Peptide dimer structure in an Aβ(1-42) fibril visualized with cryo-EM

Abstract

Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of β-amyloid (Aβ) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aβ fibrillation is extensively documented, much is still unknown about the formation of these Aβ aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aβ(1-42), a particularly pathogenic variant of Aβ peptide. Our model reveals that the individual layers of the Aβ fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal β-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aβ peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aβ peptide.

Keywords: Frealix; cross-β; protein aggregation; protein folding.

Fig. S1.

Twofold symmetrical and asymmetrical reconstruction. Comparison of the cross-sectional density of Aβ(1–42) reconstruction with (A) and without (B) imposing twofold symmetry.

Fig. 1.

Features of the reconstructed density map. (A and B) Negative-stain EM (A) and cryo-EM (B) images of the analyzed Aβ(1–42) fibril morphology. (C and D) Side view (C) and cross-section (D) of the reconstruction (rendered at 5 Å). The coloring (blue and red) reflects the presence of two leaflets and two P domains. (E) Cross-sectional view with the zipper-like density superimposed with two six-residue poly-l-Ala β-strands. The lines show the possible peptide trace in the C domain (continuous) and P domain (dashed). The solid lines and six-residue β-strands delineate the ∼7.5-nm section of the peptide forming the β-sheet leaflets at the fibril core. A–C are on the same scale.

Fig. S2.

FRC curves calculated for the reconstructed Aβ(1–42) fibril. Different curves were calculated to assess the overall resolution and the resolution of features at different radii from the fibril center. To calculate the resolution for a particular radial interval, the map was masked with a soft-edged mask to remove density outside that interval. The curve calculated for the unmasked reconstruction indicates a resolution of ∼7 Å at FRC = 0.143 (green). At the fibril core (0 ≤ radius ≤ 6 Å), this resolution is about 5 Å (red). At larger radii (16 Å ≤ radius ≤ 25 Å, purple), the resolution drops to about 7-Å resolution and drops further to about 9 Å at the periphery (25 Å ≤ radius ≤ 45 Å, blue).

Fig. S3.

Core region density of Aβ(1–42) superimposed with a steric zipper structure. C domain of the Aβ(1–42) density superimposed with a steric zipper predicted for Aβ(1–42). Out of all Aβ-derived steric zippers that have been published within the zipper database (services.mbi.ucla.edu/zipperdb) or listed within the Protein Data Bank (19, 20), this is the one that best fits our fibril density within the zipper-like segment.

Fig. 2.

Antibody Fab-based identification of the Aβ(1–42) N terminus. (A and B) Negative-stain EM images of Aβ(1–42) fibrils (A) and of fibrils decorated with 2H4 Fab (B). (C–F) Cryo-EM–based reconstructions of Aβ(1–42) fibrils (C and E) and of 2H4-Fab-decorated fibrils (D and F), shown in side views (C and D) or cross-sections (E and F) rendered at 15 Å. (G) Superimposition of the cross-section of a 2H4-Fab–decorated fibril (yellow) with the cross-section of the undecorated fibril (magenta) and ribbon diagrams of four Aβ-binding Fab fragments [Fab WO2 (17)]. Blue lines indicate the path of the peptide in the density with multiple possible conformations at the peptide N terminus to produce multiple binding sites. A–D are on the same scale.

Fig. S4.

Epitope mapping of the Aβ-binding antibody 2H4. (A) The spot peptide binding assay of all different Aβ(1–42) sequences bound on a membrane incubated with N-terminal antibody 2H4. Spots 1–30 comprise all possible 13-mer fragments of Aβ(1–42) starting at the N terminus, spots 31–63 comprise all 10-mer fragments, spots 64–98 comprise all 8-mer fragments, and spots 99–135 comprise all 6-mer fragments. Spots highlighted with red frames show antibody binding to a corresponding peptide sequence. The recognized binding sequence for the antibody found in all bound fragments is FRH (framed). (B and C) Comparison of cross-sectional density of 2H4 Fab-decorated Aβ(1–42) fibrils with (B) and without (C) imposed twofold symmetry filtered to 15 Å.

Fig. S5.

Peptide interference with fibril assembly. (A) Amino acid sequences of Aβ(1–42) and derived peptide fragments. (B) Negative-stain EM images showing the effect of different peptide fragments on Aβ(1–42) fibril formation with ratios of Aβ(1–42) peptide to fragments 1:1 and 1:10. C-terminal fragment Aβ(31–36) shows clear inhibition of fibril formation at a 1:10 ratio.

Fig. 3.

Structural model of the Aβ(1–42) fibril. (A) Density superimposed with the family of models producing the best fit to our data. Models that did not account for clear density features or that produced highly unfavorable contacts were excluded from the list of possible models (Fig. S6 and Table S2). (B) Aβ(1–42) sequence and schematic of the packing of two Aβ(1–42) peptides with the zipper-like region framed. Positively charged amino acids are shown in blue and negatively charged ones in red. (C) Side view of the fibril density (transparent) superimposed with a backbone model of the fibril core (26 residues). Color scheme from blue to red denotes the N- to C-terminal orientation of the chain. (D) Fibril cross-section with dashed lines indicating the possible peptide path in the P domain. (E) Fibril slice corresponding to a stack of seven dimers. (F and G) Distribution of charged and uncharged amino acid residues in a space-filled model displaying all 42 residues. Yellow, uncharged residue; purple, charged residue. (F) Top view. (G) Side view. All models in this figure assume a nonstaggered assembly.

Fig. S6.

Peptide dimer models superimposed with the experimental density. (A) Four starting Cα traces (red, blue, orange, and cyan) were created (each 26 residues long), all following the central ordered density but with different paths and lengths at the C terminus. (B) These traces were used as templates to create models with different peptide fragment registers (15–40, 16–41, and 17–42). Only refined nonstaggered models are shown. Models shown in Fig. 3A are Red 17–42, Blue 16–41, and Orange 15–40.

Fig. S7.

Comparison of 2D projections. (A) Fully refined Red (17–42) Aβ model from Fig. S6 with manually extended N terminus to account for as much of the reconstructed density as possible and allow direct comparison of projections calculated from the model and reconstruction with the raw images. Manual building was performed using UCSF Chimera’s Structure Editing tool, followed by Adjust Torsion (32). (B) Different projection views of the density derived from the Aβ(1–42) model in A, low-pass–filtered to 5 Å (top row); projections of the reconstruction, filtered to 5 Å (middle row); and raw cryo-EM images, low-pass–filtered to 5 Å (bottom row). Projections were calculated using SPIDER (39).

Fig. 4.

Peptide dimer conformations in Aβ(1–42) and Aβ(1–40) fibrils. Cross-sections of the single-protofilament Aβ(1–42) fibril density from the current study and of the two-protofilament Aβ(1–40) fibril (13), both rendered at 7-Å resolution. Cyan, blue, and yellow: symmetrical and asymmetrical peptide dimers. The red lines represent the dimer interfaces that extend over 7.3 nm in the Aβ(1–42) fibril and over 5.3 nm in the Aβ(1–40) protofilament.