Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity

Olfactory receptor repertoires exhibit remarkable functional diversity, but how these proteins have evolved is poorly understood. Through analysis of extant and ancestrally-reconstructed drosophilid olfactory receptors from the Ionotropic Receptor (IR) family, we investigated evolution of two organic acid-sensing receptors, IR75a and IR75b. Despite their low amino acid identity, we identify a common “hotspot” in their ligand-binding pocket that has a major effect on changing the specificity of both IRs, as well as at least two distinct functional transitions in IR75a during evolution. Ligand-docking into IR models predicts that the hotspot does not contact odor molecules, suggesting that this residue indirectly influences ligand/receptor interactions. Moreover, we show that odor specificity is refined by changes in additional, receptor-specific sites, including those outside the ligand-binding pocket. Our work reveals how a core, common determinant of ligand-tuning acts within epistatic and allosteric networks of substitutions to lead to functional evolution of olfactory receptors.