Abstract
Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3,4,5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results—combined with signalling studies and molecular dynamics simulations—delineate the structural basis for PTCH1–SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.
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Data availability
All data generated or analysed during this study are included in the published Letter and its Supplementary Information. Crystallographic coordinates and structure factors for the SMO–SAG21k–NbSmo8 complex have been deposited in the PDB under accession code 6O3C.
References
Briscoe, J. & Thérond, P. P. The mechanisms of Hedgehog signalling and its roles in development and disease. Nat. Rev. Mol. Cell Biol. 14, 416–429 (2013).
Wu, F., Zhang, Y., Sun, B., McMahon, A. P. & Wang, Y. Hedgehog signaling: from basic biology to cancer therapy. Cell Chem. Biol. 24, 252–280 (2017).
Huang, P. et al. Cellular cholesterol directly activates Smoothened in Hedgehog signaling. Cell 166, 1176–1187.e1114 (2016).
Huang, P. et al. Structural basis of Smoothened activation in Hedgehog signaling. Cell 174, 312–324.e316 (2018).
Byrne, E. F., Luchetti, G., Rohatgi, R. & Siebold, C. Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates. Curr. Opin. Cell Biol. 51, 81–88 (2018).
Taipale, J., Cooper, M. K., Maiti, T. & Beachy, P. A. Patched acts catalytically to suppress the activity of Smoothened. Nature 418, 892–896 (2002).
Byrne, E. F. X. et al. Structural basis of Smoothened regulation by its extracellular domains. Nature 535, 517–522 (2016).
Cooper, M. K. et al. A defective response to Hedgehog signaling in disorders of cholesterol biosynthesis. Nat. Genet. 33, 508–513 (2003).
Luchetti, G. et al. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling. eLife 5, e20304 (2016).
Myers, B. R., Neahring, L., Zhang, Y., Roberts, K. J. & Beachy, P. A. Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium. Proc. Natl Acad. Sci. USA 114, E11141–E11150 (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).
Zhang, Y. et al. Structural basis for cholesterol transport-like activity of the Hedgehog receptor Patched. Cell 175, 1352–1364.e1314 (2018).
Gong, X. et al. Structural basis for the recognition of Sonic Hedgehog by human Patched1. Science 361, eaas8935 (2018).
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).
Blassberg, R., Macrae, J. I., Briscoe, J. & Jacob, J. Reduced cholesterol levels impair Smoothened activation in Smith–Lemli–Opitz syndrome. Hum. Mol. Genet. 25, 693–705 (2016).
Manglik, A., Kobilka, B. K. & Steyaert, J. Nanobodies to study G protein-coupled receptor structure and function. Annu. Rev. Pharmacol. Toxicol. 57, 19–37 (2017).
Ayers, K. L. & Thérond, P. P. Evaluating Smoothened as a G-protein-coupled receptor for Hedgehog signalling. Trends Cell Biol. 20, 287–298 (2010).
McMahon, C. et al. Yeast surface display platform for rapid discovery of conformationally selective nanobodies. Nat. Struct. Mol. Biol. 25, 289–296 (2018).
Manglik, A. & Kruse, A. C. Structural basis for G protein-coupled receptor activation. Biochemistry 56, 5628–5634 (2017).
Zhang, X. et al. Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand. Nat. Commun. 8, 15383 (2017).
Wang, C. et al. Structural basis for Smoothened receptor modulation and chemoresistance to anticancer drugs. Nat. Commun. 5, 4355 (2014).
Chen, J. K., Taipale, J., Cooper, M. K. & Beachy, P. A. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, 2743–2748 (2002).
Rohatgi, R., Milenkovic, L., Corcoran, R. B. & Scott, M. P. Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process. Proc. Natl Acad. Sci. USA 106, 3196–3201 (2009).
Taipale, J. et al. Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine. Nature 406, 1005–1009 (2000).
Raleigh, D. R. et al. Cilia-associated oxysterols activate Smoothened. Mol. Cell 72, 316–327.e315 (2018).
Audet, M. & Stevens, R. C. Emerging structural biology of lipid G protein-coupled receptors. Protein Sci. 28, 292–304 (2019).
Yang, H. et al. Converse conformational control of Smoothened activity by structurally related small molecules. J. Biol. Chem. 284, 20876–20884 (2009).
Chen, J. K., Taipale, J., Young, K. E., Maiti, T. & Beachy, P. A. Small molecule modulation of Smoothened activity. Proc. Natl Acad. Sci. USA 99, 14071–14076 (2002).
