Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms1,2,3. Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers2,4,5,6. N-terminally palmitoylated HH releases the repression of Patched to the oncoprotein smoothened (SMO); however, the mechanism by which HH recognizes Patched is unclear. Here we report cryo-electron microscopy structures of human patched 1 (PTCH1) alone and in complex with the N-terminal domain of ‘native’ sonic hedgehog (native SHH-N has both a C-terminal cholesterol and an N-terminal fatty-acid modification), at resolutions of 3.5 Å and 3.8 Å, respectively. The structure of PTCH1 has internal two-fold pseudosymmetry in the transmembrane core, which features a sterol-sensing domain and two homologous extracellular domains, resembling the architecture of Niemann–Pick C1 (NPC1) protein7. The palmitoylated N terminus of SHH-N inserts into a cavity between the extracellular domains of PTCH1 and dominates the PTCH1–SHH-N interface, which is distinct from that reported for SHH-N co-receptors8. Our biochemical assays show that SHH-N may use another interface, one that is required for its co-receptor binding, to recruit PTCH1 in the absence of a covalently attached palmitate. Our work provides atomic insights into the recognition of the N-terminal domain of HH (HH-N) by PTCH1, offers a structural basis for cooperative binding of HH-N to various receptors and serves as a molecular framework for HH signalling and its malfunction in disease.
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We appreciate support and suggestions from Günter Blobel, and dedicate this manuscript to him. We thank M. Ebrahim and J. Sotiris at the Evelyn Gruss Lipper Cryo-EM Resource Center of the Rockefeller University for assistance with data collection and D. Nicastro and Z. Chen at the UT Southwestern Cryo-EM Facility (funded in part by the CPRIT Core Facility Support Award RP170644) for facility access and data acquisition; L. Beatty for help with tissue culture; A. Lemoff at the UT Southwestern Proteomics Core for mass spectrometry identification; B. Chen and J. Kim for 5E1 antibody, SHH Light II cells and Ptch1−/− MEFs; and M. Brown, E. Debler, J. Goldstein, J. Jiang, D. Rosenbaum and Z. Zhang for discussion. This work was supported by the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center and O’Donnell Junior Faculty Funds (to X.L.), by NIH grant P01 HL020948 (Tissue Culture Core), by the Rockefeller University (to E.C.) and by the National Key Research and Development Program of MOST (numbers 2016YFA0501103 and 2015CB910104 to J.W.). X.L. is the Rita C. and William P. Clements, Jr. Scholar in Biomedical Research of UT Southwestern.
Nature thanks F. de Sauvage and the other anonymous reviewer(s) for their contribution to the peer review of this work.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
The residue numbers of human PTCH1 are indicated above the protein sequence. The transmembrane helices and secondary structures of extracellular domains are labelled (structural elements of ECD-II with asterisk). Residues under the dashed lines are excluded from the 3D reconstruction.
a–c, Size-exclusion chromatogram and SDS–PAGE gel of the purified full-length PTCH1 (a), the purified PTCH1* (b) and the purified PTCH1*–SHH-N complex (c). Molecular standards are indicated on the left side of the gels and above the elution curves. The assays were reproduced at least three times with similar results.
a, The data processing workflow for PTCH1*. b, A representative electron micrograph at a defocus of −2.0 μm. c, 2D classification. d, FSC curve of the structure as a function of resolution using Frealign output. e, The FSC curves calculated between the refined structure and the half map used for refinement, the other half map and the full map. f, Density maps of PTCH1* structure coloured by local resolution estimate using Blocres.
a, TM1–TM6. b, TM7–TM12. c, ECD-I. d, ECD-II. NAG, N-acetylglucosamine.
a, NPC1 SSD. The putative pocket (indicated by the red arrow) in the SSD is created by TM3–TM5. b, PTCH1* SSD.
a–f, Same as Extended Data Fig. 3 but for PTCH1*–SHH-N.
a, TM1–TM6 at the 5σ level. b, TM7–TM12 at the 5σ level. c, Major structural elements of ECD-I at the 4.5σ level, d, Major structural elements of ECD-II at the 4.5σ level. e, Major structural elements of SHH-N at the 4.5σ level; palmitate (PLM) at the 3σ level.
a, Size-exclusion chromatogram and SDS–PAGE gel of the purified PTCH1* with Amphipol A8-35 in buffer A. Molecular standards are indicated on the left side of the gels and above the elution curves. b, 5E1 does not compete with the binding of native SHH-N to PTCH1*. 5E1 and SHH-N at a 1:1 molar ratio were incubated with PTCH1*-immobilized Flag M2 resin; the complex was eluted by Flag peptide. Protein was detected by Coomassie staining. The assay was reproduced three times with similar results.
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Qi, X., Schmiege, P., Coutavas, E. et al. Structures of human Patched and its complex with native palmitoylated sonic hedgehog. Nature 560, 128–132 (2018). https://doi.org/10.1038/s41586-018-0308-7
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