Structural basis for the phase separation of the chromosome passenger complex

  1. Nikaela W Bryan
  2. Aamir Ali
  3. Ewa Niedzialkowska
  4. Leland Mayne
  5. P Todd Stukenberg
  6. Ben E Black

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Abstract

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as ‘fuzzy’. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation – a heterotrimer of INCENP, Survivin, and Borealin – we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

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  1. Version published to 10.7554/elife.92709 on eLife
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    1. General Statements

    We were naturally pleased to read the enthusiasm coming from both reviewers. Both mentioned that an extension to experimentation in cells would increase the impact of the study, even though both recognize that the biophysical and biochemical experiments constitute a study that is significant and interesting to a broad readership.

    2. Point-by-point description of the revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    This manuscript by Bryan et al., describes the use of Hydrogen/Deuterium-exchange Mass Spectrometry (HXMS) as a powerful tool to identify key amino acid residues and associated interactions driving liquid-liquid demixing. They have particularly focused on the Chromosomal Passenger Complex (CPC), an important regulator of chromosome segregation, which has recently been shown to undergo liquid-liquid demixing in vitro. Their work presented here allowed them to identify a few key electrostatic interactions as molecular determinants driving the liquid-liquid demixing of the CPC. Their work also shows that crystal packing information of protein molecules, where available, can provide valuable insight into likely factors driving liquid-liquid demixing.

    Major comments:

    [#1] A previous study by Trivedi et al., NCB 2019 identified an unstructured region in Borealin (aa residues 139-160) as the main region driving the phase separation of CPC. Interestingly, this region only shows a moderate reduction in HX upon liquid-liquid demixing. But no experiments or discussions related to this observation are presented in the current version of the manuscript.

    In the Trivedi et al. paper, the authors were careful to state that the region of borealin between 139-160 contributed to phase separation, but there was clearly a remaining propensity to phase separate in vitro in the mutant. Thus, it is fully expected that there should be other regions in the complex that contribute to phase separation. It was satisfying that this region was independently identified in the hydrogen-deuterium exchange experiments and we suggest that a “moderate” reduction is consistent with a protein condensate having liquid properties. Since this region was already characterized we have focused our work in this paper to the new region identified by the hydrogen-deuterium exchange experiments.

    [#2] In the absence of cellular data on if and how these mutations (within the triple-helical bundle region) affect CPC's ability to phase separate in cells, the implication of this work is very limited - One can't say for sure these are interactions driving phase separation of CPC in a cellular environment. In the absence of any cellular data with the mutants described here, much of the discussion on the possible roles of CPC phase separation in cells does not appear relevant to this manuscript. I would suggest that the authors focus mainly on highlighting the power of using HXMS as a tool to characterise the molecular determinants of liquid-liquid demixing at a relatively high resolution.

    We have now added cellular data in the form of one of the key experiments used to explore CPC liquid-liquid demixing utilizing the Cry2 optogenetic system for inducible dimerization. The results of testing WT Borealin versus the mutant we identified is defective in droplet formation are shown in the all new Fig. 6. Some relation of our overall findings, encompassing observations made with purified components and now in cells, to the cellular function of the CPC is pertinent. In light of the reviewer comments, we have also reduced this aspect in the discussion (see the substantial edits on pg. 12).

    Minor comments:

    [#3] The authors should ensure that the introduction cites relevant literature thoroughly. For example, where the potential role of Borealin residues 139-160 in conferring phase separation properties to the CPC is mentioned, the authors failed to cite Abad et al., 2019, which showed the contribution of the same Borealin region in conferring nucleosome binding ability to the CPC.

    We have made this particular change on pg. 4 and also have gone through to ensure we are appropriately citing relevant literature.

    Reviewer #1 (Significance (Required)):

    This is a highly relevant and significant work, particularly considering the rapidly growing list of examples for Phase separation of proteins/protein assemblies and their potential biological roles (in spite of ongoing debates in the field about the cellular relevance of several phase separation claims). The data presented in this manuscript are solid and convincingly establish HXMS as a useful tool to characterise molecular interactions driving liquid-liquid demixing. Considering its applicability to characterise wide-ranging protein assemblies implicated in phase separation, this work will be of interest to a broad readership.

