Structure determination of GPCRs: cryo-EM compared with X-ray crystallography

Abstract

G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and β-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposited in the PDB (Jan-Jul 2021) were determined by cryo-EM. Cryo-EM has also opened up new possibilities in GPCR structural biology, such as determining structures of GPCRs embedded in a lipid nanodisc and multiple GPCR conformations from a single preparation. However, X-ray crystallography still has a number of advantages, particularly in the speed of determining many structures of the same receptor bound to different ligands, an essential prerequisite for effective SBDD. We will discuss the relative merits of cryo-EM and X-ray crystallography for the structure determination of GPCRs and the future potential of both techniques.

Keywords: G-protein-coupled receptors; cryo-electron microscopy; crystallography.

Figure 1.. GPCR structures.

(A) Cartoon of β₂AR indicating the conformational change from the inactive state (blue; PDB ID 2RH1) [20] to the active state (purple; PDB ID 3SN6) [26] when coupled to a G protein (not shown). (B) Cryo-EM density for the serotonin 5-HT_1B receptor coupled to the heterotrimeric G protein G_o (PDB ID 6G79, EMDB-4358) [79]. The detergent used for purification of the receptor was decylmaltoside. (C) Cryo-EM density for the β₁-adrenoceptor coupled to β-arrestin (PDB 6TKO, EMDB-10515) [9]. The detergent-purified receptor was reconstituted into a lipid nanodisc containing a bilayer of phosphatidylcholine and phosphatidylglycerol and has an external diameter of ∼13 nm. (D) Percentage of GPCR structures in the Protein Data Bank (PDB) determined by cryo-EM per year. (E) Cumulative number of GPCR structures determined by X-ray crystallography and cryo-EM, which includes multiple structures of the same receptor bound to different ligands, different intracellular binding partners and different species. Note that in panels D and E the data for 2021 includes only the first 7 months of the year.

Figure 2.. Artefacts in X-ray crystallography.

(A) 60° kink of H1 in chain A (brown) of β₁AR compared with chain B (green); PDB ID 2VT4 [23]. (B) Different pose of the methylphenoxy moiety of carmoterol in chain A (brown) compared with chain B (green); PDB ID 2Y02 [59]. (C) The salt bridge in the extracellular region of A_2AR determined in the crystal structure at pH 4.8 (PDB ID 5G53) [32] is absent from the cryo-EM structure determined at pH 7.5 (PDB ID 6GDG) [57]. (D) The RMSD differences (rainbow colouration) between β₁AR crystallised in detergent (PDB ID 2VT4) compared with β₁AR crystallised in lipidic cubic phase (PDB ID 4BVN) were determined and plotted on the structure (red, large differences; dark blue, no differences) [58]. Panel 2D has been reproduced from [58].