The Arthropoda-specific Tramtrack group BTB protein domains use previously unknown interface to form hexamers
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eLife assessment
This important work offers an experimental structural characterization of the Tramtrack-like BTB/POZ domains in insects, revealing that these domains form stable hexameric assemblies. The structural evidence is convincing, and validated by fold prediction and evolutionary pathway analyses. This paper would be of interest to structural and evolutionary biologists.
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Abstract
BTB (bric-a-brack, Tramtrack, and broad complex) is a diverse group of protein-protein interaction domains found within metazoan proteins. Transcription factors contain a dimerizing BTB subtype with a characteristic N-terminal extension. The Tramtrack group (TTK) is a distinct type of BTB domain, which can multimerize. Single-particle cryo-EM microscopy revealed that the TTK-type BTB domains assemble into a hexameric structure consisting of three canonical BTB dimers connected through a previously uncharacterized interface. We demonstrated that the TTK-type BTB domains are found only in Arthropods and have undergone lineage-specific expansion in modern insects. The Drosophila genome encodes 24 transcription factors with TTK-type BTB domains, whereas only four have non-TTK-type BTB domains. Yeast two-hybrid analysis revealed that the TTK-type BTB domains have an unusually broad potential for heteromeric associations presumably through a dimer-dimer interaction interface. Thus, the TTK-type BTB domains are a structurally and functionally distinct group of protein domains specific to Arthropodan transcription factors.
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Author response:
Reviewer #1 (Public Review):
Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.
I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some …
Author response:
Reviewer #1 (Public Review):
Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.
I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some additional points for consideration for the same.
Major points.
- It would be valuable to discuss alternative multimer assembly interfaces, considering the diverse ways POZs can multimerize. For instance, the Potassium channel POZ domains form tetramers. A comparison of their inter-subunit interface with that of TTK and non-TTK POZs could provide insightful contrasts.
Thanks for the suggestion, we added this important comparison, as well as comparison with recently published structures of filament-forming BTB domains.
- The so-called TTK motif, despite its unique sequence signature, essentially corresponds to the N-terminal extension observed in other "non-TTK" proteins such as Miz-1. Given Miz-1's structure, it becomes evident that the utilization of the N-terminal extension for dimerization is shared with the TTK family, suggesting a common evolutionary origin in metazoan transcription factors. Early phylogenetic trees (e.g. in PMID: 9917379) support the grouping of the TTK-like POZs with other animal Transcription factors containing POZ domains such as those with Kelch repeats further suggesting that the extension might be ancestral. Structural investigations by modeling prominent examples or comparing known structures of similar POZ domains, could support this inference. Control comparisons with POZ domains from fungi, plants and amoebozoans like Dictyostelium could offer additional insights.
We performed AlphaFold2-Multimer modeling of dimers of all BTB domains from the most ancestral metazoan clades, Placozoa and Porifera, along with BTBs from Choanoflagellates – the closest to first metazoans unicellular eukaryotes. The presence of N-terminal beta-sheet was evaluated. KLHL-BTBs are present in all eukaryotes and likely are predecessors of ZBTB domains. According to AlphaFold modeling of dimers, all KLHL-BTB domains of plants and basal metazoans have alpha1 helix, but most of these domains from do not possess additional N-terminal beta-strand (beta1) characteristic for ZBTB domains. We found only one KLHL-BTB (Uniprot ID: AA9VCT1_MONBE) with such N-terminal extension in Choanoflagellate proteome, one in Dictyostelium proteome (Q54F31_DICDI), and 7 (out of 43 BTB domains in total) and 13 (out of 81) such domains in Trichoplax and Amphimedon proteomes correspondingly. There was no significant sequence similarity of beta1 element at the level of primary sequence. However, most of these domains bear 3-box/BACK extension and represent typical KLHL-BTBs which are member of E3 ubiquitin-ligase complexes, they are often associated with protein-protein interacting MATH domain or WD40 repeats. We found only one protein in Trichoplax proteome with beta1 strand devoid of 3-box/BACK (B3RQ74_TRIAD), thus resembling ZBTB topology. Thus, likely emergence of BTB domains of this subtype occurred early in Metazoan evolution. At this point ZBTBs were not yet associated with zinc-fingers. According to our survey, actual fusion of ZBTB domain with zinc-finger domains occurred in the evolution of earlier bilaterian organisms since proteins with such domain architecture are not found in Radiata but are present in basal Protostomia and Deuterostomia clades. TTK-type sequence is characteristic only for Arthropoda and emerged early in their evolution. We added all these data to the article.
- Exploring the ancestral presence of the aforementioned extension in metazoan transcription factors could serve as a foundation for understanding the evolutionary pathway of hexamerization. This analysis could shed light on exposed structural regions that had the potential to interact post-dimerization with the N-terminal extension and also might provide insights into the evolution of multimer interfaces, as observed in the Potassium channel.
