title: Role of an Absolutely Conserved Tryptophan Pair in the Extracellular Domain of Cys-Loop Receptors.
abstract: Cys-loop receptors mediate fast synaptic transmission in the nervous system, and their dysfunction is associated with a number of diseases. While some sequence variability is essential to ensure specific recognition of a chemically diverse set of ligands, other parts of the underlying amino acid sequences show a high degree of conservation, possibly to preserve the overall structural fold across the protein family. In this study, we focus on the only two absolutely conserved residues across the Cys-loop receptor family, two Trp side chains in the WXD motif of Loop D and in the WXPD motif of Loop A. Using a combination of conventional mutagenesis, unnatural amino acid incorporation, immunohistochemistry and MD simulations, we demonstrate the crucial contributions of these two Trp residues to receptor expression and function in two prototypical Cys-loop receptors, the anion-selective GlyR α1 and the cation-selective nAChR α7. Specifically, our results rule out possible electrostatic contributions of these Trp side chains and instead suggest that the overall size and shape of this aromatic pair is required in stabilizing the Cys-loop receptor extracellular domain.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , Copenhagen 2100, Denmark.
authors.forename: Nina
authors.initials: N
authors.lastname: Braun
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , Copenhagen 2100, Denmark.
authors.forename: Timothy
authors.initials: T
authors.lastname: Lynagh
authors.affiliation: Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Rilei
authors.initials: R
authors.lastname: Yu
authors.affiliation: Department of Biochemistry, University of Oxford , South Parks Road, Oxford OX1 3QU, United Kingdom.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Biggin
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen , Copenhagen 2100, Denmark.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Pless
mesh_terms: D000818:Animals; D006801:Humans; D007150:Immunohistochemistry; D008958:Models, Molecular; D018408:Patch-Clamp Techniques; D000072417:Protein Domains; D018009:Receptors, Glycine / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry*; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor / Q000737:chemistry*
chemical_list: D018009:Receptors, Glycine; D064569:alpha7 Nicotinic Acetylcholine Receptor; D014364:Tryptophan
keywords: Cys-loop receptors; glycine receptor; nicotinic acetylcholine receptor; structure−function; tryptophan cluster; unnatural amino acids