Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors
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This manuscript addresses the mechanism of ligand specificity of odorant receptors (OR) through mutational analyses and structure prediction. The authors identify a single amino acid substitution that switches ligand specificity between two olfactory receptors. Obtaining structures of OR complexes has been challenging, so such an approach is valuable and will be of interest to scientists within the fields of chemical ecology and sensory neuroscience.
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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.
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eLife assessment
This manuscript addresses the mechanism of ligand specificity of odorant receptors (OR) through mutational analyses and structure prediction. The authors identify a single amino acid substitution that switches ligand specificity between two olfactory receptors. Obtaining structures of OR complexes has been challenging, so such an approach is valuable and will be of interest to scientists within the fields of chemical ecology and sensory neuroscience.
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Reviewer #1 (Public Review):
This manuscript uses two OR molecues as a model to understand the mechanism behind their ligand specificity. It combines a series of targeted mutations and domain swapping followed by functional analysis in Xenopus oocyte expression system to analyse functional aspects of the modified ORs. It also models the various OR structures. The authors find that a single amino acid residue is critical for ligand specificity and that this is mediated by space constraints generated in the ligand docking region. The manuscript is generally well written and the data are clear and well represented.
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Reviewer #2 (Public Review):
The antennae of insects are excellent sensors and are able to distinguish chemicals/compounds using odorant receptor proteins. Though many are promiscuous, several ORs are extremely specific and respond to only one or few related chemicals. In this study, the authors focus on two ORs from southern house mosquito, Culex quinquefasciatus namely OR10 and OR2, which respond to (show high specificity) skatole and indole respectively. Notably, these two compounds differ only by a methyl group raising the question how this is achieved. To address this question, the authors have chosen CquiOR10 (as it is more sensitive) for swapping the transmembrane domains (TMDs) from CquiOR2 and by performing heroic work, arrive at one single residue in one of the TMDs to explain the specificity in these ORs.
The major strengths …
Reviewer #2 (Public Review):
The antennae of insects are excellent sensors and are able to distinguish chemicals/compounds using odorant receptor proteins. Though many are promiscuous, several ORs are extremely specific and respond to only one or few related chemicals. In this study, the authors focus on two ORs from southern house mosquito, Culex quinquefasciatus namely OR10 and OR2, which respond to (show high specificity) skatole and indole respectively. Notably, these two compounds differ only by a methyl group raising the question how this is achieved. To address this question, the authors have chosen CquiOR10 (as it is more sensitive) for swapping the transmembrane domains (TMDs) from CquiOR2 and by performing heroic work, arrive at one single residue in one of the TMDs to explain the specificity in these ORs.
The major strengths of the manuscript include the careful design of the many different chimeric receptors (36 in total) and dissecting the importance of each TMD and zeroing on TMD2 as the specificity determinant, followed by zooming to a single residue in TMD2 that can change responsiveness of CquiOR10 to CquiOR2 and vice versa. This residue in TM2 is an alanine in CquiOR10, which when mutated to bulky residue becomes responsive to indole but when mutated to glycine remains specific to skatole and shows higher sensitivity. Similarly, mutating the equivalent residue in CquiOR2, Leucine 74 to a smaller residue makes this receptor now more responsive to skatole and making it more like CquiOR10.
Using RoseTTAfold and AlphaFold, the authors build models of CquiOR10 and CquiOR2, which gives them a platform to observe how ligands can bind using Rosettaligand both in native structures as well as mutants. They further ask how larger ligands or the methyl group at different position in the indole ring affects the response of the receptor, which follow a consistent trend on the key residue of Alanine 73. All these analysis allow authors to propose that the odorants or chemicals are accommodated/restricted due to the volume constraints by residues lining the cavity derived from the TMDs.
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Reviewer #3 (Public Review):
Franco et al. consider two mosquito olfactory receptors that have different sensitivities to two odorants: CquiOr10 is activated by skatole while CquiOr2 is activated by indole. Starting with chimeric receptors composed of pieces from each receptor, they are ultimately able to identify a single amino acid that, when mutated, switches the specificity of the receptors. When Ala73 is mutated to a Leu in CquiOr10, the mutant receptor now preferentially binds indole, while the counterpart Leu74 to Ala substitution in CquiOr2 creates a receptor that is more sensitive to skatole. To better understand why these substitutions alter ligand-binding specificities, the authors use molecular docking to identify the likely interactions between indole or skatole and the natural or mutant CquiOr10 receptors. They conclude …
Reviewer #3 (Public Review):
Franco et al. consider two mosquito olfactory receptors that have different sensitivities to two odorants: CquiOr10 is activated by skatole while CquiOr2 is activated by indole. Starting with chimeric receptors composed of pieces from each receptor, they are ultimately able to identify a single amino acid that, when mutated, switches the specificity of the receptors. When Ala73 is mutated to a Leu in CquiOr10, the mutant receptor now preferentially binds indole, while the counterpart Leu74 to Ala substitution in CquiOr2 creates a receptor that is more sensitive to skatole. To better understand why these substitutions alter ligand-binding specificities, the authors use molecular docking to identify the likely interactions between indole or skatole and the natural or mutant CquiOr10 receptors. They conclude that the size of the amino acid at position 73 affects ligand specificity by altering the amount of space available to bind ligands.
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