    We thank the reviewer for the strong praise of the significance of our study.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    In this manuscript, using the technique of hydrogen/deuterium-exchange mass spectrometry (HXMS), the authors have tried to gain insights into the structure of the chromosomal passenger complex (CPC) within the phase separated chromatin body, known to regulate chromosome segregation in mitosis. The CPC phase separated compartment comprises three regulatory and targeting subunits, INCENP, Survivin, and Borealin, forming a three-helix bundle hetero-trimer. By measuring changes in the polypeptide backbone dynamics of this trimeric INCENP/Survivin/Borealin complex, in the liquid-liquid de-mixed state in comparison to its soluble state, using HXMS measurements, the paper puts forward high-resolution structural details of the phase separated CPC. Using a step-wise mutagenesis approach in conjunction with the information from HXMS measurements and previous crystallographic data, this work also identifies distinct regions/interfaces within this complex harboring crucial salt bridges, which directly contribute toward the liquid-liquid demixing of the CPC. Comments:

    1. "The three non-catalytic subunits of the CPC (INCENP1-58, Borealin, and Survivin) form soluble homotrimers that have a propensity to undergo liquid-liquid phase separation.8 " Do the authors mean the hetero-trimeric CPC?

    Yes, we meant heterotrimers. It is now corrected.

    1. For better clarity, the authors can indicate the residue numbers of each of the components INCENP, Survivin, and Borealin in the CPC trimeric helix-bundle crystallographic structure in Fig 1.

    These are included on the revised Figure 1A.

    1. "In the condition we identified, 90% +/- 5% of the ISB protein was found within the rapidly sedimenting droplet population (Fig. 1C)." The authors should include the time-point corresponding to the gel shown in Fig 1C.

    This information is now directly labeled in Fig. 1C.

    1. Prior to the HXMS experiments on the phase-separated ISB protein complex, were the samples subjected to sedimentation to separate the dispersed from the condensed droplet phase? Since several time points after formation of phase-separated ISB complex have been characterized to compare and contrast between the dispersed and the droplet phase, the authors can consider performing a time-dependent sedimentation assay to ascertain the fraction of the ISB complex in the droplet phase.

    The HXMS experiments were not performed on sedimented samples, so this complication in our HX workflow is not necessary. We note that the sedimentation that we include in our study (Figs. 1C, 5E, and S6), involves centrifugation for 10 minutes, and that length of time presents a substantial design challenge to our HX experimentation. We considered it at the outset of our study, but, in the end, our study was facilitated by our finding early on that this separation step was unnecessary. Further, we note that we report statistically significant differences at the earliest HX timepoints in the areas prominently protected from HX upon droplet formation (10 and 100 s; see Fig. 1C for an example). Indeed, we do not observe broadening of our HXMS spectra (examples shown for all timepoints, Fig. 2B,F) that would be expected if there were a large degree of mixed states (i.e. a large population of molecules in the free protein state and a large population of molecules in the droplet state) each having different HXMS rates. One can imagine that this sort of envelope broadening behavior (“EX1-like”) could be observed in other samples where there are multiple substantially populated states of a protein present at a particular timepoint, but this is not what we observe in the experiments we performed in this study.

    1. "At the 100 s timepoint, the most prominent differences between the soluble and droplet state were located within the three-helix bundle of the ISB, with long stretches in two subunits (INCENP and Borealin) and a small region at the N-terminal portion of the impacted a-helix in Survivin (Fig. 1F)" According to Fig 1F, at the 100 s time-point, there is also another small region in Survivin (approximately residues 12-20) that exhibits slower exchange rates in the droplet state. Can the authors comment on whether this region undergoes any conformational change or if it exhibits homotypic interactions retarding the hydrogen/deuterium exchange rates in the droplet phase?

