Dissecting the phase separation and oligomerization activities of the carboxysome positioning protein McdB

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Across bacteria, protein-based organelles called bacterial microcompartments (BMCs) encapsulate key enzymes to regulate their activities. The model BMC is the carboxysome that encapsulates enzymes for CO 2 fixation to increase efficiency and is found in many autotrophic bacteria, such as cyanobacteria. Despite their importance in the global carbon cycle, little is known about how carboxysomes are spatially regulated. We recently identified the two-factor system required for the maintenance of carboxysome distribution (McdAB). McdA drives the equal spacing of carboxysomes via interactions with McdB, which associates with carboxysomes. McdA is a ParA/MinD ATPase, a protein family well-studied in positioning diverse cellular structures in bacteria. However, the adaptor proteins like McdB that connect these ATPases to their cargos are extremely diverse. In fact, McdB represents a completely unstudied class of proteins. Despite the diversity, many adaptor proteins undergo phase separation, but functional roles remain unclear. Here, we define the domain architecture of McdB from the model cyanobacterium Synechococcus elongatus PCC 7942, and dissect its mode of biomolecular condensate formation. We identify an N-terminal intrinsically disordered region (IDR) that modulates condensate solubility, a central coiled-coil dimerizing domain that drives condensate formation, and a C-terminal domain that trimerizes McdB dimers and provides increased valency for condensate formation. We then identify critical basic residues in the IDR, which we mutate to fine-tune condensate solubility. Finally, we find that a condensate-defective mutant of McdB has altered association with carboxysomes and influences carboxysome enzyme content. The results have broad implications for understanding spatial organization of BMCs and the molecular grammar of protein condensates.

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  1. Evaluation Summary:

    Carboxysomes enable the efficient fixation of carbon dioxide in specific bacteria. Phase separation has been invoked as a mechanism that drives the formation of carboxysomes. The current work focuses on the biophysical principles of how one of two essential specific protein components enable spatial regulation over carboxysomes. This important work highlights the connection between oligomerization via specific molecular interactions and phase separation. The work is of interest to the areas of biochemistry and carbon dioxide fixation as well as phase separation.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    Bacterial carboxysomes are compartments that enable the efficient fixation of carbon dioxide in certain types of bacteria. A focus of the current work is on two protein components that provide spatial regulation over carboxysomes. The McdA system is an ATPase that drives the positioning of carboxysomes. The McdB system is essential for maintaining carboxysome homeostasis, although how this role is achieved is unclear. Previous studies, by the lead author's lab, showed that the McdB system is a driver of phase separation in vitro and in cells. They proposed a putative connection between McdB phase separation and carboxysome homeostasis. The central premise of the current work is as follows: In order to understand if and how phase separation of McdB impacts carboxysome homeostasis, it is important to know how the driving forces for phase separation are encoded in the sequence and architecture of McdB. This is the central focus of the current work. The picture that emerges is of a protein that forms hexamers, which appears to be a trimer of dimers. The domains that drive that the dimerziation and trimerization appear to be essential for driving phase separation under the conditions interrogated by the authors. The N-terminal disordered region regulates the driving forces for phase separation - referred to as the solubility of McdB by the authors. To converge upon the molecular dissections, the authors use a combination of computational and biophysical methods. The work highlights the connection between oligomerization via specific interactions and emergent phase behavior that presumably derives from the concentration (and solution condition) dependent networking transitions of oligomerized McdB molecules.

    Having failed to obtain specific structural resolution for the full-length McdB as a monomer or oligomer, the authors leverage a combination of computational tools, the primary one being iTASSER. This, in conjunction with disorder predictors, is used to identify / predict the domain structure of McdB. The domain structure predictions are tested using a limited proteolysis approach and, for the most part, the predictions stand up to scrutiny affirming the PONDR predictions. SEC-MALS data are used to pin down the oligomerization states of McdB and the consensus that emerges, through the investigations that are targeted toward a series of deletion constructs, is the picture summarized above.

