Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling

Nat Chem Biol. 2020 Dec;16(12):1368-1375. doi: 10.1038/s41589-020-0646-2. Epub 2020 Sep 14.


Smoothened (SMO), a class Frizzled G protein-coupled receptor (class F GPCR), transduces the Hedgehog signal across the cell membrane. Sterols can bind to its extracellular cysteine-rich domain (CRD) and to several sites in the seven transmembrane helices (7-TMs) of SMO. However, the mechanism by which sterols regulate SMO via multiple sites is unknown. Here we determined the structures of SMO-Gi complexes bound to the synthetic SMO agonist (SAG) and to 24(S),25-epoxycholesterol (24(S),25-EC). A novel sterol-binding site in the extracellular extension of TM6 was revealed to connect other sites in 7-TMs and CRD, forming an intramolecular sterol channel from the middle side of 7-TMs to CRD. Additional structures of two gain-of-function variants, SMOD384R and SMOG111C/I496C, showed that blocking the channel at its midpoints allows sterols to occupy the binding sites in 7-TMs, thereby activating SMO. These data indicate that sterol transport through the core of SMO is a major regulator of SMO-mediated signaling.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Cholesterol / analogs & derivatives*
  • Cholesterol / chemistry
  • Cholesterol / metabolism
  • Cyclohexylamines / chemistry
  • Cyclohexylamines / pharmacology
  • GTP-Binding Protein alpha Subunits / chemistry
  • GTP-Binding Protein alpha Subunits / genetics
  • GTP-Binding Protein alpha Subunits / metabolism*
  • GTP-Binding Protein beta Subunits / chemistry
  • GTP-Binding Protein beta Subunits / genetics
  • GTP-Binding Protein beta Subunits / metabolism*
  • GTP-Binding Protein gamma Subunits / chemistry
  • GTP-Binding Protein gamma Subunits / genetics
  • GTP-Binding Protein gamma Subunits / metabolism*
  • Gene Expression Regulation
  • HEK293 Cells
  • Humans
  • Molecular Docking Simulation
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Signal Transduction*
  • Single-Chain Antibodies
  • Smoothened Receptor / agonists
  • Smoothened Receptor / chemistry
  • Smoothened Receptor / genetics
  • Smoothened Receptor / metabolism*
  • Thiophenes / chemistry
  • Thiophenes / pharmacology


  • Cyclohexylamines
  • GTP-Binding Protein alpha Subunits
  • GTP-Binding Protein beta Subunits
  • GTP-Binding Protein gamma Subunits
  • Recombinant Proteins
  • SAG compound
  • SMO protein, human
  • Single-Chain Antibodies
  • Smoothened Receptor
  • Thiophenes
  • 24,25-epoxycholesterol
  • Cholesterol