Identification of a carbonic anhydrase–Rubisco complex within the alpha-carboxysome

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 120; no. 43; p. e2308600120
• Main Authors: Blikstad, Cecilia, Dugan, Eli J., Laughlin, Thomas G., Turnšek, Julia B., Liu, Mira D., Shoemaker, Sophie R., Vogiatzi, Nikoleta, Remis, Jonathan P., Savage, David F.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 24.10.2023
• Subjects: Bacterial Proteins - metabolism; BASIC BIOLOGICAL SCIENCES; Bicarbonates; Binding sites; Biochemistry; Biological Sciences; Carbon dioxide; Carbon Dioxide - metabolism; Carbon dioxide concentration; Carbon dioxide fixation; Carbonic anhydrase; Carbonic anhydrases; Carbonic Anhydrases - metabolism; Carboxylation; Carboxysome; CO2 fixation; Cryoelectron Microscopy; Encapsulation; Hydrogen bonding; Hydrogen bonds; Organelles; Organelles - metabolism; Protein interaction; Proteins; Protein–protein interactions; Ribulose-bisphosphate carboxylase; Ribulose-Bisphosphate Carboxylase - metabolism; Scaffolding; Science & Technology; Self-assembly; protein–protein interactions; fixation; carbonic anhydrase; cryoelectron microscopy; CO; carboxysome; CO2 fixation
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.2308600120

SignificanceRubisco is responsible for the majority of inorganic carbon assimilation on Earth. To ensure efficient CO2 fixation, cyanobacteria and many autotrophic proteobacteria concentrate CO2 in the carboxysome, a bacterial organelle encapsulating Rubisco and carbonic anhydrase within a protein shell. It remains unknown exactly how this 250+ megadalton protein complex assembles with high fidelity inside cells. Here, we explore the encapsulation mechanism of the carbonic anhydrase, CsoSCA, and demonstrate that it is incorporated into the α-carboxysome via a carbonic anhydrase–Rubisco complex. Our results update the current model for carboxysome biogenesis and inform strategies for engineering CO2 concentration mechanisms into crops and industrially relevant microorganisms for improved growth and yields. Carboxysomes are proteinaceous organelles that encapsulate key enzymes of CO2 fixation—Rubisco and carbonic anhydrase—and are the centerpiece of the bacterial CO2 concentrating mechanism (CCM). In the CCM, actively accumulated cytosolic bicarbonate diffuses into the carboxysome and is converted to CO2 by carbonic anhydrase, producing a high CO2 concentration near Rubisco and ensuring efficient carboxylation. Self-assembly of the α-carboxysome is orchestrated by the intrinsically disordered scaffolding protein, CsoS2, which interacts with both Rubisco and carboxysomal shell proteins, but it is unknown how the carbonic anhydrase, CsoSCA, is incorporated into the α-carboxysome. Here, we present the structural basis of carbonic anhydrase encapsulation into α-carboxysomes from Halothiobacillus neapolitanus. We find that CsoSCA interacts directly with Rubisco via an intrinsically disordered N-terminal domain. A 1.98 Å single-particle cryoelectron microscopy structure of Rubisco in complex with this peptide reveals that CsoSCA binding is predominantly mediated by a network of hydrogen bonds. CsoSCA's binding site overlaps with that of CsoS2, but the two proteins utilize substantially different motifs and modes of binding, revealing a plasticity of the Rubisco binding site. Our results advance the understanding of carboxysome biogenesis and highlight the importance of Rubisco, not only as an enzyme but also as a central hub for mediating assembly through protein interactions.