Abstract
The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G-protein-coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor’s preference for the Gαq protein and the M2 receptor’s preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non-cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor–G protein interactions showed some cognate-complex-specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G-protein-binding receptor interface was found only in the two cognate complexes and not in the non-cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non-cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non-cognate receptor–G protein complexes fills a structural gap due to difficulties in determining non-cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.
Highlights
G-protein-coupled receptors (GPCRs) comprise the largest superfamily of integral membrane proteins, covering ~3% of the human proteome
Non-synonymous single-nucleotide polymorphisms in GPCRs that are linked with specific disease pathologies can cause pathophysiological changes due to altered signaling via conformational changes in the receptor
This work is aimed at gaining a mechanistic understanding of this specificity of receptor–G protein interactions (Figure 1) in the muscarinic receptor family because this family is the most homologous compared to any other receptor family and because if there are any clear structural determinants of G protein selectivity, they should be easy to identify for this family
Summary
G-protein-coupled receptors (GPCRs) comprise the largest superfamily of integral membrane proteins, covering ~3% of the human proteome They mediate pleiotropic transmembrane (TM) signal transduction by allosterically facilitating information transfer across the cellular membrane in response to extracellular signals [1,2] and converting them into one or more intracellular signaling cascades [3]. The extracellular signals range from photons (for rhodopsin) and small molecules (like neurotransmitters, metabolites, odorants, tastants, etc.) to large oligopeptides (like chemokines, incretins, etc.) This critical role of GPCRs in cellular signaling makes these receptors therapeutic targets in a large number of diseases (with ~50% of all modern drugs targeting GPCRs [4]), either due to their direct role in the pathophysiology of a specific disease or due to their ability to modulate a set of signaling cascades implicated in a disease [5]. In this context of ligand-induced GPCR signaling cascades [8], the receptor and the ligand play unique functional roles
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