Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit Yunxiao Zhanga,1, Wan-Jin Lua, David P. Bulkleyb, Jiahao Liangc, Arthur Ralkod, Shuo Hana, Kelsey J. Robertse, Anping Lia, Wonhwa Chod, Yifan Chengb,f, Aashish Manglikc,g,2, and Philip A. Beachya,e,h,i,2 aInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305; bDepartment of Biochemistry and Biophysics, University of California, San Francisco, CA 94158; cDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158; dDepartment of Chemistry, University of Illinois at Chicago, Chicago, IL 60607; eDepartment of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305; fHoward Hughes Medical Institute, University of California, San Francisco, CA 94158; gDepartment of Anesthesia and Perioperative Care, University of California, San Francisco, CA 94158; hDepartment of Urology, Stanford University School of Medicine, Stanford, CA 94305; and iDepartment of Chemical and Systems Biology (by courtesy), Stanford University School of Medicine, Stanford, CA 94305 Contributed by Philip A. Beachy, September 23, 2020 (sent for review June 5, 2020; reviewed by James Briscoe and Volodymyr M. Korkhov) Activation of the Hedgehog pathway may have therapeutic value for primary receptor for Hedgehog is Patched1 (PTCH1), which improved bone healing, taste receptor cell regeneration, and allevi- maintains pathway quiescence by suppressing Smoothened ation of colitis or other conditions. Systemic pathway activation, (SMO), a downstream G-protein–coupled receptor (GPCR)-like however, may be detrimental, and agents amenable to tissue protein (10–12). When bound to Hedgehog, PTCH1 is inacti- targeting for therapeutic application have been lacking. We have vated, permitting SMO to become active and trigger downstream developed an agonist, a conformation-specific nanobody against signaling events (12–15). Mechanistically, the activation of SMO the Hedgehog receptor Patched1 (PTCH1). This nanobody potently requires binding of a sterol, likely entering the 7TM bundle from activates the Hedgehog pathway in vitro and in vivo by stabilizing an the inner leaflet of the plasma membrane (16–21). PTCH1 is alternative conformation of a Patched1 “switch helix,” as revealed by proposed to prevent SMO activation by transporting sterols from our cryogenic electron microscopy structure. Nanobody-binding likely the inner leaflet of the plasma membrane, thereby limiting SMO traps Patched in one stage of its transport cycle, thus preventing access to activating sterols (22–25). A hydrophobic conduit substrate movement through the Patched1 sterol conduit. Unlike coursing through the PTCH1 extracellular domain is required for the native Hedgehog ligand, this nanobody does not require lipid this transport activity (24), and Hedgehog blocks this conduit modifications for its activity, facilitating mechanistic studies of Hedge- and inactivates PTCH1 by inserting its essential amino-terminal hog pathway activation and the engineering of pathway activating palmitoyl adduct (23–26). agents for therapeutic use. Our conformation-selective nanobody ap- Transporters typically act by moving through a repeated cycle of proach may be generally applicable to the study of other PTCH1 conformational changes. If PTCH1 transport function employs such homologs. a conformational cycle, an agent that preferentially binds and sta- bilizes a specific PTCH1 conformation would be expected to disrupt hedgehog | cryo-EM | nanobody | Patched | transporter its conformational cycle and transport activity, thus permitting ac- tivation of SMO. Such an agent thus may serve as a pathway edgehog signaling functions in embryonic tissue patterning modulator that could make lipid modifications dispensable and may Hand in postembryonic regulation of tissue homeostasis and regeneration. The postembryonic regenerative activities of the Significance Hedgehog pathway clearly suggest potential therapeutic benefits of pathway activation. The only modality of pathway modulation Precise manipulation of Hedgehog pathway activity holds tested clinically, however, is inhibition, with clear benefits for great value for biological research and clinical applications, but patients suffering from malignancies whose initiation and growth pathway agonists amenable to engineering have been lacking. depend on pathway-activating mutations in the primary cells of the We selected a nanobody that potentially targets the confor- tumor, such as medulloblastoma (1) and basal cell carcinoma (2). mational changes of the Hedgehog receptor Patched1 and The apparent lack of clinical interest in pathway-activating demonstrated that this nanobody potently activates