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Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling

Nature Chemical Biology volume 9, pages 557–564 (2013)Cite this article

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Abstract

Oxysterols bind the seven-transmembrane protein Smo (Smo) and potently activate vertebrate Hedgehog (Hh) signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, whether oxysterols are important for normal vertebrate Hh signaling and whether antagonizing oxysterols can inhibit the Hh pathway. We developed azasterols that block Hh signaling by binding the oxysterol-binding site of Smo. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smo and is completely separable from the site bound by other small-molecule modulators, located within the heptahelical bundle of Smo. Smo mutants in which oxysterol binding is abolished no longer respond to oxysterols and cannot be maximally activated by the Hh ligand. Our results show that oxysterol binding to vertebrate Smo is required for normal Hh signaling and that targeting the oxysterol-binding site is an effective strategy to inhibit Smo.

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Figure 1: 22-NHC inhibits vertebrate Hh signaling.
Figure 2: 22-NHC binds Smo at the oxysterol-binding site.
Figure 3: Oxysterols and 22-NHC bind the extracellular CRD of vertebrate Smo.
Figure 4: Structural requirements for oxysterol activation of Smo.
Figure 5: Oxysterol binding to Smo is required for high Hh signaling.
Figure 6: Conserved and divergent aspects of Smo signaling.

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Acknowledgements

We thank Y. Kishi and members of his laboratory for help with chiral chromatography and R. Rohatgi (Stanford University) for the initial gift of 20-OHC beads. A.S. is supported in part by US National Institutes of Health grant RO1 GM092924.

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  1. Daniel Nedelcu and Jing Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA

    Daniel Nedelcu, Jing Liu, Yangqing Xu, Cindy Jao & Adrian Salic

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  1. Daniel Nedelcu
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Contributions

D.N. and A.S. performed cellular and biochemical experiments. J.L., C.J. and A.S. designed and synthesized reported compounds. J.L. purified and characterized the compounds. Y.X. and D.N. developed automated image analysis software, and D.N. analyzed imaging data. All authors contributed data to the manuscript. A.S. wrote the manuscript, with input from all other authors.

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Correspondence to Adrian Salic.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–12, and Supplementary Note. (PDF 9872 kb)

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Nedelcu, D., Liu, J., Xu, Y. et al. Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat Chem Biol 9, 557–564 (2013). https://doi.org/10.1038/nchembio.1290

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