Nachtergaele, S. et al. Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling. eLife 2, e01340 (2013).
Nedelcu, D., Liu, J., Xu, Y., Jao, C. & Salic, A. Oxysterol binding to the extracellular domain of Smoothened in Hedgehog signaling. Nat. Chem. Biol. 9, 557–564 (2013).
Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).
Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).
Rodriguez, E. A. et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat. Methods 13, 763–769 (2016).
Chen, I., Dorr, B. M. & Liu, D. R. A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl Acad. Sci. USA 108, 11399–11404 (2011).
Huang, W. et al. Structural insights into μ-opioid receptor activation. Nature 524, 315–321 (2015).
Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nat. Protoc. 4, 706–731 (2009).
Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Betz, R. Dabble http://dabble.robinbetz.com/ (2017).
MacKerell, A. D. et al. All-atom empirical potential for molecular modeling and dynamics studies of proteins. J. Phys. Chem. B 102, 3586–3616 (1998).
MacKerell, A. D., Jr, Feig, M. & Brooks, C. L. III. Improved treatment of the protein backbone in empirical force fields. J. Am. Chem. Soc. 126, 698–699 (2004).
Guvench, O. et al. CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling. J. Chem. Theory Comput. 7, 3162–3180 (2011).
Best, R. B. et al. Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ1 and χ2 dihedral angles. J. Chem. Theory Comput. 8, 3257–3273 (2012).
Salomon-Ferrer, R., Götz, A. W., Poole, D., Le Grand, S. & Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh Ewald. J. Chem. Theory Comput. 9, 3878–3888 (2013).
Case, D. A. et al. AMBER 2018 (Univ. of California, San Francisco 2018).
Hopkins, C. W., Le Grand, S., Walker, R. C. & Roitberg, A. E. Long-time-step molecular dynamics through hydrogen mass repartitioning. J. Chem. Theory Comput. 11, 1864–1874 (2015).
Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. J. C. Numerical integration of the Cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes. J. Comput. Phys. 23, 327–341 (1977).
Humphrey, W., Dalke, A. & Schulten, K. VMD: visual molecular dynamics. J. Mol. Graph. 14, 33–38, 27–28 (1996).
Roe, D. R. & Cheatham, T. E. III. PTRAJ and CPPTRAJ: software for processing and analysis of molecular dynamics trajectory data. J. Chem. Theory Comput. 9, 3084–3095 (2013).
Klein, U., Gimpl, G. & Fahrenholz, F. Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34, 13784–13793 (1995).
Yauch, R. L. et al. Smoothened mutation confers resistance to a Hedgehog pathway inhibitor in medulloblastoma. Science 326, 572–574 (2009).
Buonamici, S. et al. Interfering with resistance to Smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci. Trans. Med. 2, 51ra70 (2010).
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).
Atwood, S.-X. et al. Smoothened variants explain the majority of drug resistance in basal cell carcinoma. Cancer Cell 27, 342–353 (2015).
Sharpe, H. J. et al. Genomic analysis of Smoothened inhibitor resistance in basal cell carcinoma. Cancer Cell 27, 327–341 (2015).
Acknowledgements
This work was supported by National Institutes of Health (NIH) grants DP5OD023048 (A.M.), R01GM127359 (R.O.D.) and R01GM102498 (P.A.B.). I.D. acknowledges support from the Swiss National Science Foundation. A.M. acknowledges support from the Pew Charitable Trusts. B.R.M. acknowledges support from the American Cancer Society. N.R.L. and K.J.R. were supported by National Science Foundation Graduate Research Fellowships and K.J.R. by a Gerhard Casper Stanford Graduate Fellowship. The content is solely the responsibility of the authors and does not necessarily represent the official view of the NIH. We thank A. Wang for assistance with simulation setup and analysis. We thank the staff at the GM/CA beamlines at the Advanced Photon Source, which have been funded in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The Eiger 16M detector was funded by an NIH–Office of Research Infrastructure Programs, High-End Instrumentation Grant (1S10OD012289-01A1).
