluorescence of Alexa Fluor 488 cross-linked to the Cys side chain is given for various α-syn variants relative to the A124C variant. The side chain of residue 124 is, in accordance to H/D exchange, EPR, solid-state NMR, and limited proteolysis studies, highly solvent exposed. A large fluorescence value is indicative of a high degree of solvent accessibility.

Fig. 3.

Solid-state NMR of α-syn (30–110) fibrils. (A) ¹³C–¹³C DREAM spectrum for the intraresidual assignment. (B) ¹³C–¹³C proton-driven spin diffusion (PDSD) spectrum with a mixing time of 250 ms. Long-range cross-peaks are indicated by red circles and labeled accordingly. In addition, several cross-sections with long-range cross-peaks are shown and labeled accordingly. (C) Secondary chemical shift plot of the difference of Δδ(¹³Cα) − Δδ(¹³Cβ) with Δδ(¹³Cα) and Δδ(¹³Cβ), which are the differences between experimental ¹³Cα or ¹³Cβ chemical shifts and their corresponding “random-coil” chemical shifts. Highlighted in cyan are Gly residues for which only the Δδ(¹³Cα) could be calculated. Highlighted in gray are ambiguous assignments. This secondary chemical shift plot indicates the presence of β-strand motifs as indicated. (D) 2D distance plot of α-syn (30–110) fibrils showing distance restraints < ≈6 Å between residues, which have been extracted from the ¹³C–¹³C proton-driven spin diffusion spectra. Black squares indicate unambiguous distance restraints between residues. Gray squares indicate distances, which are only unambiguous under the assumption that α-synuclein fibrils form a five-layered β-sandwich. In addition, the β-strand motifs extracted from C are shown.

Fig. 4.

Cryo-negative stain transmission electron microscopy of straight wild-type α-syn and twisted α-syn (30–110) fibrils. In both cases, different bundling states can be observed. The straight filament is composed of a basic strand formed of two denser lines of 2.0-nm width separated by a gap of 1.5 nm (A, arrows labeled with “*1”) and tends to dimerize (A, arrows labeled with “*2”). Thicker filaments can be interpreted as tilted views of dimeric strands (A Right Center and Right), or as a different aggregation state. Straight filaments allowed the calculation of particle class averages, reproduced in C from 146, 39, and 71 particles (Top to Bottom). Twisted filaments also show variable thicknesses or density (B) and allowed a helical 3D reconstruction in D. D Lower shows the cross-section of the helical filament. (Scale bars: A and B, 20 nm; C and D, 10 nm.)

Fig. 5.

Proposed fold of α-syn fibrils. The proposed fold of a monomeric α-syn within a protofilament is shown in Center. The incorporation of a protofilament into the straight (Left) and twisted (Right) fibril type is indicated by a schematic drawing.