Atomic structure and hierarchical assembly of a cross-β amyloid fibril

Abstract

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Fig. 1.

MAS NMR experiments used to probe the intermolecular arrangement of TTR(105–115) fibrils. (A and B) Shown are 2D ¹³C–¹⁵N ZF-TEDOR (τ_mix = 10.24 ms) (A) and 2D ¹³C–¹³C PDSD (τ_mix = 200 ms) (B) MAS NMR spectra of TTR(105–115) U–¹⁵N,¹³C labeled as YTIAALLSPYs and recorded at ω_0H/2π = 750 MHz and ω_r/2π = 12.5 kHz and ω_0H/2π = 900 MHz and ω_r/2π = 11 kHz, respectively, and T = 280 K. The spectra were used to constrain the intermolecular distances between the β-sheets within the TTR(105–115) protofilament. In the two spectra, intermolecular and intramolecular cross-peaks are labeled in red and black, respectively, and all cross-peaks have been assigned. (C) A cross-section of the TTR(105–115) protofilament depicting the sheet-to-sheet interface and the 11 distance constraints labeled in the spectra of A and B (red lines). The blue lines denote four additional distances between L111–¹³Cδ1 and –¹³Cδ2 and A109–¹⁵N and A108–¹³CO detected in additional spectra of a sample labeled as YTIAALLSPYS. The complete set of 23 intersheet distance restraints is shown in SI Appendix, Fig. S2B. Odd- and even-numbered side chains are shown as orange and violet sticks, respectively, with secondary structure shown in a cyan ribbon representation. (D) ¹³C–¹⁵N REDOR data obtained from a TTR(105–115) sample labeled as Y105–¹⁵N, S115–¹³CO₂H (YTIAALLSPYS) and designed to detect interprotofilament contacts in the fibril. The intermolecular ¹³C–¹⁵N interaction between the terminal ¹⁵N and ¹³C atoms corresponds to a distance of 3.57 ± 0.06 Å, consistent with a head-to-tail protofilament arrangement as illustrated in Right. The curve was fitted by using the program SPINEVOLUTION (SI Appendix, SI Materials and Methods).

Fig. 2.

Atomic-resolution structure of the TTR(105–115) protofilament determined by MAS NMR. The structures (calculated with CNSsolve; see SI Appendix, SI Materials and Methods) have a rmsd of ∼0.4 Å for the backbone and 0.7 Å for all atoms. Odd- and even-numbered side chains are shown as orange and violet sticks, respectively, with secondary structure shown in a cyan ribbon representation. (A) The β-sheet viewed perpendicularly to the fibril axis illustrating the parallel in-register β-strands and the hydrogen bonds defining the β-sheet (yellow lines). The conformation was determined from eight ¹³C=O–¹³C=O double quantum distance measurements and one ¹³C=O–¹³C=O REDOR distance measurement (SI Appendix, Table S1). (B) Cross-sectional view of the two-sheet protofilament along the peptide chain direction. There is a sheet–sheet offset of approximately one-fifth the separation of hydrogen-bonded β-strands (i.e., 0.2 × 4.67 Å = 0.93 Å) shown clearly by the interdigitation of the Y105 (orange sticks) and Y114 (violet sticks) side chains. (C) Protofilament–protofilament interface viewed looking down the long axis of the fibril showing the head-to-tail packing arrangement. Interprotofilament hydrogen bonds between the terminal C=O and N–H groups and between the Y105 OH atoms and the S115 Oγ atoms are depicted as yellow dashes.

Fig. 3.

Representative cryo-EM images of averaged fibrils (class averages), surface representations of reconstructions, 2D projections, and contoured density cross-sections of the three types of fibril formed by TTR(105–115). (A) Doublet class average (Left), 3D reconstruction (Center; orange), and 2D projection of the fibril reconstruction (Right). (B) Triplet class average (Left), 3D reconstruction (Center; yellow), and 2D projection of the fibril reconstruction (Right). (C) Quadruplet class average (Left), 3D reconstruction (Center; purple), and 2D projection of the fibril reconstruction (Right). (D–F) Contoured density cross-sections of the doublet (D), triplet (E), and quadruplet (F) fibrils. Contours represent density levels of multiples of 0.5σ above the average density of the background (outermost contour). Measurements of dimensions were determined at 1.0σ above the mean density.

