Elucidating the Unique J-Shaped Protomer Structure of Amyloid-β(1-40) Fibril with Cryo-Electron Microscopy

Abstract

Although the structural diversity of amyloid-β (Aβ) fibrils plays a critical role in the pathology of Alzheimer's disease (AD), the mechanisms underlying this diversity remain poorly understood. In this study, we report the discovery of a novel J-shaped protomer structure of Aβ40 fibrils, resolved at 3.3 Å resolution using cryo-electron microscopy. Under controlled conditions (20 mM sodium phosphate buffer, pH 8.0) designed to emphasize intra-protomer interactions and slow fibril elongation, the J-shaped structure revealed distinct salt bridges (e.g., D1-K28, R5-E22) that stabilize the fibril core. These findings expand our understanding of the free energy landscape of fibril formation, shedding light on how specific environmental factors, such as pH and ionic strength, may influence fibril polymorphism. Importantly, the unique features of the J-shaped protomer provide insights into the structural basis of amyloid plaque diversity in AD and suggest potential therapeutic strategies targeting intra-protomer interactions. This study underscores the importance of fibril polymorphism in AD pathology and offers a foundation for future research into fibril-targeted therapies.

Keywords: Alzheimer’s disease; amyloid-β; cryo-electron microscopy; fibril.

Figure 1

ThT fluorescence assay of Aβ40. Aβ40 solutions (0.1 mM) were prepared in sodium phosphate buffers and incubated at 37 °C. The buffers used were 100 mM pH 7.4 (red), 100 mM pH 8.0 (orange), 20 mM pH 7.4 (green), and 20 mM pH 8.0 (blue). Each intensity value represents the mean ± SD of the three values.

Figure 2

Cryo-EM structure of J-shaped Aβ40 fibrils: (a) Sequence of Aβ40, with the observed three β-strands colored blue. (b) Top view of the cryo-EM density map of Aβ40 fibrils, with the core layer model overlaid. The ball-and-stick model represents the atomic structure of the fibril backbone within the cryo-EM density. The map is displayed at a contour level of 9 σ, with the scale bar indicating 1 nm. (c) Packing scheme of one molecular layer of the fibril. (d) Ribbon representation of the top (upper) and side views (lower) of the J-shaped Aβ40 fibril core, containing five molecular layers with a dimer.

Figure 3

Schematic view of Aβ40 fibril core: (a) J-shaped fibril structure (this research, PDB: 9IIO). (b) I-shaped fibril structure (PDB: 6W0O). (c) C-shaped fibril structure (PDB: 6SHS).

Figure 4

Intra-protomer interactions of Aβ40 fibrils: (a) J-shaped fibril structure (this research, PDB: 9IIO). (b) I-shaped fibril structure (PDB: 6W0O). (c) C-shaped fibril structure (PDB: 6SHS). The side chains of amino acid residues forming salt bridges are displayed in stick representation, with acidic amino acids in red and basic amino acids in cyan.

Figure 5

Inter-protomer interactions of Aβ40 fibrils. Cross-sectional view of ((a), left) the J-shaped fibril (this research, PDB: 9IIO), ((b), left) the I-shaped fibril (PDB: 6W0O), and ((c), left) the C-shaped fibril (PDB: 6SHS). Hydrophobic residues involved in inter-protomer interactions are highlighted in green, and residue S26 is marked in yellow. The acidic amino acid residues and basic amino acid residues involved in intra-protomer interactions are shown in red and cyan, respectively. Hydrogen bonding network in ((a), right) the segment from V12 to V40 in the β-sheet of the J-shaped fibril, ((b), right) from Y10 to V40 in the I-shaped fibril, and ((c), right) from H14 to M35 in the C-shaped fibril.