BPS2026 – Unraveling metabolite transport pathways in bacterial microcompartments using enhanced sampling

Bacterial microcompartments (BMCs) are protein-based organelles that enhance metabolic efficiency by sequestering enzymes and preventing the diffusion of toxic intermediates. A defining feature of BMCs is their selectively permeable protein shell, composed of hexameric and trimeric units that regulate flux of reactants, products, and cofactors. Therefore, understanding the transport process across the BMC shell provides insight into how shell architecture causes permeability defects, or enhances BMC function and how it can be modified to create new functionalities. While prior studies have demonstrated selective transport of Calvin cycle metabolites into CB hexameric pores, the precise mechanisms governing molecular influx and efflux through both the pores remain unclear. In the case of the Pdu and Eut BMC, the enzymes encapsulated within require ATP and NAD+/NADH cofactors. The exchange of oxidized and reduced NAD+/NADH cofactors across the microcompartment shell has been suggested, but another possibility is that those cofactors are regenerated in situ by redox interconversions, thereby obviating their need for transport. To date, quantitative data for metabolite flux across any BMC shell have not been obtained. Here, we employed enhanced sampling molecular dynamics simulations to quantify the permeability coefficients of key Calvin cycle metabolites (HCO 3 − , CO 2 , O 2 , ribulose bisphosphate, and 3-phosphoglycerate), essential cofactors (NAD(H) and NADP(H)), abiotic molecules (Ru(bpy) 3 ), and ATP through both hexameric and trimeric pores. The resulting free-energy profiles revealed that (1) most metabolites encounter relatively low free-energy barriers, suggesting limited restrictions on transport, although the shell still functions as a barrier to preserve enzyme-generated gradients, and (2) transport is strongly modulated by the electrostatic properties of gating residues within the pores. The results highlight the importance of substrate permeability across BMCs in a biological context and step forward toward generating synthetic shells or bioengineer novel nanoreactors.