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Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling


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.

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Fig. 1: Structure of the SMO–Gi–SAG complex reveals an endogenous sterol in 7-TMs.
Fig. 2: Structure of SMO–Gi–24(S),25-EC reveals a novel sterol-binding site.
Fig. 3: SMOD384R is a gain-of-function SMO variant.
Fig. 4: Structure of the SMOD384R–Gi complex reveals an endogenous sterol in site 1.
Fig. 5: Structure of the SMOG111C/I496C–Gi complex suggests site 3 as a sterol gate to control sterol access to CRD.
Fig. 6: A working model of the effect of sterols in the SMO channel.

Data availability

The data that support the findings of this study are available from the corresponding author upon request. The 3D cryo-EM density maps have been deposited in the Electron Microscopy Data Bank under accession nos. EMD-22119, EMD-22120, EMD-22117 and EMD-22118. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under accession nos. 6XBL, 6XBM, 6XBJ and 6XBK. Source data are provided with this paper.


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We thank D. Stoddard at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award no. RP170644) for assistance in data collection. We thank B. Kobilka for sharing the plasmids of Gi and scFv16; L. Beatty, L. Esparza and C. Lee for tissue culture; A. Lemoff at the UT Southwestern Proteomics Core for mass spectrometry identification; and M. Brown, B. Chen, E. Debler, J. Goldstein, J. Jiang, Y. Yu and C. Zhang for discussions. This work was supported by the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center, NIH grant nos. P01 HL020948 and R01 GM135343 and the Welch Foundation (no. I-1957, to X.L.). X.Q. is the recipient of a DDBrown Fellow of the Life Sciences Research Foundation. X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (no. DRR-53-19) and a Rita C. and William P. Clements, Jr Scholar in Biomedical Research at UT Southwestern Medical Center.

Author information




X.L. conceived the project and designed the research with X.Q. X.Q. purified protein for cryo-EM study and performed functional characterization with L.F., R.D.B.-B. and T.L. X.Q. carried out cryo-EM work, built the model and refined the structure. X.Q., T.L. and X.L. analyzed the data. All authors contributed to manuscript preparation and X.L. wrote the manuscript.

Corresponding author

Correspondence to Xiaochun Li.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Functional characterization of the C-terminal tail of SMO in HH signaling.

The domain structure of SMO is shown above. HH signaling in Smo−/− mouse embryonic fibroblasts (MEFs) transfected with pcDNA3.1 and SMO variants was measured via luciferase activity. Error bars denote s.d. (n = 3 biologically independent experiments).

Source data

Extended Data Fig. 2 The overall cryo-EM maps of SMO–Gi complexes.

a, The map of SMO–Gi–SAG complex. b, The map of SMO–Gi–24(S),25-EC complex. c, The map of SMOD384R–Gi complex. d, The map of SMOG111C/I496C–Gi complex. Each subunit is displayed in different colors. The α1 and α2 of CRD are labeled and the position of CRD is indicated by red circles.

Extended Data Fig. 3 Structural comparison of SMO and Gi protein in the different states.

a, Structural comparison of SMO–Gi–SAG complex with the previously reported structure of SMO–Gi–24(S),25-EC complex (PDB: 6OT0). b, structural comparison of SMO in SMO–Gi– SAG complex with inactive SMO (PDB: 5L7D). c, Structural comparison of 3.1-Å SMO–Gi–24(S),25-EC complex with the previously reported structure of SMO–Gi–24(S),25-EC complex (PDB: 6OT0). d, Structural comparison of Gi proteins in these reported four complexes.

Extended Data Fig. 4 Sequence alignment of hSMO-CRD with xSMO-CRD.

The cholesterol binding residues are indicated by blue circles in both of hCRD and xCRD. The secondary structures of both CRDs are indicated above the sequences.

Extended Data Fig. 5 24(S),25-EC binding assay.

The construct of the xCRD domain structure is shown. The protein was detected by Western blotting. The assay was reproduced three times with similar results.

Source data

Extended Data Fig. 6 Expression levels of SMO and its variants in this study.

The transiently expressed SMO proteins were detected by western blotting. Calnexin served as an internal control and was detected via anti-calnexin antibody. Similar results were obtained in three biologically independent experiments.

Source data

Extended Data Fig. 7 Structural comparison of SMOV329F with wild type SMO.

a, Structural comparison of Vismodegib-bound SMOV329F with SANT1-bound SMO. The similar conformations of both structures indicate that V329F could not abolish the folding of SMO. b, Structural comparison reveals that V329F could abolish the endogenous cholesterol binding in site 1. The ligand is shown as sticks. The CRD and 7-TMs of SMO have been indicated by circles.

Extended Data Fig. 8 Identification of the designed disulfide bond in SMOG111C/I496C by mass spectrometry.

SMOG111C/I496C and SMOWT proteins were treated with or without 5mM TCEP at room temperature for 40 minutes, followed by the incubation of 150 mM iodoacetamide for an additional 40 minutes. The corresponding bands of SMO were cropped from the SDS-PAGE gel and sent for mass spectrometry analysis. Annotated Sequences of SMO were detected by Liquid Chromatography-Mass Spectrometry (LC-MS). After normalizing the amount of the samples, the results show that the abundance of peptide 106-113 and peptide 483-501, containing either Cys111 or Cys496, after TCEP treatment increase 2.0~3.7 fold than without treatment, suggesting that the majority of SMOG111C/I496C contains the designed disulfide bond between Cys111 and Cys496. Both peptides could not be found in the TCEP-treated SMOWT sample. The experiment was re-produced twice in different weeks with similar results.

Extended Data Fig. 9 Cartoon model of the SMO–Gi complexes in this work.

Sterols have been blocked in site-1 in panel a; while sterols have been observed in site-2 and site-3 in panel b. TM6 is labeled.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8 and Table 1.

Reporting Summary

Source data

Source Data Fig. 3

Statistical Source data.

Source Data Fig. 5

Statistical Source data.

Source Data Extended Data Fig. 1

Statistical Source data.

Source Data Extended Data Fig. 5

Unprocessed immunoblots.

Source Data Extended Data Fig. 6

Unprocessed immunoblots.

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Qi, X., Friedberg, L., De Bose-Boyd, R. et al. Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling. Nat Chem Biol 16, 1368–1375 (2020).

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