Characterisation of the Insect Odorant Receptor Complex

Abstract

Insect odorant receptors (ORs) are members of a novel family of seven-transmembrane proteins that are thought to form ligand-gated non-selective cation channels. One highly conserved insect OR, Orco, is thought to form the active channel in vivo, with odorant specificity conferred by a panel of ligand-binding OR subunits (OrX). However, little is known about the structural nature of the Orco/OrX complex. A protein biochemistry approach was applied to directly investigate the oligomeric structure of these receptors without the need for downstream indicators such as changes in fluorescence or electrical current. As a first step to address these questions, a protocol was developed to overexpress, and purify the Drosophila melanogaster (Dm) Orco and six ligandbinding subunits (OrX). Escherichia coli cell-based and cell-free expression systems were not capable of producing these subunits. In contrast, three eukaryotic systems (wheat germ cellfree, baculovirus-mediated Sf9 expression, and HEK-293 cells) expressed each of the subunits, with baculovirus-mediated expression producing the most at 4-6 mg of protein per litre of Sf9 cells. The receptors were tested for solubility in a panel of 19 detergents, of which Zwittergent 3- 16 was the only non-denaturing detergent that could solubilise all OR subunits produced in each expression system. The subunits were purified to homogeneity using Ni-NTA and size exclusion chromatography (SEC) with this detergent. Circular dichroism (CD) analyses demonstrated that the purified subunits were highly structured, having a predominantly - helical secondary structure. The purified subunits were reconstituted into preformed liposomes using a wheat germ cell-free expression system, and receptors purified from Sf9 cells. Each of the OR subunits was analysed in a range of detergents, and the presence of higher order structures was determined by Native PAGE. Depending on the OR subunit, monomers, dimers, trimers, tetramers, hexamers, and potentially hexadecamers (16-mers), could be detected. Some oligomers could also be detected by denaturing SDS-PAGE analysis. In contrast, size exclusion chromatography - multi angle light scattering (SEC-MALS) analysis of the molecular weights of the subunits demonstrated that they were predominantly monomeric with a small proportion forming dimers. Preliminary surface plasmon resonance (SPR) experiments indicated a potential heteromeric interaction between DmOr22a and DmOrco. Furthermore the DmOrco and DmOr10a subunits were shown to respond to their respective ligands in HEK 293 cells using a calcium sensitive dye assay indicating the potential for these subunits to interact in heterologous cells. However, pull-down assays using His10-tagged subunits and Ni-NTA resin did not provide evidence of an interaction between the subunits in any of the expression systems. Similarly incubating the OR subunits with cross-linking reagents did not produce an observable interaction by SDS-PAGE. Single molecule fluorescence photobleaching experiments did not provide any information on the oligomeric structure of these receptor subunits. However, this is likely due to the limitations of the available microscopy equipment. Finally, each of the six subunits was screened against a panel of 192-480 different crystallisation conditions. This resulted in crystal formation for the DmOrco subunit; however these crystals have not yet been tested for diffraction. This study is the first to describe the purification of insect ORs and their analysis using a purely biochemical approach. The results obtained provide initial steps towards understanding the structure of the OR subunits and the complexes they from. This research also paves the way for the application of insect ORs in receptor-based biosensors and the generation of novel pest management strategies.