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The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism

Abstract

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids—a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.

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Fig. 1: Structural and functional characterization of PTCH1–nanobody interactions.
Fig. 2: Structure of the PTCH1–pShhNc complex.
Fig. 3: Structural and biophysical characterization of the PTCH1 ECD1-cholesterol complex.
Fig. 4: The cholesterol attached to pShhNc inactivates PTCH1.
Fig. 5: The PTCH1 SBD can bind cholesterol in two opposite orientations.
Fig. 6: Tunnel analysis of PTCH1 structures.

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Data availability

Atomic coordinates and structure factors for PTCH1ECD1–NB64, PTCH1ECD1–NB64–cholesterol-HS, apo-PTCH1ECD1 and PTCH1ECD2–NB75, as well as the coordinates for the revised PTCH1–SHH complex have been deposited in the PDB under accession numbers 6RTY, 6RTW, 6RTX, 6RVC and 6RVD.

References

  1. Kong, J. H., Siebold, C. & Rohatgi, R. Biochemical mechanisms of vertebrate hedgehog signaling Development 146, dev166892 (2019).

  2. Porter, J. A., Young, K. E. & Beachy, P. A. Cholesterol modification of hedgehog signaling proteins in animal development. Science 274, 255–259 (1996).

    Article  CAS  Google Scholar 

  3. Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998).

    Article  CAS  Google Scholar 

  4. Luchetti, G. et al. Cholesterol activates the G-protein coupled receptor Smoothened to promote morphogenetic signaling. Elife 5, 20304 (2016).

  5. Huang, P. et al. Cellular cholesterol directly activates smoothened in Hedgehog signaling. Cell 166, 1176–1187 e1114 (2016).

    Article  CAS  Google Scholar 

  6. Bidet, M. et al. The hedgehog receptor patched is involved in cholesterol transport. PLoS One 6, e23834 (2011).

    Article  CAS  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  Google Scholar 

  8. Marigo, V., Davey, R. A., Zuo, Y., Cunningham, J. M. & Tabin, C. J. Biochemical evidence that patched is the Hedgehog receptor. Nature 384, 176–179 (1996).

    Article  CAS  Google Scholar 

  9. Stone, D. M. et al. The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature 384, 129–134 (1996).

    Article  CAS  Google Scholar 

  10. Carstea, E. D. et al. Niemann–Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277, 228–231 (1997).

    Article  CAS  Google Scholar 

  11. Loftus, S. K. et al. Murine model of Niemann-Pick C disease: mutation in a cholesterol homeostasis gene. Science 277, 232–235 (1997).

    Article  CAS  Google Scholar 

  12. Tseng, T. T. et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J. Mol. Microbiol. Biotechnol. 1, 107–125 (1999).

    CAS  PubMed  Google Scholar 

  13. Davies, J. P. & Ioannou, Y. A. Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein. J. Biol. Chem. 275, 24367–24374 (2000).

    Article  CAS  Google Scholar 

  14. 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  Google Scholar 

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

    Article  Google Scholar 

  16. 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).

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

    Article  Google Scholar 

  18. Bishop, B. et al. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat. Struct. Mol. Biol. 16, 698–703 (2009).

    Article  CAS  Google Scholar 

  19. 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  Google Scholar 

  20. Peters, C., Wolf, A., Wagner, M., Kuhlmann, J. & Waldmann, H. The cholesterol membrane anchor of the Hedgehog protein confers stable membrane association to lipid-modified proteins. Proc. Natl Acad. Sci. USA 101, 8531–8536 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Lewis, P. M. et al. Cholesterol modification of sonic hedgehog is required for long-range signaling activity and effective modulation of signaling by Ptc1. Cell 105, 599–612 (2001).

    Article  CAS  Google Scholar 

  23. Chen, M. H., Li, Y. J., Kawakami, T., Xu, S. M. & Chuang, P. T. Palmitoylation is required for the production of a soluble multimeric Hedgehog protein complex and long-range signaling in vertebrates. Genes Dev. 18, 641–659 (2004).

    Article  CAS  Google Scholar 

  24. Burke, R. et al. Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99, 803–815 (1999).

    Article  CAS  Google Scholar 

  25. Chen, Y. & Struhl, G. Dual roles for patched in sequestering and transducing Hedgehog. Cell 87, 553–563 (1996).

    Article  CAS  Google Scholar 

  26. McLellan, J. S. et al. The mode of Hedgehog binding to Ihog homologues is not conserved across different phyla. Nature 455, 979–983 (2008).

    Article  CAS  Google Scholar 

  27. Bosanac, I. et al. The structure of SHH in complex with HHIP reveals a recognition role for the Shh pseudo active site in signaling. Nat. Struct. Mol. Biol. 16, 691–697 (2009).

    Article  CAS  Google Scholar 

  28. Whalen, D. M., Malinauskas, T., Gilbert, R. J. & Siebold, C. Structural insights into proteoglycan-shaped Hedgehog signaling. Proc. Natl Acad. Sci. USA 110, 16420–16425 (2013).

    Article  CAS  Google Scholar 

  29. Kavran, J. M., Ward, M. D., Oladosu, O. O., Mulepati, S. & Leahy, D. J. All mammalian Hedgehog proteins interact with cell adhesion molecule, down-regulated by oncogenes (CDO) and brother of CDO (BOC) in a conserved manner. J. Biol. Chem. 285, 24584–24590 (2010).

    Article  CAS  Google Scholar 

  30. Chuang, P. T. & McMahon, A. P. Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature 397, 617–621 (1999).

    Article  CAS  Google Scholar 

  31. Allen, B. L. et al. Overlapping roles and collective requirement for the coreceptors GAS1, CDO, and BOC in SHH pathway function. Dev. Cell 20, 775–787 (2011).