    Our general approach in the Black lab over the past decade-plus of HXMS has been to restrict our conclusions whenever practical to do so to the consensus behavior. This permits multiple partially overlapping peptides to be used to generate confidence in the changes that drive our conclusions. The reviewer carefully recognizes the behavior of a single peptide (in 2 different charge states) that might have actual changes relative to some of the longer peptides that it partially overlaps with, and smaller changes can yield larger percentage changes on small peptides. We have chosen to not include this single peptide in the text describing our main conclusions from the work to be consistent with our longstanding strategy for rigorous interpretation of HXMS data. Our conclusion is that this region of not substantially changed upon droplet formation.

    1. The authors mention that: "By the latest timepoint, 3000 s, there was some diminution in the number of droplets which may indicate the start of a transition of the droplets to a more solid state (i.e., gel-like)." As a result of this time points beyond 3000 s have not been used for comparing Hydrogen/Deuterium exchange rates in the condensed droplet phase with the soluble state. Can the authors comment on what happens to the nature of these specific interactions between the components of the CPC in the 'gel-like state'? A combination of both non-specific weak interactions as well as strong site-specific interactions between macromolecular components has been widely known to contribute towards the formation of several phase-separated compartments. It will be interesting to know the perspective of the authors on what sort of interactions get populated within these compartments to give rise to a more solid gel-like state. At this later time points, do the droplets exhibit reversibility under higher ionic strength conditions? Do the authors have some data to show how the material property of these droplets evolve as a function of time?

    We offered the idea of a transition to a more solid state to the reader because it was a reasonable conclusion, although challenging to prove (something the Stukenberg lab is actively working on, though, see our response to point #9, below). The vast majority of our conclusions in the paper, and essentially all of what we emphasize are the important ones, are based on earlier timepoints where this is not an issue. Thus, we find an extended study of the late-developing features in our droplets something more appropriate for separate studies outside the scope of the current one.

    1. "Examination of the entire time course shows that during intermediate levels of HX (i.e., between 100-1000 s), this region takes about three times as long to undergo the same amount of exchange when the ISB is in the droplet state relative to when it's in the free protein state (Figs. 2B, C and Supplemental Fig. 2). Upon droplet formation, HX protection within Borealin is primarily located in the interacting a-helix and is less pronounced at any given peptide when compared to INCENP peptides (Fig. 2E). Nonetheless, similar to INCENP peptides, it still takes about twice as long to achieve the same level of deuteration for this region of Borealin in the droplet state as compared to the free state." How do the hydrogen/deuterium exchange rates and extent of deuteration in the N-terminal part (residues 98-142) of the Survivin polypeptide chain, constituting the three-helix bundle core, evolve as a function of time? Also, how do the exchange rates for peptides in this region compare with those of the other protein subunits Borealin and INCENP and what inference can be drawn from these differences?

    The peptides from a.a. 98-142 of Survivin exhibit HX protection through the timecourse (and before and after droplet formation) consistent with a folded a-helix (and comparable to the overall HX behavior of the other helices in the 3-helix bundle of the ISB)(Fig. S2). There is subtly slower HX in the droplet state for this region at later timepoints for this portion of Survivin (Fig. S4), and this is explicitly highlighted in the Results section on pg. 6.

    1. The authors mention that mutating either all the glutamate residues or combinations of these residues on the acidic patch on the INCENP subunit, to positively charged residues, causes a decrease in the propensity of phase separation, as formation of salt bridges with Borealin subunit from adjacent hetero-trimeric complexes appears to be the major driving force for phase separation. Can the authors elaborate on how the reduction in the phase separation propensity of these salt-bridge inhibiting mutants might be directly affecting the subsequent localization of the CPC to the inner centromeres? Can the authors supplement their existing in vitro data with further in vivo characterization of CPC recruitment or localization to the centromeres, for each of the constructs exhibiting reduced propensity of phase separation?

