Structure and encapsulation of carbonic anhydrase within the α-carboxysome

Carboxysomes in cyanobacteria and certain proteobacteria enable efficient CO₂ fixation by encapsulating ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a semipermeable shell. Sequestered CA catalyze the rapid interconversion of CO₂ and HCO₃^-, supplying elevated levels of CO₂ to boost Rubisco carboxylation. Despite its essential role, the structure and encapsulation of CA within carboxysomes remain poorly understood. Here, we determined the molecular structure of α-carboxysomal CA from the model chemoautotrophic bacterium <i>Halothiobacillus neapolitanus</i> (<i>Hn</i>CsoSCA). <i>Hn</i>CsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that <i>Hn</i>CsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. <i>Hn</i>CsoSCA truncations suggest nonspecific interactions between <i>Hn</i>CsoSCA and CsoS1A. We further show that <i>Hn</i>CsoSCA bridges Rubisco and the shell facets. Our study offers insights into the assembly and encapsulation mechanisms of α-carboxysomes and provides the framework for reprogramming carboxysome structures for synthetic biology and biotechnological applications.