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• W4313290141 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Appendix 4 Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This article addresses the mechanism of ligand specificity of odorant receptors (OR) through mutational analyses and structure prediction. Through solid data, 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. https://doi.org/10.7554/eLife.82922.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Insects perceive the world with a sophisticated olfactory system essential for survival and reproduction. Their antennae are biosensors par excellence, allowing detection of a plethora of compounds, some with extraordinary sensitivity and selectivity (Kaissling, 2014). The insect olfactory system is comprised mainly of odorant-binding proteins, odorant-degrading enzymes, ionotropic receptors, and the ultimate gatekeepers of selectivity (Leal, 2013; Leal, 2016; Leal, 2020) – the odorant receptors (ORs) (Clyne et al., 1999; Gao and Chess, 1999; Vosshall et al., 1999). The ORs are the binding units in functional heteromeric cation channels (Neuhaus et al., 2005) formed with an odorant receptor coreceptor (Orco) (Larsson et al., 2004). Unlike mammalian olfactory receptors, insect ORs and Orco have inverse topologies compared to G-protein-coupled receptors (GPCRs), with a cytosolic N-terminus and an extracellular C-terminus (Benton et al., 2006; Lundin et al., 2007). One of the major breakthroughs in the fields of insect olfaction in the last two decades since the discovery of ORs (Leal, 2020) was the determination of the cryo-electron microscopy (cryo-EM) structure of the Orco homomer from the parasitic fig wasp, Apocrypta bakeri, AbakOrco (Butterwick et al., 2018). Subsequently, the structure for a promiscuous OR from the evolutionarily primitive (Apterygota, wingless) jumping bristletail, Machilis hrabei, MhraOR5, was solved (DelMarmol et al., 2021). Although structures of ORs from winged insects (Pterygota) have not been solved to date, amino acid residues critical for OR specificity have been reported (Auer et al., 2020; Cao et al., 2021; Hughes et al., 2014; Leary et al., 2012; Pellegrino et al., 2011; Yang et al., 2017; Yuvaraj et al., 2021). These studies focused on one-way alteration of specificity but did not examine how an insect detects two odorants with reverse specificity. Mosquitoes are vectors of pathogens that cause tremendous harm to public health. Male and female mosquitoes visit plants to obtain nutrients for flight. For reproduction and survival of the species, females must acquire a blood meal to fertilize their eggs and, subsequently, oviposit in an aquatic environment suitable for the offspring to flourish. While feeding on hosts, females transmit viruses and other pathogens. The southern house mosquito, Culex quinquefasciatus, transmits pathogens causing filariasis and various encephalitis (Nasci and Miller, 1996). In the United States, mosquitoes belonging to the Culex pipiens complex transmit the West Nile virus (Andreadis, 2012). Due to its opportunistic feeding on avian and mammalian hosts, Cx. quinquefasciatus is a significant bridge vector in urbanized centers in the Western United States, particularly southern California (Andreadis, 2012; Syed and Leal, 2009). Female mosquitoes rely on multiple sensory modalities, including olfaction, to find plants, vertebrate hosts, and suitable environments for oviposition. The genome of the southern house mosquito, Cx. quinquefasciatus (Arensburger et al., 2010), has the most extensive repertoire of OR genes (Leal et al., 2013) among mosquito species. The genomes of Anopheles darlingi, the malaria mosquito, Anopheles gambiae, the yellow fever mosquito, Aedes aegypti, the Asian tiger mosquito, Aedes albopictus, and the southern house mosquito contain 18 (Marinotti et al., 2013), 79 (Hill et al., 2002), 117–131 (Bohbot et al., 2007; Nene et al., 2007), 158 (Chen et al., 2015), and 180 (Arensburger et al., 2010) OR genes, respectively. Of