Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi

Abstract

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway^1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins³. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric G_i protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a G_i-coupled activation mechanism of human SMO. Notably, the G_i protein presents a different arrangement from that of class-A GPCR-G_i complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.

Extended Data Fig. 1. Assembly of hSMO–G_i–Fab complex

a, GTPγS causes the dissociation of 24(S),25-EC mediated hSMO–G_i complex. b, Size-exclusion chromatogram and SDS–PAGE gel of the purified hSMO–G_i–Fab complex. Molecular standards are indicated on left side of the gel.

Extended Data Fig. 2. Data processing.

a, A representative electron micrograph at −2.0 μm defocus. b, The data processing work-flow for the complex with the full map. The cryo-EM 2D classification from RELION is shown. The subtracted parts were indicated by dash circles. c, The data processing work-flow for the complex with the subtracted map. Class 3 of the full map and Class 4 of the subtracted map were used for the final refinement; Class 4 of the full map and Class 1 of the subtracted map failed to have sufficient structural features in the final refinement. Masks used for the refinement are shown. The cryo-EM map after Frealign refinement sharpened using BFACTOR.EXE (author: Nikolaus Grigorieff) with a resolution limit of 4 Å or 3.9 Å and a B-factor value of −100 Ų. Each subunit is colored.

Extended Data Fig. 3. The model quality assessment.

a, Fourier shell correlation (FSC) curve of the structure without the CRD and half Fab with FSC as a function of resolution using Frealign output. b, The FSC curves calculated between the refined structure and the half map used for refinement (blue), the other half map (red) and the full map (black). c, Density maps of structure colored by local resolution estimation using Blocres.

Extended Data Fig. 4. cryo-EM map of structural elements in the complex.

a, The major helices of hSMO. b, The major structural elements of G_i protein. EM density map and model of the complex are shown in mesh and cartoon. c, the putative ligand.

Extended Data Fig. 5. Comparison of the maps in the ligand-binding pocket of hSMO.

a, The extra density within the TMD ligand-binding pocket in the hSMO crystal structure (PDB: 5L7D). The density is shown in green at 3σ level and indicated by arrow. b, The density of the ligand in the G_i–hSMO complex. The density is shown in purple mesh at 5σ level at 3.9Å and indicated by arrow.

Extended Data Fig. 6. Comparison of the binding sites of different SMO ligands.

a, SAG1.5 bound hSMO (PDB:4QIN). b, Cyclopamine bound hSMO (PDB: 4O9R). c, Vismodegib bound hSMO (PDB: 5L7I). d, SANT1 bound hSMO (PDB:4N4W). e, LY2940680 bound hSMO (PDB:4JKV). Structures of hSMO with different ligands viewed from the side of the membrane. f, Superimposition of the ligands that bind the pocket in the transmembrane domain of hSMO.

Extended Data Fig. 7. Comparisons of the TM6s and cytosolic sites of hSMO and μOR.

a, Structural comparison of TM6s of hSMO, μOR and GLP-1R in the inactive and G protein-bound states. Left: hSMO, inactive SMO in pink (PDB: 5L7D); Middle: μOR, inactive μOR in light orange (PDB: 4DKL), G_i-μOR in light cyan (PDB: 6DDE); Right: GLP-1R, inactive GLP-1R in red (PDB: 5VEW), G_s-GLP-1R (PDB: 6B3J) in dark blue. b, Electrostatic surface representations of the cytosolic side of SMO and μOR complex with Gα_i-α5.

Extended Data Fig. 8. The structures of G_i-bound Class-A GPCRs.

a, Rhodopsin-G_i complex (PDB: 6CMO). b, A₁R-G_i complex (PDB: 6D9H). c, μOR-G_i complex (PDB: 6DDE). d, CB1-G_i complex (PDB: 6N4B).

Extended Data Fig. 9. Comparison of the G_i coupled hSMO, inactive hSMO and apo hFZD4.

The G_i coupled hSMO is in blue, the inactive hSMO is in pink (PDB: 5L7D) and apo hFZD4 is in yellow (PDB: 6BD4). Structures are viewed from the side of the membrane.

