The ligand binding site of
The ligand-binding site of cys-loop receptors is located at the interface of two adjacent subunits, which are loops A-C on the principal subunit and loops D-G on the complementary subunit (Hibbs and Gouaux, 2011). Each receptor has a different ligand-binding domain that contain various residues, which allow for the selection of different molecular agonists. There are key aromatic residues in the binding site of LGICs that have shown to be involved in ligand binding (Beene et al., 2004). Many neurotransmitters contain a cationic center that acts to stabilize the interaction between a cation and the negative electrostatic potential on the face of the aromatic ring. This is known as cation-π interactions (Beene et al., 2002). These cation- π interactions involve aromatic amino acids (either phenylalanine, tyrosine, or tryptophan) in the ligand binding region (Dougherty, 1996). While the importance of aromatic residues for the function of the agonist binding pocket is widely shared across the phyla, there is variability across different receptors with respect the type of aromatic residues that contribute to binding and their location within the binding loops (Lynagh and Pless, 2014). This highlights the promiscuous nature of neurotransmitter binding across a variety of receptors in animals.
Early studies from the mollusk, Aplysia, reported the presence of a unique class of sik inhibitor receptors in neurons, the acetylcholine-gated chloride channels (ACCs) (Kehoe and McIntosh, 1998). Later, studies identified these receptors in the model nematode Caenorhabditis elegans (Putrenko et al., 2005; Wever et al., 2015). In C. elegans the ACC-1 family is made up of eight receptor subunit genes, acc-1, -2, -3, and -4, and lgc-46, -47, -48, and -49 (Jones and Sattelle, 2008). The sheep parasite, Haemonchus contortus, contains homologues for seven of the ACC-1 gene members, with the absence of lgc-48 (Laing et al., 2013). As a whole, this family of receptors has potential to be novel antiparasitic drug targets. This is primarily due to the fact that they appear to be expressed in tissues that are sensitive to anthelmintic action, and as sequence analysis suggests, they are not present in mammals and exhibit a unique acetylcholine binding site (Putrenko et al., 2005; Wever et al., 2015). However, the structural components that are important for agonist recognition of this class of cholinergic receptors has not been explored.
Here we have isolated a novel member if the ACC-1 family (Hco-ACC-2) from the parasitic nematode H. contortus and investigated the binding site through site-directed mutagenesis and pharmacological analysis. Several introduced point mutations that changed key aromatic residues at the binding site revealed some interesting pharmacological properties. Molecular modelling was used to visualize the structure of the binding pocket and the interaction of key residues with a variety of agonists.