The Role of a Tryptophan Cluster in the Extracelluar Domain of Cys-Loop Receptors

In order to accommodate a wide variety of chemically diverse ligands, Cys-loop receptors display significant sequence variability in their extracellular domain (ECD). By contrast, and as predicted by the high structural homology, other parts of the underlying amino acid sequences show a higher degree of conservation in order to preserve a similar structural fold across the protein family. Here, we set out to determine some of the key structural features for this common structural theme. To this end, we focused on the only two absolutely conserved side chains in the ECD of Cys-loop receptors, Trp side chains in the loop D Trp-X-Asp and the Loop A Trp-X-Pro motif. Focusing on the glycine receptor α1 (GlyR α1) and the nicotinic acetylcholine receptor α7 (nAChR α7), we employed unnatural amino acids, immunohistochemistry and molecular dynamics simulations to study the two Trp side chains. Although we could rule out cation-pi interactions with positively charged moieties, our findings indicate that in the GlyR α1 and the nAChR α7, an aromatic side chain is absolutely required for channel function in both the Trp-X-Asp motif in loop D and the Trp-X-Pro motif in loop A. While the indole nitrogen of the Trp in the Trp-X-Asp motif in loop D of GlyR α1 may contribute to a hydrogen bond, we propose the main function of the two side chains to be to serve as the core of a hydrophobic cluster that fulfills a crucial structural role in the ECD across all Cys-loop receptors.