Molecular dynamics simulations illuminate the role of sequence context in the ELF3-PrD-based temperature sensing mechanism in plants
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eLife Assessment
In this potentially valuable computational study, the authors conducted atomistic and coarse-grained simulations to probe the temperature-dependent phase behaviors of ELF3, a disordered component of the evening complex in plant. The results aim to highlight the role of polyQ tracts in modulating the temperature sensitivity. The level of evidence is considered incomplete, due to the lack of systematic calibration of the coarse-grained model and limited statistical uncertainty analysis, especially considering the relatively subtle nature of the differences due to temperature change.
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Abstract
The evening complex (EC) is a tripartite DNA repressor and a core component of the circadian clock that provides a mechanism for temperature-responsive growth and development of many plants. ELF3, a component of the EC, is a disordered scaffolding protein that blocks transcription of growth genes at low temperature. At increased temperature EC DNA binding is disrupted and ELF3 is sequestered in a reversible nuclear condensate, allowing transcription and growth to proceed. The condensation is driven by a low complexity prion-like domain (PrD), and the sensitivity of the temperature response is modulated by the length of a variable polyQ tract, with a longer polyQ tract corresponding to enhanced condensate formation and hypocotyl growth at increased temperature. Here, a series of computational studies provides evidence that polyQ tracts promote formation of temperature-sensitive helices in flanking residues with potential impacts for EC stability under increasing temperature. REST2 simulations uncover a heat-induced population of condensation-prone conformations that results from the exposure of ‘sticky’ aromatic residues by temperature-responsive breaking of long-range contacts. Coarse-grained Martini simulations reveal both polyQ tract length and sequence context modulate the temperature dependence of cluster formation. Understanding the molecular mechanism underlying the ELF3-PrD temperature response in plants has implications for technologies including modular temperature-response elements for heat-responsive protein design and agricultural advances to enable optimization of crop yields and allow plants to thrive in increasingly inhospitable environments.
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eLife Assessment
In this potentially valuable computational study, the authors conducted atomistic and coarse-grained simulations to probe the temperature-dependent phase behaviors of ELF3, a disordered component of the evening complex in plant. The results aim to highlight the role of polyQ tracts in modulating the temperature sensitivity. The level of evidence is considered incomplete, due to the lack of systematic calibration of the coarse-grained model and limited statistical uncertainty analysis, especially considering the relatively subtle nature of the differences due to temperature change.
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Reviewer #1 (Public review):
Summary:
This manuscript explores the role of the Evening Complex (EC), specifically focusing on ELF3, a disordered protein component of the EC, and its temperature-dependent phase behavior. The study highlights the role of polyQ tracts in modulating temperature-sensitive condensate formation and provides a combination of computational approaches, including REST2 simulations and coarse-grained Martini simulations, to investigate how polyQ tract length and sequence context influence this behavior.
Strengths:
The study addresses a key question in plant biology - how temperature influences circadian clock-mediated growth regulation through protein phase behavior. The manuscript introduces the novel finding that polyQ tract length modulates the temperature-dependent formation of helices and condensates.
Weaknesse…
Reviewer #1 (Public review):
Summary:
This manuscript explores the role of the Evening Complex (EC), specifically focusing on ELF3, a disordered protein component of the EC, and its temperature-dependent phase behavior. The study highlights the role of polyQ tracts in modulating temperature-sensitive condensate formation and provides a combination of computational approaches, including REST2 simulations and coarse-grained Martini simulations, to investigate how polyQ tract length and sequence context influence this behavior.
Strengths:
The study addresses a key question in plant biology - how temperature influences circadian clock-mediated growth regulation through protein phase behavior. The manuscript introduces the novel finding that polyQ tract length modulates the temperature-dependent formation of helices and condensates.
Weaknesses:
(1) Coarse-Grained Simulation Results Not Supported by Data:
The results presented in Figure 6A of the manuscript do not seem to show a clear trend in the number of clusters formed as a function of polyQ tract length. This is particularly evident in the comparison between 0Q and 7Q polyQ lengths, which display statistically similar values in terms of the number of clusters. The lack of distinction between these values raises questions about the sensitivity of the coarse-grained simulations to polyQ tract length, which the authors claim as a key modulator of condensate formation. This discrepancy weakens the argument that polyQ length directly impacts the clustering behavior in the simulations.
