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.
Subscribe to Journal
Get full journal access for 1 year
only $14.08 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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.
Arensdorf, A. M., Marada, S. & Ogden, S. K. Smoothened regulation: a tale of two signals. Trends Pharmacol. Sci. 37, 62–72 (2016).
Hu, A. & Song, B. L. The interplay of Patched, Smoothened and cholesterol in hedgehog signaling. Curr. Opin. Cell Biol. 61, 31–38 (2019).
Kozielewicz, P., Turku, A. & Schulte, G. Molecular pharmacology of class F receptor activation. Mol. Pharmacol. 97, 62–71 (2020).
Qi, X. & Li, X. Mechanistic insights into the generation and transduction of Hedgehog signaling. Trends Biochem. Sci. 45, 397–410 (2020).
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).
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).
Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched. Cell 175, 1352–1364 (2018).
Qian, H. et al. Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm. Nat. Commun. 10, 2320 (2019).
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).
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).
Pak, E. & Segal, R. A. Hedgehog signal transduction: key players, oncogenic drivers, and cancer therapy. Dev. Cell 38, 333–344 (2016).
Nachtergaele, S. et al. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2, e01340 (2013).
Huang, P. et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling. Cell 166, 1176–1187 (2016).
Qi, X. et al. Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi. Nature 571, 279–283 (2019).
Deshpande, I. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019).
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).
Huang, P. et al. Structural basis of Smoothened activation in Hedgehog signaling. Cell 175, 295–297 (2018).
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).
Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).
Rana, R. et al. Structural insights into the role of the Smoothened cysteine-rich domain in Hedgehog signalling. Nat. Commun. 4, 2965 (2013).
Xiao, X. et al. Cholesterol modification of Smoothened is required for Hedgehog signaling. Mol. Cell 66, 154–162 (2017).
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).
Wang, C. et al. Structure of the human Smoothened receptor bound to an antitumour agent. Nature 497, 338–343 (2013).
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).
Kim, J. et al. The role of ciliary trafficking in Hedgehog receptor signaling. Sci. Signal. 8, ra55 (2015).
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).
Marada, S. et al. Functional divergence in the role of N-Linked glycosylation in Smoothened signaling. PLoS Genet. 11, e1005473 (2015).
Pandit, T. & Ogden, S. K. Contributions of noncanonical Smoothened signaling during embryonic development. J. Dev. Biol. 5, https://doi.org/10.3390/jdb5040011 (2017).
Koehl, A. et al. Structure of the micro-opioid receptor-Gi protein complex. Nature 558, 547–552 (2018).
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).
Yang, H. et al. Converse conformational control of Smoothened activity by structurally related small molecules. J. Biol. Chem. 284, 20876–20884 (2009).
Raleigh, D. R. et al. Cilia-associated oxysterols activate Smoothened. Mol. Cell 72, 316–327 (2018).
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).
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).
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).
Gong, X. et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science 361, eaas8935 (2018).
Winkler, M. B. L. et al. Structural insight into eukaryotic sterol transport through Niemann–Pick Type C proteins. Cell 179, 485–497 (2019).
Long, T. et al. Structural basis for itraconazole-mediated NPC1 inhibition. Nat. Commun. 11, 152 (2020).
Zhang, Y. Hedgehog pathway activation through conformational blockade of the Patched sterol conduit. Preprint at bioRxiv https://doi.org/10.1101/783290 (2019).
Kinnebrew, M. et al. Cholesterol accessibility at the ciliary membrane controls Hedgehog signaling. eLife 8, e50051 (2019).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60, 2126–2132 (2004).
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).
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).
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).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 66, 12–21 (2010).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
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).
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).
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.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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
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.
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.
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.
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
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
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.
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.
About this article
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