Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sterols in an intramolecular channel of Smoothened mediate Hedgehog signaling

Nature Chemical Biology volume 16, pages 1368–1375 (2020)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

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.

References

  1. Arensdorf, A. M., Marada, S. & Ogden, S. K. Smoothened regulation: a tale of two signals. Trends Pharmacol. Sci. 37, 62–72 (2016).

    Article  CAS  PubMed  Google Scholar 

  2. Hu, A. & Song, B. L. The interplay of Patched, Smoothened and cholesterol in hedgehog signaling. Curr. Opin. Cell Biol. 61, 31–38 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Kozielewicz, P., Turku, A. & Schulte, G. Molecular pharmacology of class F receptor activation. Mol. Pharmacol. 97, 62–71 (2020).

    Article  CAS  PubMed  Google Scholar 

  4. Qi, X. & Li, X. Mechanistic insights into the generation and transduction of Hedgehog signaling. Trends Biochem. Sci. 45, 397–410 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Qi, X., Schmiege, P., Coutavas, E., Wang, J. & Li, X. Structures of human Patched and its complex with native palmitoylated Sonic Hedgehog. Nature 560, 128–132 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Qi, X., Schmiege, P., Coutavas, E. & Li, X. Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex. Science 362, eaas8843 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched. Cell 175, 1352–1364 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Qian, H. et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm. Nat. Commun. 10, 2320 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Rudolf, A. F. et al. The morphogen Sonic Hedgehog inhibits its receptor Ppatched by a pincer grasp mechanism. Nat. Chem. Biol. 15, 975–982 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tukachinsky, H., Petrov, K., Watanabe, M. & Salic, A. Mechanism of inhibition of the tumor suppressor Patched by Sonic Hedgehog. Proc. Natl Acad. Sci. USA 113, E5866–E5875 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Pak, E. & Segal, R. A. Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy. Dev. Cell 38, 333–344 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nachtergaele, S. et al. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2, e01340 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Huang, P. et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling. Cell 166, 1176–1187 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Deshpande, I. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Sharpe, H. J., Wang, W., Hannoush, R. N. & de Sauvage, F. J. Regulation of the oncoprotein Smoothened by small molecules. Nat. Chem. Biol. 11, 246–255 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Huang, P. et al. Structural basis of Smoothened activation in Hedgehog signaling. Cell 175, 295–297 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Luchetti, G. et al. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling. eLife 5, https://doi.org/10.7554/eLife.20304 (2016).

  19. Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Rana, R. et al. Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling. Nat. Commun. 4, 2965 (2013).

    Article  PubMed  Google Scholar 

  21. Xiao, X. et al. Cholesterol modification of Smoothened is required for Hedgehog signaling. Mol. Cell 66, 154–162 (2017).

    Article  CAS  PubMed  Google Scholar 

  22. Myers, B. R. et al. Hedgehog pathway modulation by multiple lipid binding sites on the Smoothened effector of signal response. Dev. Cell 26, 346–357 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Wang, C. et al. Structure of the human Smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Li, S., Ma, G., Wang, B. & Jiang, J. Hedgehog induces formation of PKA-Smoothened complexes to promote Smoothened phosphorylation and pathway activation. Sci. Signal. 7, ra62 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Kim, J. et al. The role of ciliary trafficking in Hedgehog receptor signaling. Sci. Signal. 8, ra55 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Riobo, N. A., Saucy, B., Dilizio, C. & Manning, D. R. Activation of heterotrimeric G proteins by Smoothened. Proc. Natl Acad. Sci. USA 103, 12607–12612 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Marada, S. et al. Functional divergence in the role of N-Linked glycosylation in Smoothened signaling. PLoS Genet. 11, e1005473 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Pandit, T. & Ogden, S. K. Contributions of noncanonical Smoothened signaling during embryonic development. J. Dev. Biol. 5, https://doi.org/10.3390/jdb5040011 (2017).

  29. Koehl, A. et al. Structure of the micro-opioid receptor-Gi protein complex. Nature 558, 547–552 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Chen, Y. et al. Sonic Hedgehog dependent phosphorylation by CK1alpha and GRK2 is required for ciliary accumulation and activation of Smoothened. PLoS Biol. 9, e1001083 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yang, H. et al. Converse conformational control of Smoothened activity by structurally related small molecules. J. Biol. Chem. 284, 20876–20884 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Raleigh, D. R. et al. Cilia-associated oxysterols activate Smoothened. Mol. Cell 72, 316–327 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kowatsch, C., Woolley, R. E., Kinnebrew, M., Rohatgi, R. & Siebold, C. Structures of vertebrate Patched and Smoothened reveal intimate links between cholesterol and Hedgehog signalling. Curr. Opin. Struct. Biol. 57, 204–214 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Dijkgraaf, G. J. et al. Small molecule inhibition of GDC-0449 refractory Smoothened mutants and downstream mechanisms of drug resistance. Cancer Res. 71, 435–444 (2011).

    Article  CAS  PubMed  Google Scholar 

  35. Qi, C., Di Minin, G., Vercellino, I., Wutz, A. & Korkhov, V. M. Structural basis of sterol recognition by human Hedgehog receptor PTCH1. Sci. Adv. 5, eaaw6490 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gong, X. et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science 361, eaas8935 (2018).

    Article  PubMed  Google Scholar 

  37. Winkler, M. B. L. et al. Structural insight into eukaryotic sterol transport through Niemann–Pick Type C proteins. Cell 179, 485–497 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Long, T. et al. Structural basis for itraconazole-mediated NPC1 inhibition. Nat. Commun. 11, 152 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang, Y. Hedgehog pathway activation through conformational blockade of the Patched sterol conduit. Preprint at bioRxiv https://doi.org/10.1101/783290 (2019).

  40. Kinnebrew, M. et al. Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling. eLife 8, e50051 (2019).

  41. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).

  44. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  Google Scholar 

  45. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  46. Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Sect. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  Google Scholar 

  47. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. Sect. D Biol. Crystallogr. 71, 136–153 (2015).

    Article  CAS  Google Scholar 

  48. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  49. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Metherall, J. E., Goldstein, J. L., Luskey, K. L. & Brown, M. S. Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells. J. Biol. Chem. 264, 15634–15641 (1989).

    Article  CAS  PubMed  Google Scholar 

  51. Chen, B. et al. Posaconazole, a second-generation triazole antifungal drug, inhibits the Hedgehog signaling pathway and progression of basal cell carcinoma. Mol. Cancer Ther. 15, 866–876 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

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

Authors and Affiliations

  1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiaofeng Qi, Lucas Friedberg, Ryan De Bose-Boyd, Tao Long & Xiaochun Li

  2. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Xiaochun Li

Authors
  1. Xiaofeng Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lucas Friedberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryan De Bose-Boyd
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tao Long
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiaochun Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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). https://doi.org/10.1038/s41589-020-0646-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-020-0646-2

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing