Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand

Abstract

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

“…Recently two additional structures of Xenopus laevis SMO emerged, bound to cholesterol and to cyclopamine at the CRD (Huang et al). Both structures exhibited an alternative 7TMD conformation in which the intracellular portion of TM6 adopts an arrangement in which the intracellular tip moves outward by several Ångstrom compared to previous SMO structures Wang et al, 2014;Wang et al, 2013;Weierstall et al, 2014;Zhang et al, 2017). We subjected the 7TMD of the cyclopamine bound structure (PDB id: 6D32) to 8 x 10 μ s of CGMD, performed in the same manner already described.…”

“…SMO is a member of the Frizzled-class of G-protein coupled receptors (GPCRs) and is found at the plasma membrane. Its structural architecture consists of an extracellular cysteine rich domain (CRD), stacked on top of a short linker domain (LD), and a hepta-helical transmembrane domain (7TMD) Zhang et al, 2017), (Figure 1). To date, eleven crystal structures containing the SMO 7TMD have been solved (Byrne et al, 2018), revealing structural similarity to the presumed inactive state of Class A GPCRs (Wang et al, 2013).…”

Assembly and architecture of the Wnt/β‐catenin signalosome at the membrane

“…The full-length structure of FZD receptors is currently only known from smoothened, the most divergent family member. The extracellular and transmembrane domains (TMDs) of smoothened have been crystallized (Figure 2A), revealing a structured CRD very similar to that of the FZD 4 and FZD 8 receptors a flexible and largely unstructured CRD-to-TMD linker, a TMD bundle very reminiscent of class B GPCRs and a long extracellular loop 3 (ECL3) which coordinates with the CRD and linker to encase the CRD sterol-binding pocket (Byrne et al, 2016;Zhang et al, 2017). Since FZD receptor TMDs and CRDs are very similar with regard to sequence to smoothened ( Figure 2C), it is probable that the most substantial structural differences will be due to the linker and ECL3 length and minor variations in CRD structure ( Figure 2D).…”

Recent achievements in developing selective Gq inhibitors

“…They all share a common macrostructure with an extracellular N-terminal region, seven transmembrane (TM) α-helices connected by intracellular (ICL1-3) and extracellular (ECL1-3) loop regions, and an intracellular C-terminus (Figure 1 A).Moreover, X-ray crystal structures indicate that GPCRs share an overall conserved protein folding of TM1-7 domains, resulting in overall related intracellular regions 11-13 while the major differences are found in the extracellular region. [14][15][16][17]…”

“…Moreover, X-ray crystal structures indicate that GPCRs share an overall conserved protein folding of TM1-7 domains, resulting in overall related intracellular regions 11-13 while the major differences are found in the extracellular region. [14][15][16][17]…”