Direct and Indirect Salt Effects on Homotypic Phase Separation

Reviewer #1 (Public Review):

Summary:

The authors examined the salt-dependent phase separation of the low-complexity domain of hnRN-PA1 (A1-LCD). Using all-atom molecular dynamics simulations, they identified four distinct classes of salt dependence in the phase separation of intrinsically disordered proteins (IDPs), which can be predicted based on their amino acid composition. However, the simulations and analysis, in their current form, are inadequate and incomplete.

Strengths:

The authors attempt to unravel the mechanistic insights into the interplay between salt and protein phase separation, which is important given the complex behavior of salt effects on this process. Their effort to correlate the influence of salt on the low-complexity domain of hnRNPA1 (A1-LCD) with a range of other proteins known to undergo salt-dependent phase separation is an interesting and valuable topic.

Weaknesses:

(1) The simulations performed are not sufficiently long (Figure 2A) to accurately comment on phase separation behavior. The simulations do not appear to have converged well, indicating that the system has not reached a steady state, rendering the analysis of the trajectories unreliable.

(2) The majority of the data presented shows no significant alteration with changes in salt concentration. However, the authors have based conclusions and made significant comments regarding salt activities. The absence of error bars in the data representation raises questions about its reliability. Additionally, the manuscript lacks sufficient scientific details of the calculations.

(3) In Figures 2B and 2C, the changes in the radius of gyration and the number of contacts do not display significant variations with changes in salt concentration. The change in the radius of gyration with salt concentration is less than 1 Å, and the number of contacts does not change by at least 1. The authors' conclusions based on these minor changes seem unfounded.

Reviewer #2 (Public Review):

This is an interesting computational study addressing how salt affects the assembly of biomolecular condensates. The simulation data are valuable as they provide a degree of atomistic details regarding how small salt ions modulate interactions among intrinsically disordered proteins with charged residues, namely via Debye-like screening that weakens the effective electrostatic interactions among the polymers, or through bridging interactions that allow interactions between like charges from different polymer chains to become effectively attractive (as illustrated, e.g., by the radial distribution functions in Supplementary Information). However, this manuscript has several shortcomings: (i) Connotations of the manuscript notwithstanding, many of the authors' concepts about salt effects on biomolecular condensates have been put forth by theoretical models, at least back in 2020 and even earlier. Those earlier works afford extensive information such as considerations of salt concentrations inside and outside the condensate (tie-lines). But the authors do not appear to be aware of this body of prior works and therefore missed the opportunity to build on these previous advances and put the present work with its complementary advantages in structural details in the proper context. (ii) There are significant experimental findings regarding salt effects on condensate formation [which have been modeled more recently] that predate the A1-LCD system (ref.19) addressed by the present manuscript. This information should be included, e.g., in Table 1, for sound scholarship and completeness. (iii) The strengths and limitations of the authors' approach vis-à-vis other theoretical approaches should be discussed with some degree of thoroughness (e.g., how the smallness of the authors' simulation system may affect the nature of the "phase transition" and the information that can be gathered regarding salt concentration inside vs. outside the "condensate" etc.). Accordingly, this manuscript should be revised to address the following. In particular, the discussion in the manuscript should be significantly expanded by including references mentioned below as well as other references pertinent to the issues raised.

(1) The ability to use atomistic models to address the questions at hand is a strength of the present work. However, presumably because of the computational cost of such models, the "phase-separated" "condensates" in this manuscript are extremely small (only 8 chains). An inspection of Fig.1 indicates that while the high-salt configuration (snapshot, bottom right) is more compact and droplet-like than the low-salt configuration (top right), it is not clear that the 50 mM NaCl configuration can reasonably correspond to a dilute or homogeneous phase (without phase separation) or just a condensate with a lower protein concentration because the chains are still highly associated. One may argue that they become two droplets touching each other (the chains are not fully dispersed throughout the simulation box, unlike in typical coarse-grained simulations of biomolecular phase separation). While it may not be unfair to argue from this observation that the condensed phase is less stable at low salt, this raises critical questions about the adequacy of the approach as a stand-alone source of theoretical information. Accordingly, an informative discussion of the limitation of the authors' approach and comparisons with results from complementary approaches such as analytical theories and coarse-grained molecular dynamics will be instructive-even imperative, especially since such results exist in the literature (please see below).

(2) The aforementioned limitation is reflected by the authors' choice of using Dmax as a sort of phase-separation order parameter. However, no evidence was shown to indicate that Dmax exhibits a two-state-like distribution expected of phase separation. It is also not clear whether a Dmax value corresponding to the linear dimension of the simulation box was ever encountered in the authors' simulated trajectories such that the chains can be reliably considered to be essentially fully dispersed as would be expected for the dilute phase. Moreover, as the authors have noted in the second paragraph of the Results, the variation of Dmax with simulation time does not show a monotonic rank order with salt concentration. The authors' explanation is equivalent to stipulating that the simulation system has not fully equilibrated, inevitably casting doubt on at least some of the conclusions drawn from the simulation data.