Fig. 4.

Atomic-resolution cross-sections of the three types of TTR(105–115) amyloid fibrils determined by combining MAS NMR and cryo-EM. Reconstructed cross-section density maps are shown as two electron density isosurfaces (1.0σ and 2.2σ above the mean density) with the secondary structure of the constituent NMR-derived protofilaments shown in a cyan ribbon representation. (A–C) The doublet (A), triplet (B), and quadruplet (C) fibril cross-sections can accommodate pairs of two, three, and four interconnected protofilaments, respectively. (D–F) All-atom representation of the doublet (D), triplet (E), and quadruplet (F) cross-sections with cryo-EM envelopes superimposed. Cryo-EM envelopes are shown as orange, yellow, and purple contours at 1.0σ above the mean density for doublet, triplet, and quadruplet fibrils, respectively. For a discussion on the resolution of the cryo-EM maps, see SI Appendix, Table S4.

Fig. 5.

Five diverse biophysical techniques were integrated to determine unambiguously the structures of each of the motifs that make up the TTR(105–115) amyloid fibrils. Spanning five orders of magnitude, the overlapping length scales of MAS NMR (0.1–10 Å), X-ray diffraction (3–100 Å), cryo-EM (8–1,000 Å), AFM (30–1,000 Å), and STEM (80–1,000 Å) enabled us to derive self-consistent, high-precision structural restraints on the secondary (β-strand and -sheet; distance restraints of <6 Å), tertiary (protofilament; distance restraints of 4.5–37 Å), and quaternary structure (filament and fibril; distance restraints of 16–1,000 Å) of the TTR(105–115) amyloid fibrils. (A) Histogram of STEM MPL measurements of TTR(105–115) fibrils, which reveals three populations of fibrils, with a best fit (gray solid line) being the sum of three Gaussian curves with values of 2.5 ± 0.3 kDa/Å (orange solid line), 3.3 ± 0.3 kDa/Å (yellow solid line), and 4.1 ± 0.3 kDa/Å (purple solid line). The orange, yellow, and purple dashed lines refer to the number of TTR(105–115) peptides per 4.67-Å repeat in the doublet (8 peptides), triplet (12 peptides), and quadruplet (16 peptides) fibrils, respectively. (B) Comparison of the high-resolution experimental X-ray diffraction pattern from TTR(105–115) fibrils (34) (Left) and the simulated X-ray diffraction pattern for TTR(105–115) fibrils (Right). The fibril axis is vertical, with the incident beam directed orthogonally to this axis. The meridional reflection at 4.67 Å and the equatorial reflection at 8.86 Å are characteristic of cross-β structure. (C) High-resolution AFM image of fibrils (pink and purple) and filaments (green) formed by TTR(105–115). (Scale bar, 1 μm.) Fibrils (pink and purple) have heights ranging from 70 to 160 Å and pitches of 950 ± 100 Å. The filament has an average height of 38.7 ± 4.4 Å. (D) Hierarchy of atomic-resolution motifs involved in the self-assembly of the amyloid fibrils and their polymorphism.

Fig. 6.

Close-up view of the MAS NMR atomic-resolution structure of the triplet fibril fitted into the cryo-EM reconstruction (Center). The background image of the fibril (Left) was taken using TEM. (Scale bar, 50 nm.) The fibril surfaces (Right) are shown at 1.0σ (white) and 2.2σ (yellow) above the mean density, respectively, and the constituent β-sheets are shown in a ribbon representation; oxygen, carbon, and nitrogen atoms are shown in red, gray, and blue, respectively.