Abstract
Many polypeptides can self-associate into linear, aggregated assemblies termed amyloid fibers. High-resolution structural insights into the mechanism of fibrillogenesis are elusive owing to the transient and mixed oligomeric nature of assembly intermediates. Here, we report the conformational changes that initiate fiber formation by β-2-microglobulin (β2m) in dialysis-related amyloidosis. Access of β2m to amyloidogenic conformations is catalyzed by selective binding of divalent cations. The chemical basis of this process was determined to be backbone isomerization of a conserved proline. On the basis of this finding, we designed a β2m variant that closely adopts this intermediate state. The variant has kinetic, thermodynamic and catalytic properties consistent with its being a fibrillogenic intermediate of wild-type β2m. Furthermore, it is stable and folded, enabling us to unambiguously determine the initiating conformational changes for amyloid assembly at atomic resolution.
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Acknowledgements
We thank L. J. Regan and T. A. Steitz for careful reading of this work and D. E. Engelman and S. A. Strobel for the use of their instruments. We also thank D. Blaho, M. Calabrese, J. Cochrane, S. Kamtekar, M. Strickler, A. Valentine and the staff of the Center for Structural Biology and National Synchrotron Light Source beamline X25 for assistance and helpful discussions. This work was supported by the US National Institutes of Health (grants DK54899 and 1F31NS046937).
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Supplementary information
Supplementary Fig. 1
Effect of Cu2+:β2m stoichiometry on fraction of protein in oligomeric state. (PDF 73 kb)
Supplementary Fig. 2
Effects of protein stability and intrinsic metal affinity on oligomer formation. (PDF 82 kb)
Supplementary Fig. 3
Determination of P32A Kd for Cu2+ by global analysis. (PDF 99 kb)
Supplementary Fig. 4
Relationship between aromatic changes in WT apo- and holo-β2m. (PDF 109 kb)
Supplementary Fig. 5
Representative electron density of β2m P32A mutant (PDB entry 2F8O). (PDF 567 kb)
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Eakin, C., Berman, A. & Miranker, A. A native to amyloidogenic transition regulated by a backbone trigger. Nat Struct Mol Biol 13, 202–208 (2006). https://doi.org/10.1038/nsmb1068
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DOI: https://doi.org/10.1038/nsmb1068
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