Structural basis for chemosensation by insect olfactory ionotropic receptors

Project Abstract/Summary Insects are guided by their chemosensory systems of smell and tase. A multitude of behaviors, such as foraging, mating, navigation, and oviposition rely on smell and taste. For disease vectors such as mosquitoes, their chemosensory systems guide host-seeking (attraction to humans) and biting. As such, the chemosensory systems of insects are excellent targets for behavioral control, and strategies that target insect smell and taste could have significant and widespread benefits from reducing mosquito biting to preventing crop destruction by invasive pests. The chemosensory systems of insects rely on the expression of 3 different receptor family members: the Odorant Receptors (ORs), the chemosensory Ionotropic Receptors (IRs) and the Gustatory Receptors (GRs). Of these three, IRs represent one of the most ancient and abundant chemosensory receptors on the planet. IRs likely diverged ~600 million years ago from an ancestral ionotropic glutamate receptor (iGluRs) to take on new roles as a multi-functional chemosensory receptor family. Chemosensory insect IRs are a complex between an IR co-receptor and a ‘tuning’ IR that binds to a chemical ligand. The IR co-receptors Ir8a and Ir25a share protein homology to iGluRs which contain a large amino- terminal domain. In contrast, the tuning IRs lack this domain. The structure of mammalian iGluR complexes has been solved by the Twomey group using cryo-EM. The structure of chemosensory IRs remains unknown. A key limitation has been harvesting functional chemosensory IR-complexes for cryo-EM analyses. The expression of IR complexes in cell culture systems does not lead to functional complexes, likely reflecting defects in IR complex formation or cellular trafficking. To address these limitations, we assembled a team of insect sensory biologists and structural biologists. Using transgenic Drosophila that express an N-terminal tagged functional Ir8a co- receptor (EGFP:Ir8a), we will: Specific Aim 1, develop methods to harvest functional Ir8a receptor complexes directly from ~10,000,000 Drosophila antenna, and Specific Aim 2, purify EGFP:Ir8a complexes from Drosophila tissues using nanobodies to EGFP followed by cryo-EM analyses on the complexes as previously done for iGluR complexes. These experiments have the potential to establish Drosophila as a viable in vivo tissue source for chemosensory receptor purification. This work will also reveal the cryo-EM structure of chemosensory IRs, their stoichiometry, and provide clues as to how this receptor complex evolved from iGluRs to be gated by a variety of chemicals. This could lead to innovative new strategies to target these receptor complexes to control insect behaviors.