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

Abstract

All photosynthetic bacteria and some chemoautotrophic bacteria fix CO2into sugars in specialized proteinaceous compartments called carboxysomes. Carboxysomes enclose the enzymes Rubisco and carbonic anhydrase inside a layer of shell proteins to increase the CO2concentration 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-carboxysomesin vivo. These highly conserved and repetitive residues, VTG and Y, contribute to the interaction between CsoS2 and shell proteins. We also demonstratein vitroreconstitution 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.

α-Carboxysome Size Is Controlled by the Disordered Scaffold Protein CsoS2

“…In a recent study of the MR, we observed significant effects of the conserved tyrosine on the biochemical interactions with the shell and sufficiency of resulting carboxysomes for atmospheric growth in H. neapolitanus, highlighting a distinct yet crucial role of the M-peptides . The AlphaFold predictions for the C-peptides are of low confidence and lack a common structure.…”

“…It is now well established that the CTD forms interactions with the carboxysome shell via both the C-peptides and CTP. , Our recent study found that the MR also specifically interacts with the shell hexamers and that the VTG triplets are an essential component of that interaction . To further test this interaction in the context of carboxysome assembly, we designed a series of MR-GFP fusion proteins having different numbers of M-peptides.…”

“… − CsoS2 is unique among the carboxysome proteins as it is predicted to be largely structurally disordered along its entire length. ,, It does, however, contain several distinct repeated sequence motifs that collectively define a three-part domain structure: the N-terminal domain (NTD) with N-peptide motifs, the middle region (MR) with M-peptide motifs, and the C-terminal domain (CTD) with C-peptide motifs and a conserved C-terminal peptide (CTP). Previous work has shown that CsoS2 is essential to growth and acts as a central node with interactions to both Rubisco and CsoS1. ,, Specifically, the NTD binds to and encapsulates Rubisco via interactions with the N-peptides, , and the CTD has been shown to associate with the shell. , We recently showed that MR also interacts with the shell, though what distinguishes its functional role from the CTD is not yet clear …”

“… 20 , 21 We recently showed that MR also interacts with the shell, though what distinguishes its functional role from the CTD is not yet clear. 29 …”

A carboxysome‐based CO2 concentrating mechanism for C3 crop chloroplasts: advances and the road ahead

“…To successfully assemble a functional carboxysome in plants, it is necessary to have a comprehensive understanding of both the carboxysome lumen and the shell, and how these two components interconnect. The supramolecular structure of the microcompartment interior has been subject of study for some time and recent reports have provided molecular-level detail as to the forces governing internal lumen organisation and interaction networks (Blikstad et al, 2023;Flecken et al, 2020;Metskas et al, 2022;Ni et al, 2022Ni et al, , 2023Oltrogge et al, 2020Turnšek et al, 2023;Wang, Yan, et al, 2019). The intrinsically disordered proteins (IDPs) CsoS2 and CcmM in αand β-carboxysomes, respectively, are the primary Rubisco interaction partners (Oltrogge et al, 2020;Wang, Yan, et al, 2019;Zang et al, 2021).…”