those, 61 transcripts were found in An. gambiae (Pitts et al., 2011), 107 in Ae. aegypti (Matthews et al., 2018), and 177 in Cx. quinquefasciatus (Leal et al., 2013). Given that the number of odorants in the environment is larger than the number of OR genes even in Cx. quinquefasciatus, it is not surprising that many ORs from insects are promiscuous (Leal, 2013; Pask, 2020). However, ORs detecting behaviorally critical compounds (semiochemicals) may be narrowly tuned (Hughes et al., 2010; Nakagawa et al., 2005; Stensmyr et al., 2012). ORs narrowly tuned to 3-methylindole (=skatole) and indole have been found in mosquito species in the subfamilies Culicinae and Anophelinae. Specifically, OR10 and OR2 have been de-orphanized in the southern house mosquito (Hughes et al., 2010; Pelletier et al., 2010), the yellow fever mosquito (Bohbot et al., 2011), and the malaria mosquito (Carey et al., 2010; Wang et al., 2010). Recently, these so-called indolergic receptors Bohbot and Pitts, 2015 have also been found in the housefly, Musca domestica (Pitts et al., 2021). Skatole and indole are fecal products that have been identified as oviposition attractants for the southern house mosquito (Blackwell et al., 1993; Mboera et al., 2000; Millar et al., 1992). Skatole is a potent oviposition attractant in minute doses, whereas indole is active only at high doses (Millar et al., 1992). To the human nose, skatole has a pungent fecal odor. In contrast, indole has an almost floral odor when highly purified and presented at low doses (Fenaroli, 1975), but a fecal odor at high doses. Remarkably, in all mosquito species and the housefly, OR10s are narrowly tuned to skatole and respond with lower sensitivity to indole, whereas indole is the most potent ligand for OR2s, which give lower responses to skatole. In Cx. quinquefasciatus, these receptor genes were initially named CquiOR10 and CquiOR2, respectively, renamed CquiOR21 and CquiOR121, but a proposition to restore the original names is under consideration (Carolyn McBride, personal communication). Here, we refer to these receptors from Cx. quinquefasciatus as CquiOR10 (VectorBase and GenBank IDs, CPIJ002479 and GU945397, respectively) and CquiOR2 (CPIJ014392, GU945396.1). These oviposition attractant-detecting (Chen and Luetje, 2014) receptors in Cx. quinquefasciatus, CquiOR10 and CquiOR2, provide a suitable model to identify specific determinants as they respond to skatole and indole with reverse specificity. CquiOR10 and CquiOR2 proteins have 377 and 375 amino acid residues, respectively, and share only 49.5% amino acid identity, whereas 61.9% of the amino acids in the predicted transmembrane domains are identical (Supplementary file 1, Table 1). To identify the specificity determinants of these receptors, we swapped transmembrane (TM) domains and tested the chimeric receptors using the Xenopus oocyte recording system. Given that CquiOR10 is the second most sensitive Cx. quinquefasciatus OR (second only to CquiOR36; Choo et al., 2018), we replaced the predicted (Reynolds et al., 2008) transmembrane domains from CquiOR2 into CquiOR10 and measured the specificity of the chimeric receptors. With this approach, we identified TM2 as a specificity determinant. Next, we tested mutations of chimeric CquiOR10 and identified a single amino acid residue in TM2 (Ala-73) that determines the receptor’s specificity. We then directly mutated the wildtype receptor and observed that CquiOR10A73L is specific to indole as CquiOR2. Additionally, CquiOR2L74A emulated the response profile of CquiOR10. To better understand the structural basis of this single-point specificity determinant, we generated structural models of CquiOR10 and CquiOR10A73L using RoseTTAFold (Baek et al., 2021) and AlphaFold (Jumper et al., 2021). We identified the binding poses for skatole and indole using RosettaLigand molecular docking and observed a finely tuned volumetric space to accommodate specific oviposition attractants. Results Chimeric OR with reversed specificity