    Is the characterization of the oligomerization landscape complete and likely perfect? Quite possibly, the answer is no. Deletion constructs pose numerous challenges because they delete interactions and inevitably impose a modularity to the interpretation of the totality of the data. Accordingly, we are led to believe that the N-terminal IDR plays no role whatsoever in the oligomerization. Close scrutiny, driven by the puzzling choice of nomenclature and the Lys to Gln titrations in the N-terminal IDR raise certain unresolved issues. First, the central dimerization domain is referred to as being Q-rich. This does not square with the compositional biases of this region. If anything is Q/L or just L-rich. This in fact makes more sense because the region does have the architecture of canonical Leu-zippers, which do often feature Gln residues. However, there is nothing about the sequence features that mandates the designation of being Q-rich nor are there any meaningful connections to proteins with Q-rich or polyQ tracts. This aspect of the analysis and discussion is a serious and erroneous distraction. Back to the middle region that drives dimerization, the missing piece of the puzzle is the orientation of the dimers. One presumes these are canonical, antiparallel dimers. However, this issue is not addressed even though it is directly relevant to the topic of how the trimer of dimers is assembled. If the trimer is such that all binding sites are fully satisfied (with the binding sites presumably being on the C-terminal pseudo-IDR), then the hexamer should be a network terminating structure, which it does not seem to be based on the data. Instead, we find that only the full-length protein can undergo phase separation (albeit at rather high concentrations) in the absence of crowder. We also find that the driving forces for phase separation are pH dependent, with pH values above 8.5 being sufficient to dissolve condensates. Substitution of Lys to Gln in the N-terminal IDR leads to a graded weakening of the driving forces for phase separation. The totality of these data suggest a more complex interplay of the regions than is being advocated by the authors. Almost certainly, there are complementary electrostatic interactions among the N-terminal IDR and C-terminal pseudo IDR that are important and responsible for the networking transition that drives phase separation, even if these interactions do not contribute to hexamer formation. The net charge per residue of the 18-residue N-terminal IDR is +0.22 and the NCPR of the remainder is ≈ -0.1. To understand how the N-terminal IDR is essential, in the context of the full-length protein, to enable phase separation (in the absence of crowder), it is imperative that a model be constructed for the topology of the hexamer. It is also likely that the oligomer does not have a fixed stoichiometry.

    Therefore, the central weakness of the current work is that it is too preliminary. A set of interesting findings are emerging but by fixating on Lys to Gln titrations within the N-terminal IDR and referring to these titrations as impacting solubility, a premature modular and confused picture emerges from the narrative that leaves too many questions unanswered.

    The work itself is very important given the growing interest in bacterial condensates. However, given that the focus is on understanding the molecular interactions that govern McdB phase behavior - a necessary pre-requisite in the authors minds for understanding if and how phase separation impacts carboxysome homeostasis - it becomes imperative that the model that emerges be reasonably robust and complete. At this juncture, the model raises far too many questions. The MoRF analysis is distraction away from the central focus.

    The problem, as I see it, is that the authors have gone down the wrong road in terms of how they have interpreted the preliminary set of results. Further, the methods used do not have the resolution to answer all the questions that need to be answered. Another issue is that a lot of standard tropes are erected and they become a distraction. For example, it is simply not true that in a protein featuring folded domains and IDRs it almost always is the case that the IDR is the driver of phase transitions. This depends on the context, the sequence details of the IDRs, and whether the interactions that contribute to the driving forces for phase separation are localized within the IDR or distributed throughout the sequence. In McdB it appears to be the latter, and much of the nuance is lost through the use of specific types of deletion constructs.

    Overall, the work represents a good beginning but the data do not permit a clear denouement that allows one to connect the molecular and mesoscales to fully describe McdB phase behavior. Significantly more work needs to be done for such a picture to emerge.

