Globular tetramers of beta(2)-microglobulin assemble into elaborate amyloid fibrils

Abstract

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-beta-strands and have revealed some details of local beta-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length beta(2)-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.

Fig. 1

Helical and subunit repeats of β₂m amyloid fibrils. (a) Negative-stain EM showing the helical twist in β₂m fibrils. One cross-over repeat (180° turn) is labelled. (b) Cryo-EM class average (upper) and reprojection (lower) of the 3D reconstruction for a type A fibril. (c) Cryo-EM class average (upper) and reprojection (lower) for a type B fibril. (d) Negative-stain image of a cross-over with a marked region of subunit repeats. (e) Diffraction pattern from negatively stained fibrils with single sided staining. The marked layer line (arrows) indicates a spacing of 5.88 nm, corresponding to the subunit repeat. The layer line lies on an axis tilted by ∼ 7° from the fibre (vertical) axis, corresponding to the long-range helical twist of the fibrils. The intensity of the strong central diffraction was reduced by a factor of 10 so that it could be displayed together with the subunit layer lines. Scale bars represent 100 nm (a) and 50 nm (b–d).

Fig. 2

Variability in β₂m fibril structure. (a) Class averages of C-type fibrils. (b) Three class averages of type A fibrils showing the large variation in cross-over spacing. (c) Histogram showing the frequency of cross-over repeats in the classes analysed (85% of the data). The A-type classes are shown in lilac, and the B-type classes are shown in green. (d and e) Cross sections of maps generated from different classes of (d) A-type and (e) B-type fibrils. Scale bar represents 50 nm (a and b). Map widths in (d) and (e) are ∼ 18–20 nm.

Fig. 3

Selection of A-type (lilac) and B-type (green) maps showing side views, their cross-over repeat lengths, and resolutions at 0.5 Fourier shell correlation.

Fig. 4

Three-dimensional reconstructions of the type A and type B forms of β₂m fibrils. Side views of an A-type fibril (a) and a B-type fibril (b). The maps are contoured at a volume corresponding to an MPL of 53 kDa/nm. One dimeric density unit is indicated by a red box in (b). The directions of the half-fibrils are indicated by arrows below the maps. Cross sections of the A-type (c) and B-type fibrils (d) show that the structures are formed of crescent-shaped units stacked back-to-back. (e) Superposed contour plots of the A (lilac) and B (green) repeat units, showing that the two fibril types have the same underlying organisation that differs only in the orientation of the two stacks, either parallel or anti-parallel.

Fig. 5

MPL measured by STEM. (a) STEM image showing fibrils with different masses per unit length. The boxes highlight 53-kDa/nm (lower left) and 27-kDa/nm (upper right) fibrils, respectively. Scale bar represents 57 nm. (b) Histogram showing the pooled data from 1030 STEM MPL measurements, with the component peaks fitted. Minor components in the samples with different morphologies may account for the smaller peaks at higher MPL.

Fig. 6

Schematic of subunit packing and interfaces in a B-type fibril. (a and b) Cross section and side view of a B-type fibril. (c and d) Schematic representation of the dimer-of-dimers subunit packing for the same fibril orientation; each elliptical cylinder corresponds to two β₂m monomers. The outermost (orange) protofilaments in the model are disordered in the map, so that their full density is not reconstructed. It is possible, although unlikely, that pairs of protein subunits are threaded through all three protofilaments in one crescent with flexible hinge regions between the protofilaments.