Introduction
Despite the first identification of proteinaceous amyloid fibrils as the defining feature of clinical disease over 50 years ago,1 the full structure of amyloid fibrils remains unresolved. Although more than 25 protein and peptide sequences are involved in human amyloid diseases2 or in the formation of functional amyloid fibrils in prokaryotes and eukaryotes,3 it is now widely accepted that most polypeptide sequences can assemble into amyloid fibrils under suitable experimental conditions.4,5 The resulting fibrils appear to be built of a common core structure in which two or more long ribbons of β-sheet (protofilaments) assemble into flat or twisted fibrils.6 Accordingly, amyloid fibrils can be identified by generic properties such as the ability to bind dyes like Congo red and thioflavin T, with the former resulting in the characteristic green birefringence of amyloid fibres.7 In addition, all amyloid fibrils give rise to characteristic low-frequency absorption bands indicative of highly organised β-sheets visualised by Fourier transform infrared (FTIR)8 and a cross-β fibre diffraction pattern indicating that the constituent β-strands are oriented perpendicular to the fibril long axis.9 Consistent with a generic fold for amyloid, all such fibrils are recognised by the antibody WO1 and other ligands commonly found associated with amyloid fibrils in disease,10,11 irrespective of the length or sequence of the constituent polypeptide chains.
More recent studies of amyloid fibrils have focused on defining local structural information on β-sheet packing using X-ray fibre diffraction, solid-state NMR, and electron paramagnetic resonance spectroscopy.12–15 High resolution models for fibril core structures have recently been provided based on tightly packed β-sheets in three-dimensional (3D) crystals of short peptides,16,17 as well as assemblies built from parallel arrays of in-register β-strands in β-helical or serpentine arrangements.6 While these studies provide detailed information about local packing within the cross-β core, the larger scale assembly of amyloid fibrils is not well understood. For example, it is still unclear how protein subunits assemble into protofilaments and what determines protofilament assembly into fibrils. Because of the structural heterogeneity of fibril preparations seen by electron microscopy (EM) and atomic force microscopy, it is unlikely that these questions can be addressed by measurements on bulk samples. Different fibrils within a population contain different numbers of protofilaments and variable twists ranging from flat ribbons to tubular arrangements of protofilaments. This range of structures is typically seen within a single sample.18–20
In its native soluble form, the human protein β2-microglobulin (β2m) has a classical immunoglobulin fold and forms the non-covalently bound light chain of the class 1 major histocompatibility complex. During its normal catabolic cycle, β2m is degraded by the kidney. In patients with renal failure, the concentration of β2m in serum is increased by up to 60-fold, whereupon the full-length disulfide bonded protein self-associates into amyloid fibrils that accumulate in the musculo-skeletal system, causing dialysis-related amyloidosis.21 Like other soluble globular proteins involved in amyloidosis, native β2m is stable as a monomer in solution, and partial or full unfolding is required to initiate assembly into amyloid fibrils in vitro.22 While amyloid formation at neutral pH is a slow and inefficient process, seeding and/or addition of copper ions, detergents, or organic co-solvents can induce fibril formation at neutral pH.23–25 Under highly acidic conditions, the precursor monomeric protein is unfolded,26 and amyloid fibrils are rapidly formed in a nucleation-dependent mechanism.27,28 These fibrils display all the hallmarks of authentic amyloid, including nucleation-dependent polymerisation kinetics, Congo red birefringence, a cross-β fibre diffraction pattern, and the ability to bind serum amyloid P component, apolipoprotein E, glycosaminoglycans, and the generic anti-amyloid antibody WO1.24,27,29 They also show the same characteristic amide I band in FTIR spectra as fibrils formed in vitro at neutral pH, as well as those extracted from patient tissues, confirming their structural authenticity.30 FTIR analysis indicates a high β-sheet content in the fibrils, most likely involving a predominantly parallel arrangement of β-strands, in contrast with the anti-parallel arrangement of β-strands in the immunoglobulin fold of the native monomer.31
Here we describe the 3D structures of β2m amyloid fibrils obtained by cryo-EM and image processing by sorting fibril segments into homogeneous subsets. The 3D maps and mass analysis by scanning transmission electron microscopy (STEM) reveal a novel dimer-of-dimers repeat unit in a complex, hierarchical fibril assembly. We suggest that a globular repeat unit may be a common feature of amyloid fibrils formed from larger protein precursors. The results reveal a new view for amyloid, with a more elaborate superstructure than any previously described.