  3. Reviewer #2 (Public Review):

    In this work, Basalla et al. study the biochemical properties of the carboxysome positioning protein, McdB. Using in vitro experiments, the authors characterize McdB oligomeric states and the domains driving and modulating its phase separation. Based on bioinformatics analysis, the authors identify a putative binding recognition motif between McdB and its two-component system counterpart McdA. As McdAB-like systems emerge as spatial regulators of bacterial compartments, the data presented here may be of general interest. The study is well executed and provides exciting hypotheses to be tested in vivo.

    The authors found that McdB from S. elongatus PCC 7942 consists of three domains: an N-terminal 18 aa disordered region, a Q-rich helical domain, and a helical C-terminal domain (CTD). Analyzing these domains, the authors present three key results: (i) The Q-rich domains form dimers, and the CTD drives the formation of trimers of dimers (ii) Phase separation is pH sensitive, driven by the Q-rich domain, and modulated by basic residues in the IDR, (iii) The IDR contains a putative recognition motif that binds McdA. While these three sets of results are rich in data, they are disjointed. Relating the three datasets (oligomeric states of the protein, its phase separation behavior, and its ability to bind McdA) is required to provide a complete picture of the molecular mechanism driving McdB condensation.

    Specific comments:

    1. The main limitation of this manuscript is the lack of integration between the three areas of results. In particular: how do the IDR basic residues disrupt phase separation? Is that through interference with either the dimer or timer interface?
    Does the McdB IDR regulate phase separation behavior when bound to McdA? or in other words, is the MoRF acting both as a binding interface and as a solubility regulator, and if so, can both functions be achieved simultaneously? It seems like the MoRF includes at least three basic residues. Finally, what is the effective concentration of McdB in cells, and how does that translate to the in vitro studies?

    2. How general are the conclusions made here to other McdB? The authors have published nice work surveying the commonalities and differences between homologous McdB proteins. Can you comment on the applicability of your findings to other McdB proteins?

    Additional issues:

    3. Using SEC and SEC-MALS, the authors demonstrated that the Q-rich domain forms a stable dimer and that the full-length protein forms hexamers, suggesting trimers of dimers assembly. The authors also suggest that the CTD is responsible for forming those trimers of dimers based on SEC-MALS measurements. However, Figure 2D shows that while the full length runs at 6.6x the monomer, the Q-rich+CTD runs at 5.4x the monomer. First, I could not find SEC-MALS of the full-length protein, and it is not clear whether SEC-MALS was used for all or a fraction of the constructs discussed in Figure 2D. Second, could it be that the Q-rich domain+CTD is an ensemble of hexamers and dimers? Perhaps the IDR is playing a secondary role in stabilizing the hexamer?

    4. The analysis of the phase separation results needs to have some extra quantification. The authors show that at 100 uM protein with 10% PEG the full-length phase separates as well as IDR+Q-rich. Lines 176-178: "The CTD, on the other hand, has no effect on the Q-rich domain condensates; Q-rich+CTD condensates formed at the same protein concentration and with identical droplet morphologies at the Q-rich domain alone." It is hard to draw this conclusion solely based on the data presented in Figure 3. An alternative interpretation might be that Q-rich+CTD reduces csat. I suggest the authors include turbidity assays (as shown for pH effect) to quantitively determine csat for these different constructs and perhaps perform FRAP to determine the mobility of these different constructs. In addition, how long after the addition of PEG were these droplets imaged?

    5. Solubility assays shown in Figures 4A, B, D, and 5C are missing error bars. Without replicates, it is difficult to assess, for example, the effect of KCl. Also, please indicate the physiological ranges of KCl and pH in Figure 6. The phase separation sensitivity to pH is intriguing. By changing basic residues to glutamines, the authors conclude that the positive charge of the IDR modulates solubility. The Q-rich domain, however, is negatively charged. Can the authors comment on the role of acidic residues in the Q-rich domain? Are they required for phase separation? Also - based on your previous bioinformatics analysis, are the charges of the IDR and the Q-rich domains conserved across McdB homologs?

