Phase-plate cryo-EM structure of a class B GPCR–G-protein complex

Abstract

SUMMARY Class B G protein-coupled receptors are major targets for treatment of chronic diseases, including osteoporosis, diabetes and obesity. Here we report the structure of a full-length class B receptor, the calcitonin receptor, in complex with peptide ligand and heterotrimeric Gαβγs protein determined by Volta phase plate single-particle cryo-electron microscopy. The peptide agonist engages the receptor through binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helices 6 and 7. This conformation is accompanied by a 60° kink in helix 6 and large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also observed is an extended intracellular helix 8 that contributes to both receptor stability and functional G protein coupling via interaction with the Gβ subunit. This structure provides a new framework for understanding G protein-coupled receptor function.

Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

“…Recent technological advancements, including novel protein engineering (3), in meso crystallization (4), and micro-focus beamlines at synchrotron facilities (5), have ushered in significant progress in the determination of high-resolution GPCR structures. However, only a few of those structures are of activated GPCRs, and thus far, only two GPCR-G protein complex structures have been solved, namely that of the ␤ 2 -adrenergic receptor-G S protein complex (6) and the calcitonin receptor-G S protein complex (7). Furthermore, virtually all of these structures, with the exception of rhodopsin, have been obtained with GPCRs that were heavily modified so as to facilitate crystallization.…”

Electron cryomicroscopy as a powerful tool in biomedical research

“…The development and application of the Volta phase plate (see above) [75] was key to solve the structure of these relatively small receptors that were recalcitrant to crystallization so far.…”

Shining a light on GPCR complexes

“…However, the degree of this interdomain opening, when it can be observed, varies for different complexes from relatively small movements or rotations up to a maximum of a 127°rotation in the crystal structure of ␤ 2 AR-G S . CryoEM studies of the ␤ 2 AR-G S complex and low resolution images of the CTR-G S complex further indicate the helical domain can occupy multiple conformations and has a high degree of mobility (4, 8 (1,4,5). However, early low resolution structures of the rhodopsin-G T complex indicated a 2:1 stoichiometry, suggesting the dimeric receptor was the minimal functional unit for G T activation (3).…”

“…Although rhodopsin was the first GPCR crystal structure to be solved in 2000 (3), issues with expression of the receptor in heterologous systems and stabilization of rhodopsin-G T hampered efforts to crystallize the complex, and the 2011 report was at relatively low resolution. It was not until June 2017 that additional higher resolution structures of GPCR-G protein complexes of two class B GPCRs emerged (4,5). The structures of calcitonin receptor (CTR) and glucagonlike peptide 1 receptor (GLP-1R) with G S were solved by cryoelectron microscopy (cryoEM) using tagged and/or truncated receptors expressed and purified from insect cells.…”