Smoothened stimulation by membrane sterols drives Hedgehog pathway activity

Abstract

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models3–5, a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results—combined with signalling studies and molecular dynamics simulations—delineate the structural basis for PTCH1 -SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors.

A NanoBRET-based binding assay for Smoothened allows real time analysis of small-molecule ligand binding and distinction of two separate ligand binding sites for BODIPY-cyclopamine

“…The development of a NanoBRET-based ligand binding assay provides an interesting complement to GPCR pharmacology enabling ligand binding studies on living cells in real time with simplified protocols (Stoddart et al, 2015;Stoddart, Kilpatrick & Hill, 2018 Recent insight into the molecular mechanisms of drug action on SMO by crystallography and CryoEM provide somewhat controversial yet intriguing information regarding cyclopamine and cholesterol interaction with the 7TM ligandbinding site and the CRD (Deshpande et al, 2019;Huang et al, 2016;Huang et al, 2018;Qi, Liu, Thompson, McDonald, Zhang & Li, 2019;Weierstall et al, 2014). Here, we have been able to pharmacologically separate two BODIPY-cyclopamine binding sites on the 7TM core of SMO.…”

“…Along these lines, different binding poses of cyclopamine and related sterols were reported in several crystal structures using SMO from various species. In summary, there is a cyclopamine/sterol binding site in the lipid groove of the CRD (Byrne et al, 2016;Deshpande et al, 2019;Huang et al, 2016;Huang et al, 2018), one in the upper pocket of the 7TM core (Huang et al, 2018;Qi, Liu, Thompson, McDonald, Zhang & Li, 2019) and one in the lower position (Deshpande et al, 2019), which overlaps with the SANT-1 binding pocket (Fig. 7).…”

“…The residual binding of BODIPY-cyclopamine to CRD Nluc-SMO above 10 -7 M identifies a SANT-1-insensitive fraction suggesting an additional binding pocket for BODIPY-cyclopamine. Given the simultaneous binding of SAG21k and cholesterol to SMO in the recent crystal structure (PDB 6O3C) of active, nanobody NbSmo8-bound SMO (Deshpande et al, 2019), and the fact that SANT-1 and cyclopamine occupy two different parts of the small molecule binding space in SMO (Huang et al, 2018;Wang et al, 2014;Weierstall et al, 2014) it could also be possible that BODIPYcyclopamine and SANT-1 bind the 7TM course simultaneously. It remains to be verified by structural studies if binding modes of cyclopamine and BODIPYcyclopamine are indeed the same.…”

Sphingomyelin suppresses Hedgehog signaling by restricting cholesterol accessibility at the ciliary membrane

“…Sharpe et al 2015) . Another sterol binding site has also been recently described deep in the TMD (Deshpande et al 2019) . Mutations in the CRD site abolish sterol binding and responses to SHH; mutations in the more superficial TMD site prevent SMO activation by synthetic agonists like SAG but do not affect responses to SHH ( Fig.4B ) (E. F. Byrne et al 2016;Luchetti et al 2016;P.…”

“…Mutations in either site prevent the activation of signaling by HH ligands, implicating both in the regulation of SMO by PTCH1. The extracellular CRD site is closer to the outer leaflet of the plasma membrane,while the TMD site has been proposed to obtain cholesterol from the inner leafter through an opening between transmembrane helices 5 and 6 (E. F Byrne et al 2016;Pengxiang Huang et al 2018;Deshpande et al 2019…”