title: Chemical-scale studies on the role of a conserved aspartate in preorganizing the agonist binding site of the nicotinic acetylcholine receptor.
abstract: The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site but does not directly contact the ligand plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently, the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function) or if a compensating backbone mutation is introduced to relieve unfavorable electrostatics.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Cashin
authors.forename: Michael M
authors.initials: MM
authors.lastname: Torrice
authors.forename: Kathryn A
authors.initials: KA
authors.lastname: McMenimen
authors.forename: Henry A
authors.initials: HA
authors.lastname: Lester
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000613:Aminobutyrates / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D001665:Binding Sites; D003198:Computer Simulation; D004594:Electrophysiology; D006860:Hydrogen Bonding; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics; D040681:Structural Homology, Protein; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: C517545:3-(3-indolyl)-2-hydroxypropanoic acid cyanomethyl ester; D000596:Amino Acids; D000613:Aminobutyrates; D008024:Ligands; D011978:Receptors, Nicotinic; C083213:4-nitro-2-aminobutyric acid; D001224:Aspartic Acid; C006547:2-amino-4-ketopentanoate; D014364:Tryptophan