Suggested Analysis:
- A more detailed statistical analysis should be performed to assess whether the observed differences between polyQ lengths are significant. This could involve hypothesis testing or the use of error bars in the graphs to better communicate the variability in the data.
- Additionally, the authors should examine whether there are other features, such as cluster shape or internal structure, that might differentiate between different polyQ lengths, even if the total number of clusters is similar.(2) Inconsistency in Cluster Size Across Temperatures (Figure 6B):
The results in Figure 6B show a striking difference in the size of the largest cluster between temperatures of 290K and 300K. This abrupt shift in behavior lacks a clear mechanistic explanation. Typically, phase transitions driven by temperature are more gradual, unless there is some underlying structural or chemical shift that the authors have not accounted for. Without a clear explanation, this sudden change in behavior reduces confidence in the simulation results.
Suggested Analysis:
- The authors should explore possible explanations for the dramatic difference in cluster size between 290K and 300K. For example, they could investigate whether specific interactions (such as the breaking or formation of hydrogen bonds or hydrophobic contacts) might explain the behavior at higher temperatures.
- It is important to check whether the coarse-grained simulation model has been adequately parameterized and scaled for accurate temperature dependence. Atomistic simulations of monomers and dimers with varying polyQ tract lengths could be used to fine-tune the coarse-grained model, ensuring it accurately reflects molecular behavior. The gross estimate of a 10% scaling factor might be insufficient and could lead to inaccurate representations of cluster formation.(3) Scaling of Coarse-Grained Model with Atomistic Simulations:
As mentioned, the coarse-grained model used in the study may not have been properly scaled against atomistic data. A simple scaling factor of 10% may not be appropriate for accurately capturing the behavior of polyQ tracts across different lengths, especially considering their sensitivity to subtle changes in temperature. Without rigorous validation against atomistic simulations, the coarse-grained model's predictions could be skewed.
Suggested Analysis:(4) To address this, the authors should compare the coarse-grained model with atomistic simulations of monomeric and dimeric forms of ELF3 with different polyQ tract lengths. By comparing key structural parameters (e.g., radius of gyration, contact maps, and clustering propensity), the authors could adjust the coarse-grained model to more accurately reflect the atomistic behavior. The authors have wealth of atomistic simulation data that could afford such benchmarking and identification of scaling factor
o Additionally, the authors should investigate whether the assumed scaling factor of 10% is appropriate for each polyQ length or whether it needs to be refined based on specific properties, such as the number of hydrophobic interactions or secondary structure stability.(5) Lack of Analysis for Liquid-Like Behavior in Phase Separation:
The simulations presented in the manuscript do not analyze the liquid-like behavior of ELF3 condensates, which is a key characteristic of liquid-liquid phase separation (LLPS). In LLPS systems, condensates are often dynamic, with chains exchanging between clusters, indicating liquid-like rather than solid-like behavior. The authors fail to probe this crucial aspect, which is necessary to support the claim that ELF3 undergoes phase separation.
Suggested Analysis:
- The authors should conduct additional analyses to probe the liquid-like nature of the clusters formed by ELF3. One approach would be to analyze the dynamics of chain exchange between clusters, measuring how frequently chains leave one cluster and join another over time. This analysis would reveal whether the condensates behave as liquid-like, dynamic structures or more static, solid-like aggregates.
- Additionally, the temperature dependence of these exchange dynamics should be investigated. In true liquid-liquid phase separation, the rate of chain exchange is often sensitive to temperature. Observing how this rate changes between 290K and 300K, for instance, could help explain the abrupt shift in cluster size seen in Figure 6B.
- The authors should also analyze whether the internal structures of the condensates are consistent with a liquid-like phase. For example, radial distribution functions and contact lifetimes could be calculated to reveal whether the clusters exhibit liquid-like organization.(6) Lack of justification of polydispersity of polyQ:
The authors don't provide any rationale for choice of different copies of polyQ used in the manuscript for their chain-growth simulation studies. It will be more apt if it can be motivated via some precedent experimental observations.(7) Lack of initiative to connect to Experiments:
While the computational models and simulations provide robust theoretical insights, the absence of direct experimental validation weakens the overall impact of the manuscript. For example, experimental data on how specific mutations in the polyQ tract influence ELF3 behavior in vivo would significantly bolster the authors' claims. The manuscript would benefit from either citing existing experimental studies that corroborate these findings or from suggesting future experimental directions. -
Reviewer #2 (Public review):
Summary:
The authors aimed to explore how a key protein in the circadian clock of plants, ELF3, responds to temperature changes by forming molecular condensates. They focused on understanding the role of a specific region of the protein, a polyQ tract, in promoting temperature-sensitive structural changes and regulating the formation of condensates. Through a series of computational simulations, they sought to uncover the molecular basis for ELF3's temperature responsiveness and its broader implications for plant growth and adaptation to environmental conditions.
