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.

Protein Design: From the Aspect of Water Solubility and Stability

“…The number increased rapidly and was higher than that from X-ray crystallography in 2019. Seventy-seven out of the ninety-nine GPCR structures deposited in the PDB last year (Jan–Jul 2021) were determined by Cryo-EM (Figure A,B) …”

“…The data for 2021 includes only the first seven months of the year. A, B: Reprinted with permission from ref under Creative Commons licenses. (C) Schematic of the activation of a class B GPCR by extracellular peptide agonist via a “two-domain” binding mechanism.…”

“…Although Cryo-EM has proven to be a suitable approach for determining the structure of membrane proteins, technical limitations such as the protein size limit still posed difficulties in its applications. It is challenging to determine 35–45 kDa GPCRs in the inactive state using the technique . Hence, a fusion strategy between GPCRs and binding partners was commonly adopted to prepare protein complexes with molecular weight high enough for Cryo-EM SPA.…”

Angiotensin and Endothelin Receptor Structures With Implications for Signaling Regulation and Pharmacological Targeting

“…Moreover, reflecting the high number of GPCR heteromer reports for ATRs and ETRs with functional impact, it also appears necessary to intensify further means of exploring ways to elucidate heteromer arrangements, both structurally and functionally for these receptors and binding partners. In addition, this is an area of utmost pharmacological importance ( 165 , 166 ) and, therefore, must be of structural interest, especially given the increasing possibilities in the determination of complex structures ( 167 ). Finally, the relevance of autoantibody binding to both receptor groups require questions on antibody binding and its functional significance to be explored in-depth, intending to use improved understanding to tailor the design of optimal ligands useful for pharmacological intervention strategies or to recruit these receptors (as monomers or dimers) as hubs for precisely sought specific responses.…”

Plasmalogenic Lipid Analogs as Platelet-Activating Factor Antagonists: A Potential Novel Class of Anti-inflammatory Compounds

“…The canonical view of how GPCRs may modulate cellular physiology is that the binding of ligands (such as hormones, neurotransmitters or sensory stimuli) induces the conformational changes of transmembrane and intracellular domains of the receptor, further allowing interactions with heterotrimeric G proteins ( Ritter and Hall, 2009 ). Up to July 2021, there are total 99 GPCR structures deposited in the Protein Data Bank (PDB: www.pdbus.org ), and most of them were determined by the cryo-EM method ( García-Nafría and Tate, 2021 ). After binding to PAF-R, PAF may activate intracellular signaling pathways, including NF-κB and MAPK pathways ( Lordan et al, 2019 ).…”