"Air-Lock" gating mechanism of CsoS1D for metabolite translocation through the α-carboxysome shell

Carboxysomes are specialized bacterial microcompartments (BMCs) for CO2 assimilation in cyanobacteria and many chemoautotrophs. Selective transport of gas molecules and metabolites across the carboxysome shell plays an essential role in creating a high-CO2 environment around Rubisco and ensuring efficient metabolite flux. However, the molecular mechanisms underlying this specific permeability remain elusive. Using integrated computational approaches, including all-atom molecular dynamics (MD) simulations, self-random acceleration MD simulations, umbrella sampling and targeted MD simulations, we systematically investigated the permeation pathways of large anionic metabolites ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA) through the α-carboxysome shell protein CsoS1D, which exhibits a trimer-of-dimer architecture and an enlarged central pore compared with hexameric and pentameric shell proteins. The results indicate that the central pore of CsoS1D serves as the primary conduit for the translocation of bulky metabolites RuBP and 3-PGA and reveal a 3-stage "air-lock" transport mechanism driven by electrostatic interactions. Moreover, the shallow free-energy landscape for channel gating enables the pore to undergo frequent, thermally driven transitions between open and closed states, implementing a conformational selection transport model independent of ligand binding. Our analysis further revealed 5 conserved residues that establish an electrostatic transport pathway within trimeric shell proteins, suggesting that this permeability mechanism represents a generalizable design principle across diverse BMCs. By elucidating shell protein permeability mechanisms at atomic resolution, this study lays the framework for understanding carboxysome physiology and guides the rational engineering of carboxysome permeability to facilitate system-level metabolic modeling and optimization of synthetic carboxysomes for biotechnological applications.