    As we state in the introduction, the recruitment to centromeres requires established ‘conventional’ targeting via the specific histone marks to which we refer. We also cite the correlations demonstrated between prior mutations in Borealin (impacting aa 139-160) that both disrupt phase separation in vitro and reduce CPC levels at the centromere. In our revision, we have added what we feel are the most critical cell-based experiments to relate to our HX studies in the new Fig. 6. We are preparing for future studies to study mutants arising from our HX studies, and our plans are to pursue gene replacement approaches that will rigorously test the impact on the mitotic function of the CPC. In the process of these future studies, the impact on localization will be measured, too. As others in the field are investigating the correlations between observations made with purified components and those made in the cell, and where there are nuances at play in how the actual experiments are conducted, we are certain our cell-based studies will extend far beyond the timeframe appropriate for our HX-focused study. Rigorous cell-based studies of mitotic functions are what is needed, however, and we have made our plans with that in mind.

    1. It might be really interesting for the authors to look at the recent preprint from Hedtfeld et al. 2023 Molecular Cell, (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4472737). In this preprint they have recombinantly purified a stoichiometric trimer (referred to as CPC-TARGWT) comprising full length survivin, borealin, and a 1-350 residue fragment of INCENP (instead of 1-58 used in this study) and have tried to assess if any correlation exists between the in-vitro phase behaviour of CPC-TARGWT mutants and their corresponding recruitment to the inner centromere, to form a phase separated compartment. Targeting residues in the BIR domain of Survivin involved in interactions with the N-terminus of the Histone H3, Shugosin 1 or in the recognition of H3T3phos, and substituting them with Alanine or completely deleting C-terminal domain of Borealin (a region implicated in CPC dimerization and centromere recruitment), was found to result in poor centromere localization, although the in vitro phase separation properties of these constructs were found to be indistinguishable, suggesting no evident correlation between the two phenomena. Thus it might be a useful piece of data to correlate the phase separation propensities of the ISB complex variants used in this current study with the extents of their in vivo recruitment to the inner centromere. This maybe beyond the scope of the paper, but it would be good to comment on this.

    For the correlation studies, please refer to our response to point #8, above. From our reading of the June 2023 preprint that the reviewer mentions, the main concern raised by the authors is questioning whether the region first identified in the Trivedi et al paper in Borealin (aa 139-160) has a role in phase separation. As the reviewer noted, Hedtfeld et al report using a complex that includes more of the INCENP protein than used in the Trivedi et al study, complicating the direct comparison between studies. Using the data in figure 5E of the Hedtfeld et al preprint, the authors suggest that the condensate formation of their version of the Borealin mutant D139-160 in vitro complex has similar phase separation properties as the wild type. However, we note that in our inspection of these data we see numerous differences. The mutant forms rounder, and larger condensates than WT and have reduced concentration of protein (less bright intensity). Finally only the WT protein has a “grape bunch” morphology. We note that unpublished data in the Stukenberg lab show these same differences can represent a defect in liquid demixing properties of a version of the purified CPC. While it is intuitive that larger condensates represent more phase separation, the unpublished data mentioned above suggests the opposite is true for the CPC. In particular, the data from the Stukenberg lab suggest the size of a droplet is mostly governed by the amount of droplet fusion in the first minutes after dilution and thus is limited by relatively rapid hardening of the complex. We note that in the course of discussions with the corresponding author of the preprint mentioned by the reviewer we did apprise them of the unpublished observations mentioned, above, in case they saw fit to include in their ongoing studies what would seem to be critical measurements (e.g. measuring circularity, droplet size, droplet intensity, and FRAP) to assess our suspicion that their construct contains a portion of INCENP that can accelerate condensate formation. If true, the Hedtfeld et al data are fully consistent with the Borealin mutant D139-160 having a significant condensate formation potential than the WT protein.

