Molecular reconstruction of recurrent evolutionary switching in olfactory receptor specificity
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Evaluation Summary:
This study investigates evolutionary changes in ligand preference that occur in an olfactory receptor (IR75a) across the Drosophila phylogeny. The authors find that IR75a displays different odor preferences, for acetic acid or butyric acid, across Drosophila species, and link odor preference to particular protein mutations in the receptor. Reconstruction of a putative ancestral IR75a revises the timeline for IR75a evolution, and structural modeling suggests how mutations alter odor preference.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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. 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.
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Author Response
Reviewer #1 (Public Review):
This manuscript provides an interesting analysis of the evolution of the IR75a protein across the Drosophila phylogeny. As reported by the authors previously, IR75a in D melanogaster and several related species preferentially recognize the odor acetic acid, while IR75a in D sechellia instead prefers butyric acid. Here, the authors report that more distant Drosophila species, as well as the putative ancestral IR75a, also prefer butyrate, suggesting that IR75a changed its preference from butyrate to acetate within the melanogaster/obscura group, with reversion to butyrate subsequently occurring in D sechellia. Moreover, the authors identify a key site (position 538) whose identity as Phe or Leu tracks with odor preference. They also identify other secondary lineage-specific mutations that …
Author Response
Reviewer #1 (Public Review):
This manuscript provides an interesting analysis of the evolution of the IR75a protein across the Drosophila phylogeny. As reported by the authors previously, IR75a in D melanogaster and several related species preferentially recognize the odor acetic acid, while IR75a in D sechellia instead prefers butyric acid. Here, the authors report that more distant Drosophila species, as well as the putative ancestral IR75a, also prefer butyrate, suggesting that IR75a changed its preference from butyrate to acetate within the melanogaster/obscura group, with reversion to butyrate subsequently occurring in D sechellia. Moreover, the authors identify a key site (position 538) whose identity as Phe or Leu tracks with odor preference. They also identify other secondary lineage-specific mutations that presumably provide structural support to help optimize ligand preference. Interestingly, different solutions for secondary optimization were observed across lineages, suggesting multiple evolutionary and structural paths for tweaking the ligand pocket. These data are generally solid and expertly generated, but I do note that there is substantial speculation based on molecular modeling (which the authors acknowledge) as well as speculation of mutational timeline which should be trimmed or removed. There are many ways to extend these findings, such as linking odor recognition properties to behavior, which would substantially increase the impact of this study.
We fully agree that understanding the behavioral significance of the changes in odor recognition of these receptors is of high interest, but as we do not yet clearly know the behavioral function(s) of these receptors even in D. melanogaster, such an effort is likely to take months (if not years) and goes well beyond the current study. We hope that our identification of tuning changes of these Irs across the Drosophila genus might provide some additional clues to the contributions of these receptors to the odor-guided behaviors of these species.
Reviewer #2 (Public Review):
[...] Weaknesses: The authors attempt to link the characterized molecular events to the ecological needs that might have played a significant role in the linage's evolution, and to the structural aspects of receptor-ligand interaction. These two aspects are on the more speculative side, which the authors themselves acknowledge as limitations of the study.
- In terms of the ecological context, this study focuses on a narrow set of ligands that are probed at concentrations that are quite high and thus of unclear physiological relevance. While their results are very exciting, they are restricted to the inverse relationship of the responses to C2-C4 carboxylic acids. Although data in the literature shows that these ligands, in particular acetic acid, are likely relevant, other data support that ligands from the same series may also be relevant. In particular, noni volatiles are dominated by octanoic acid (Auer et al., 2020; Pino et al., 2010), and Ir75a responds very strongly to propionic acid as well as acetic acid (Pietro Godino et al., 2016, Silbering et al., 2011). While the C2-C4 relationship captures most of the variance in PCA (which leads the authors to focus on these ligands in the first place), perhaps at other more physiological concentrations the relationship between other ligands in the series becomes more prominent, which would be interesting to explore.