Fig. 1. Functional characterization of PTCH1-associated oxysterols in HH signaling.

a, The sterol-like densities in the SHH-N mediated PTCH1 dimer. Sterol-like densities at 5s level at 3.5 Å resolution in the domains of ECD-I, SSDs and near TM-12 are colored in green, red and purple, respectively. b, HPLC-MS quantitation of oxysterols extracted from purified PTCH1 protein. Data are mean ± s.d. (n = 3 biologically independent experiments). Oxysterol structures are shown. c, Oxysterol-mediated HH signaling. The SHH-light II cells were treated with vehicle (0.3 mM MCD), MCD complexed with 30μM sterol or SHH-N conditioned media. d, GTPγS binding assay. Basal represents the hSMO basal activity without ligand. All ligands were used at a saturating concentration of 50 μM. e, PTX decreases the 24(S),25-EC mediated HH signaling. HH activity was measured by dual-luciferase assay. Each assay in c-e was repeated at least three times with similar results and data are mean ± s.d. (n = 3 biologically independent experiments). *P ≤ 0.05, **P ≤ 0.01, two-sided t-test using GraphPad Prism 7.

Fig. 2. Structure of hSMO–G_i–Fab complex.

a, Ribbon representation of the complex structure. Primary structure of hSMO is on the top. Residues 556–787 of hSMO were removed for protein expression and the CRD domain (gray) was not determined in the cryo-EM map. hSMO, Gα, Gβ, Gγ and Fab-G50 are colored in blue, green, magenta, dark teal and orange; the putative 24(S),25-EC is shown as yellow sticks. b, The ligand-binding pocket. The putative ligand and its bound residue are shown as sticks. c, HH signaling in Smo^−/− MEFs transfected with pcDNA3.1, full-length hSMO-wild type (WT) or full-length hSMO-N521A mutant and response to SAG or 24(S),25-EC via luciferase activity. d, The GTPγS binding competition assay using cells overexpressing hSMO-ΔCRD. The assays were set up as Fig. 1d with various ligand concentrations. Each assay in c-d was repeated at least three times with similar results and data are mean ± s.d. (n = 3 biologically independent experiments). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-sided t-test using GraphPad Prism 7.

Fig. 3. Structural comparison of the G_i-bound hSMO with inactive hSMO and cyclopamine bound xSMO.

a, Superimposition of TMs of hSMO molecules. G_i-coupled hSMO is colored in blue, inactive hSMO (PDB: 5L7D) is colored in pink. The movements of structural elements are indicated. b, Movement of TM6 in the G_i-coupled hSMO due to ligand binding. c, Movements of TMs 5–7 in the G_i-coupled hSMO compared with the inactive hSMO. The related residues are shown as sticks. d, Superimposition of 7-TMs of hSMO (blue) and xSMO (PDB: 6D32, gray). e, Comparison of R451 and W535 in G_i-coupled hSMO with the corresponding residues in xSMO. f, Comparison of the ICL1 of hSMO and xSMO. R261 and its corresponding residue in xSMO are shown. g, The putative tunnel in hSMO is shown as red mesh.

Fig. 4. Conformational changes in G_i upon coupling to hSMO.

a, The interaction details between hSMO (blue) and Gα_i (green). The structural elements involved in the interaction are indicated. b, R257 in hSMO-ICL1 binds D312 of Gβ. Both residues are labeled and shown. c and d, Comparison of GDP-bound Gα_i (PDB: 1GP2, gray) and nucleotide-free Gα_i (green) from hSMO–G_i complex. GDP is shown as yellow sticks. e, Structural rearrangement of Gα_i-AHD domain after coupling to hSMO.

Fig. 5. Distinct orientations of heterotrimeric G_i proteins after coupling to hSMO and Class-A GPCRs.

a, Structural comparison of hSMO-G_i complex with μOR-G_i complex (PDB: 6DDE, light cyan). The Gα_i-α5 and Gα_i-αN are indicated. hSMO, Gα, Gβ and Gγ in hSMO-G_i complex are colored as Fig. 2a. b, The comparison of the ICL2 and ICL3 among the five GPCR-G_i complexes. The structural elements from Rhodopsin-G_i (PDB: 6CMO) are in orange; from A₁R-G_i (PDB: 6D9H) are in pink, from μOR-G_i are in light cyan and from CB1-G_i (PDB: 6N4B) are in cyan. c, The comparison of the Gα_i-α5 among the five GPCR-G_i complexes. d, The structural comparison of G_i proteins after coupling μOR and hSMO. e, The structural comparison of G_i proteins after coupling rhodopsin and hSMO. The major differences of G_i protein orientations are indicated.