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Ligand binding to distinct states diverts aggregation of an amyloid-forming protein

Nature Chemical Biology volume 7, pages 730–739 (2011)Cite this article

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Abstract

Although small molecules that modulate amyloid formation in vitro have been identified, significant challenges remain in determining precisely how these species act. Here we describe the identification of rifamycin SV as a potent inhibitor of β2 microglobulin (β2m) fibrillogenesis when added during the lag time of assembly or early during fibril elongation. Biochemical experiments demonstrate that the small molecule does not act by a colloidal mechanism. Exploiting the ability of electrospray ionization–ion mobility spectrometry–mass spectrometry (ESI-IMS-MS) to resolve intermediates of amyloid assembly, we show instead that rifamycin SV inhibits β2m fibrillation by binding distinct monomeric conformers, disfavoring oligomer formation and diverting the course of assembly to the formation of spherical aggregates. The results demonstrate the power of ESI-IMS-MS to identify specific protein conformers as targets for intervention in fibrillogenesis using small molecules and reveal a mechanism of action in which ligand binding diverts unfolded protein monomers toward alternative assembly pathways.

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Figure 1: Fibrillogenesis of β2m at pH 2.5 in the presence of different small molecules.
Figure 2: Inhibition of fibril formation of β2m by rifamycin SV does not occur by a colloidal mechanism.
Figure 3: Structural properties of spherical aggregates of β2m formed in the presence of rifamycin SV.
Figure 4: ESI-IMS-MS 3D Driftscope plots of β2m monomeric conformers and their ligand binding capabilities at pH 2.5.
Figure 5: 1H-15N HSQC NMR spectra of β2m and its variant F62A,Y63A,Y67A in the presence of rifamycin SV.
Figure 6: β2m assembly at pH 2.5, 37 °C monitored using ThT fluorescence and ESI-IMS-MS.
Figure 7: Rifamycin SV dissociates β2m oligomers when added at different times throughout the lag phase.
Figure 8: Model of the inhibition of β2m aggregation by rifamycin SV at pH 2.5.

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Acknowledgements

We thank A. Kalverda for advice on NMR spectroscopy, D.P. Smith for advice about α-synuclein, K. Ainley for technical assistance, C. Glabe for the gift of OC and A11 antibodies and Aβ1-40 oligomers, J. Rushworth for creating fibrillar oligomers of Aβ1-42, R. Wetzel for the gift of WO1 antibodies and the Radford and Ashcroft groups for helpful discussions and advice. We acknowledge the Wellcome Trust (grant numbers 075675 and 062164) and BBSRC (Grant Number BB/526502/1) for funding. The Synapt HDMS mass spectrometer was purchased with funding from the Biotechnology and Biological Sciences Research Council (Research Equipment Initiative grant BB/E012558/1).

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  1. Lucy A Woods and Geoffrey W Platt: These authors contributed equally to this work.

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom

    Lucy A Woods, Geoffrey W Platt, Andrew L Hellewell, Eric W Hewitt, Steve W Homans, Alison E Ashcroft & Sheena E Radford

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Contributions

L.A.W. designed and carried out MS experiments, performed MS data analysis, carried out colloidal inhibition assays and wrote the paper. G.W.P. designed and carried out small-molecule screen, designed NMR experiments and performed NMR data analysis and wrote the paper. A.L.H. carried out immunological assays, cell toxicity assays and wrote the paper. S.E.R., A.E.A., S.W.H. and E.W.H. designed experiments, performed data interpretation and wrote the paper.

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Correspondence to Alison E Ashcroft or Sheena E Radford.

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Woods, L., Platt, G., Hellewell, A. et al. Ligand binding to distinct states diverts aggregation of an amyloid-forming protein. Nat Chem Biol 7, 730–739 (2011). https://doi.org/10.1038/nchembio.635

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