A new structural model of Alzheimer's Aβ42 fibrils based on electron paramagnetic resonance data and Rosetta modeling

Abstract

Brain deposition of Aβ in the form of amyloid plaques is a pathological hallmark of Alzheimer's disease. There are two major species of Aβ in the brain: Aβ42 and Aβ40. Although Aβ40 is several-fold more abundant than Aβ42 in soluble form, Aβ42 is the major component of amyloid plaques. Structural knowledge of Aβ42 fibrils is important both for understanding the process of Aβ aggregation and for designing fibril-targeting drugs. Here we report site-specific structural information of Aβ42 fibrils at 22 residue positions based on electron paramagnetic resonance data. In combination with structure prediction program Rosetta, we modeled Aβ42 fibril structure at atomic resolution. Our Aβ42 fibril model consists of four parallel in-register β-sheets: βN (residues ∼7-13), β1 (residues ∼17-20), β2 (residues ∼32-36), and βC (residues 39-41). The region of β1-loop-β2 in Aβ42 fibrils adopts similar structure as that in Aβ40 fibrils. This is consistent with our cross seeding data that Aβ42 fibril seeds shortened the lag phase of Aβ40 fibrillization. On the other hand, Aβ42 fibrils contain a C-terminal β-arc-β motif with a special turn, termed "arc", at residues 37-38, which is absent in Aβ40 fibrils. Our results can explain both the higher aggregation propensity of Aβ42 and the importance of Aβ42 to Aβ40 ratio in the pathogenesis of Alzheimer's disease.

Keywords: Alzheimer disease; Amyloid; Aβ; Protein aggregation; Spin labeling.

Figure 1. EPR spectra of Aβ42 fibrils spin-labeled at indicated residue positions

(A) Experimental spectra (black) are superimposed with best fits from spectral simulations (red). From spectral simulations we can extract spin exchange frequency as a measure of the strength of spin exchange interactions. (B) Individual spectral components of the two-component fit for indicated positions are shown in blue and magenta. The rest of the labeled positions were fitted with just the exchange component. Note that most EPR spectra are characterized by the single-line feature, resulting from strong spin exchange interactions between spin labels. The single-line feature is a signature of parallel in-register β-sheet structure in amyloid fibrils. R1 represents the spin label. Scan width is 200 G.

Figure 2. Plot of spin exchange frequencies reveals secondary structure in Aβ42 fibrils

Four regions with strong spin exchange interactions (>120 MHz) are categorized as β-strands.

Figure 3. Transmission electron microscopy images of wild-type and spin-labeled Aβ42 fibrils

Similar morphology from wild-type and spin-labeled Aβ42 fibrils suggest that spin labeling does not perturb the process of fibril formation.

Figure 4. The structural model of Aβ42 fibrils

A model of Aβ42 protofilament was built based on EPR restraints and Rosetta prediction. The side chains of residues 17-42 are shown in sticks, elucidating possible side chain interactions in the fibril core. The four stretches of strong exchange residues are colored based on how well they are structurally ordered. The three ordered regions, β₁ (residues 17-20), β₂ (residues 32-36) and β_C (residues 39-41), are colored in cyan, while the less ordered region β_N (residues 7-13) is in purple. The salt bridges between Asp23 and Lys28 are shown as black dashed lines.

Figure 5. Aβ42 fibrils seed the aggregation of Aβ40

Aggregation of Aβ40 was followed with thioflavin T fluorescence. Three repeats of Aβ40 in the absence of fibril seeds (black traces) and in the presence of 2% Aβ42 fibril seeds (red traces) are shown. Note that the lag time is shortened by the presence of seeds, suggesting that Aβ42 fibril seeds promote the aggregation of Aβ40.