We envisioned that studying a pair of ORs with reverse specificities, like CquiOR10 and CquiOR2, could lead us to specificity determinants. CquiOR10 is activated by the oviposition attractant skatole (Blackwell et al., 1993; Mboera et al., 2000; Millar et al., 1992) with high specificity (Figure 1A), whereas CquiOR2 is specific to indole (Figure 1B). Figure 1 with 1 supplement see all Download asset Open asset Concentration–response analysis for activation of wildtype odorant receptors (ORs) by skatole and indole. (A) CquiOR10 and (B) CquiOR2. Lines were obtained with nonlinear fit. Bars represent SEM. n = 4–5. Figure 1—source data 1 Concentration–response analysis for activation of wildtype odorant receptors (ORs) by skatole and indole. https://cdn.elifesciences.org/articles/82922/elife-82922-fig1-data1-v2.xlsx Download elife-82922-fig1-data1-v2.xlsx Our approach was designed to swap TM domains using the more sensitive receptor, CquiOR10, as the acceptor. Specifically, we generated chimeric receptors by replacing CquiOR10 TM domains with related domains from CquiOR2 (Figure 2A). During the life of this project, the cryo-EM structure of an odorant receptor coreceptor AbakOrco from the parasitic fig wasp, Aprocrypta bakeri, was reported (Butterwick et al., 2018). We then compared the experimental structure (Butterwick et al., 2018) with the predicted topology for AbakOrco using the same OCTOPUS method (Viklund and Elofsson, 2008) we used to identify CquiOR10 and CquiOR2 TMs (Figure 2A; Viklund and Elofsson, 2008). The almost perfect overlap between OCTOPUS prediction and the AbakOrco structure (Figure 2B) validated not only our TM predictions (Figure 2A), but also the 21 chimeric ORs already tested when the structure of the coreceptor AbakOrco (Butterwick et al., 2018) was reported. Figure 2 Download asset Open asset Alignment of the amino acid sequences of CquiOR10 and CquiOR2 highlighting the predicted transmembrane (TM) domains and a comparison of predicted and experimentally determined TM domains of the odorant receptor coreceptor, AbakOrco. (A) CqOR10 and CqOR2 are abbreviations for CquiOR10 and CquiOR2, respectively. The TM domains, predicted by OCTOPUS, are displayed in red and blue for CquiOR10 and CquiOR2, respectively. The sequences of the N-terminus and the intracellular loops are displayed in black, and the C-terminus and extracellular loops in green. (B) Left: the cryo-EM structure of AbakOrco (PDB, 6C70) displayed in rainbow color using UCSF Chimera (Pettersen et al., 2004). Right: the predicted TM domains (right) are displayed in gray. The dashed lines represent the membrane boundaries. We referred to these chimeric receptors as CquiOR10Mx, where Mx refers to TMx from CquiOR2. We performed functional assays of these CquiOR10Mx receptors using the Xenopus oocyte recording system. This long-term project could require as many as 127 possible chimeric receptors. We started by swapping all seven TM domains. We envisioned that this chimeric receptor would have a reverse specificity. If so, we would restore one TM at a time to identify critical domains. It turned out that CquiOR10M1,2,3,4,5,6,7 was silent (see Appendix 1, Supplementary file 1, Table 2). To minimize the number of tested mutants, we changed the strategy to start from single mutations to obtain educated guess for the subsequent design of mutants. With this approach, we generated and tested only 8 of the required 99 mutants with 7–3 TMs swapped. We tested 36 chimeric receptors (see Supplementary file 1, Table 2, and Figure 1—figure supplement 1). Fourteen chimeric receptors did not respond to skatole or indole, and 21 receptors retained the specificity to skatole (Supplementary file 1, Table 2, and Figure 1—figure supplement 1). Lastly, CquiOR10M2,7/CquiOrco-expressing oocytes responded to both skatole and indole with a reverse profile (Figure 3A). This dataset shows that CquiOR10M2,7 emulated the profile of the indole receptor CquiOR2 (Figure 1B). Figure 3 with 1 supplement see all Download asset