Abstract
The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC) and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS). Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.
Highlights
Muscarinic acetylcholine receptors are integral membrane proteins that belong to the rhodopsin family of G-protein-coupled receptors (GPCRs)
Binding of agonists and competitive antagonists to GPCRs occurs at the orthosteric site, a hydrophobic pocket located at the extracellular side of the helix bundle that is highly conserved among the members of a subfamily [4]
We focus on the human M3 muscarinic acetylcholine receptor (M3R), which is involved in modulation of neurotransmitter release, temperature homeostasis, and food intake in the central nervous system, as well as in the induction of smooth muscle contraction, gland secretion, indirect relaxation of vascular smooth muscle in the peripheral nervous system [1]
Summary
Muscarinic acetylcholine receptors (mAchRs) are integral membrane proteins that belong to the rhodopsin family of G-protein-coupled receptors (GPCRs). These proteins play a pivotal role in the regulation of many physiological functions both in the central and in the peripheral nervous systems [1]. The physiological actions the natural agonist acetylcholine are mediated by at least five subtypes of receptors known as M1M5 that exhibit high sequence identity across mammalian species and exhibit different tissue distribution [2, 3]. While residues defining the orthosteric binding site are well conserved within the different GPCR subfamilies, those of the extracellular loops are remarkably diverse within a subfamily, providing a good possibility to design selective allosteric antagonist [6]. A deeper understanding of the structure-activity relationships of the different subtypes will help designing new selective
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