Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors

Abstract

The southern house mosquito, Culex quinquefasciatus, utilizes two odorant receptors, CquiOR10 and CquiOR2, narrowly tuned to oviposition attractants and well conserved among mosquito species. They detect skatole and indole, respectively, with reciprocal specificity. We swapped the 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. 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 more robust response than the wildtype receptor CquiOR10. On the other hand, Leu to Gly mutation of the indole receptor CquiOR2 reverted the specificity to skatole. Lastly, CquiOR10A73L, CquiOR2, and CquiOR2L74I were insensitive to 3-ethylindole, whereas CquiOR2L74A and CquiOR2L74G gained activity. Additionally, CquiOR10A73G gave more robust responses to 3-ethylindole than CquiOR10. Thus, we suggest the specificity of these receptors is mediated by a single amino acid substitution, leading to finely tuned volumetric space to accommodate specific oviposition attractants.

A tale of two copies: Evolutionary trajectories of moth pheromone receptors

“…What remains puzzling is that, despite the short evolutionary time, we showed here that multiple amino acid substitutions seem to be necessary for the shift of ligand specificity between the ancestors and the actual OR5 receptors. This is an original finding since previous studies identified that only one or two amino acid substitutions are sufficient for a shift of function in other ecologically important receptors ( 13 – 15 , 37 – 41 ). Our study clearly demonstrated that in the case of OR5, one amino acid substitution does not have a drastic impact on ligand specificity.…”

A potential link between aromatics‐induced oviposition repellency behaviors and specific odorant receptor of Aedes albopictus

“…We speculated that this discrepancy might be related to differences in the intracellular epitope tags of two heterogeneous expression systems, which may influence the selectivity of the receptor, whereas several studies indicated that the in vivo transgenic Drosophila system is generally more accurate 19,27,45 . In addition, we found that the odorant response profiles of Or10s in different mosquito species did not correspond exactly to homologous receptors in identical heterogeneous expression system 17,27,46,47 . In the Drosophila melanogaster ‘empty neuron’ system, 3‐methylinole and a further six aromatics consistently activated AgamOr10 and AalbOr10, whereas two phenolics, indole and phenol, and two heterocyclics, 2‐acetylthiophene and 4‐methylthiazole, could activate Agam Or10 but not Aalb Or10 27 .…”

“…19,27,45 In addition, we found that the odorant response profiles of Or10s in different mosquito species did not correspond exactly to homologous receptors in identical heterogeneous expression system. 17,27,46,47 In the Drosophila melanogaster 'empty neuron' system, 3-methylinole and a further six aromatics consistently activated AgamOr10 and AalbOr10, whereas two phenolics, indole and phenol, and two heterocyclics, 2-acetylthiophene and 4-methylthiazole, could activate AgamOr10 but not Aal-bOr10. 27 In addition, 3-methylindole strongly activated both CquiOr10 and AgamOr10 expressed in the Xenopus oocytes system, whereas 4-methylphenol and 4-ethylphenol activated only AgamOr10 and not CquiOr10.…”

“…24 Because of the difficulty in obtaining the cryo-electron microscopy structure of insect odorant receptors, homology modeling and molecular docking have been used to predict the interaction between Ors and ligands. Recently, studies have shown that the substitution of key residues predicted by molecular docking resulted in reduced or no odorant detection, 11,46,[60][61][62] suggesting that structural modeling and molecular docking are reliable in decoding the Or-ligand interaction. Based on a reverse chemical ecology approach, assessing the binding affinity of Ors for natural compounds conducted via multiple docking approaches could be a new method to select efficient repellents or attractants.…”

Structural basis of odor sensing by insect heteromeric odorant receptors

“…The cryo-EM structure of an OR-Orco complex has a 1:3 subunit stoichiometry We first focused on the conserved OR10 receptor family, which detects indole and derivatives in various mosquito species (25)(26)(27). Coexpression of Ae.…”