Open asset Concentration–response curves obtained with chimeric odorant receptors (ORs) stimulated with skatole and indole. (A) CquiOR10M2,7; (B) CquiOR10M2,5,6,7; (C) CquiOR10M2,5,6,7_Outer; (D) CquiOR10M2,5,6,7_Mid;Inner; (E) CquiOR10M2,5,6,7_Inner; (F) CquiOR10M2,5,6,7T78I; (G) CquiOR10M2,5,6,7L73A; (H) CquiOR10M7A73L; (I) CquiOR10A73L; (J) CquiOR2L74A. Lines were obtained with nonlinear fit. Bars represent SEM. The number of replicates (n) were 7, 4, 5, 5, 4, 3, 9, 7, 6, and 5, respectively. Figure 3—source data 1 Concentration–response curves obtained with chimeric odorant receptors (ORs) stimulated with skatole and indole. https://cdn.elifesciences.org/articles/82922/elife-82922-fig3-data1-v2.xlsx Download elife-82922-fig3-data1-v2.xlsx A single-point mutation that reverses the specificity of the skatole and indole receptors We proceeded to identify the amino acid residues in the swapped domains of CquiOR10M2,7, directly affecting the specificity of the chimeric and wildtype receptors. Given the observation that, by and large, chimeric receptors with TM5 and TM6 from CquiOR2 gave stronger responses (Figure 1—figure supplement 1), we asked whether CquiOR10M2,7 responses with these two additional TM domains swapped would give more robust responses while keeping the same specificity to indole. CquiOR10M2,5,6,7/CquiOrco-expressing oocytes were indeed more sensitive while maintaining the selectivity to indole (Figure 3B). We then used CquiOR10M2,5,6,7 and designed various mutants to rescue single or multiple residues in TM2 at a time (Figure 4). We focused on TM2 because swapping TM7 did not affect the specificity of the receptor (Figure 1—figure supplement 1E). We divided TM2 into outer, middle, and inner segments based on the topology predicted by OCTOPUS (Viklund and Elofsson, 2008). Figure 4 Download asset Open asset Partial sequences of CquiOR10 and chimeric odorant receptors (ORs) highlighting transmembrane domain-2 (TM2). The two last residues of the extracellular loop-1 (Ile-57 and Asp-58) appear in the N-terminus. The TM2 was divided into the arbitrary segments outer, middle (mid), and inner to identify specificity determinants. It has been postulated that the extracellular halves of TM domains form an odorant-binding pocket (Guo and Kim, 2010); thus, we first examined a mutant (CquiOR10M2,5,6,7_Outer) having the residues in the outer segment at 59, 60, 63, and 64 restored to Glu, Val, Asn, and Ala, respectively, as in the wildtype receptor (Figure 4). CquiOR10M2,5,6,7_Outer/CquiOrco-expressing oocytes retained the specificity of CquiOR10M2,5,6,7 with a more robust response to indole than skatole (Figure 3C). These findings indicated that the residues in the outer segment of TM2 are not specificity determinants. After that, we tested a chimeric receptor with the residues in the middle and inner part of the TM2 domain rescued to match those in the wildtype receptor (Figure 4). CquiOR10M2,5,6,7_Mid;Inner restored the skatole-specific profile of CquiOR10 (Figure 3D), thus suggesting that amino acid residues in these segments are specificity determinants. Then, we tested CquiOR10M2,5,6,7_Inner (=CquiOR10M2,5,6,7L73A;T78I), which had only residues at 73 and 78 rescued to the wildtype Ala and Ile, respectively. Oocytes co-expressing CquiOR10M2,5,6,7_Inner and CquiOrco reverted the specificity to skatole (Figure 3E). As a result, we concluded that residues in the predicted inner part of TM2 are critical for the chimeric receptor’s specificity. We further probed the chimeric receptor CquiOR10M2,5,6,7 with single-point mutations to identify the residue(s) determining specificity. CquiOR10M2,5,6,7T78I/CquiOrco-expressing oocytes showed the same specificity as the chimeric receptor CquiOR10M2,5,6,7 (Figure 3F). Specifically, CquiOR10M2,5,6,7T78I gave a more robust response to indole than skatole, suggesting that rescuing the residue at 78 did not affect CquiOR10M2,5,6,7 specificity. By contrast, CquiOR10M2,5,6,7L73A/CquiOrco-expressing oocytes reverted the specificity