title: Curariform antagonists bind in different orientations to acetylcholine-binding protein.
abstract: 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 approximately 170 degrees rotation and approximately 30 degrees degree 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.
authors.affiliation: Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Clinic Department of Pharmacology, Mayo Graduate School, Rochester, Minnesota 55905, USA.
authors.forename: Fan
authors.initials: F
authors.lastname: Gao
authors.forename: Nina
authors.initials: N
authors.lastname: Bern
authors.forename: Alicia
authors.initials: A
authors.lastname: Little
authors.forename: Hai-Long
authors.initials: HL
authors.lastname: Wang
authors.forename: Scott B
authors.initials: SB
authors.lastname: Hansen
authors.forename: Todd T
authors.initials: TT
authors.lastname: Talley
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
authors.forename: Steven M
authors.initials: SM
authors.lastname: Sine
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D001667:Binding, Competitive; D002038:Bungarotoxins / Q000493:pharmacokinetics; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014403:Tubocurarine / Q000031:analogs & derivatives / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: C428944:AChBP protein, Lymnaea; D002038:Bungarotoxins; D002352:Carrier Proteins; D008024:Ligands; C032943:metocurine; D014403:Tubocurarine