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.
Cholesterol/analogs & derivatives , GTP-Binding Protein alpha Subunits/metabolism , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Signal Transduction , Smoothened Receptor/metabolism , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Cholesterol/chemistry , Cholesterol/metabolism , Cyclohexylamines/chemistry , Cyclohexylamines/pharmacology , GTP-Binding Protein alpha Subunits/chemistry , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/chemistry , GTP-Binding Protein gamma Subunits/genetics , 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 , Single-Chain Antibodies , Smoothened Receptor/agonists , Smoothened Receptor/chemistry , Smoothened Receptor/genetics , Thiophenes/chemistry , Thiophenes/pharmacology
