All amyloid fibrils contain a cross-β fold. How this structure differs in fibrils formed from proteins associated with different diseases remains unclear. Here, we combine cryo-EM and MAS-NMR to determine the structure of an amyloid fibril formed in vitro from β₂-microglobulin (β₂m), the culprit protein of dialysis-related amyloidosis. The fibril is composed of two identical protofilaments assembled from subunits that do not share β₂m’s native tertiary fold, but are formed from similar β-strands. The fibrils share motifs with other amyloid fibrils, but also contain unique features including π-stacking interactions perpendicular to the fibril axis and an intramolecular disulfide that stabilises the subunit fold. We also describe a structural model for a second fibril morphology and show that it is built from the same subunit fold. The results provide insights into the mechanisms of fibril formation and the commonalities and differences within the amyloid fold in different protein sequences.

The presence of amyloid plaques composed of amyloid beta (Aβ) fibrils is a hallmark of Alzheimer’s disease (AD). The Aβ peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aβ_1–40 and Aβ_1–42, respectively. While there have been numerous reports that structurally characterize fibrils of Aβ_1–40, very little is known about the structure of amyloid fibrils of Aβ_1–42, which are considered the more toxic alloform involved in AD. We have prepared isotopically ¹³C/¹⁵N labeled Aβ_M01–42 fibrils in vitro from recombinant protein and examined their ¹³C–¹³C and ¹³C–¹⁵N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aβ fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of Aβ_M01–42 fibrils utilizing ¹³C and ¹⁵N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D ¹³C–¹³C, ¹³C–¹⁵N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16–42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 β-strands are present in the fibril’s secondary structure.

Metal complexes based on Gd(iii) and Mn(ii) spin-labels can be utilized for dynamic nuclear polarization of ¹H, ¹³C, and ¹⁵N.