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Nature thanks Raymond Stevens and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Contributions
I.D. developed purification approaches for wild-type mSMO, characterized the effect of NbSmo8 on SAG21k affinity, performed crystallization trials, collected diffraction data, determined and refined the structure, and performed fluorescence size-exclusion experiments. J.L. assisted with yeast surface display selections, expressed and purified NbSmo8, and performed fluorescence size-exclusion experiments. D.H. performed NbSmo8 imaging experiments. K.J.R. and Y.Z. performed signalling studies for SMO mutants under guidance from P.A.B. Y.Z. also assisted with yeast surface display selections. B.H. and N.R.L. performed and analysed molecular dynamics simulations under the guidance of R.O.D. B.R.M. performed preliminary experiments establishing SMO nanobodies as intracellular biosensors as a postdoctoral fellow with input and support from P.A.B. B.F. and I.D. performed surface plasmon resonance experiments. I.D., B.R.M. and A.M. wrote the manuscript with revisions provided by P.A.B. A.M. purified SMO and performed yeast selection, identified individual nanobodies, refined the structure and supervised the overall project.
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Extended data figures and tables
Extended Data Fig. 1 NbSmo8 is selective for active SMO.
a, Gating scheme for flow cytometry experiments in Fig. 1b with selection of yeast singlets for analysis. b, Fluorescent size-exclusion chromatography traces of 2 μM NbSmo8–Alexa647 incubated with 0.9 μM SMO incubated with or without 25 μM SAG21k. NbSmo8 co-elutes with SMO–SAG21k with an elution volume of 12.5 ml. c, Fluorescent size-exclusion chromatography traces of NbSmo8–Alexa647 as in b, with SMO purified in the presence of cholesterol–LMNG micelles (yellow). Dashed traces depict apo (black) and SAG21k (green) conditions shown in b. Cholesterol potentiates NbSmo8 binding. SANT-1 (25 μM) (red) completely prevents NbSmo8 binding. SAG21k is cooperative with cholesterol and increases NbSmo8 binding (blue). d, Representative line scans (pixel intensity versus distance, both in arbitrary units) for cells in Fig. 1d. Dashed white line indicates the location of the scan. Scale bar, 10 μm. e, Co-immunoprecipitation of NbSmo8 is dependent on SMO activation. Flag–SMO was co-expressed in suspension HEK293 cells with GFP-tagged versions of NbSmo8 or Nb80, a control nanobody specific to the β2-adrenergic receptor with no affinity for SMO. After stimulation with DMSO (vehicle), KAAD-cyclopamine (KAAD-cyc), SAG21k or MβCD, cells were solubilized in LMNG and SMO–nanobody complexes were isolated using M1 Flag affinity resin. SMO (anti-Flag antibody) and NbSmo8 or Nb80 (anti-GFP antibody) were evaluated via immunoblotting.
Extended Data Fig. 2 NbSmo8 recognition of active SMO.
a, Overall structure of active SMO bound to NbSmo8. The three highly variable nanobody CDRs are highlighted in different colours. b, Close-up view of the interface. NbSmo8 CDRs recognize a large intracellular cavity in active SMO. c, Intracellular view of active SMO cavity stabilized by NbSmo8. Surface is coloured by regions that are contacted by a specific NbSmo8 CDR. NbSmo8 recognizes a unique three-dimensional epitope, with each CDR making important contributions to overall binding. d, CDR1 makes important contacts with SMO intracellular loop (ICL) 3. e, CDR2 residues interact with ICL2 and ICL3. f, The long CDR3 has a complex fold that reaches deep inside the active SMO cavity and stabilizes an outwardly displaced conformation of TM6.
Extended Data Fig. 3 Activation-induced changes in SMO.
a–c, Cytoplasmic view of 7TM helix rearrangements of active SMO–SAG21k–NbSmo8 (green) compared with apo hSMO (PDB code 5L7D) (grey) (a), inactive hSMO (PDB code 5L7I) (salmon) (b) and intermediate active xSMO (PDB code 6D32) (cyan) (c). In all panels, the TM6 outward movement and TM5 inward movement are highlighted with a red arrow.
Extended Data Fig. 4 Unique binding mode of 7TM cholesterol.
a, All experimentally determined GPCR structures in the PDB with a modelled cholesterol (60 individual structures) are shown overlaid with receptors shown in light grey. In each case, cholesterol binds outside of the 7TM bundle. b, c, By contrast, the 7TM cholesterol in active SMO binds within the 7TM bundle (b), which is highly similar to the binding mode of agonists bound to active GPCRs (c) (eight individual structures; agonists are shown in unique colours, and GPCRs in light green transparency).
Extended Data Fig. 5 Molecular dynamics of active SMO.
