Polyphosphate Discriminates Protein Conformational Ensembles More Efficiently than DNA Promoting Diverse Assembly and Maturation Behaviors
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eLife Assessment
This manuscript offers important insights into how polyphosphate (polyP) influences protein phase separation differently from DNA. The authors present compelling evidence that polyP distinguishes between protein conformational states, leading to diverse condensate behaviors. However, differences in charge density between polyP and DNA complicate direct comparisons, and the extent to which polyP-driven phase transitions reveal initial protein states remains unclear. Addressing these concerns would strengthen the manuscript's impact for researchers interested in biomolecular condensates, protein dynamics, and stress response mechanisms.
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Abstract
Disordered proteins and domains often assemble into condensates with polyanionic nucleic acids, primarily via charge complementarity, regulating numerous cellular functions. However, the assembly mechanisms associated with the other abundant and ubiquitous, anionic, stress-response regulating polymer, polyphosphate (polyP), is less understood. Here, we employ the intrinsically disordered DNA binding domain (DBD) of cytidine repressor (CytR) from E.coli to study the nature of assembly processes with polyP and DNA. CytR forms metastable liquid-like condensates with polyP and DNA, while undergoing liquid-to-solid transition in the former and solubilizing in the latter. On mutationally engineering the ensemble to exhibit more or less structure and dimensions than the WT, the assembly process with polyP is directed to either condensates with partial time-dependent solubilization or spontaneous aggregation, respectively. On the other hand, the CytR variants form only liquid-like but metastable droplets with DNA which solubilize within a few hours. Polyphosphate induces large secondary-structure changes, with two of the mutants adopting polyproline II-like structures within droplets, while DNA has only minimal structural effects. Our findings reveal how polyphosphate can more efficiently discern conformational heterogeneity in the starting protein ensemble, its structure, and compactness, with broad implications in assembly mechanisms involving polyP and stress response in bacterial systems.
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eLife Assessment
This manuscript offers important insights into how polyphosphate (polyP) influences protein phase separation differently from DNA. The authors present compelling evidence that polyP distinguishes between protein conformational states, leading to diverse condensate behaviors. However, differences in charge density between polyP and DNA complicate direct comparisons, and the extent to which polyP-driven phase transitions reveal initial protein states remains unclear. Addressing these concerns would strengthen the manuscript's impact for researchers interested in biomolecular condensates, protein dynamics, and stress response mechanisms.
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Reviewer #1 (Public review):
Summary:
In the article titled "Polyphosphate discriminates protein conformational ensembles more efficiently than DNA promoting diverse assembly and maturation behaviors," Goyal and colleagues investigate the role of negatively charged biopolymers, i.e., polyphosphate (polyP) and DNA, play in phase separation of cytidine repressor (CytR) and fructose repressor (FruR). The authors find that both negative polymers drive the formation of metastable protein/polymer condensates. However, polyP-driven condensates form more gel- or solid-like structures over time while DNA-driven condensates tend to dissipate over time. The authors link this disparate condensate behavior to polyP-induced structures within the enzymes. Specifically, they observe the formation of polyproline II-like structures within two tested …
Reviewer #1 (Public review):
Summary:
In the article titled "Polyphosphate discriminates protein conformational ensembles more efficiently than DNA promoting diverse assembly and maturation behaviors," Goyal and colleagues investigate the role of negatively charged biopolymers, i.e., polyphosphate (polyP) and DNA, play in phase separation of cytidine repressor (CytR) and fructose repressor (FruR). The authors find that both negative polymers drive the formation of metastable protein/polymer condensates. However, polyP-driven condensates form more gel- or solid-like structures over time while DNA-driven condensates tend to dissipate over time. The authors link this disparate condensate behavior to polyP-induced structures within the enzymes. Specifically, they observe the formation of polyproline II-like structures within two tested enzyme variants in the presence of polyP. Together their results provide a unique insight into the physical and structural mechanism by which two unique negatively charged polymers can induce distinct phase transitions with the same protein. This study will be a welcomed addition to the condensate field and provide new molecular insights into how binding partner-induced structural changes within a given protein can affect the mesoscale behavior of condensates. The concerns outlined below are meant to strengthen the manuscript.
