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


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 pathway1,2. 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 Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR–Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.

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Fig. 1: Functional characterization of PTCH1-associated oxysterols in Hedgehog signalling.
Fig. 2: Structure of hSMO–Gi–Fab complex.
Fig. 3: Structural comparison of the Gi-bound hSMO with inactive hSMO and cyclopamine-bound xSMO.
Fig. 4: Conformational changes in Gi upon coupling to hSMO.
Fig. 5: Distinct orientations of heterotrimeric Gi proteins after coupling to hSMO and class-A GPCRs.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. The 3D cryo-EM density map has been deposited in the Electron Microscopy Data Bank under the accession number EMD-20190. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under the accession number 6OT0.


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The data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644); we thank D. Stoddard for assistance in data collection; E. Xu for sharing the materials for making Fab-G50; L. Beatty, L. Friedberg, A. Hassan and P. Schmiege for technical help; M. Brown, E. Debler, J. Goldstein and J. Jiang for discussion during manuscript preparation; B. Chen and J. Kim for Smo−/− mouse embryonic fibroblasts. This work was supported by the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center (to X.L.), O’Donnell Junior Faculty Funds (to X.L.), Welch Foundation (I-1957) (to X.L.), NIH grant P01 HL020948 (to J.M. and X.L.) and NIH grant 1R35GM128641 (to C.Z.). X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53-19) and a Rita C. and William P. Clements Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.

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Authors and Affiliations



X.Q. and X.L. conceived the project and designed the research with H.L and C.Z. B.T. and J.M. performed the sterol analysis by mass spectrometry. X.Q. purified the protein for mass spectrometry analysis, and H.L. purified the protein for the cryo-EM study. X.Q. carried out cryo-EM work, built the model and refined the structure. X.Q. and H.L. performed the functional characterization. All the authors analysed the data. X.Q., H.L., C.Z. and X.L. contributed to manuscript preparation. X.L. wrote the manuscript. Z.C. and X.L. supervised the project.

Corresponding authors

Correspondence to Cheng Zhang or Xiaochun Li.

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The authors declare no competing interests.

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Extended data figures and tables

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

a, GTPγS causes the dissociation of the 24(S),25-EC-mediated hSMO–Gi complex. b, Size-exclusion chromatogram and SDS–PAGE gel of the purified hSMO–Gi–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 workflow for the complex with the full map. The cryo-EM 2D classification from RELION is shown. The subtracted parts are indicated by dashed circles. c, The data-processing workflow 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 did not show sufficient structural features in the final refinement. Masks used for the refinement are shown. The cryo-EM map after FREALIGN refinement sharpened using BFACTOR with a resolution limit of 4 Å or 3.9 Å, and a B-factor value of −100 Å2. Each subunit is coloured in a different colour.

Extended Data Fig. 3 Assessment of model quality.

a, FSC curve of the structure without the CRD and half of the 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, coloured 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 Gi protein. Electron microscopy 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 transmembrane-domain ligand-binding pocket in the hSMO crystal structure (PDB 5L7D). The density is shown in green at the 3σ level, and is indicated by an arrow. b, The density of the ligand in the Gi–hSMO complex. The density is shown in purple mesh at the 5σ level at 3.9 Å, and is indicated by an 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). In ae, structures of hSMO with different ligands are 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 TM6 and cytosolic sites of hSMO and μOR.

a, Structural comparison of TM6 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), Gi–μOR in light cyan (PDB 6DDE). Right, GLP-1R, inactive GLP-1R in red (PDB 5VEW), Gs–GLP-1R (PDB 6B3J) in dark blue. b, Electrostatic surface representations of the cytosolic side of SMO and μOR complex with Gαi–α5.

Extended Data Fig. 8 The structures of Gi-bound class-A GPCRs.

a, Rhodopsin–Gi complex (PDB 6CMO). b, A1R–Gi complex (PDB 6D9H). c, μOR–Gi complex (PDB 6DDE). d, CB1–Gi complex (PDB 6N4B).

Extended Data Fig. 9 Comparison of the Gi-coupled hSMO, inactive hSMO and apo human FZD4.

The Gi-coupled hSMO is in blue, the inactive hSMO is in pink (PDB 5L7D) and apo human FZD4 is in yellow (PDB 6BD4). Structures are viewed from the side of the membrane.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Qi, X., Liu, H., Thompson, B. et al. Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi. Nature 571, 279–283 (2019).

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