It seems unlikely that odors that do not evoke robust responses at 10^-2 will stimulate biologically meaningful responses if tested at higher concentrations. Regarding the abundant noni volatile octanoic acid, we have previously shown that this odor (surprisingly) does not evoke strong responses in any acid-sensing Ir neuron, nor does it provoke strong olfactory behavioral attraction of D. sechellia (PMID 28111079, Figure 1). These results contrast with the physiological and behavioral responsiveness to hexanoic acid, and suggest octanoic acid may act principally via the gustatory system.
The universe of odors is enormous, so for practical reasons for this study we focused on a simple series of ligands to extract principles of molecular evolution of olfactory receptor specificity. Acknowledging the limitations of our study, we have taken care to state in the Discussion that our findings may only capture part changes of the response properties of individual receptors, and that we can only speculate on the behavioral/ecological significance of at least some particular odor tuning properties.
Regarding the quantitative analyses in our manuscript, the vast majority of the results are not restricted to C2-C4, but rather the whole linear series (C1-C6). In Figure 1E, we present the clustering of species on a C2 vs C4 response plot because we show that these two odors are the major contributors of variance in the data by PCA (Figure 1D) and because this allows easy visualization in two dimensions. When we analyze the statistical significance of the epistatic interactions between different mutations (Figure 3F), we use PC1 from the dataset, to encompass all of the main variance in the data and avoid biasing towards specific odors.
- The authors don't discuss whether the proposed polymorphisms are found in population genomic data, which is available at least for Dmel and Dsec. Mining these datasets and looking at intraspecific variation (or lack thereof) has the potential to support their speculations on the evolutionary trajectories of mutations with empirical data and offer complementary insight.
We mentioned this briefly in our original submission (there is no informative population genetic variation) and have now added additional information to the manuscript on the results of our survey of intraspecific genetic variation in these drosophilids.
- A more critical limitation is the use of docking onto homology models. Modeling techniques are incredibly powerful as they can provide solid hypotheses for how protein-ligand interactions might occur. However, much caution should be taken to interpreting modeling results without experimental validation. The reliability of homology models scales substantially with sequence identity, which turns this protein family into rather poor substrates for extracting atomic-scale conclusions from these models. In this case, homology models are combined with docking and very little support is offered. For example, the docking scores presented for the homology models are relatively low, and there is no significant difference between the docking score of acetic and butyric acid onto Dmel Ir75a. Although it is well known that docking results will in general only qualitatively match the behavior of a receptor-ligand pair, in the absence of alternative validation of the modeling procedures, these results fail to convince the reader that the homology models and docking results are reasonably likely. It should be noted that the proposed mode of action is entirely plausible and an interesting possibility, but as it is it appears too speculative and without validation.
We now present what we consider are better protein models to indicate the relative position of different residues in the LBD but have removed all docking analyses.
