Structural Basis of Smoothened Activation in Hedgehog Signaling

Cell. 2018 Jul 12;174(2):312-324.e16. doi: 10.1016/j.cell.2018.04.029. Epub 2018 May 24.


The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.

Keywords: Hedgehog; Smoothened; cancer; cholesterol; cilia; membrane; oncogene; signaling.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Allosteric Regulation
  • Animals
  • Binding Sites
  • Cell Line
  • Cholesterol / chemistry
  • Cholesterol / metabolism
  • Crystallography, X-Ray
  • Flow Cytometry
  • Hedgehog Proteins / genetics
  • Hedgehog Proteins / metabolism*
  • Humans
  • Mice
  • Molecular Dynamics Simulation
  • Protein Binding
  • Protein Domains
  • Protein Structure, Tertiary
  • Signal Transduction
  • Small Molecule Libraries / chemical synthesis
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / metabolism
  • Smoothened Receptor / antagonists & inhibitors
  • Smoothened Receptor / chemistry*
  • Smoothened Receptor / metabolism
  • Veratrum Alkaloids / chemistry
  • Veratrum Alkaloids / metabolism
  • Xenopus Proteins / antagonists & inhibitors
  • Xenopus Proteins / chemistry*
  • Xenopus Proteins / metabolism
  • Xenopus laevis / metabolism


  • Hedgehog Proteins
  • Small Molecule Libraries
  • Smoothened Receptor
  • Veratrum Alkaloids
  • Xenopus Proteins
  • Cholesterol
  • cyclopamine