Rubisco packaging and stoichiometric composition of a native β-carboxysome

Abstract

Carboxysomes are anabolic bacterial microcompartments that play an essential role in carbon fixation in cyanobacteria. This self-assembling proteinaceous organelle encapsulates the key CO₂-fixing enzymes, Rubisco and carbonic anhydrase, using a polyhedral shell constructed by hundreds of shell protein paralogs. Deciphering the precise arrangement and structural organization of Rubisco enzymes within carboxysomes is crucial for understanding the formation process and overall functionality of carboxysomes. Here, we employed cryo-electron tomography and subtomogram averaging to delineate the three-dimensional packaging of Rubiscos within β-carboxysomes in the freshwater cyanobacterium Synechococcus elongatus PCC7942 that were grown under low light. Our results revealed that Rubiscos are arranged in multiple concentric layers parallel to the shell within the β-carboxysome lumen. We also identified the binding of Rubisco with the scaffolding protein CcmM in β-carboxysomes, which is instrumental for Rubisco encapsulation and β-carboxysome assembly. Using QconCAT-based quantitative mass spectrometry, we further determined the absolute stoichiometric composition of the entire β-carboxysome. This study and recent findings on the β-carboxysome structure provide insights into the assembly principles and structural variation of β-carboxysomes, which will aid in the rational design and repurposing of carboxysome nanostructures for diverse bioengineering applications.

Keywords: Carboxysome; Rubisco packaging; assembly; carbon fixation; cyanobacteria; protein stoichiometry.

Figure 1.. Purification and characterization of Syn7942 β-carboxysomes.

(A) Thin-section electron microscopy of a Syn7942 cell with β-carboxysomes indicated by yellow arrows (top) and the organization of genes expressing β-carboxysome proteins in the Syn7942 genome (bottom); (B) Functional diagrams of β-carboxysomes; (C) SDS-PAGE of purified β-carboxysomes from three biological replicates; (D) Electron microscopy image of isolated β-carboxysomes from Syn7942.

Figure 2.. CryoEM structures and organization of Rubisco within β-carboxysomes.

(A) A tomogram slice showing a typical beta-carboxysome. Scale bar: 50 nm. (B) The position and orientation of individual rubisco mapped back to the tomogram in (a). It contains a total of 6 layers and the layer number is defined from the core to the shell, as shown. (C) Histogram of Rubisco numbers in β-carboxysomes (n = 185). (D) The cryoET subtomogram averaged structures of Rubisco at 3.5 Å, overlapped with refined atomic model (PDB ID: 8BCM), shown in two views. (E) Zoomed-in view showing the density from CcmM, the linker protein which binds Rubisco, and zoomed-in views fitted with atomic model of CcmM SSUL (PDB ID: 6HBC) in two binding modes (upper groove and lower groove). (F) The overall atomic models of Rubisco along with 4 SSUL domains in two views. Only one binding mode is shown (upper groove). The diamond and dashed black lines indicate the fourfold axis.

Figure 3.. In situ cryo-electron tomography (cryoET) of frozen-hydrated Syn7942 cell lamella.

Yellow arrows indicate the β-carboxysome structures which possess regularly packed Rubisco in the β-carboxysome lumen. G represents large electron-dense granule. Scale bar, 200 nm.

Figure 4.. Absolute quantification of the subunit stoichiometry of Syn7942 β-carboxysomes using QconCAT standardization.

See also the content of protein oligomers per β-carboxysome in Table 1. Data are presented as means ± SD from four independent biological replicates.

Figure 5.. Schematic model of the Syn7942 β-carboxysome structure.

The model illustrates the interior organisation and shell facet composition determined by QconCAT-determined stoichiometry and cryoET. Rubiscos (green) are organized in multiple layers in the carboxysome. CcmN (cyan) and the N-terminus of CcmM58 (red fragment) mediate the Rubisco-shell binding. Rubiscos are crosslinked by SSUL of CcmM58 (red) and CcmM35 (orange). CcaA (brown) and RbcX (purple) are co-encapsulated with the Rubisco matrix. On the shell, CcmK3, CcmK4, CcmO, and CcmP are integrated into the facets formed predominantly by CcmK2 hexamers. The vertexes are partially capped by CcmL pentamers.