We added this important comparison as well as comparison with recent structures of filament-forming BTB domains.
- Considering the role of conserved residues in the multimer interface is crucial. Reference to conserved residues involved in multimer formation, such as discussed in PMID: 9917379, would enrich the discussion.
We updated our description of multimer interface with respect to conservation of residues.
Reviewer #2 (Public Review):
BTB domains are protein-protein interaction domains found in diverse eukaryotic proteins, including transcription factors. It was previously known that many of the Drosophila transcription factor BTB domains are of the TTK-type - these are defined as having a highly-conserved motif, FxLRWN, at their N-terminus, and they thereby differ from the mammalian BTB domains. Whereas the well-characterised mammalian BTB domains are dimeric, several Drosophila TTK-BTB domains notably form multimers and function as chromosome architectural proteins. The aims of this work were (i) to determine the structural basis of multimerisation of the Drosophila TTK-BTB domains, (ii) to determine how different Drosophila TTK-BTB domains interact with each other, and (iii) to investigate the evolution of this subtype of BTB domain.
The work significantly advances our understanding of the biology of BTB domains. The conclusions of the paper are mostly well-supported, although some aspects need clarification:
Hexameric organisation of the TTK-type BTB domains:
Using cryo-EM, the authors showed that the CG6765 TTK-type BTB domain forms a hexameric assembly in which three "classic" BTB dimers interact via a beta-sheet interface involving the B3 strand. This is particularly interesting, as this region of the BTB domain has recently been implicated in protein-protein interactions in a mammalian BTB-transcription factor, MIZ1. SEC-MALS analysis indicated that the LOLA TTK-type BTB domain is also hexameric, and SAXS data was consistent with a hexameric assembly of the CG6765- and LOLA BTB domains.
The data regarding the hexameric organisation is convincing. However, interpreting the role of specific regions of the BTB domain is difficult because the description of the molecular contacts lacks depth.
Heteromeric interactions between TTK-type BTB domains:
The authors use yeast two-hybrid assays to study heteromeric interactions between various Drosophila TTK-type BTB domains. Such assays are notorious for producing false positives, and this needs to be mentioned. Although the authors suggest that the heteromeric interactions are mediated via the newly-identify B3 interaction interface, there is no evidence to support this, since mutation of B3 yielded insoluble proteins.
We are aware that Y2H can give false positive results in cases where the BTB domain fused to the DNA binding domain can activate reporter genes. Therefore, all tested BTB domains were examined for their ability to activate transcription. Furthermore, in our study, assays with non-TTK-type BTB domains, which showed almost no interactions, provide additional negative control. We have added a corresponding disclaimer in the text. We agree that our data do not explain the basis for heteromeric interactions. Design of mutations in B3 beta-sheet proved to be complicated, using of biochemical methods to study the principles of heteromer assembly also does not seem to be feasible since most TTK-type BTBs tend to form aggregates and are difficult to be expressed and purified. But most important issue is that demonstrated ability of heteromer assembly through B3 in few tested pairs cannot be applied for all pairs, some of them still may use different mechanism. We used AlphaFold to predict possible mechanisms of heteromer assemblies. AlphaFold suggested that usage of both B3 and conventional dimerization interfaces for heteromeric interactions are possible in various cases, with preference of one over another in different pairs. Thus, most likely the presence of two potential heteromerization interfaces extends the heteromerization capability of these domains. We changed the text accordingly.
Evolution of the TTK-type BTB domains:
The authors carried out a bioinformatics analysis of BTB proteins and showed that most of the Drosophila BTB transcription factors (24 out of 28) are of the TTK-type. They investigated how the TTK-type BTB domains emerged during evolution, and showed that these are only found in Arthropoda, and underwent lineage-specific expansion in the modern phylogenetic groups of insects. These findings are well-supported by the evidence.
-
eLife assessment
This important work offers an experimental structural characterization of the Tramtrack-like BTB/POZ domains in insects, revealing that these domains form stable hexameric assemblies. The structural evidence is convincing, and validated by fold prediction and evolutionary pathway analyses. This paper would be of interest to structural and evolutionary biologists.
-
Reviewer #1 (Public Review):
Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.
I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some additional points for …
Reviewer #1 (Public Review):
Using structural analysis, Bonchuk and colleagues demonstrate that the TTK-like BTB/POZs of insects form stable hexameric assemblies composed of trimers of POZ dimers, a configuration observed consistently across both homomultimers and heteromultimers, which are known to be formed by TTK-like BTB/POZ domains. The structural data is comprehensive, unambiguous, and further supported by theoretical fold prediction analyses. In particular the judicious complementation of experiments and fold prediction is commendable. This study now adds an important cog that might help generalize the general principles of the evolution of multimerization in members of this fold family.