    6. In previous work, the authors showed a conserved RKR segment in the IDR is highly conserved and missing in S. elongatus PCC 7942 (MacCready et al., Mol Biol Evol. 2020). Given the current finding, it would be important to understand whether the RKR deletion carries functional implications for phase separation behavior.

    7. McdB proteins with 2Q left mutated vs. 2Q middle and 2Q right seem to result in condensates with different material properties (e.g., DIC pictures show different droplet morphologies for the different constructs). Is that the case? And if so, can you comment on that?

  4. Reviewer #3 (Public Review):

    Through a series of rigorous in vitro studies, the authors determined McdB's domain architecture, its oligomerization domains, the regions required for phase separation, and how to fine-tune its phase separation activity. The SEC-MALS study provides clear evidence that the α-helical domains of McdB form a trimer-of-dimers hexamer. Through analysis of a small library of domain deletions by microscopy and SDS-PAGE gels of soluble and pellet fractions, the authors conclude that the Q-rich domain of McdB drives phase separation while the N-terminal IDR modulates solubility. A nicely executed study in Figure 4 demonstrated that McdB phase separation is highly sensitive to pH and is influenced by basic residues in the N terminal IDR. The study demonstrates that net charge, as opposed to specific residues, is critical for phase separation at 100 micromolar. In addition, the experimental design included analysis of McdB constructs that lack fluorescent proteins or organic dyes that may influence phase separation. Therefore, the observed material properties have full dependence on the McdB sequence.

    Studies of proteins often neglect short, disordered segments at the N- or C- terminus due to unclear models for their potential role. This study was interesting because it revealed a short IDR as a critical regulator of phase separation. This includes experiments that remove the IDR (Fig 2 & 3) and mutate the basic residues to show their importance towards McdB phase separation. In a nice set of SDS-PAGE experiments, the authors showed that as the net charge of the IDR decreased the construct became more soluble.

    One challenge is in the experimental design when mutating residues is to assess their impact on phase separation. The author's avoided substitutions to alanine, as alanine substitutions have synthetically stimulated phase separation in other systems. The authors, therefore, have a good rationale for selecting potentially milder mutations of lysine/arginine to glutamine. A potential caveat of mutation to glutamine is that stretches of glutamines have been associated with amyloid/prion formation. So, the introductions of glutamines into the IDR may also have unexpected effects on material properties. Despite these caveats, the authors show mutation of six basic residues in the short IDR abolished phase separation at 100 mM.

    Computational studies (Fig 7) also suggest that this short N-IDR region may play a role as a MORF upon potential binding to a second protein McdA. The formulation of this hypothesis is strengthened by the fact that for other ParA/MinD-family ATPases, the associated partner proteins have also been shown to interact with their cognate ATPase via positively charged and disordered N-termini. This aspect of understanding McdB's N-IDR as a MORF is at a very early stage. This study lacks experimental evidence for an N-IDR: McdA interaction and experimental data showing conformational change upon McdA binding. However, the computation study sets up the future to consider whether and how the phase separation activity of McdB is related to its structural dynamics and interactions with McdA.

    In summary, this study provides a strong foundation for the contribution of domains to McdB's in vitro phase separation. This knowledge will inform and impact future studies on McdB regulating carboxysomes and how the related family of ParA/MinD-family ATPases and their cognate regulatory proteins. For example, it is unknown if and how McdB's phase separation is utilized in vivo for carboxysome regulation. However, the revealed roles of the Q-rich domain and N-IDR will provide valuable knowledge in developing future research. In addition, the systematic domain analysis of McdB can be combined with a similar analysis of a broad range of other biomolecular condensates in bacteria and eukaryotes to understand the design principles of phase separating proteins.