    Article  CAS  Google Scholar 

  32. Izzi, L. et al. Boc and Gas1 each form distinct Shh receptor complexes with Ptch1 and are required for Shh-mediated cell proliferation. Dev. Cell 20, 788–801 (2011).

    Article  CAS  Google Scholar 

  33. Rubin, J. B., Choi, Y. & Segal, R. A. Cerebellar proteoglycans regulate sonic hedgehog responses during development. Development 129, 2223–2232 (2002).

    CAS  PubMed  Google Scholar 

  34. Hausmann, G., von Mering, C. & Basler, K. The hedgehog signaling pathway: where did it come from? PLoS Biol. 7, e1000146 (2009).

    Article  Google Scholar 

  35. Bazan, J. F. & de Sauvage, F. J. Structural ties between cholesterol transport and morphogen signaling. Cell 138, 1055–1056 (2009).

    Article  CAS  Google Scholar 

  36. Molday, R. S. & MacKenzie, D. Monoclonal antibodies to rhodopsin: characterization, cross-reactivity, and application as structural probes. Biochemistry 22, 653–660 (1983).

    Article  CAS  Google Scholar 

  37. Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D Biol. Crystallogr. 62, 1243–1250 (2006).

    Article  Google Scholar 

  38. Taylor, F. R. et al. Enhanced potency of human Sonic hedgehog by hydrophobic modification. Biochemistry 40, 4359–4371 (2001).

    Article  CAS  Google Scholar 

  39. Tukachinsky, H., Kuzmickas, R. P., Jao, C. Y., Liu, J. & Salic, A. Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand. Cell Rep. 2, 308–320 (2012).

    Article  CAS  Google Scholar 

  40. Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nat. Protoc. 9, 674–693 (2014).

    Article  CAS  Google Scholar 

  41. Walter, T. S. et al. A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr. D Biol. Crystallogr. 61, 651–657 (2005).

    Article  Google Scholar 

  42. Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).

    Article  CAS  Google Scholar 

  43. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D. Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  Google Scholar 

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

    Google Scholar 

  45. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  46. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  47. Murshudov, G. N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D. Biol. Crystallogr. 67, 355–367 (2011).

    Article  CAS  Google Scholar 

  48. Stansfeld, P. J. & Sansom, M. S. From coarse grained to atomistic: a serial multiscale approach to membrane protein simulations. J. Chem. Theory Comput. 7, 1157–1166 (2011).

    Article  CAS  Google Scholar 

  49. Krone, M. et al. Visual analysis of biomolecular cavities: state of the art. Comput. Graph. Forum 35, 527–551 (2016).

    Article  Google Scholar 

  50. Jussupow, A., Di Luca, A. & Kaila, V. R. I. How cardiolipin modulates the dynamics of respiratory complex I. Sci. Adv. 5, eaav1850 (2019).

    Article  Google Scholar 

  51. Stock, C. et al. Cryo-EM structures of KdpFABC suggest a K+ transport mechanism via two inter-subunit half-channels. Nat. Commun. 9, 4971 (2018).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Waldmann and S. Sievers (Max Planck Institute of Molecular Physiology) for the gift of the cholesteroylated SHH peptide (ShhN7-chol), N. Buys for technical assistance during Nanobody discovery, T. Walter and K. Harlos for help with crystallization and O. Fedorov for help with the biolayer interference measurements. C.S. was supported by grants from Cancer Research UK (C20724/A14414 and C20724/A26752) and a European Research Council grant (647278). D.F.C. was supported by a grant from the National Institutes of Health (HL067773) and the Taylor Family Institute for Innovative Psychiatric Research. R.R. was supported by grants from the National Institutes of Health (GM118082 and GM106078). C.K. was supported by a Cancer Research UK studentship (C20724/A16135). M.K. was supported by a pre-doctoral fellowship from the National Science Foundation. M.S.P.S. was supported by the Wellcome Trust (208361/Z/17/Z and 102164/B/13/Z), BBSRC (BB/R00126X/1) and EPSRC (EP/L000253/1). This project made use of time on ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim (http://www.hecbiosim.ac.uk/), supported by EPSRC (EP/R029407/1). This work benefited from access to the Nanobodies4Instruct center (PID1129), we acknowledge the support and use of resources of Instruct-ERIC, part of the European Strategy Forum on Research Infrastructures (ESFRI). We acknowledge the Research Foundation Flanders (FWO) for their support of the Nanobody discovery. The Wellcome Centre for Human Genetics, Oxford, is funded by Wellcome Trust Core Award 203852/Z/16/2.

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C.S. and R.R. designed the project. A.F.R., C.K. and B.B. expressed and purified the proteins for crystallization and biophysical experiments. A.F.R. and B.B. carried out SPR, ITC and thermostability analyses, C.K. carried out biolayer interference measurements. A.F.R. crystallized the proteins. A.F.R., C.K., K.E.O., R.D., A.W. and C.S. collected and processed the X-ray data. K.E.O., T.M. and C.S solved and refined the crystal structures. K.E.O. and C.S rerefined the cryo-EM structure. M.K. and R.R. expressed SHH proteins for cellular assays and performed the HH signaling assays. R.A.S. performed the HH signaling assay for PTC1ECD1-ECD2. T.B.A. and M.S.P.S. performed molecular dynamics and tunnel analysis. E.P. and J.S. produced the nanobodies. M.Q. and D.F.C. synthesized the PEG-cholesterol. C.K., R.R. and C.S. wrote the paper, and all authors commented on the paper.

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Correspondence to Rajat Rohatgi or Christian Siebold.

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Rudolf, A.F., Kinnebrew, M., Kowatsch, C. et al. The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism. Nat Chem Biol 15, 975–982 (2019). https://doi.org/10.1038/s41589-019-0370-y

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