Acetylcholine-binding protein (AChBP) recently emerged as a prototype for relating structure to function of the ligand binding domain of nicotinic acetylcholine receptors (AChRs). To understand interactions of competitive antagonists at the atomic structural level, we studied binding of the curare derivatives d-tubocurarine (d-TC) and metocurine to AChBP using computational methods, mutagenesis, and ligand binding measurements. To account for protein flexibility, we used a 2-ns molecular dynamics simulation of AChBP to generate multiple snapshots of the equilibrated dynamic structure to which optimal docking orientations were determined. Our results predict a predominant docking orientation for both d-TC and metocurine, but unexpectedly, the bound orientations differ fundamentally for each ligand. At one subunit interface of AChBP, the side chain of Tyr-89 closely approaches a positively charged nitrogen in d-TC but is farther away from the equivalent nitrogen in metocurine, whereas, at the opposing interface, side chains of Trp-53 and Gln-55 closely approach the metocurine scaffold but not that of d-TC. The different orientations correspond to ϳ170°rotation and ϳ30°d egree tilt of the curare scaffold within the binding pocket. Mutagenesis of binding site residues in AChBP, combined with measurements of ligand binding, confirms the different docking orientations. Thus structurally similar ligands can adopt distinct orientations at receptor binding sites, posing challenges for interpreting structure-activity relationships for many drugs.The superfamily of pentameric ligand-gated ion channels activated by acetylcholine (ACh), 1 ␥-aminobutyric acid, glycine, and serotonin mediate rapid synaptic transmission throughout the nervous system. Their strategic position in the pathway of information flow makes them strategic loci for disease processes as well as logical targets for drugs used clinically. The synaptic protrusion of these channels contains a ligand binding domain, which harbors structural components specialized for binding agonists and competitive antagonists. The ligand binding domain is formed at interfaces between subunits where conserved aromatic and hydrophobic residues are clustered (1-3). In the nicotinic receptor found at the motor endplate, the alpha subunit forms one face of the ligand binding domain, whereas a non-alpha subunit, gamma, delta or epsilon, forms the other face. Studies of subunit chimeras and site-directed mutations have pinpointed key residues critical for stabilizing bound agonists and antagonists. In particular, residues on both alpha and non-alpha subunits are critical for binding competitive antagonists of the curare family, suggesting that these antagonists prevent ACh binding by bridging the subunit interface (4).The potential for understanding ligand binding at the atomic structural level recently arose with the discovery of acetylcholine-binding protein (AChBP) (5), a soluble protein homologous to the ligand binding domains of pentameric ligand-gated ion channels. Moreover, ...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.