    10[A]) "Our data also provide an important clue about the previously identified region on Borealin that is required for liquid-demixing in vitro and proper CPC assembly in cells 8. Specifically, our data (Fig. 1F, Supplementary Figs. 2, 4A) suggest this region of Borealin adopts secondary structure that undergoes additional HX protection in the liquid-liquid demixed state" This data fits perfectly with previous studies from Trivedi et al. (2019), which states that deletion of the Borealin 139-160 fragment obliterates its phase separation in vitro and also reduces the accumulation of CPC at the centromere. On the contrary, in the recent preprint from Hedtfeld et al. 2023 Molecular Cell, they have shown that the phase separation behaviour of their reconstituted CPC-TARGWT harboring the Borealin 139-160 deletion mutant was found to be indistinguishable from the WT. Can the authors comment on what might be the reason for this difference? Is it possible that this central Borealin region is involved in interactions with the additional fragment of INCENP subunit used in the helical bundle reconstitution, or with other centromere component proteins, whereby the deletion of region is causing inefficient recruitment to the inner centromere? This can be elaborated in the discussion section of the manuscript.

    This is discussed in the response to #9, above. Through this format (the Review Commons procedure for public posting of author responses before submission of the study to a journal), our comments herein will be made public for those with the most interest in comparing our data to what is has been posted on preprint servers. We think that is the most appropriate for now, with more to surely come when the aforementioned results from the Stukenberg lab are posted/published and, hopefully when there is more information about the nature of the droplets reported in the Hedtfeld et al., study.

    10 [B]) It is also well known that in addition to these electrostatic interactions, the core of the ISB helical bundle is formed by an extensive network of hydrophobic interactions. Have the authors ever looked into how perturbing any of these intra-trimeric complex hydrophobic interactions affect their ability to phase separate and perform their subsequent function?

    We think there is some confusion, here. The electrostatics we focus on are between heterotrimers rather than within them. We certainly would predict that disrupting the hydrophobic surface that generates a stable heterotrimer would, in turn, disrupt individual heterotrimers. Our study assumes a stable heterotrimer as a starting point, so we view this type of perturbation as unrelated to our conclusions.

    1. The phase separated CPC compartment is known to enrich several other inner centromere proteins such as the Histone H3, Sgo1, the histone H3T3phos, among others. Have the authors tried to increase the complexity of the reconstituted CPC scaffold by incorporating more components to look into whether that changes any of the interaction interfaces between the ISB trimeric complexes within the condensed phase? Can this CPC compartment be reconstituted using a bottom-up approach?

    We are glad that our studies with a reductionist biochemical reconstitution approach have inspired the questions that require increased complexity. They are now warranted based on the advance we have made in the present study, and hopefully will form the basis for future, separate studies.

    Overall, this paper brings forward a useful technique to probe the conformational landscape of proteins in the condensed droplet phase and compare it with its dispersed phase. This paper serves as an interesting read showing how specific salt-bridge interactions between multiple stoichiometric protein complexes can be the driving force for phase separation.

    __Reviewer #2 __(Significance (Required)):

    In this manuscript, using the technique of hydrogen/deuterium-exchange mass spectrometry (HXMS), the authors have tried to gain insights into the structure of the chromosomal passenger complex (CPC) within the phase separated chromatin body, known to regulate chromosome segregation in mitosis. The CPC phase separated compartment comprises three regulatory and targeting subunits, INCENP, Survivin, and Borealin, forming a three-helix bundle hetero-trimer. By measuring changes in the polypeptide backbone dynamics of this trimeric INCENP/Survivin/Borealin complex, in the liquid-liquid de-mixed state in comparison to its soluble state, using HXMS measurements, the paper puts forward high-resolution structural details of the phase separated CPC. Using a step-wise mutagenesis approach in conjunction with the information from HXMS measurements and previous crystallographic data, this work also identifies distinct regions/interfaces within this complex harboring crucial salt bridges, which directly contribute toward the liquid-liquid demixing of the CPC.

    Overall, this paper brings forward a useful technique to probe the conformational landscape of proteins in the condensed droplet phase and compare it with its dispersed phase. This paper serves as an interesting read showing how specific salt-bridge interactions between multiple stoichiometric protein complexes can be the driving force for phase separation

    We thank the reviewer for the positive comments on the significance of our study.

