Decoding Phase Separation of Prion-Like Domains through Data-Driven Scaling Laws

Reviewer #1 (Public Review):

Summary:

In this preprint, the authors systematically and rigorously investigate how specific classes of residue mutations alter the critical temperature as a proxy for the driving forces for phase separation. The work is well executed, the manuscript well-written, and the results reasonable and insightful.

Strengths:

The introductory material does an excellent job of being precise in language and ideas while summarizing the state of the art. The simulation design, execution, and analysis are exceptional and set the standard for these types of large-scale simulation studies. The results, interpretations, and Discussion are largely nuanced, clear, and well-motivated.

Weaknesses:

This is not exactly a weakness, but I think it would future-proof the authors' conclusions to clarify a few key caveats associated with this work. Most notably, given the underlying implementation of the Mpipi model, temperature dependencies for intermolecular interactions driven by solvent effects (e.g., hydrophobic effect and charge-mediated interactions facilitated by desolvation penalties) are not captured. This itself is not a "weakness" per se, but it means I would imagine CERTAIN types of features would not be well-captured; notably, my expectation is that at higher temperatures, proline-rich sequences drive intermolecular interactions, but at lower temperatures, they do not. This is likely also true for the aliphatic residues, although these are found less frequently in IDRs. As such, it may be worth the authors explicitly discussing.

Similarly, prior work has established the importance of an alpha-helical region in TDP-43, as well as the role of aliphatic residues in driving TDP-43's assembly (see Schmidt et al 2019). I recognize the authors have focussed here on a specific set of mutations, so it may be worth (in the Discussion) mentioning [1] what impact, if any, they expect transient or persistent secondary structure to have on their conclusions and [2] how they expect aliphatic residues to contribute. These can and probably should be speculative as opposed to definitive.

Again - these are not raised as weaknesses in terms of this work, but the fact they are not discussed is a minor weakness, and the preprint's use and impact would be improved on such a discussion.

Reviewer #2 (Public Review):

This is an interesting manuscript where a CA-only CG model (Mpipi) was used to examine the critical temperature (Tc) of phase separation of a set of 140 variants of prion-like low complexity domains (PLDs). The key result is that Tc of these PLDs seems to have a linear dependence on substitutions of various sticker and space residues. This is potentially useful for estimating the Tc shift when making novel mutations of a PLD. However, I have strong reservations about the significance of this observation as well as some aspects of the technical detail and writing of the manuscript.

(1) Writing of the manuscript: The manuscript can be significantly shortened with more concise discussions. The current text reads as very wordy in places. It even appears that the authors may be trying a bit too hard to make a big deal out of the observed linear dependence.

The manuscript needs to be toned done to minimize self-promotion throughout the text. Some of the glaring examples include the wording "unprecedented", "our research marks a significant milestone in the field of computational studies of protein phase behavior ..", "Our work explores a new framework to describe, quantitatively, the phase behavior ...", and others.

There is really little need to emphasize the need to manage a large number of simulations for all 140 variants. Yes, some thoughts need to go into designing and managing the jobs and organizing the data, but it is pretty standard in computational studies. For example, large-scale protein ligand-free energy calculations can require one to a few orders of magnitude larger number of runs, and it is pretty routine.

When discussing the agreement with experimental results on Tm, it should be noted that the values of R > 0.93 and RMSD < 14 K are based on only 16 data points. I am not sure that one should refer to this as "extended validation". It is more like a limited validation given the small data size.

Results of linear fitting shown in Eq 4-12 should be summarized in a single table instead of scattering across multiple pages.

The title may also be toned down a bit given the limited significance of the observed linear dependence.

(2) Significance and reliability of Tc: Given the simplicity of Mpipi (a CA-only model that can only describe polymer chain dimension) and the low complexity nature of PLDs, the sequence composition itself is expected to be the key determinant of Tc. This is also reflected in various mean-field theories. It is well known that other factors will contribute, such as patterning (examined in this work as well), residual structures, and conformational preferences in dilute and dense phases. The observed roughly linear dependence is a nice confirmation but really unsurprising by itself. It appears how many of the constructs deviate from the expected linear dependence (e.g., Figure 4A) may be more interesting to explore.

The assumption that all systems investigated here belong to the same universality class as a 3D Ising model and the use of Eqn 20 and 21 to derive Tc is poorly justified. Several papers have discussed this issue, e.g., see Pappu Chem Rev 2023 and others. Muthukumar and coworkers further showed that the scaling of the relevant order parameters, including the conserved order parameter, does not follow the 3D Ising model. More appropriate theoretical models including various mean field theories can be used to derive binodal from their data, such as using Rohit Pappu's FIREBALL toolset. Imposing the physics of the 3D Ising model as done in the current work creates challenges for equivalence relationships that are likely unjustified.

While it has been a common practice to extract Tc when fitting the coexistence densities, it is not a parameter that is directly relevant physiologically. Instead, Csat would be much more relevant to think about if phase separation could occur in cells.

Reviewer #3 (Public Review):

Summary:

"Decoding Phase Separation of Prion-Like Domains through Data-Driven Scaling Laws" by Maristany et al. offers a significant contribution to the understanding of phase separation in prion-like domains (PLDs). The study investigates the phase separation behavior of PLDs, which are intrinsically disordered regions within proteins that have a propensity to undergo liquid-liquid phase separation (LLPS). This phenomenon is crucial in forming biomolecular condensates, which play essential roles in cellular organization and function. The authors employ a data-driven approach to establish predictive scaling laws that describe the phase behavior of these domains.

Strengths:

The study benefits from a robust dataset encompassing a wide range of PLDs, which enhances the generalizability of the findings. The authors' meticulous curation and analysis of this data add to the study's robustness. The scaling laws derived from the data provide predictive insights into the phase behavior of PLDs, which can be useful in the future for the design of synthetic biomolecular condensates.

Weaknesses:

While the data-driven approach is powerful, the study could benefit from more experimental validation. Experimental studies confirming the predictions of the scaling laws would strengthen the conclusions. For example, in Figure 1, the Tc of TDP-43 is below 300 K even though it can undergo LLPS under standard conditions. Figure 2 clearly highlights the quantitative accuracy of the model for hnRNPA1 PLD mutants, but its applicability to other systems such as TDP-43, FUS, TIA1, EWSR1, etc., may be questionable.

The authors may wish to consider checking if the scaling behavior is only observed for Tc or if other experimentally relevant quantities such as Csat also show similar behavior. Additionally, providing more intuitive explanations could make the findings more broadly accessible.

The study focuses on a particular subset of intrinsically disordered regions. While this is necessary for depth, it may limit the applicability of the findings to other types of phase-separating biomolecules. The authors may wish to discuss why this is not a concern. Some statements in the paper may require careful evaluation for general applicability, and I encourage the authors to exercise caution while making general conclusions. For example, "Therefore, our results reveal that it is almost twice more destabilizing to mutate Arg to Lys than to replace Arg with any uncharged, non-aromatic amino acid..." This may not be true if the protein has a lot of negative charges.

I am surprised that a quarter of a million CPU hours are described as staggering in terms of computational requirements.