Structures of human Patched and its complex with native palmitoylated sonic hedgehog

Abstract

Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms. Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers. 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) protein. 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-receptors. 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.

Patched regulates lipid homeostasis by controlling cellular cholesterol levels

“…The mechanism by which PTCH inhibits SMO was enigmatic for a long time because PTCH represses SMO without direct contact 3 . PTCH1 was shown to be able to transport cholesterol [4][5][6] which in turn will directly activate SMO 7 , a finding, which was supported by recent structural analyses 1,[8][9][10] . The structures suggest that Hh inhibits PTCH transporter function and hence plasma membrane cholesterol levels could increase.…”

“…Recent data suggested that mammalian PTCH1 acts as a cholesterol transporter 1,5,6,[8][9][10] . To investigate, whether PTC-3 shares the function of PTCH1 as cholesterol transporter, we first expressed PTC-3 in Saccharomyces cerevisiae, which does not contain any cholesterol 29 and measured cholesterol efflux from cells using TopFluor cholesterol in pulse-chase experiment ( Fig.…”

Association of Sonic Hedgehog with the extracellular matrix requires its zinc-coordination fold

“…Our observations that the catalytic domain of ShhN is required for its localization to the ECM provides further evidence that Shh is a peptidase, and that this activity is required for non-cell autonomous signaling. Distinct from the peptidase domain is the extreme N-terminal end of ShhN which binds to Ptch1 (Gong et al, 2018;Qi et al, 2018a;2018b) and suffices to alter Ptch1 activity (Tukachinsky et al, 2016), further demonstrating the dispensability of the peptidase activity to activate the Shh response.…”

“…showing that the N-terminal 22 residues of Shh that are not part of the peptidase domain, mediate binding to Ptch1 (Gong et al, 2018;Qi et al, 2018a;2018b) and suffice to regulate Ptch1 activity (Tukachinsky et al, 2016). Some bacteria have genes coding for peptidases that coordinate zinc and calcium identically to Shh (Rebollido-Rios et al, 2014;Roelink, 2018).…”

Hedgehog pathway activation through conformational blockade of the Patched sterol conduit

“…For example, in the ternary complex of a single native Shh ligand bound to two human PTCH1 molecules 32, PTCH1 from chain a, the molecule whose sterol conduit is occluded by interaction with the N-terminal palmitoyl moiety of the SHH ligand, adopts pose 2, whereas PTCH1 from chain b adopts pose 1 32. Indeed, in all published structures of PTCH1 the switch helix adopts one or the other of these two poses15, [31][32][33][34][35]42, suggesting that they represent discrete alternative conformations preferentially populated within the PTCH1 activity cycle ( Fig. 4b).…”