Strengths:
Throughout the article, the authors used the correct techniques to probe physical changes within proteins that can be directly linked to phase transition behaviors. Their rigorous experiments create a clear picture of what occurs at the molecular level with CytR and FruR are exposed to either DNA or polyP, which are unique, highly negatively charged biopolymers found within bacteria. This work provides a new view of mechanisms by which bacteria can regulate the cytoplasmic organization upon the induction of stress. Furthermore, this is likely applicable to mammalian and plant cells and likely to numerous proteins that undergo condensation with nucleic acids and other charged biopolymers.
Weaknesses:
The biggest weakness of this study is that compares the phase behavior of enzymes driven by negatively charged polymers that have intrinsic differences in net charge and charge density. Because these properties are extremely important for controlling phase separation, any differences may result in the observed phase transitions driven by DNA and polyP. The authors should perform an additional experiment to control for these differences as best they can. The results from these experiments will provide additional insight into the importance of charge-based properties for controlling phase transitions.
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Reviewer #2 (Public review):
Summary:
In this study, Goyal et al demonstrate that the assembly of proteins with polyphosphate into either condensates or aggregates can reveal information on the initial protein ensemble. They show that, unlike DNA, polyphosphate is able to effectively discriminate against initial protein ensembles with different conformational heterogeneity, structure, and compactness. The authors further show that the protein native ensemble is vital on whether polyphosphate induces phase separation or aggregation, whereas DNA induces a similar outcome regardless of the initial protein ensemble. This work provides a way to improve our mechanistic understanding of how conformational transitions of proteins may regulate or drive LLPS condensate and aggregate assemblies within biological systems.
Strengths:
This is a …
Reviewer #2 (Public review):
Summary:
In this study, Goyal et al demonstrate that the assembly of proteins with polyphosphate into either condensates or aggregates can reveal information on the initial protein ensemble. They show that, unlike DNA, polyphosphate is able to effectively discriminate against initial protein ensembles with different conformational heterogeneity, structure, and compactness. The authors further show that the protein native ensemble is vital on whether polyphosphate induces phase separation or aggregation, whereas DNA induces a similar outcome regardless of the initial protein ensemble. This work provides a way to improve our mechanistic understanding of how conformational transitions of proteins may regulate or drive LLPS condensate and aggregate assemblies within biological systems.
Strengths:
This is a thoroughly conducted study that provides an alternative route for inducing phase separation that is more informative on the initial protein ensemble involved. This is particularly useful and a complementary means to investigate the role played by protein dynamics and plasticity in phase transitions. The authors use an appropriate set of techniques to investigate unique phase transitions within proteins induced by polyphosphates. An alternative protein system is used to corroborate their findings that the unique assemblies induced by polyphosphates when compared to DNA are not restricted to a single system. The work here is well-documented, easy to interpret, and of relevance for the condensate community.
Weaknesses:
The major weakness of this manuscript is that it is unclear if the information on the initial protein conformational ensemble can be determined solely from the assembly and maturation behavior and the discrimination abilities of polyphosphates. In both systems studied (CytR and FruR), polyphosphate discriminates and results in unique assemblies and maturation behaviors based on the initial protein ensemble. However, it seems the assembly and maturation behavior are not a direct result of the degree of conformational dynamics and plasticity in the initial protein. In the case of CytR, the fully-folded system forms condensates that resolubilize, while the highly disordered state immediately aggregates. Whereas, in the case of FruR, the folded state induces spontaneous aggregation, and the more dynamic, molten globular, system results in short-lived condensates. These results seem to suggest the polyphosphates' ability to discriminate between the initial protein ensemble may not be able to reveal what that initial protein ensemble is unless it is already known.
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