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    Referee #2

    Evidence, reproducibility and clarity

    Structural Basis for the Phase Separation of the Chromosome Passenger Complex Nikaela W. Bryan, Ewa Niedzialkowska, Leland Mayne, P. Todd Stukenberg, and Ben E. Black# Reviewer Comments Manuscript Number: RC-2023-02017

    In this manuscript, using the technique of hydrogen/deuterium-exchange mass spectrometry (HXMS), the authors have tried to gain insights into the structure of the chromosomal passenger complex (CPC) within the phase separated chromatin body, known to regulate chromosome segregation in mitosis. The CPC phase separated compartment comprises three regulatory and targeting subunits, INCENP, Survivin, and Borealin, forming a three-helix bundle hetero-trimer. By measuring changes in the polypeptide backbone dynamics of this trimeric INCENP/Survivin/Borealin complex, in the liquid-liquid de-mixed state in comparison to its soluble state, using HXMS measurements, the paper puts forward high-resolution structural details of the phase separated CPC. Using a step-wise mutagenesis approach in conjunction with the information from HXMS measurements and previous crystallographic data, this work also identifies distinct regions/interfaces within this complex harboring crucial salt bridges, which directly contribute toward the liquid-liquid demixing of the CPC.

    Comments:

    1. "The three non-catalytic subunits of the CPC (INCENP1-58, Borealin, and Survivin) form soluble homotrimers that have a propensity to undergo liquid-liquid phase separation.8 " Do the authors mean the hetero-trimeric CPC?
    2. For better clarity, the authors can indicate the residue numbers of each of the components INCENP, Survivin, and Borealin in the CPC trimeric helix-bundle crystallographic structure in Fig 1.
    3. "In the condition we identified, 90% +/- 5% of the ISB protein was found within the rapidly sedimenting droplet population (Fig. 1C)." The authors should include the time-point corresponding to the gel shown in Fig 1C.
    4. Prior to the HXMS experiments on the phase-separated ISB protein complex, were the samples subjected to sedimentation to separate the dispersed from the condensed droplet phase? Since several time points after formation of phase-separated ISB complex have been characterized to compare and contrast between the dispersed and the droplet phase, the authors can consider performing a time-dependent sedimentation assay to ascertain the fraction of the ISB complex in the droplet phase.
    5. "At the 100 s timepoint, the most prominent differences between the soluble and droplet state were located within the three-helix bundle of the ISB, with long stretches in two subunits (INCENP and Borealin) and a small region at the N-terminal portion of the impacted a-helix in Survivin (Fig. 1F)" According to Fig 1F, at the 100 s time-point, there is also another small region in Survivin (approximately residues 12-20) that exhibits slower exchange rates in the droplet state. Can the authors comment on whether this region undergoes any conformational change or if it exhibits homotypic interactions retarding the hydrogen/deuterium exchange rates in the droplet phase?
    6. The authors mention that: "By the latest timepoint, 3000 s, there was some diminution in the number of droplets which may indicate the start of a transition of the droplets to a more solid state (i.e., gel-like)." As a result of this time points beyond 3000 s have not been used for comparing Hydrogen/Deuterium exchange rates in the condensed droplet phase with the soluble state. Can the authors comment on what happens to the nature of these specific interactions between the components of the CPC in the 'gel-like state'? A combination of both non-specific weak interactions as well as strong site-specific interactions between macromolecular components has been widely known to contribute towards the formation of several phase-separated compartments. It will be interesting to know the perspective of the authors on what sort of interactions get populated within these compartments to give rise to a more solid gel-like state. At this later time points, do the droplets exhibit reversibility under higher ionic strength conditions? Do the authors have some data to show how the material property of these droplets evolve as a function of time?
    7. "Examination of the entire time course shows that during intermediate levels of HX (i.e., between 100-1000 s), this region takes about three times as long to undergo the same amount of exchange when the ISB is in the droplet state relative to when it's in the free protein state (Figs. 2B, C and Supplemental Fig. 2). Upon droplet formation, HX protection within Borealin is primarily located in the interacting a-helix and is less pronounced at any given peptide when compared to INCENP peptides (Fig. 2E). Nonetheless, similar to INCENP peptides, it still takes about twice as long to achieve the same level of deuteration for this region of Borealin in the droplet state as compared to the free state." How do the hydrogen/deuterium exchange rates and extent of deuteration in the N-terminal part (residues 98-142) of the Survivin polypeptide chain, constituting the three-helix bundle core, evolve as a function of time? Also, how do the exchange rates for peptides in this region compare with those of the other protein subunits Borealin and INCENP and what inference can be drawn from these differences?
