Arthropod repellent interactions with olfactory receptors and ionotropic receptors analyzed by molecular modeling

The main insect chemoreceptors are olfactory receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs). The odorant binding sites of many insect ORs appear to be occluded and inaccessible from the surface of the receptor protein, based on the three-dimensional structure of OR5 from the jumping bristletail Machilis hrabei (MhraOR5) and a survey of a sample of vinegar fly (Drosophila melanogaster) OR structures obtained from artificial intellegence (A.I.) modeling. Molecular dynamics simulations revealed that the occluded site can become accessible through tunnels that transiently open and close. The present study extends this analysis to examine seventeen ORs and one GR docking with ligands that have known valence: nine that signal attraction and nine that signal aversion. All but one of the receptors displayed occluded ligand binding sites analogous to MhraOR5, and docking software predicted the known attractant and repellent ligands will bind to the occluded sites. Docking of the repellent DEET was examined, and more than half of the OR ligand sites were predicted to bind DEET, including receptors that signal aversion as well as those that signal attraction. However, DEET may not actually have access to all the attractant binding sites. The larger size and lower flexibility of repellent molecules may restrict their passage through the tunnel bottlenecks, which could act as filters to select access to the ligand binding sites. In contrast to ORs and GRs, the IR ligand binding site is in an extracellular domain known to undergo a large conformational change from an open to a closed state. A.I. models of two D. melanogaster IRs of known valence and two blacklegged tick (Ixodes scapularis) IRs having unknown ligands were computationally tested for attractant and repellent binding. The ligand-binding sites in the closed state appear inaccessible to the protein surface, so attractants and repellents must bind initially at an accessible site in the open state before triggering the conformational change. In some IRs, repellent binding sites were identified at exterior sites adjacent to the ligand-binding site. These may be allosteric sites that, when occupied by repellents, can stabilize the open state of an attractant IR, or stabilize the closed state of an IR in the absence of its activating ligand. The model of D. melanogaster IR64a suggests a possible molecular mechanism for the activation of this IR by H⁺. The amino acids involved in this proposed mechanism are conserved in IR64a from several Dipteran pest species and disease vectors, potentially offering a route to discovery of new repellents that act via the allosteric site.