Sequence and structural conservation reveal fingerprint residues in TRP channels
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Evaluation Summary:
Transient Receptor Potential (TRP) cation channels, related to voltage-gated channels, appeared before plants and animals diverged in evolution and expanded in vertebrates into seven major subfamilies and took multiple essential physiological functions encoding chemical and physical information into electrical signals. In this manuscript, Deny Cabezas-Bratesco and co-workers draw from multiple sequence alignments and available structural information to identify highly conserved features in the transmembrane domains across several major TRP subfamilies in vertebrate and invertebrate animals and even in unicellular organisms. By systematically analyzing their findings, the authors propose a structural framework hinting at common mechanisms utilized by TRP channels to integrate stimuli into electric signals, which has major implications for a wide range of biological processes where TRP channels play a role.
(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. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Transient receptor potential (TRP) proteins are a large family of cation-selective channels, surpassed in variety only by voltage-gated potassium channels. Detailed molecular mechanisms governing how membrane voltage, ligand binding, or temperature can induce conformational changes promoting the open state in TRP channels are still a matter of debate. Aiming to unveil distinctive structural features common to the transmembrane domains within the TRP family, we performed phylogenetic reconstruction, sequence statistics, and structural analysis over a large set of TRP channel genes. Here, we report an exceptionally conserved set of residues. This fingerprint is composed of twelve residues localized at equivalent three-dimensional positions in TRP channels from the different subtypes. Moreover, these amino acids are arranged in three groups, connected by a set of aromatics located at the core of the transmembrane structure. We hypothesize that differences in the connectivity between these different groups of residues harbor the apparent differences in coupling strategies used by TRP subgroups.
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Evaluation Summary:
Transient Receptor Potential (TRP) cation channels, related to voltage-gated channels, appeared before plants and animals diverged in evolution and expanded in vertebrates into seven major subfamilies and took multiple essential physiological functions encoding chemical and physical information into electrical signals. In this manuscript, Deny Cabezas-Bratesco and co-workers draw from multiple sequence alignments and available structural information to identify highly conserved features in the transmembrane domains across several major TRP subfamilies in vertebrate and invertebrate animals and even in unicellular organisms. By systematically analyzing their findings, the authors propose a structural framework hinting at common mechanisms utilized by TRP channels to integrate stimuli into electric signals, which has …
Evaluation Summary:
Transient Receptor Potential (TRP) cation channels, related to voltage-gated channels, appeared before plants and animals diverged in evolution and expanded in vertebrates into seven major subfamilies and took multiple essential physiological functions encoding chemical and physical information into electrical signals. In this manuscript, Deny Cabezas-Bratesco and co-workers draw from multiple sequence alignments and available structural information to identify highly conserved features in the transmembrane domains across several major TRP subfamilies in vertebrate and invertebrate animals and even in unicellular organisms. By systematically analyzing their findings, the authors propose a structural framework hinting at common mechanisms utilized by TRP channels to integrate stimuli into electric signals, which has major implications for a wide range of biological processes where TRP channels play a role.
(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. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In the present manuscript, Deny Cabezas-Bratesco curate a set of sequences of the transmembrane domain (TM) and TM-proximal regions from group I Transient Receptor Potential (TRP) channels (including subfamilies A, V, C, M and N) belonging to vertebrate and invertebrate animals as well as unicellular organisms. The authors generate a multiple sequence alignment, which reveals subfamily-specific patterns in the extra- and intracellular regions that connect each of the six transmembrane helices, as well as a set of 11 residues that are highly conserved across subfamilies in TRP channels from animals. Interestingly, these residues appear to be conserved, albeit to a lower extent, in TRP channels from unicellular organisms and even in voltage-gated channels that are evolutionary related to TRP channels. This …
Reviewer #1 (Public Review):
In the present manuscript, Deny Cabezas-Bratesco curate a set of sequences of the transmembrane domain (TM) and TM-proximal regions from group I Transient Receptor Potential (TRP) channels (including subfamilies A, V, C, M and N) belonging to vertebrate and invertebrate animals as well as unicellular organisms. The authors generate a multiple sequence alignment, which reveals subfamily-specific patterns in the extra- and intracellular regions that connect each of the six transmembrane helices, as well as a set of 11 residues that are highly conserved across subfamilies in TRP channels from animals. Interestingly, these residues appear to be conserved, albeit to a lower extent, in TRP channels from unicellular organisms and even in voltage-gated channels that are evolutionary related to TRP channels. This suggests that the degree of conservation of these residues provides information about the evolution of TRP channels in different organisms. Importantly, the positions of these 11 conserved 'signature' residues appear to also be conserved at a structural level, and are grouped into three separate 'patches' at key regions that are known to be important for channel function. Applying direct-coupling analysis the authors find evidence that supports the conservation across subfamilies of interactions between the different channel regions included in patch 1, and suggest that interactions with lipids in this region might also be conserved. This is consistent with a wealth of information pointing to this region as a central hub for allosteric signaling in TRP channels. The authors also find that TRP channels from group I all have a cluster of aromatic residues at the core of the ligand-binding S1-S4 domain that is very different from the equivalent domains in voltage-activated channels and that would be expected to confer rigidity to that region in TRP channels. Finally, the authors find further conserved aromatic residues connecting the hydrophobic cluster in the S1-S4 domain with conserved patch 2 in the extracellular pore - this points to the existence of a conserved mechanism that communicates the ligand-binding domains with the extracellular half of the pore. Differences in the hydrophobic cluster between subfamilies and the in the connection between the cluster and conserved patches 1 and 2 could be related to mechanistic differences in signal integration between TRP channel members of each subfamily. Together, the data presented highlights a set of robustly conserved features across evolutionary related proteins that together, offer an intriguing framework for interpreting further structural information and data on channel function based on a core mechanism of function that appears to be conserved in the TRP family of ion channels.