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Evaluation Summary:
This study investigates evolutionary changes in ligand preference that occur in an olfactory receptor (IR75a) across the Drosophila phylogeny. The authors find that IR75a displays different odor preferences, for acetic acid or butyric acid, across Drosophila species, and link odor preference to particular protein mutations in the receptor. Reconstruction of a putative ancestral IR75a revises the timeline for IR75a evolution, and structural modeling suggests how mutations alter odor preference.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This manuscript provides an interesting analysis of the evolution of the IR75a protein across the Drosophila phylogeny. As reported by the authors previously, IR75a in D melanogaster and several related species preferentially recognize the odor acetic acid, while IR75a in D sechellia instead prefers butyric acid. Here, the authors report that more distant Drosophila species, as well as the putative ancestral IR75a, also prefer butyrate, suggesting that IR75a changed its preference from butyrate to acetate within the melanogaster/obscura group, with reversion to butyrate subsequently occurring in D sechellia. Moreover, the authors identify a key site (position 538) whose identity as Phe or Leu tracks with odor preference. They also identify other secondary lineage-specific mutations that presumably provide …
Reviewer #1 (Public Review):
This manuscript provides an interesting analysis of the evolution of the IR75a protein across the Drosophila phylogeny. As reported by the authors previously, IR75a in D melanogaster and several related species preferentially recognize the odor acetic acid, while IR75a in D sechellia instead prefers butyric acid. Here, the authors report that more distant Drosophila species, as well as the putative ancestral IR75a, also prefer butyrate, suggesting that IR75a changed its preference from butyrate to acetate within the melanogaster/obscura group, with reversion to butyrate subsequently occurring in D sechellia. Moreover, the authors identify a key site (position 538) whose identity as Phe or Leu tracks with odor preference. They also identify other secondary lineage-specific mutations that presumably provide structural support to help optimize ligand preference. Interestingly, different solutions for secondary optimization were observed across lineages, suggesting multiple evolutionary and structural paths for tweaking the ligand pocket. These data are generally solid and expertly generated, but I do note that there is substantial speculation based on molecular modeling (which the authors acknowledge) as well as speculation of mutational timeline which should be trimmed or removed. There are many ways to extend these findings, such as linking odor recognition properties to behavior, which would substantially increase the impact of this study.
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Reviewer #2 (Public Review):
While the tuning of sensory receptors is thought to have an important role in shaping ecological fitness, how tuning shifts occur through evolution is poorly understood, in part due to the difficulties in comparing receptor sequences and receptive fields in highly diversified families of receptors.
This work seeks to identify the molecular trajectory that led to the functional divergence of a previously characterized insect olfactory receptor, Ir75a, providing an unusual opportunity to hone in on individual amino-acid residues that are causative of functional divergence.
The authors first compare the receptor responses of 10 orthologous receptors in a Drosophila lineage spanning 40 million years of evolution and find that the response profiles can be classified into two main categories. This functional …Reviewer #2 (Public Review):
While the tuning of sensory receptors is thought to have an important role in shaping ecological fitness, how tuning shifts occur through evolution is poorly understood, in part due to the difficulties in comparing receptor sequences and receptive fields in highly diversified families of receptors.
This work seeks to identify the molecular trajectory that led to the functional divergence of a previously characterized insect olfactory receptor, Ir75a, providing an unusual opportunity to hone in on individual amino-acid residues that are causative of functional divergence.
The authors first compare the receptor responses of 10 orthologous receptors in a Drosophila lineage spanning 40 million years of evolution and find that the response profiles can be classified into two main categories. This functional clustering largely matches the evolutionary relationships between the studied species and allows the authors to hypothesize the evolutionary trajectory that the ancestral receptor followed to give rise to differences in function. Based on the extant receptor sequences in the group they predict two ancestral sequences at key positions in the phylogeny using maximum likelihood estimates, and then 'resurrect' these receptors by synthesizing the genes and expressing them in a well characterized in vivo expression system. They find that these hypothetical ancestral proteins functionally behave as predicted, and, using sequence alignments and previous experimental observations, they narrow down the candidate amino-acid positions that cause functional variation to 3 residues. Mutation of these 3 residues in the Dmel Ir75a into the Dsec variant shifts its tuning to perfectly match the tuning of the Dsec receptor, and the converse experiment yields the analogous result. The authors then synthesize the 8 possible combinations of these 3 mutations (single, double, and triple mutants) and functionally test their responses in vivo. This set of experiments allows them to analyze the phenotypic contribution of each mutation, which they then use to hypothesize on the most likely evolutionary trajectory from the ancestral Dmel-like receptor variant to the Dsec variant.