    8. The authors mention that mutating either all the glutamate residues or combinations of these residues on the acidic patch on the INCENP subunit, to positively charged residues, causes a decrease in the propensity of phase separation, as formation of salt bridges with Borealin subunit from adjacent hetero-trimeric complexes appears to be the major driving force for phase separation. Can the authors elaborate on how the reduction in the phase separation propensity of these salt-bridge inhibiting mutants might be directly affecting the subsequent localization of the CPC to the inner centromeres? Can the authors supplement their existing in vitro data with further in vivo characterization of CPC recruitment or localization to the centromeres, for each of the constructs exhibiting reduced propensity of phase separation?
    9. It might be really interesting for the authors to look at the recent preprint from Hedtfeld et al. 2023 Molecular Cell, (https://papers.ssrn.com/sol3/papers.cfm?abstract_id=4472737). In this preprint they have recombinantly purified a stoichiometric trimer (referred to as CPC-TARGWT) comprising full length survivin, borealin, and a 1-350 residue fragment of INCENP (instead of 1-58 used in this study) and have tried to assess if any correlation exists between the in-vitro phase behaviour of CPC-TARGWT mutants and their corresponding recruitment to the inner centromere, to form a phase separated compartment. Targeting residues in the BIR domain of Survivin involved in interactions with the N-terminus of the Histone H3, Shugosin 1 or in the recognition of H3T3phos, and substituting them with Alanine or completely deleting C-terminal domain of Borealin (a region implicated in CPC dimerization and centromere recruitment), was found to result in poor centromere localization, although the in vitro phase separation properties of these constructs were found to be indistinguishable, suggesting no evident correlation between the two phenomena. Thus it might be a useful piece of data to correlate the phase separation propensities of the ISB complex variants used in this current study with the extents of their in vivo recruitment to the inner centromere. This maybe beyond the scope of the paper, but it would be good to comment on this.
    10. "Our data also provide an important clue about the previously identified region on Borealin that is required for liquid-demixing in vitro and proper CPC assembly in cells 8. Specifically, our data (Fig. 1F, Supplementary Figs. 2, 4A) suggest this region of Borealin adopts secondary structure that undergoes additional HX protection in the liquid-liquid demixed state" This data fits perfectly with previous studies from Trivedi et al. (2019), which states that deletion of the Borealin 139-160 fragment obliterates its phase separation in vitro and also reduces the accumulation of CPC at the centromere. On the contrary, in the recent preprint from Hedtfeld et al. 2023 Molecular Cell, they have shown that the phase separation behaviour of their reconstituted CPC-TARGWT harboring the Borealin 139-160 deletion mutant was found to be indistinguishable from the WT. Can the authors comment on what might be the reason for this difference? Is it possible that this central Borealin region is involved in interactions with the additional fragment of INCENP subunit used in the helical bundle reconstitution, or with other centromere component proteins, whereby the deletion of region is causing inefficient recruitment to the inner centromere? This can be elaborated in the discussion section of the manuscript.
    11. It is also well known that in addition to these electrostatic interactions, the core of the ISB helical bundle is formed by an extensive network of hydrophobic interactions. Have the authors ever looked into how perturbing any of these intra-trimeric complex hydrophobic interactions affect their ability to phase separate and perform their subsequent function?
    12. The phase separated CPC compartment is known to enrich several other inner centromere proteins such as the Histone H3, Sgo1, the histone H3T3phos, among others. Have the authors tried to increase the complexity of the reconstituted CPC scaffold by incorporating more components to look into whether that changes any of the interaction interfaces between the ISB trimeric complexes within the condensed phase? Can this CPC compartment be reconstituted using a bottom-up approach?

