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An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase

Nature Biotechnology volume 28, pages 733–737 (2010)Cite this article

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Abstract

The specificity of SCF ubiquitin ligase–mediated protein degradation is determined by F-box proteins1,2. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCFCdc4. A co-crystal structure reveals that SCF-I2 inserts itself between the β-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 Å away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

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Figure 1: Small-molecule inhibitors of the Cdc4-substrate interaction.
Figure 2: Structure analysis of the SCF-I2–Skp1-Cdc4 complex.
Figure 3: Inhibition and allosteric modulation of human WD40 domains.

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Acknowledgements

We thank M. Auer, J. Walton and M. Bradley for stimulating discussions. This work was supported by grants to F.S. and M.T. from the Canadian Institutes of Health Research (MOP-57795), to E.D.B. from the Ontario Research and Development Challenge Fund and to M.T. from the National Cancer Institute of Canada and the European Research Council. F.S. is supported by a Canada Research Chair in Structural Biology of Signal Transduction and M.T. is supported by a Research Chair of the Scottish Universities Life Sciences Alliance and a Royal Society Wolfson Research Merit Award.

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Authors and Affiliations

  1. Center for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada

    Stephen Orlicky, Xiaojing Tang, Frank Sicheri & Mike Tyers

  2. Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Mayfield Road, Edinburgh, Scotland

    Victor Neduva & Mike Tyers

  3. Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada

    Nadine Elowe & Eric D Brown

  4. Department of Molecular Genetics, University of Toronto, Toronto, Canada

    Frank Sicheri & Mike Tyers

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  1. Stephen Orlicky
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Contributions

S.O., small-molecule library screen, affinity determinations and structural analysis; X.T., in vitro substrate binding and ubiquitination assays; V.N., bioinformatic analysis and sequence alignments; N.E. and E.D.B., small-molecule library screen; F.S. and M.T. conceived and directed the project, interpreted results and wrote the manuscript.

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Correspondence to Frank Sicheri or Mike Tyers.

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Supplementary Tables 1–3 and Supplementary Figs. 1–5 (PDF 931 kb)

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Orlicky, S., Tang, X., Neduva, V. et al. An allosteric inhibitor of substrate recognition by the SCFCdc4 ubiquitin ligase. Nat Biotechnol 28, 733–737 (2010). https://doi.org/10.1038/nbt.1646

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