(3) With these limitations, is it realistic to estimate possible differences in salt concentration between the dilute and condensed phases in the present work? These features, including tie-lines, were shown to be amenable to analytical theory and coarse-grained molecular dynamics simulation (please see below).

(4) In the comparison in Fig.2B between experimental and simulated radius of gyration as a function of [NaCl], there is an outlier among the simulated radii of gyration at [NaCl] ~ 250 mM. An explanation should be offered.

(5) The phenomenon of no phase separation at zero and low salt and phase separation at higher salt has been observed for the IDP Caprin1 and several of its mutants [Wong et al., J Am Chem Soc 142, 2471-2489 (2020) [https://pubs.acs.org/doi/full/10.1021/jacs.9b12208], see especially Fig.9 of this reference]. This work should be included in the discussion and added to Table 1.

(6) The authors stated in the Introduction that "A unifying understanding of how salt affects the phase separation of IDPs is still lacking". While it is definitely true that much remains to be learned about salt effects on IDP phase separation, the advances that have already been made regarding salt effects on IDP phase separation is more abundant than that conveyed by this narrative. For instance, an analytical theory termed rG-RPA was put forth in 2020 to provide a uniform (unified) treatment of salt, pH, and sequence-charge-pattern effects on polyampholytes and polyelectrolytes (corresponding to the authors' low net charge and high net charge cases). This theory offers a means to predict salt-IDP tie-lines and a comprehensive account of salt effect on polyelectrolytes resulting in a lack of phase separation at extremely low salt and subsequent salt-enhanced phase separation (similar to the case the authors studied here) and in some cases re-entrant phase separation or dissolution [Lin et al., J Chem Phys 152. 045102 (2020) [https://doi.org/10.1063/1.5139661]]. This work is highly relevant and it already provided a conceptual framework for the authors' atomistic results and subsequent discussion. As such, it should definitely be a part of the authors' discussion.

(7) Bridging interactions by small ions resulting in effective attractive interactions among polyelectrolytes leading to their phase separation have been demonstrated computationally by Orkoulas et al., Phys Rev Lett 90, 048303 (2003) [https://journals.aps.org/prl/abstract/10.1103/PhysRevLett.90.048303]. This result should also be included in the discussion.

(8) More recently, the salt-dependent phase separations of Caprin1, its RtoK variants and phosphorylated variant (see item #5 above) were modeled (and rationalized) quite comprehensively using rG-RPA, field-theoretic simulation, and coarse-grained molecular dynamics [Lin et al., arXiv:2401.04873 [https://arxiv.org/abs/2401.04873]], providing additional data supporting a conceptual perspective put forth in Lin et al. J Chem Phys 2020 (e.g., salt-IDP tie-lines, bridging interactions, re-entrance behaviors etc.) as well as in the authors' current manuscript. It will be very helpful to the readers of eLife to include this preprint in the authors' discussion, perhaps as per the authors' discretion-along the manner in which other preprints are referenced and discussed in the current version of the manuscript.

Reviewer #3 (Public Review):

Summary:

This study investigates the salt-dependent phase separation of A1-LCD, an intrinsically disordered region of hnRNPA1 implicated in neurodegenerative diseases. The authors employ all-atom molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which salt influences A1-LCD phase separation. Contrary to typical intrinsically disordered protein (IDP) behavior, A1-LCD phase separation is enhanced by NaCl concentrations above 100 mM. The authors identify two direct effects of salt: neutralization of the protein's net charge and bridging between protein chains, both promoting condensation. They also uncover an indirect effect, where high salt concentrations strengthen pi-type interactions by reducing water availability. These findings provide a detailed molecular picture of the complex interplay between electrostatic interactions, ion binding, and hydration in IDP phase separation.

Strengths:

• Novel Insight: The study challenges the prevailing view that salt generally suppresses IDP phase separation, highlighting A1-LCD's unique behavior.
• Rigorous Methodology: The authors utilize all-atom MD simulations, a powerful computational tool, to investigate the molecular details of salt-protein interactions.
• Comprehensive Analysis: The study systematically explores a wide range of salt concentrations, revealing a nuanced picture of salt effects on phase separation.
• Clear Presentation: The manuscript is well-written and logically structured, making the findings accessible to a broad audience.

Weaknesses:

• Limited Scope: The study focuses solely on the truncated A1-LCD, omitting simulations of the full-length protein. This limitation reduces the study's comparative value, as the authors note that the full-length protein exhibits typical salt-dependent behavior. A comparative analysis would strengthen the manuscript's conclusions and broaden its impact.

Overall, this manuscript represents a significant contribution to the field of IDP phase separation. The authors' findings provide valuable insights into the molecular mechanisms by which salt modulates this process, with potential implications for understanding and treating neurodegenerative diseases. While the study is well-conducted and clearly presented, further research is needed to validate the findings and explore their broader applicability.