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

Xianjun Zhang,Mark S Hunter,Fei Zhao,Xi Lin,Venkatasubramanian Dharmarajan,Suwen Zhao,Vadim Cherezov,Houchao Tao,Patrick R Griffin,Lintao Ye,Wenfu Tan,Andrii Ishchenko,Jun Yang,Michael A Hanson,Cornelius Gati,Yiran Wu,Garrett Nelson,Raymond C Stevens,Kang Ding,Gye Won Han,Fei Xu

doi:10.1038/ncomms15383

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.

Highlights

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
The mechanism of how the binding of an agonist to the cysteine-rich domain (CRD) triggers a conformational change in the transmembrane helices domain (TMD) remains obscure
As noted, binding of cholesterol to the multi-domain SMO induces an B7° tilt of the CRD, which is associated with three major conformational changes: the extracellular extension of helix VI tilts outward by B5°, extracellular loop 3 (ECL3) forms an extra a-helical turn and shifts outward by 7 Å, and the hinge domain (HD) is displaced towards the TMD by 2 Å compared to the TC114-bound structure (Fig. 3a–c)

Summary

Introduction

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. 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

Methods

Results

Conclusion