to skatole (Figure 3G), thus behaving like the wildtype receptor CquiOR10 (Figure 1A). To further examine the role of Ala-73 as a specificity determinant residue, we obtained a single-point mutation of CquiOR10M7, which is specific to skatole (Figure 1—figure supplement 1E). The responses recorded from CquiOR10M7A73L/CquiOrco-expressing oocytes showed a reverse, indole-specific profile (Figure 3H), like the CquiOR2 profile (Figure 1B). Having identified a single amino acid residue in the chimeric receptor that switches the skatole/indole specificity, we tested the effect of single-point mutation on the specificity of the wildtype receptor CquiOR10 (EC50: skatole, 3.6 µM; indole 29.9 µM). CquiOR10A73L showed a reverse specificity, with dose-dependent responses to indole (Figure 3I) (EC50: indole, 3.4 µM; skatole 53.7 µM). Collectively, these findings suggest that a single amino acid residue in CquiOR10 determines the specificity of this receptor. Additionally, we obtained an equivalent single-point mutation in the indole-specific CquiOR2 (Figure 1B) (EC50: indole, 7.7 µM; skatole 16.4 µM). Thus, CquiOR2L74A/CquiOrco-expressing oocytes gave robust and specific responses to skatole (Figure 3J) (EC50: skatole, 8.5 µM; indole 27.6 µM). As summarized in a graphical representation (Figure 3—figure supplement 1), these findings demonstrate that these two mosquito odorant receptors, CquiOR10 and CquiOR2, have reciprocal specificity mediated by a single amino acid residue, Ala-73 and Leu-74, respectively. We also recorded the response of these ORs to other phenolic ligands that activate indolic receptors, albeit generating small currents. While CquiOR10 and CquiOR2 responded to phenol, 3,5-dimethylphenol activated only CquiOR10 (Figure 5A). A single-point mutation in CquiOR10 rendered the chimeric receptor insensitive to 3,5-dimethylphenol. By contrast, an equivalent mutation in CquiOR2 recapitulated the profile of CquiOR10 (Figure 5A). Figure 5 Download asset Open asset Quantification of wildtype and chimeric receptors to phenol and 2,3-dimethylphenol, and methylindoles. (A) Each receptor was co-expressed with CquiOrco in Xenopus oocytes and stimulated with the phenolic compounds at 1 mM. n = 3–5. (B) CquiOR10/CquiOrco-, (C) CquiOR2/CquiOrco-, and (D)-CquiOR2L74A-expressing oocytes were stimulated with 100 µM of the specified methylindoles. n = 9–11. Bars represent SEM. Figure 5—source data 1 Quantification of wildtype and chimeric receptors to phenol and 2,3-dimethylphenol, and methylindoles. https://cdn.elifesciences.org/articles/82922/elife-82922-fig5-data1-v2.xlsx Download elife-82922-fig5-data1-v2.xlsx Additionally, we recorded responses elicited by methylindoles. Specifically, we challenged oocytes with 1-methylindole, 2-methylindole, 4-methylindole, 5-methylindole, 6-methylindole, and 7-methylindole. In these analyses, we did not stimulate the oocyte preparations with 3-methylindole to avoid possible desensitization. CquiOR10/CquiOrco-expressing oocytes elicited stronger responses when challenged with 1-methylindole and 5-methylindole than when stimulated with the other methylindoles (Figure 5B). By contrast, CquiOR2/CquiOrco-expressing oocytes elicited similarly lower responses when stimulated with methylindoles (Figure 5C). CquiOR2L74A with a single-point mutation to mimic OR10 receptor recapitulated CquiOR10 response profile (Figure 5D). These data suggest that a single amino acid residue determines a receptor’s specificity toward ligands eliciting robust or small responses. CquiOR10 computational modeling suggests space-filling constraints for indole-based odorants around A73 To structurally hypothesize the above-described reciprocal specificity, we generated structural models of CquiOr10, CquiOR2, CquiOR10A73L, and CquiOR2L74A using RoseTTAFold (Baek et al., 2021) and a structural model of CquiOR10 using AlphaFold (Jumper et al., 2021; Figure 6). Figure 6 with 4 supplements see all Download asset Open asset AlphaFold and RoseTTAFold models. Structural models of