This analysis identifies a single position that has the largest epistatic effect. Across the clade, two residues can be found occupying this position, and the identity of this residue perfectly segregates with the functional phenotype of this receptor in the 10 species under study. Through this analysis the authors arrive to the conclusion that this particular site in this receptor has undergone at least 2 critical retuning mutations in a timespan of 40 million years. Further sequence alignments and chimeric experiments show that the analogous position in a related receptor Ir75b is in part responsible for adaptive change, leading the authors to conclude that this is a 'hotspot' for evolutionary variation, repeatedly tweaked across the lineage to achieve species-specific olfactory capabilities. The authors then use homology models and docking studies to attempt to shed light on the possible role of this amino-acid position in receptor function. Overall, the study is rigorous, detailed, and elegantly designed. The experimental layout is carefully explained, and the conclusions are cautiously presented.Strengths: A main contribution of this work is to provide a plausible evolutionary path of discrete molecular events that led to the adaptive tuning of a receptor, and substantiate this analysis with an unusual high level of experimental support. Indeed, these studies are often hindered by difficulties in expressing receptors for functional studies as well as a high level of sequence divergence that prevents the identification of potential sites of causal change. In that light, the functional analysis of receptors across a lineage, together with the reconstruction of ancestral variants and dissection of the evolutionary trajectory of this receptor's tuning represents an important contribution to the field. This set of experiments and analyses offers an elegant example of a rigorous way to approach the challenging topic of the genetic basis of sensory adaptation.
Weaknesses: The authors attempt to link the characterized molecular events to the ecological needs that might have played a significant role in the linage's evolution, and to the structural aspects of receptor-ligand interaction. These two aspects are on the more speculative side, which the authors themselves acknowledge as limitations of the study.
1. In terms of the ecological context, this study focuses on a narrow set of ligands that are probed at concentrations that are quite high and thus of unclear physiological relevance. While their results are very exciting, they are restricted to the inverse relationship of the responses to C2-C4 carboxylic acids. Although data in the literature shows that these ligands, in particular acetic acid, are likely relevant, other data support that ligands from the same series may also be relevant. In particular, noni volatiles are dominated by octanoic acid (Auer et al., 2020; Pino et al., 2010), and Ir75a responds very strongly to propionic acid as well as acetic acid (Pietro Godino et al., 2016, Silbering et al., 2011). While the C2-C4 relationship captures most of the variance in PCA (which leads the authors to focus on these ligands in the first place), perhaps at other more physiological concentrations the relationship between other ligands in the series becomes more prominent, which would be interesting to explore.
2. The authors don't discuss whether the proposed polymorphisms are found in population genomic data, which is available at least for Dmel and Dsec. Mining these datasets and looking at intraspecific variation (or lack thereof) has the potential to support their speculations on the evolutionary trajectories of mutations with empirical data and offer complementary insight.
3. A more critical limitation is the use of docking onto homology models. Modeling techniques are incredibly powerful as they can provide solid hypotheses for how protein-ligand interactions might occur. However, much caution should be taken to interpreting modeling results without experimental validation. The reliability of homology models scales substantially with sequence identity, which turns this protein family into rather poor substrates for extracting atomic-scale conclusions from these models. In this case, homology models are combined with docking and very little support is offered. For example, the docking scores presented for the homology models are relatively low, and there is no significant difference between the docking score of acetic and butyric acid onto Dmel Ir75a. Although it is well known that docking results will in general only qualitatively match the behavior of a receptor-ligand pair, in the absence of alternative validation of the modeling procedures, these results fail to convince the reader that the homology models and docking results are reasonably likely. It should be noted that the proposed mode of action is entirely plausible and an interesting possibility, but as it is it appears too speculative and without validation.
These weaknesses do not detract from the overall value of the study and the authors take considerable care in acknowledging some of these caveats, which helps interpret these results and weigh the various aspects of it in full light.
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Reviewer #3 (Public Review):
Although genomic information has been increasingly accumulated in closely related insect species, a molecular basis for functional changes in the olfactory receptors and the relationship with a change in food preference during the course of evolution has not been fully resolved. The authors successfully identified a 'hotspot' amino acid in an olfactory receptor, IR75a, that plays a crucial role in the selectivity of acid odors that appears to affect feeding behavior in Drosophila species. The finding provides an insight into how a molecular evolution occurred in an IR in correlation with the biological significance.
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