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 Halothiobacillus neapolitanus (HnCsoSCA). HnCsoSCA adopts a trimer-of-dimers oligomeric structure without the incorporation of a zinc ion at its symmetric center. Using synthetic minishells, we demonstrate that HnCsoSCA interacts with the CsoS1A shell hexamer and is incorporated into the minishells at the inner surface, independent of the CsoS2 linker protein. HnCsoSCA truncations suggest nonspecific interactions between HnCsoSCA and CsoS1A. We further show that HnCsoSCA 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.