Conserved and repetitive motifs in an intrinsically disordered protein drive α-carboxysome assembly

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All photosynthetic bacteria and some chemoautotrophic bacteria fix CO 2 into sugars in specialized proteinaceous compartments called carboxysomes. Carboxysomes enclose the enzymes Rubisco and carbonic anhydrase inside a layer of shell proteins to increase the CO 2 concentration for efficient carbon fixation by Rubisco. In the α-carboxysome lineage, a disordered and highly repetitive protein named CsoS2 is essential for carboxysome formation and function. Without it, the bacteria are unable to fix enough carbon to grow in air. How a protein lacking structure serves as the architectural scaffold for such a vital cellular compartment remains unknown. In this study, we identify key residues in CsoS2 that are necessary for building functional α-carboxysomes in vivo . These highly conserved and repetitive residues, VTG and Y, contribute to the interaction between CsoS2 and shell proteins. We also demonstrate in vitro reconstitution of the α-carboxysome into spherical condensates with CsoS2, Rubisco, and shell proteins, and show the utility of reconstitution as a biochemical tool to study carboxysome biogenesis. The precise self-assembly of thousands of proteins is crucial for carboxysome formation, and understanding this process could enable their use in alternative biological hosts or industrial processes as effective tools to fix carbon.

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  1. Although both carboxysome lineages contain scaffolding proteins, these proteins are related in function alone; they have no sequence or structural similarity

    I'd be interested to see this compared for the three structural components of the carboxysome. Do the hexameric/pentameric shell proteins show more evidence of shared origin? Is there significantly lower conservation of sequence/structure in the scaffolding proteins?

  2. The presence or absence of the CTD in the CsoS2 and wtMR constructs is a proxy for CsoS2A and CsoS2B, suggesting that these two proteins may contribute differently to the physical properties of the nascent carboxysome.

    Could it also be related to the lack on NTD in the wtMR construct?

  3. Repeat 7 was left out of the wtMR construct because it occurs after the ribosomal slip site in Repeat 6, in an effort to eliminate potential confounding variables between the CsoS2A and CsoS2B isoforms.

    I understand the effort to eliminate confounding variables. However, based on the western blots in Fig S3, it seems like in most cases in your strains, you're ending up with CsoS2B (which I think is the long isoform?), but here you're forcing all mutants to the short version? Or maybe I'm misunderstanding! Either way, this part is a bit confusing and I'd love a bit of clarification here.

  4. ysteines play a non-essential structural role that strengthens the overall integrity of the complex, but may not be necessary for its assembly or function.

    Do you think this would be the case if you had mutated the cysteine to an alanine?

  5. Fig. S2

    This is super helpful and it might be worth moving some version to the main text or adding a table of your mutants or something since you have so many and it can be a little tricky to keep track of.

  6. All strains expressed similar amounts of CsoS2 (Fig. S3), though it should be noted that only CsoS2B was detected; it is likely that expression from the neutral site instead of the native operon reduced ribosomal frameshifting responsible for the production of non-essential CsoS2A.

    Would be great if you could quantify these results? I also think it could be helpful to check all mutants with both antibodies to compare 2B to 2A for all instead of just the VTG mutants.

  7. C to S

    I totally see the reasoning behind mutating to C to S instead of A. However, I think it would also be interesting to see if mutating this to an A would cause further functional changes, especially because you changed the rest to A.

  8. Although both carboxysome lineages contain scaffolding proteins, these proteins are related in function alone; they have no sequence or structural similarity.

    This is super interesting! Would love some more information - could you provide citations and/or data to demonstrate this?

  9. Overall super cool work! A super fascinating system with so much amazing biology! I truly loved the variety of techniques you have used to make your points. I’m a huge fan of clever and unique assays so i really enjoyed everything, from the native gels and turbity readings to the FRAP assays. Thank you for uncovering this novel biology for the rest of us to learn from! I have a few questions/comments. Please check them out below.

    Regarding this statement "Although both carboxysome lineages contain scaffolding proteins, these proteins are related in function alone; they have no sequence or structural similarity.", what do you mean by “no sequence or structural similarity”? do you mean low? may be 50%? or lower?

    Regarding CsoS2A and CsoS2B in H. neapolitanus, would these be considered isoforms? Are proteins with isoforms common in this species (or among species in this particular proteobacteria clade)?

    Regarding this statement "(1) (V/I)(T/S)G triplets spaced ~8-11 amino acids apart (hereafter VTG repeats), (2) cysteine pairs, (3) a highly conserved lysine, and (4) a highly conserved tyrosine", are these motifs conserved across alpha and beta carboxysome lineages?

    Regarding this statement "CsoS2, like most IDPs, stymies AI structure prediction programs - AlphaFold yields a disordered coil and low confidence scores", what about the AlphaFold prediction for the complex between the shell protein and CsoS2?

  10. motifs stand out in particular

    It would be nice to understand how you identified the repeats. Valine also appears to be conserved in the second position of the MR and I could imagine considering a "KV" repeat rather than just the conserved lysine (K). Was there a reason you didn’t highlight it?

  11. (A)

    I love this illustration! It does a great job at summarizing the role of each part of the CsoS2 protein. What is the difference between the teal and purple shell components? I assumed the teal component refers to the CsoS1A shell protein. Does the purple component refer to other CsoS2 proteins, the truncated CsoS2A protein or something else?