All atom molecular dynamics simulations of SMO were performed in the absence of SAG21k and NbSmo8 to assess the stability of 7TM cholesterol and the overall stability of the active conformation. a, A model of active SMO derived from lower-resolution data had initially placed cholesterol in an opposite ‘flipped’ orientation (right panel and graph), with the hydroxyl pointing towards the intracellular side. In five out of six simulations starting from such a pose, cholesterol displayed substantial movement deeper into the 7TM pocket. By contrast, the correctly modelled cholesterol (left panel and graph) was stable over all six simulations, showing an overall root mean squared deviation of 1 Å from the starting position. Close-up view of the binding pockets shows simulation frames sampled every 50 ns from a representative simulation as transparent sticks. b, Even after removal of NbSmo8 and SAG21k, TM6 remains close to its initial position but can adopt slightly outward conformations. The displacement of Cα D259 in TM6 from Cα K448 in TM1 is used to track TM6 movement (Methods); these atoms are shown as spheres. Individual structural snapshots from a representative simulation are shown with the conformation of TM6 highlighted in blue. For comparison, the structure of inactive SMO is shown in the intracellular view (salmon cartoon). TM6 spontaneously returns to the crystallographic conformation after outward displacements, which provides confidence in the observed state stabilized by NbSmo8.
Extended Data Fig. 6 SMO mutants bind BODIPY-cyclopamine.
a, Structural modelling of SMO mutations that line the deep 7TM sterol pocket. Wild-type residues are shown as green sticks. Grey sticks show the most-common rotamer of each engineered mutation that does not clash with neighbouring residues in SMO. Red regions show clashes between engineered mutations and cholesterol. b, Gating scheme for BODIPY-cyclopamine (BODIPY-cyc) binding flow cytometry experiments with selection of single cells for analysis. c, Example of flow cytometry plots showing percentage of BODIPY-cyclopamine+ cells with and without competition with KAAD-cyclopamine. Plots shown are for cells that express wild-type SMO. d, Engineered mutations in SMO used to probe the 7TM sterol site and the hydrophobic tunnel are fully competent to bind BODIPY-cyclopamine, which indicates that these mutations fold and traffic to the cellular membrane. Some mutants (including V333F, I412F and T470Q) show increased BODIPY-cyclopamine binding compared to wild-type SMO, which suggests that they stabilize the inactive conformation. Mean ± s.e.m., n = 3 biological replicates. e, Fluorescence size-exclusion chromatography traces of 0.5 μM SMO and SMO(V333F) incubated with 2.5 μM BODIPY-cyclopamine. Both preparations bind BODIPY-cyclopamine, which indicates that the purified proteins are functionally capable of binding ligands.
Extended Data Fig. 7 Comparison of hydrophobic tunnel in mSMO and xSMO.
Cutaway view of active mSMO (green surface) and cyclopamine-bound xSMO (PDB code 6D32) (cyan surface) in similar orientations. In both cases, a hydrophobic tunnel opening to the inner leaflet of the plasma membrane is observed (outlined in red).
Extended Data Fig. 8 SMO structural pharmacology at the 7TM site.
a, Comparison of cholesterol bound to active SMO (green cartoon) and SANT-1 bound to inactive SMO (PDB code 4N4W) (grey cartoon). A hydrogen bond between H470 of hSMO (H474 of mSMO) and SANT-1 (dotted black line) stabilizes an inward conformation of TM6. By comparison, the bulky cholesterol methyl groups outwardly displace TM6. Both cholesterol and SANT-1 form a hydrogen bond with Y398. b, Structure–activity relationship of SAG (adapted from ref. 27). Bulkier replacements to the 4-amino group (orange circle) that are predicted to clash with the 7TM cholesterol switch the molecular efficacy of SAG from an agonist into an antagonist.
Extended Data Fig. 9 SMO-inhibitor resistance mutations.
Previously identified54,55,56,57,58 hSMO-inhibitor resistance mutations are shown as sticks mapped onto the active mSMO structure. Numbering is for hSMO. Residues highlighted in light violet are within 5 Å of SAG21k. Mutations in this upper site probably directly disrupt inhibitor binding. Residues in yellow are within 5 Å of the 7TM cholesterol. Mutations in these sites probably alter sterol affinity or directly stabilize the active SMO conformation. Residues in green are outside of the 7TM binding pocket and probably stabilize the SMO active state.
Supplementary information
Supplementary Information
This file contains Supplementary Figure 1 and Supplementary Table 1. These include uncropped immunoblots and a table summarizing molecular dynamics simulations.
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Deshpande, I., Liang, J., Hedeen, D. et al. Smoothened stimulation by membrane sterols drives Hedgehog pathway activity. Nature 571, 284–288 (2019). https://doi.org/10.1038/s41586-019-1355-4
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DOI: https://doi.org/10.1038/s41586-019-1355-4
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