Smith, HI, Guthertz, N, Cawood, EE et al. (3 more authors) (2020) The role of the IT-state in D76N β2-microglobulin amyloid assembly: a crucial intermediate or an innocuous bystander? Journal of Biological Chemistry. jbc.RA120.014901. ISSN 0021-9258
Abstract
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.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © the Author(s). Reproduced in accordance with the publisher's self-archiving policy. |
Keywords: | NMR; CD; β2-microglobulin; real-time folding; amyloid; amyloid; protein folding; aggregation; protein aggregation; biophysics |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 15 Jul 2020 11:45 |
Last Modified: | 13 Jul 2021 00:38 |
Status: | Published online |
Publisher: | American Society for Biochemistry & Molecular Biology (ASBMB) |
Identification Number: | 10.1074/jbc.ra120.014901 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:163223 |