Atomic Resolution Structure of Monomorphic Aβ42 Amyloid Fibrils

Abstract

Amyloid-β (Aβ) is a 39-42 residue protein produced by the cleavage of the amyloid precursor protein (APP), which subsequently aggregates to form cross-β amyloid fibrils that are a hallmark of Alzheimer's disease (AD). The most prominent forms of Aβ are Aβ1-40 and Aβ1-42, which differ by two amino acids (I and A) at the C-terminus. However, Aβ42 is more neurotoxic and essential to the etiology of AD. Here, we present an atomic resolution structure of a monomorphic form of AβM01-42 amyloid fibrils derived from over 500 (13)C-(13)C, (13)C-(15)N distance and backbone angle structural constraints obtained from high field magic angle spinning NMR spectra. The structure (PDB ID: 5KK3 ) shows that the fibril core consists of a dimer of Aβ42 molecules, each containing four β-strands in a S-shaped amyloid fold, and arranged in a manner that generates two hydrophobic cores that are capped at the end of the chain by a salt bridge. The outer surface of the monomers presents hydrophilic side chains to the solvent. The interface between the monomers of the dimer shows clear contacts between M35 of one molecule and L17 and Q15 of the second. Intermolecular (13)C-(15)N constraints demonstrate that the amyloid fibrils are parallel in register. The RMSD of the backbone structure (Q15-A42) is 0.71 ± 0.12 Å and of all heavy atoms is 1.07 ± 0.08 Å. The structure provides a point of departure for the design of drugs that bind to the fibril surface and therefore interfere with secondary nucleation and for other therapeutic approaches to mitigate Aβ42 aggregation.

Figure 1

2D ¹³C−¹³C MAS PAR spectrum of U−¹³C/¹⁵N-Aβ_M01–42 fibrils recorded at ω_0H/2π = 800 MHz, T = 277 K, ω_r/2π = 20 kHz. τ_mix = 20 ms, and ω_1H/2π = 83 kHz decoupling field. For optimal PAR mixing, the radio frequency (RF) fields were set to ω_1C/2π = 62.5 kHz and ω_1H/2π = 55 kHz on the ¹³C and ¹H channels, respectively. Several important inter-residue cross-peaks are denoted with red labels in the expanded region of the spectrum. The inset shows several important intermolecular contacts including Q15–M35 and L17–M35, while the main panel shows numerous intramolecular contacts used in calculating the structure, including F19–I32, F19–A30, V24–F20, V24–G29, I41–G29, and K28–A42.

Figure 2

Slices from two 20 ms PAR spectra illustrating the presence of intermolecular contacts at the interface between the two members of the Aβ₄₂ dimer. The top slice shows a total of 13 inter- and intramolecular cross-peaks involving M35CE from the 100% U−¹³C/¹⁵N labeled sample. By diluting the sample to 30% with natural abundance material (lower slice), 10 of these cross-peaks, shown in red in the top slice and assigned to contacts between M35CE and Q15 and L17, are no longer present, confirming that they are intermolecular in origin.

Figure 3

(A) 2D ¹³C−¹⁵N MAS PAIN spectrum of U−¹³C/¹⁵N-Aβ_M01–42 fibrils recorded at ω_0H/2π = 750 MHz, T = 277 K, ω_r/2π = 20 kHz, τ_mix = 30 ms, with ω_1H/2π = 83 kHz ¹H decoupling field. Particularly relevant intramolecular contacts include F20–G25, G29–I41, V24–A30, and I31–V36, and intermolecular contacts include V18–L34 and L17–L34. (B) 2D ¹³C−¹⁵N ZF-TEDOR (τ_mix = 16 ms) spectrum of 2−¹³C₁-glycerol/15N mixed sample recorded at 600 MHz, ω_r/2π = 12.5 kHz, VT gas regulated to 105 K with 83 kHz TPPM during acquisition. The cross-peaks observed in this spectrum confirm that the fibrils are PIR. A total of 24 cross-peaks are observed, with the most relevant cross-peaks observed being I32N–I32CA, F20N–F20CA, G29N–G29CA, G33N–G33CA, L34N–L34CO, V24N–V24CO, and V36N–G37CO.

Figure 4

FS-REDOR of Aβ_M01–42 fibrils recorded at 750 MHz, T = 277 K, and ω_r/2π = 8 kHz with ω_1H/2π = 83 kHz ¹H decoupling field applied during acquisition. The Gaussian selective π pulse on ¹³C was 0.6 ms long and set on the resonance of A42−¹³COO⁻. For ¹⁵N, ω_1S/2π = 33 kHz during REDOR and set to the resonance of the N^ζ of K28. The S and S0 signals were measured with and without the 15N selective pulse, respectively. The curve fits show that a salt bridge exists between K28 and A42 with a distance of 4.0 Å in the 100% labeled sample and 4.5 Å in the 30% sample. Intermolecular contacts between the PIR fibrils are responsible for the discrepancy in the dephasing observed.

Figure 5

Schematic representation of unique constraints used for structure calculation. There were a total of 487 unique distance constraints of which 264 were sequential contacts, 93 medium range, 104 long-range, and 26 intermolecular distance constraints.

Figure 6

(A) STEM micrograph showing the ∼2.5 kDa/Å and ∼4.5 kDa/Å fibrils present in the Aβ_M01–42 samples. The numbers adjacent to the particle indicate the molecular weight in kD observed for that segment. In addition, there are two particles present that are the standard TMV (13.1 kDa/Å) used in STEM measurements. Note that the length of the Aβ_M01–42 fibrils is ∼50–200 nm, which is shorter than found for Aβ_1–40. In other micrographs (Figure S7), we observe similar fibril masses and lengths. (B) Distribution of fibril masses determined from the STEM measurements on 894 different segments and Gaussian fits to the distributions. The distributions are centered at 2.474 and 4.880 kDa/Å with widths of 0.273 and 0.449 kDa/Å, respectively. The lower and higher molecular weights we associate with dimeric and tetrameric fibrils, respectively. The vertical lines indicate the theoretical MPL for integer numbers of molecules of MW = 4909.2 Da, which is weight expected for U-13C/15N-Aβ_M01–42.

Figure 7

(A) Stick model representation of the 10 lowest energy structures. Shown is the central dimer; one monomer is in bright and one in pale colors. The structures were aligned using the backbone heavy atoms of residues Q15–A42. The structures converged to a heavy atom backbone RMSD of 0.77 ± 0.17 Å and an RMSD of 1.11 ± 0.14 Å for all heavy atoms. (B) CPK model showing the two center monomers from the top (top image) and from the side of a fibril (bottom image). One monomer is in bright and one in pale colors. Only residues Q15–A42 are shown. The hydrophobic side chain of one buried cluster (I31, V36, V39, I41) is shown in orange, and of the other buried cluster (L17, F19, F20, V24, A30, I32, L34) in gold. M35 is shown in dark red. E22 and D23 are shown in red and K16 and K28 in blue. Hydrophobic side chains facing the solvent are shown in yellow (V18, A21 and V40, A42). (C) Surface representation of the lowest energy structure. Carbon atoms are shown in gray, oxygen atoms in red, nitrogen atoms in blue, and sulfur atoms in yellow. Only residues Q15–A42 are shown. (D) Ribbon representation of the lowest energy structure showing the alignment of the dimers along the fibril axis. Only residues Q15–A42 are shown. All figures were generated using the PyMOL software package.