the therapies, despite availability of potent small-molecule pathway pathway in vivo. This nanobody can serve as the basis for activators (3), is likely due to the expectation that such systemic mechanistic studies of Hedgehog pathway activation and for treatments may cause overgrowth of mesenchyme and potential potential therapeutic applications. Our method may further initiation or exacerbation of fibrosis in multiple organs (4, 5). apply to the investigation of other related transporters in the These dangerous side effects might be avoided by restricting Resistance-Nodulation-Division superfamily. pathway activation to specific cell types. A pathway agonist conjugated to targeting agents would fulfill Author contributions: Y.Z., W.C., Y.C., A.M., and P.A.B. designed research; Y.Z., W.-J.L., D.P.B., J.L., A.R., S.H., K.J.R., A.L., and A.M. performed research; Y.Z., W.-J.L., D.P.B., and this purpose, but the native Hedgehog protein is difficult to engi- P.A.B. analyzed data; and Y.Z. and P.A.B. wrote the paper. neer for cell-type specificity. Mature Hedgehog protein contains Reviewers: J.B., The Francis Crick Institute; and V.M.K., Paul Scherrer Institute. two lipid modifications (6, 7), including a cholesteryl moiety on its The authors declare no competing interest. carboxy-terminus (8) and a palmitoyl adduct on its amino terminus Published under the PNAS license. (9), which is especially critical for signaling activity. The require- 1Present address: Howard Hughes Medical Institute, Department of Neuroscience, Dorris ment for lipid modification in signaling poses a challenge for large- Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037. scale production, storage, and further derivatization for tissue tar- 2To whom correspondence may be addressed. Email: [email protected] or geting. Other synthetic or genetically encoded peptides that could [email protected]. easily be conjugated to targeting agents are currently lacking. This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Recent insights into the mechanism of Hedgehog signal doi:10.1073/pnas.2011560117/-/DCSupplemental. transduction suggest a route to remedying this deficiency. The First published November 2, 2020. 28838–28846 | PNAS | November 17, 2020 | vol. 117 | no. 46 www.pnas.org/cgi/doi/10.1073/pnas.2011560117 Downloaded by guest on September 26, 2021 shed light on conformational changes that occur during the PTCH1 embryonic palatal mesenchymal (HEPM) (32) (Fig. 1G). In working cycle. comparison with ShhNp, TI23 exhibited similar potency, but consistently lower efficacy. The maximum response induced by Results TI23 is ∼75% of that from ShhNp, suggesting that TI23 is a Development of a Conformation-Specific Nanobody that Activates partial agonist (Fig. 1H). Pathway. Nanobodies are single-chain antibody fragments (27) that have frequently been used to stabilize specific GPCR pro- Structure of the PTCH1::TI23 Complex. To determine the confor- tein conformations (28) and are amenable to genetic engineer- mational effects of TI23 binding to PTCH1, we prepared the ing. We have chosen as a starting point a synthetic yeast display PTCH1::TI23 complex for structure determination by cryogenic library to select for conformation-specific nanobodies against electron microscopy (cryo-EM). The complex was clearly visu- PTCH1 (29). To select conformation-specific nanobodies, we first alized in cryo-EM micrographs (SI Appendix, Fig. S2A) with well- introduced conformational bias in PTCH1 by altering three acidic fitted contrast transfer function parameters (SI Appendix, Fig. residues buried within its transmembrane domain (D499N, D500N, S2B) and two-dimensional class averages (SI Appendix, Fig. and E1081Q, termed PTCH1-NNQ). These acidic residues, con- S2C). Three-dimensional reconstruction of a cryo-EM dataset served within the Resistance-Nodulation-Division (RND) trans- yielded a high quality map (Fig. 2A; procedure in SI Appendix, porter family, are required for PTCH1 activity in sterol transport Fig. S2D) at a resolution of 3.4 Å (SI Appendix, Fig. S2F and and SMO regulation (17, 24, 30) and are more generally proposed Table S1). All 12 transmembrane (TM) helices and two major to drive conformational changes in RND transporters in response extracellular domains (ECDs) were resolved (Fig. 2A), and an to cation influx (Fig. 1A). We thus reasoned that alteration of these atomic model of the PTCH1::TI23 complex was built based on residues in PTCH1 might affect the relative representation of its this map and the previously determined murine PTCH1 struc- conformational states. ture (24). Most of the intracellular sequence was unresolved, and We used