CquiOR10 (A, B), CquiOR10A73L (C), CquiOR2 (D), and CquiOR2L74A (E) with AlphaFold (A) and RoseTTAFold (B–E) structure prediction methods. Superposition of all RoseTTAFold models (F) resulted in transmembrane helix root mean square deviation (RMSD) of 0.8 Å when aligned with RoseTTAFold CquiOR10. (G) The transmembrane helix RMSD of CquiOR10 RoseTTAFold (rainbow) vs. AlphaFold (gray) was 1.7 Å. Loops were not included in RMSD calculation due to inherent flexibility during structure prediction. CquiOR10 models of one homotetramer subunit were generated with AlphaFold and RoseTTAFold, each producing five models. RoseTTAFold models of CquiOR10, CquiOR10A73L, CquiOR2, and CquiOR2A73L produced a transmembrane helix root mean square deviation (RMSD) between α-Carbon atoms less than 1 Å across all 20 models (five models per odorant receptor); this suggests that the homologous CquiOR10 and CquiOR2 are structurally similar and that single-point mutations should not cause great structural deviation from wildtype. Comparing CquiOR10 models, RoseTTAFold and AlphaFold produced a transmembrane helix RMSD of 1.7 Å, with the transmembrane helices at the extracellular membrane face having the largest structural deviation. Considering there is little structural knowledge of insect odorant receptors, their binding mechanisms, and their conformational changes, we suspected that homologous odorant receptors would have similar binding modes. Further, pairwise sequence alignment suggests that a series of residues in MhraOR5 TM4 aligns with CquiOR10 TM2, which contains CquiOR10A73 (CquiOR10: 59EVI-INAYFAMIFFNAV74. MhraOR5: 199EVIAIYEAVAMIFLITA215.; Figure 6—figure supplement 1) while AlphaFold and RoseTTAFold models of CquiOR10 were broadly similar to MhraOR5 (Figure 6—figure supplement 2). Using transmembrane helix 7b (TM7b), we superimposed the top-ranking CquiOR10 RoseTTAFold and AlphaFold models with an experimentally resolved structure, M. hrabei (MhraOR5) in complex with eugenol (PDB ID: 7LID; DelMarmol et al., 2021) to identify which of our models resembled an odorant-bound conformation. With this selection criteria, we proceeded with RoseTTAFold models of the odorant receptors for Rosetta-based small-molecule docking method RosettaLigand (Davis and Baker, 2009; DeLuca et al., 2015) as the structural similarity around the hypothesized binding pocket was greater than the AlphaFold models of the odorant receptors compared with the MhraOR5 structure. We chose to select conformationally similar models over modeling and docking an apo structure into a bound conformation because it is a more cautious approach when there is little structural information. We perceived modeling an apo structure into a bound conformation to potentially yield more biologically implausible conformations than docking of a structurally comparative model. To verify that RosettaLigand could effectively sample odorants in receptors homologous to CquiOR10 and CquiOR2, we used the structure of eugenol in complex with the insect odorant receptor OR5 from MhraOR5 (PDB ID: 7LID) as a control (DelMarmol et al., 2021). Rosetta protein-ligand docking employs energy-based analyses, such as the interface energy between protein and ligand, to select the representative models (Bidula et al., 2022). With this selection method, the RMSD of our MhraOR5-eugenol models relative to the experimental structure ranged from 0.5 to 5.0 Å (Supplementary file 1, Table 3). RMSD values equal or below 2.0 Å are considered an appropriate range for validation (Park et al., 2021). After using hdbscan cluster analysis (McInnes et al., 2017) to group structurally similar models, the largest cluster had a RMSD of 0.75 Å while the lowest interface-energy model had a RMSD of 2.4 Å. Collectively, these data demonstrate that RosettaLigand paired with the hdbscan clustering method can recapitulate the MhraOR5 structure and blindly select a near-native model, thus is suitable for structural predictions of odorants with CquiOR10A73L and CquiOR2L74A (Figure 