Abstract
The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. Here we use attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to examine the ligand-dependent conformational changes in the human M2 muscarinic acetylcholine receptor (M2R). We show that different ligands affect conformational alteration appearing at the C=O stretch of amide-I band in M2R. Notably, ATR-FTIR signals strongly correlated with G-protein activation levels in cells. Together, we propose that amide-I band serves as an infrared probe to distinguish the ligand efficacy in M2R and paves the path to rationally design ligands with varied efficacy towards the target GPCR.
Highlights
The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect
Recent advance of single particle analyses using cryo electron microscopy provided the inactive structures bound with either antagonist or inverse agonist, and active structures bound with agonists and signal transducers[10,11,12,13]
This is partly because efficacy of a ligand is thought to be reflected in changes to conformational equilibria, and the presence of multiple states. These structural methods of X-ray crystallography and cryo electron microscopy (cryoEM) analysis capture only a snapshot, low-energy conformation, which lack the conformational heterogeneity, these methods cannot fully explain the mechanism of the efficacies. Spectroscopic techniques such as nuclear magnetic resonance (NMR) and double electron-electron resonance (DEER) have provided insights into the dynamic nature of GPCRs underpinning the conformational plasticity of different efficacy ligand binding[18,19,20,21,22,23]
Summary
The intrinsic efficacy of ligand binding to G protein-coupled receptors (GPCRs) reflects the ability of the ligand to differentially activate its receptor to cause a physiological effect. The muscarinic acetylcholine receptor 2 (M2R), one of the most extensively studied GPCR has been crystallized with its inverse agonist 3-quinuclidinyl-benzilate (QNB)[14] or N-methylscopolamine (NMS)[15], full agonist Iperoxo (Ixo)[16], and effector Goprotein[17] These studies have provided important insights into the structural changes including the ligand pocket and TM6 movement mediated by the two classes of ligands between inverse agonist and full agonist at the atomic level, its application to a broad variety of ligands with different efficacies, especially partial agonists and neutral antagonists, is extremely challenging.
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