I strongly feel that enhancing the inclusivity of the discussion would strengthen the paper. Below, I suggest some additional points for consideration for the same.
Major points.
(1) It would be valuable to discuss alternative multimer assembly interfaces, considering the diverse ways POZs can multimerize. For instance, the Potassium channel POZ domains form tetramers. A comparison of their inter-subunit interface with that of TTK and non-TTK POZs could provide insightful contrasts.(2) The so-called TTK motif, despite its unique sequence signature, essentially corresponds to the N-terminal extension observed in other "non-TTK" proteins such as Miz-1. Given Miz-1's structure, it becomes evident that the utilization of the N-terminal extension for dimerization is shared with the TTK family, suggesting a common evolutionary origin in metazoan transcription factors. Early phylogenetic trees (e.g. in PMID: 9917379) support the grouping of the TTK-like POZs with other animal Transcription factors containing POZ domains such as those with Kelch repeats further suggesting that the extension might be ancestral. Structural investigations by modeling prominent examples or comparing known structures of similar POZ domains, could support this inference. Control comparisons with POZ domains from fungi, plants and amoebozoans like Dictyostelium could offer additional insights.
(3) Exploring the ancestral presence of the aforementioned extension in metazoan transcription factors could serve as a foundation for understanding the evolutionary pathway of hexamerization. This analysis could shed light on exposed structural regions that had the potential to interact post-dimerization with the N-terminal extension and also might provide insights into the evolution of multimer interfaces, as observed in the Potassium channel.
(4) Considering the role of conserved residues in the multimer interface is crucial. Reference to conserved residues involved in multimer formation, such as discussed in PMID: 9917379, would enrich the discussion.
-
Reviewer #2 (Public Review):
BTB domains are protein-protein interaction domains found in diverse eukaryotic proteins, including transcription factors. It was previously known that many of the Drosophila transcription factor BTB domains are of the TTK-type - these are defined as having a highly-conserved motif, FxLRWN, at their N-terminus, and they thereby differ from the mammalian BTB domains. Whereas the well-characterised mammalian BTB domains are dimeric, several Drosophila TTK-BTB domains notably form multimers and function as chromosome architectural proteins. The aims of this work were (i) to determine the structural basis of multimerisation of the Drosophila TTK-BTB domains, (ii) to determine how different Drosophila TTK-BTB domains interact with each other, and (iii) to investigate the evolution of this subtype of BTB domain.
Th…
Reviewer #2 (Public Review):
BTB domains are protein-protein interaction domains found in diverse eukaryotic proteins, including transcription factors. It was previously known that many of the Drosophila transcription factor BTB domains are of the TTK-type - these are defined as having a highly-conserved motif, FxLRWN, at their N-terminus, and they thereby differ from the mammalian BTB domains. Whereas the well-characterised mammalian BTB domains are dimeric, several Drosophila TTK-BTB domains notably form multimers and function as chromosome architectural proteins. The aims of this work were (i) to determine the structural basis of multimerisation of the Drosophila TTK-BTB domains, (ii) to determine how different Drosophila TTK-BTB domains interact with each other, and (iii) to investigate the evolution of this subtype of BTB domain.
The work significantly advances our understanding of the biology of BTB domains. The conclusions of the paper are mostly well-supported, although some aspects need clarification:
Hexameric organisation of the TTK-type BTB domains:
Using cryo-EM, the authors showed that the CG6765 TTK-type BTB domain forms a hexameric assembly in which three "classic" BTB dimers interact via a beta-sheet interface involving the B3 strand. This is particularly interesting, as this region of the BTB domain has recently been implicated in protein-protein interactions in a mammalian BTB-transcription factor, MIZ1. SEC-MALS analysis indicated that the LOLA TTK-type BTB domain is also hexameric, and SAXS data was consistent with a hexameric assembly of the CG6765- and LOLA BTB domains.The data regarding the hexameric organisation is convincing. However, interpreting the role of specific regions of the BTB domain is difficult because the description of the molecular contacts lacks depth.
Heteromeric interactions between TTK-type BTB domains:
The authors use yeast two-hybrid assays to study heteromeric interactions between various Drosophila TTK-type BTB domains. Such assays are notorious for producing false positives, and this needs to be mentioned. Although the authors suggest that the heteromeric interactions are mediated via the newly-identify B3 interaction interface, there is no evidence to support this, since mutation of B3 yielded insoluble proteins.Evolution of the TTK-type BTB domains:
The authors carried out a bioinformatics analysis of BTB proteins and showed that most of the Drosophila BTB transcription factors (24 out of 28) are of the TTK-type. They investigated how the TTK-type BTB domains emerged during evolution, and showed that these are only found in Arthropoda, and underwent lineage-specific expansion in the modern phylogenetic groups of insects. These findings are well-supported by the evidence. -
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