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.
CORE (COnnecting REpositories)
CORE (COnnecting REpositories)