6—figure supplements 3 and 4). For each receptor–ligand complex (CquiOR10-skatole, CquiOR10A73L-skatole, CquiOR10-indole, and CquiOR10A73L-indole), we generated 100,000 docking models using RosettaLigand, clustered the 10,000 lowest interface-energy models, and selected the lowest interface-energy model from the 10 largest clusters, resulting in 10 models per receptor–ligand complex from which to draw structural hypotheses (Supplementary file 1, Tables 4 and 5). Our modeling suggests that both indole and skatole can readily reorient themselves in a similar pore depth near residue 73, regardless of mutant or wildtype receptor. Comparing the lowest interface-scoring model from each receptor–ligand complex, indole and skatole are positioned in the membrane-embedded pore, flanked by transmembrane helices S2, S4, S5, and S6, and show positional overlap in both and CquiOR10-A73L CquiOR10 (Figure 7, Figure 7—figure supplements 1–5). Figure 7 with 8 supplements see all Download asset Open asset Representative models of docked skatole and indole in complex with CquiOR10 and CquiOR10A73L using RosettaLigand. Each model shown is the lowest interface-energy model from the 10 largest clusters of each docking study. CquiOR10 – skatole (forest green), CquiOR10 – indole (brown), CquiOR10A73L – skatole (light blue), and CquiOR10A73L – indole (purple). Atoms that are not indole/skatole carbon atoms are color-coded by atom type: carbon (gray), nitrogen (dark blue), and oxygen (red). Ala-73 and Leu-73 indicated with space-filling representation. (A, B) and (C, D) Mebrane and extracellular views for CquiOR10 and CquiOR10A73L, respectively. In most models, skatole and indole form contacts with CquiOR10 and CquiOr10A73L in a similar plane about a center of rotation. These observations are supported by skatole and indole not containing rotatable bonds, thus relying on rigid translational movements and rotation to form favorable contacts with the rotatable and repackable receptor residues. Additionally, our models position indole and skatole within a series of nonpolar, polar-uncharged, and aromatic amino acids. Protein–ligand interaction profiler (PLIP) analysis (Adasme et al., 2021) suggests that the bulk of favorable interactions are nonpolar, occasional hydrogen bonding with the odorant NH group, and occasional parallel pi stacking, with the ligand-binding pocket formed by TMs 2, 4, 5, and 6 (Figure 7—figure supplement 6). Akin to eugenol forming hydrophobic contacts with MhraOR5/Ile-213 from TM4 (Figure 6—figure supplement 3), skatole and indole formed hydrophobic contacts with CquiOR10/Asn-72 from TM2 (Supplementary file 1, Table 8), which are matched pairs form Needleman–Wunsch pairwise alignment (Figure 6—figure supplement 1). We find of most importance skatole and indole not forming contacts with Ala-73 in CquiOR10 models (Supplementary file 1, Tables 6–14). By contrast, in the CquiOR17A73L models, skatole formed hydrophobic contacts with Leu-73 in 5 of the 10 representative models, while indole formed contacts with Leu-73 in 2 representative models (Supplementary file 1, Table 8). This suggests that Ala-73 may indirectly affect specificity by modulating the volume of the binding pocket (see Appendix 3). Structurally aligning CquiOR10 and CquiOR10A73L receptors by TM7b (Supplementary file 1, Table 15), demonstrates approximately a 1 Å α-carbon outward shift of A73L (Supplementary file 1, Table 16), suggesting a tightly constrained space in CquiOR10 and an expanded space in CquiOR10A73L relative to the protein backbone (Figure 8). Figure 8" @default.
• W4313290141 created "2023-01-06" @default.
• W4313290141 creator A5076618806 @default.
• W4313290141 date "2022-11-02" @default.
• W4313290141 modified "2023-10-14" @default.
• W4313290141 title "Editor's evaluation: Single amino acid residue mediates reciprocal specificity in two mosquito odorant receptors" @default.
• W4313290141 doi "https://doi.org/10.7554/elife.82922.sa0" @default.
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