Protonation-dependent substrate release in a bacterial homolog of vesicular glutamate.

The SLC17 family contains diverse organic anion transporters with various stoichiometries and ion coupling mechanisms. A bacterial protein of this family, the D-galactonate transporter DgoT, co-transports two protons per substrate molecule. Although the overall transport cycle of DgoT has been proposed, the role of substrate protonation during its release remains unclear. Galactonate is expected to bind in a deprotonated form due to its low pKa; however, it can be released from the transporter in either a protonated or deprotonated state. In this study, we used well-tempered funnel metadynamics simulations to investigate the microscopic mechanisms underlying protonated and deprotonated galactonate dissociation from the inward-facing, gate-open conformation of DgoT. Our free energy profiles reveal that, although substrate protonation lowers the energy barrier for release and may enhance dissociation kinetics, deprotonated galactonate can also dissociate, albeit less frequently. These findings indicate that galactonate protonation facilitates, but is not strictly required for, substrate release by the bacterial organic anion transporter DgoT.