The figures in the manuscript do not provide information to assess the diversity of organisms from which TRP channel sequences were analyzed - this is important because biases in this selection could influence some of the results, such as the extent of conservation at certain positions. Some of the patterns from the multiple sequence alignments are also difficult to assess from the figures, such as the relation between the gaps in the alignments and the assignment of each channel to one of the TRP subfamilies. In the absence of a quantitative analysis of these features, it is difficult for readers to assess the robustness of these findings.
It is also difficult to determine the validity of the proposed patterns of signature-residue conservation in non-TRP channels, because the considerable structural and sequence-level differences between these and TRP channels make it non-trivial to identify structurally equivalent positions - structural information to support these assignments is not shown for non-TRP channels.
In regards to the structural conservation of 'signature' positions in different TRP channels, only select structures are displayed showing only one TRP channel per subfamily - this introduces some uncertainty in the extent to which each of these positions is conserved at a structural level for all channels included in the multiple sequence alignment. For many TRP channel subtypes structures in different conformations have been determined, often showing considerable differences in conformation - it is unclear from the text in the manuscript whether the structural-level analysis that was performed was limited to the structures included in the figures, or whether a more systematic analysis was carried out that included a larger sample of TRP channel structures.
The data shown supporting the analysis of state-dependent changes in residue connectivity does not provide much insight - the changes in interactions between the signature residues and other regions of the channel are difficult to see, and the changes observed in the figure are largely dependent on the way each pair of structures were aligned, yielding limited mechanistic information. Furthermore, the functional state of most structures is not known, making it difficult to compare conformational changes between different TRP channel types because it is uncertain whether the pairs of structures being compared truly reflect equivalent conformations. Finally, the main text refers to analysis of TRPV1/2/3/5/6, TRPC3/4/6, TRPM2/4/8 and TRPA1, but only data for two conformations of TRPV1, TRPM8 and TRPC3 is shown.
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Reviewer #2 (Public Review):
This paper highlighted a set of conserved amino acids in the larger TRP channel family and ascribed various functions to them, especially to the "aromatic core," in channel behavior.
The major strength of this paper is that it demonstrates a number of deeply conserved residues in TRP channels and proposes some ways in which these may influence TRP channel activity. Its main weaknesses are that its evolutionary analysis is lacking by the absence of some important TRP channel families and, most importantly, its lack of functional tests of the proposed hypotheses of the role of these conserved residues in channel function.
The finding of a conserved interaction between residues on the TM4 and TM5 (W549 and F589) is significant to those interested in TRP mechanisms, but there is no functional data for F589 or …
Reviewer #2 (Public Review):
This paper highlighted a set of conserved amino acids in the larger TRP channel family and ascribed various functions to them, especially to the "aromatic core," in channel behavior.
The major strength of this paper is that it demonstrates a number of deeply conserved residues in TRP channels and proposes some ways in which these may influence TRP channel activity. Its main weaknesses are that its evolutionary analysis is lacking by the absence of some important TRP channel families and, most importantly, its lack of functional tests of the proposed hypotheses of the role of these conserved residues in channel function.
The finding of a conserved interaction between residues on the TM4 and TM5 (W549 and F589) is significant to those interested in TRP mechanisms, but there is no functional data for F589 or structural comparison analysis of the two sites in the apo and bound states.
In addition, without access to the supplementary data, the reviewers cannot adequately assess the quality of the evolutionary analyses. Specifically, the authors did not include reviewer links to the Dryad repositories, and the DOIs have not yet been activated, so I cannot access the data or attest to its quality.
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Reviewer #3 (Public Review):
This study focuses on finding common evolutionary and structural characteristics of the wide and elusive superfamily of transient receptor potential (TRP) ion channels, present in almost all animal species. The aim is to use standard sequence- and structure-based bioinformatics techniques to find conserved residues and analyze their structural and functional importance in the TRP families.
The manuscript is written in a clear and accessible way, and provides an interesting overview on the different pieces of information about structural and functional relevance of TRP proteins. The references to the literature are not only helpful for the reader, but also important for the comparative analysis performed. The bioinformatics techniques used are very well-established and applied with care, which guarantees the …
Reviewer #3 (Public Review):
This study focuses on finding common evolutionary and structural characteristics of the wide and elusive superfamily of transient receptor potential (TRP) ion channels, present in almost all animal species. The aim is to use standard sequence- and structure-based bioinformatics techniques to find conserved residues and analyze their structural and functional importance in the TRP families.
The manuscript is written in a clear and accessible way, and provides an interesting overview on the different pieces of information about structural and functional relevance of TRP proteins. The references to the literature are not only helpful for the reader, but also important for the comparative analysis performed. The bioinformatics techniques used are very well-established and applied with care, which guarantees the reliability of the analysis. On the other hand, more recent and advanced methodologies that could have offered even more insights have been disregarded.
Through database cross-referencing, multiple sequence alignment, phylogeny studies and comparison with literature, the authors identify a group of 11 (mostly) aromatic amino acids with >90% conservation. Structurally, they are grouped in three regions (patches) of functional relevance. Structural properties of each patch are identified and compared with previous studies.
This work is an important reference to any researcher who studies structural and evolutionary aspects of TRP families. It includes an impressive amount of well-organized information, which the authors are able to complete with their own original analyses.
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