Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human β2-microglobulin (β2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-β structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.

Oligomers which form during amyloid fibril assembly are considered to be key contributors towards amyloid disease. However, understanding how such intermediates form, their structure, and mechanisms of toxicity presents significant challenges due to their transient and heterogeneous nature. Here, we discuss two different strategies for addressing these challenges: use of (1) methods capable of detecting lowly-populated species within complex mixtures, such as NMR, single particle methods (including fluorescence and force spectroscopy), and mass spectrometry; and (2) chemical and biological tools to bias the amyloid energy landscape towards specific oligomeric states. While the former methods are well suited to following the kinetics of amyloid assembly and obtaining low-resolution structural information, the latter are capable of producing oligomer samples for high-resolution structural studies and inferring structure-toxicity relationships. Together, these different approaches should enable a clearer picture to be gained of the nature and role of oligomeric intermediates in amyloid formation and disease.

The D76N variant of human β2-microglobulin (β2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of wild-type (WT) β2m which occurs in dialysis-related amyloidosis. A folding intermediate of WT-β2m, known as the IT-state, which contains a non-native trans Pro32, has been shown to be a key precursor of WT-β2m aggregation in vitro. However, how a single amino acid substitution enhances the rate of aggregation of D76N-β2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-β2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-β2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analogue of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-β2m, but slows aggregation of D76N-β2m, supporting the view that while the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the cause of the rapid aggregation of D76N-β2m, suggesting that other non-native states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease.