Abstract
Insect olfactory receptors are routinely expressed in heterologous systems for functional characterisation. It was recently discovered that the essential olfactory receptor co-receptor (Orco) of the Hessian fly, Mayetiola destructor (Mdes), does not respond to the agonist VUAA1, which activates Orco in all other insects analysed to date. Here, using a mutagenesis-based approach we identified three residues in MdesOrco, located in different transmembrane helices as supported by 3D modelling, that confer sensitivity to VUAA1. Reciprocal mutations in Drosophila melanogaster (Dmel) and the noctuid moth Agrotis segetum (Aseg) Orcos diminish sensitivity of these proteins to VUAA1. Additionally, mutating these residues in DmelOrco and AsegOrco compromised odourant receptor (OR) dependent ligand-induced Orco activation. In contrast, both wild-type and VUAA1-sensitive MdesOrco were capable of forming functional receptor complexes when coupled to ORs from all three species, suggesting unique complex properties in M. destructor, and that not all olfactory receptor complexes are “created” equal.
Highlights
The sense of smell in insects has evolved to detect and discriminate between a vast number of odourants as a prerequisite to find suitable food, mates and oviposition sites, and to avoid dangerous situations and non-preferred habitats
Our findings suggest that in the lepidopteran, A. segetum, and the dipteran, D. melanogaster, mutation of three olfactory receptor co-receptor (Orco) residues to the WT sequence of MdesOrco causes structural changes that significantly diminish the ability of AsegOrco and DmelOrco to form functional receptor complexes with ligand-binding odourant receptor (OR)
In contrast to co-expression of AsegOR7 and DmelOR56a with DmelOrco and AsegOrco, respectively, MdesOR115 did not respond well to its ligand when coupled to AsegOrco or DmelOrco, indicating that this receptor does not efficiently form a functional Orco-OR complex with these Orcos, yet both forms of MdesOrco were capable of forming functional channels with AsegOR7 and DmelOR56a
Summary
The sense of smell in insects has evolved to detect and discriminate between a vast number of odourants as a prerequisite to find suitable food, mates and oviposition sites, and to avoid dangerous situations and non-preferred habitats. In light of these limitations, and as a rapidly increasing number of OR sequences have become available, a modelling technique that relies on amino acid covariation within proteins has been developed[33,34] This method is based on large multiple sequence alignments (MSA), in the case of Orcos and ORs, using sequences from a wide range of insect species, and allows the calculation of proximity restraints of amino acid pairs that appear to co-evolve, which guides the subsequent folding of the polypeptide to produce a more accurate theoretical structure. In efforts to identify functionally important regions of the proteins, mutational analyses have identified residues that affect OR sensitivity[39] and selectivity to ligands[40,41], as well as Orco channel activity and ion selectivity[42,43,44]
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