Antiparallel β-sheet architecture in Iowa-mutant β-amyloid fibrils

Abstract

Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ(1-40)), which is associated with early onset neurodegeneration, indicate that D23N-Aβ(1-40) fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ(1-40) fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ(1-40) fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ(1-40) fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ(1-40) fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ(1-40) fibril model represents a specific "toxic intermediate" in the aggregation process of a disease-associated Aβ mutant.

Fig. 1.

(A–C) TEM images of negatively stained D23N-Aβ_1–40 fibrils. Images are shown for parent fibrils (A), fibrils in their thermodynamic equilibrium (TE) state after 500 h incubation with intermittent sonication (B), and fibrils prepared by two generations of the seeding/filtration (SFg2) protocol (C). The TE and SFg2 protocols select different fibril morphologies from the polymorphic parent sample. (D) Measurements of intermolecular dipole-dipole couplings among ¹³C labels at A21 methyl carbons in parent (squares), TE (circles) and SFg2 (triangles) fibrils, obtained with the PITHIRDS-CT SSNMR technique. Error bars represent uncertainty due to root-mean-squared noise in the ¹³C NMR spectra. Dashed and solid curves are simulated data for linear chains of ¹³C nuclei with the indicated spacings. Average intermolecular ¹³C-¹³C distances decrease in TE fibrils and increase in SFg2 fibrils, relative to distances in the parent fibrils.

Fig. 2.

(A–E) 2D ¹³C-¹³C correlation spectra of SFg2 fibril samples A–E, with color-coded chemical shift assignment paths. (F) Secondary ¹³C chemical shifts obtained from the 2D ¹³C-¹³C spectra. Shaded areas highlight residues with non-β-strand secondary shifts.

Fig. 3.

(A, B) 2D CHHC spectra of SFg2 fibril samples A and B, with 1D slices through chemical shifts of I31 and V18 (dashed lines). Strong I31 C_α/M35 C_α and V18 C_α/F20 C_α crosspeaks indicate antiparallel β-sheets. (C) 2D RAD spectrum of SFg2 fibril sample C with 1D slices at the I32 , L34 , and A21 chemical shifts (dashed lines). Arrows indicate interresidue crosspeaks.

Fig. 4.

Structure of residues 15–40 in antiparallel D23N-Aβ_1–40 (SFg2) fibrils. (A) Backbone atoms of the full eight-molecule system used for structure development, viewed parallel (Top) and perpendicular (Bottom) to the fibril axis. Carbon atoms are colored orange or cyan in molecules with alternating orientations within the antiparallel cross-β motif. This is one example of 10 similar structures determined from the experimental restraints (PDB file 2LNQ). See Fig. S6 for superpositions of the 10 structures. (B) Central pair of molecules, showing all non-hydrogen atoms and viewed parallel to the fibril axis. (C) Schematic representation of the double-layered antiparallel cross-β motif (Left), showing β-strands in yellow, the intervening loop in red, and groups of hydrophobic side chains as light green and dark green blocks. A schematic representation of the double-layered parallel cross-β motif identified in earlier studies of WT-Aβ_1–40 fibrils (Right) is shown for comparison.