Two mosquito odorant receptors with reciprocal specificity mediated by a single amino acid residue

Abstract

Insects perceive the environment with a repertoire of promiscuous and narrowly tuned odorant receptors (ORs). The southern house mosquito, Culex quinquefasciatus, utilizes two ORs, CquiOR10 and CquiOR2, which are narrowly tuned and well conserved among mosquito species. They detect the oviposition attractants skatole and indole, respectively, with reciprocal specificity. We envisioned that by reverting specificity with chimeric receptors, we could gain a better insight into the specificity determinants of insect ORs. We swapped the seven transmembrane (TM) domains of CquiOR10 and CquiOR2 and identified TM2 as a specificity determinant. With additional mutations, we showed that CquiOR10A73L behaved like CquiOR2. Conversely, CquiOR2L74A recapitulated CquiOR10 specificity. Next, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold and AlphaFold and docked skatole and indole using RosettaLigand. These modeling studies suggested space-filling constraints around A73, although this residue in CquiOR10 did not directly form contacts with skatole or indole. Consistent with this hypothesis, CquiOR10 mutants with a bulkier residue (Ile, Val) were insensitive to skatole and indole, whereas CquiOR10A73G retained the specificity to skatole and showed a higher sensitivity than the wild-type receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10 was insensitive to 3-ethylindole, whereas CquiOR10A73G responded to the three ligands with 3-ethylindole eliciting a slightly higher response than indole. We then concluded that the specificity of these receptors is mediated by a single amino acid substitution leading to dilation or contraction of the binding pocket.