Intrinsically disordered CsoS2 acts as a general molecular thread for α-carboxysome shell assembly

Carboxysomes are a paradigm of self-assembling proteinaceous organelles found in nature, offering compartmentalisation of enzymes and pathways to enhance carbon fixation. In α-carboxysomes, the disordered linker protein CsoS2 plays an essential role in carboxysome assembly and Rubisco encapsulation. Its mechanism of action, however, is not fully understood. Here we synthetically engineer α-carboxysome shells using minimal shell components and determine cryoEM structures of these to decipher the principle of shell assembly and encapsulation. The structures reveal that the intrinsically disordered CsoS2 C-terminus is well-structured and acts as a universal “molecular thread” stitching through multiple shell protein interfaces. We further uncover in CsoS2 a highly conserved repetitive key interaction motif, [IV]TG, which is critical to the shell assembly and architecture. Our study provides a general mechanism for the CsoS2-governed carboxysome shell assembly and cargo encapsulation and further advances synthetic engineering of carboxysomes for diverse biotechnological applications.


Data collection and processing
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Construction of the different mini-shell forms.(a) The genetic arrangements of mini-shell-1 to mini-shell-6 constructs generated in this study.(b) PCR verification of the mini-shell constructs using the primers listed in Supplementary Table 3

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. The genes in PCR products are indicated at the bottom of the gel image.Two sets of primers are used in the mini-shell 2 and 3 constructs.The sizes (bp) of PCR products are labelled in orange.This experiment was repeated and confirmed by further plasmid sequencing results.(c) The protein sequence of CsoS2 C-terminal domain.The three interaction fragments in the C-terminal region (F1, F2, F3) newly identified in the T = 9 shell are shown in red.The three additional repeats in the C-terminal region (R7, R8, R9) previously identified (1) are represented in blue.*** indicates the I(V)TG motif, which was replaced by AAA in the CsoS2-Cm mutant.Characteristics of shells generated from mini-shell 1 (CsoS4A-CsoS1A) and mini-shell 2 (CsoS2-CsoS4A-CsoS1A) constructs.(a) SDS-PAGE results revealed the major protein components of purified shells from mini-shell-1 and mini-shell-2.The purification was performed at least three times and a representative purification result is shown.(b) Immunoblot analysis using anti-CsoS2 antibody (GenScript, USA) revealed that both CsoS2A and CsoS2B were expressed in the E. coli mini-shell 2 construct, but only CsoS2B was incorporated into the mini-shells and CsoS2A was not detectable.WC: whole cell lysate of the CsoS2-CsoS4A-CsoS1A mini-shell 2 construct; S2: isolated CsoS2-CsoS4A-CsoS1A mini-shells.The experiment was performed at least three times and a representative blot is shown.(c) Dynamic light scattering (DLS) analysis of shell sizes from mini-shell 1 and mini-shell 2. (d) Electron microscopy (EM) images of negatively stained purified shells from mini-shell 1 and mini-shell 2. The experiments were repeated at least three times with two representative micrographs shown here.CryoEM data processing.(a-b) Representative micrographs and 2D class averages of shells produced from mini-shell 1 (a) and mini-shell 2 (b), respectively.Boxed particles have different sizes: blue, large shells (T = 9); red, medium shell (T = 4); and green, small shell (T = 3).The cryo-EM imaging and analysis for these two samples were performed only once.Scale bars: 50 nm.(c-e) CryoEM maps of T = 9 (c), T = 4 (d) and T = 3 (e), shown in top view (left) and central slice (right).Maps are coloured according to their local resolutions.(f) Fourier Shell Correlation (FSC) of shells, T = 9 in magenta, T = 4 in blue (from mini-shell 2) and brown (from mini-shell 1), and T = 3 in black, with resolutions indicated at FSC=0.143 cut-off.loop β4-α2 loop β4-α2 loop β4-α2 loop Supplementary Fig. 4 | Comparison of T = 4 shells with and without CsoS2 from different mini-shell constructs.(a) Comparison of T = 4 shell hexamer maps (gray) from mini-shell-1, mini-shell-2, and a GFP-CsoS2-CTD/4A-1A (EMD-30385).The major difference in hexamer density is located in the loop between β4 and α2 in S1A (red arrows), which could not be resolved in the shell without CsoS2.(b) Density maps of T = 4 shell hexamers from different CsoS2 truncation and I(V)TG mutation constructs.The map contour level is set to 4.5σ for all the maps except EMD-30385 (2σ).Supplementary Fig. 5 | Sequence and structural analysis of CsoS1A and CsoS4A.(a) Conservation of CsoS1A/B/C (990 sequences) and CsoS4A (970 sequences) from the Uniprot database, presented using Weblogo.CsoS1A and CsoS1C have only two residues that are distinct from each other, and CsoS1A and CsoS1B are 91% identical.Only 40% similarity was found between CsoS4A and CsoS4B.The red line indicates the conserved βstrand interacting with the I(V)TG motif of CsoS2.(b) Structural comparison of CsoS1A hexamer (top) and CsoS4A pentamers (bottom) from mini-shell assemblies and X-ray crystallography structures in two orthogonal views, indicating very little deviations among these structures.Two quasi-equivalent hexamers from T = 9 shell are shown in blue (close to pentamer) and green (at the 3-fold), respectively (see Figure 2A).The hexamers in the T = 3 shell have the maximum curvature (~8°) compared with the crystal structure (PDB: 2EWH).

Table 2 .
Cryo-EM data collection, refinement and validation statistics of mini-shell mutants

Table 2 .
Cryo-EM data collection, refinement and validation statistics of mini-shell mutants (continued)

Table 3 .
ssDNA oligonucleotides used in this study.The overlapping sequences for Gibson assembly are underlined.