Strengths:
The study's strength lies in its focus on an important biological question: how plants sense and respond to temperature changes at the molecular level. The authors employed a variety of computational techniques, including coarse-grained …
Reviewer #2 (Public review):
Summary:
The authors aimed to explore how a key protein in the circadian clock of plants, ELF3, responds to temperature changes by forming molecular condensates. They focused on understanding the role of a specific region of the protein, a polyQ tract, in promoting temperature-sensitive structural changes and regulating the formation of condensates. Through a series of computational simulations, they sought to uncover the molecular basis for ELF3's temperature responsiveness and its broader implications for plant growth and adaptation to environmental conditions.
Strengths:
The study's strength lies in its focus on an important biological question: how plants sense and respond to temperature changes at the molecular level. The authors employed a variety of computational techniques, including coarse-grained simulations, to explore the role of specific molecular features in this process. These methods provide a multi-scale view of protein behavior and offer valuable insights into how molecular structures may influence biological function.
Weaknesses:
However, there are notable weaknesses in the evidence provided. While the authors present trends in molecular changes, such as shifts in helical propensity and the formation of condensates, these results seem subtle and are not strongly substantiated by statistical analysis. The lack of error bars in the figures makes it difficult to distinguish between meaningful signals and potential noise in the data. Furthermore, the temperature-sensitive behavior appears to be influenced more by chain length than by sequence-specific effects of the polyQ region, raising questions about whether the findings truly capture the molecular mechanisms responsible for temperature sensing. Additionally, some simulations, particularly those related to the formation of condensates, do not appear fully converged, which casts further doubt on the robustness of the results.
Additional Context for Readers:
Readers should interpret the results with caution, especially regarding the molecular mechanisms proposed for temperature sensing. While the study presents interesting trends, the evidence is not definitive, and the findings may be more reflective of general protein behavior (such as the effect of chain length on condensate formation) than specific sequence-driven responses to temperature. Further experimental studies and more converged simulations will be necessary to fully understand the role of ELF3 in temperature regulation.
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Author response:
We sincerely thank the reviewers for their constructive feedback and the editor for facilitating this thorough review. We found the suggestions insightful and valuable for refining our manuscript. We would like to clarify a few points in an initial response before presenting the fully updated manuscript. First of all, we would like to emphasize the multi-scale nature of our approach, where we derived insights from both atomistic and coarse-grained simulations. Reviewers focused mostly on the coarse-grained simulations, the drawbacks of which we are aware and were a strong motivation for starting with the atomistic approach. Reviewer 1 mentioned a lack of a proposed mechanism for the increased condensate forming propensity at 300K vs. 290K, and we feel we had clearly pointed to the aromatic contacts as a mechanism for …
Author response:
We sincerely thank the reviewers for their constructive feedback and the editor for facilitating this thorough review. We found the suggestions insightful and valuable for refining our manuscript. We would like to clarify a few points in an initial response before presenting the fully updated manuscript. First of all, we would like to emphasize the multi-scale nature of our approach, where we derived insights from both atomistic and coarse-grained simulations. Reviewers focused mostly on the coarse-grained simulations, the drawbacks of which we are aware and were a strong motivation for starting with the atomistic approach. Reviewer 1 mentioned a lack of a proposed mechanism for the increased condensate forming propensity at 300K vs. 290K, and we feel we had clearly pointed to the aromatic contacts as a mechanism for this, but we will make sure to clarify this further in the revision. Furthermore, reviewer 1 was critical of our use of the 10% adjustment to Martini protein-water interactions, which has previously been thoroughly presented and assessed in the literature (see for example Tesei et al JCTC 2022). Furthermore, for our specific system we were encouraged by the favorable comparison of our Martini simulations to the atomistic simulations, e.g. for radius of gyration, contact propensity, and solvent accessibility. We will make sure to emphasize this more clearly in the revision. Finally, we are grateful for the feedback from both reviewers and will use their comments as a guide to incorporate additional analyses and extended simulations to strengthen our conclusions in an upcoming revision.
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