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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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.
The authors declare no competing interests.
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