Decoding the Absolute Stoichiometric Composition and Structural Plasticity of α-Carboxysomes

Abstract

A wide range of bacteria use special protein-based organelles, termed bacterial microcompartments, to encase enzymes and reactions to increase the efficiency of biological processes. As a model bacterial microcompartment, the carboxysome contains a protein shell filled with the primary carbon fixation enzyme Rubisco.

Structure and assembly of cargo Rubisco in two native α-carboxysomes

“…It is yet unclear which segment(s) is bound to the native Rubisco, as the amino acid side chains are not resolved in the subtomogram averaged map. The ratio of Rubisco (CbbL 8 /CbbS 8 ) to CsoS2 is roughly 1:1.2 in a native Halo carboxysome 12 and 1:0.6 in a native Cyanobium carboxysome (Supplementary Fig. 1a ).…”

“…1e , magenta), as observed in the crystal structure of Halo Rubisco in a complex with a synthetic CsoS2 N-peptide (PDB 6UEW ) 19 . The density is unlikely from CsoSCA, since CsoSCA is inserted deeply into the cleft between two CbbL, underneath the CsoS2 binding site 31 , and the occupancy of CsoSCA on Rubisco is too low (1.6%, the ratio of CsoSCA: Rubisco is 58:447), to contribute strongly to the subtomogram average 12 . The intrinsically disordered but highly conserved CsoS2 is thought to serve as the scaffolding protein connecting the carboxysome shell using its C-terminal region to Rubisco through its N-terminus 19 , 20 .…”

“…The carboxysome encapsulates carbonic anhydrase and the primary CO 2 -fixing enzyme, ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), within a protein shell that structurally resembles a virus capsid 10 . Rubisco is among the most abundant components of carboxysomes 11 , 12 . It is generally understood that Rubiscos are densely encapsulated within the carboxysome to overcome its slow turnover rate and off-pathway oxygen reaction.…”

Tales of Two α-Carboxysomes: the Structure and Assembly of Cargo Rubisco

“…The Halothiobacillus neapolitanus ( H. neapolitanus ) strain used in this work was acquired from ATCC (The American Type Culture Collection). Cell cultivation and carboxysome purification were performed as described previously 39 . Seeding cells were maintained in liquid ATCC medium 290 or on ATCC 290 1.5% agar plates and inoculate in the Vishniac and Santer medium 40 in a 5-liter fermenter (BioFlo 115, New Brunswick Scientific, US) and were kept at constant pH 7.6 through supplement of 3 M KOH.…”

“…Negative staining electron microscopy was carried out as described previously 39,42,43 . The carboxysomes (∼4 mg mL -1 ) were stained with 3% uranyl acetate on glow-discharged carbon-coated grids and then inspected with FEI 120 kV Tecnai G2 Spirit BioTWIN TEM equipped with a Gatan Rio 16 camera.…”