    Overall, this paper brings forward a useful technique to probe the conformational landscape of proteins in the condensed droplet phase and compare it with its dispersed phase. This paper serves as an interesting read showing how specific salt-bridge interactions between multiple stoichiometric protein complexes can be the driving force for phase separation.

    Significance

    In this manuscript, using the technique of hydrogen/deuterium-exchange mass spectrometry (HXMS), the authors have tried to gain insights into the structure of the chromosomal passenger complex (CPC) within the phase separated chromatin body, known to regulate chromosome segregation in mitosis. The CPC phase separated compartment comprises three regulatory and targeting subunits, INCENP, Survivin, and Borealin, forming a three-helix bundle hetero-trimer. By measuring changes in the polypeptide backbone dynamics of this trimeric INCENP/Survivin/Borealin complex, in the liquid-liquid de-mixed state in comparison to its soluble state, using HXMS measurements, the paper puts forward high-resolution structural details of the phase separated CPC. Using a step-wise mutagenesis approach in conjunction with the information from HXMS measurements and previous crystallographic data, this work also identifies distinct regions/interfaces within this complex harboring crucial salt bridges, which directly contribute toward the liquid-liquid demixing of the CPC.

    Overall, this paper brings forward a useful technique to probe the conformational landscape of proteins in the condensed droplet phase and compare it with its dispersed phase. This paper serves as an interesting read showing how specific salt-bridge interactions between multiple stoichiometric protein complexes can be the driving force for phase separation

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    Referee #1

    Evidence, reproducibility and clarity

    This manuscript by Bryan et al., describes the use of Hydrogen/Deuterium-exchange Mass Spectrometry (HXMS) as a powerful tool to identify key amino acid residues and associated interactions driving liquid-liquid demixing. They have particularly focused on the Chromosomal Passenger Complex (CPC), an important regulator of chromosome segregation, which has recently been shown to undergo liquid-liquid demixing in vitro. Their work presented here allowed them to identify a few key electrostatic interactions as molecular determinants driving the liquid-liquid demixing of the CPC. Their work also shows that crystal packing information of protein molecules, where available, can provide valuable insight into likely factors driving liquid-liquid demixing.

    Major comments:

    A previous study by Trivedi et al., NCB 2019 identified an unstructured region in Borealin (aa residues 139-160) as the main region driving the phase separation of CPC. Interestingly, this region only shows a moderate reduction in HX upon liquid-liquid demixing. But no experiments or discussions related to this observation are presented in the current version of the manuscript.

    In the absence of cellular data on if and how these mutations (within the triple-helical bundle region) affect CPC's ability to phase separate in cells, the implication of this work is very limited - One can't say for sure these are interactions driving phase separation of CPC in a cellular environment.

    In the absence of any cellular data with the mutants described here, much of the discussion on the possible roles of CPC phase separation in cells does not appear relevant to this manuscript. I would suggest that the authors focus mainly on highlighting the power of using HXMS as a tool to characterise the molecular determinants of liquid-liquid demixing at a relatively high resolution.

    Minor comments:

    The authors should ensure that the introduction cites relevant literature thoroughly. For example, where the potential role of Borealin residues 139-160 in conferring phase separation properties to the CPC is mentioned, the authors failed to cite Abad et al., 2019, which showed the contribution of the same Borealin region in conferring nucleosome binding ability to the CPC.

    Significance

    This is a highly relevant and significant work, particularly considering the rapidly growing list of examples for Phase separation of proteins/protein assemblies and their potential biological roles (in spite of ongoing debates in the field about the cellular relevance of several phase separation claims). The data presented in this manuscript are solid and convincingly establish HXMS as a useful tool to characterise molecular interactions driving liquid-liquid demixing. Considering its applicability to characterise wide-ranging protein assemblies implicated in phase separation, this work will be of interest to a broad readership.

  8. Version published to 10.1101/2023.05.22.541822v2 on bioRxiv
  9. Version published to 10.1101/2023.05.22.541822v1 on bioRxiv