Proton Transfer Mechanisms in the MmpL3 Transporter of <i>Mycobacterium tuberculosis</i> Studied by Computer Simulations

The mycobacterial membrane exporter MmpL3 transports trehalose monomycolates (TMM) from the cytosol to the outer membrane of Mycobacterium tuberculosis, making it a potential drug target. Proton influx is believed to drive TMM efflux, suggesting that disrupting proton transfer (PT) could be therapeutic. However, the PT mechanism and its relation to function remain unclear. Recent MmpL3 structures reveal a potential proton channel in its hydrophobic core, which also binds potential antituberculosis compounds. We investigated the PT process using hybrid quantum-mechanical/molecular-mechanical and classical molecular dynamics simulations. We show that transient water chains form in two connected transmembrane cavities that act as proton conduits. Four consecutive PT events are necessary to alter the protonation states of acidic residues in the protein core, triggering conformational changes that affect the TMM binding site. The process begins with the tandem movement of two protons through an upper cavity, protonating two aspartate residues via a classical hydronium migration. After conformational shifts, PT proceeds through a lower cavity, protonating two glutamate residues near the cytosolic opening and inducing further conformational shifts; here, PT occurs sequentially via hydronium and proton-hole migration. The cycle ends with the release of protons into the cytosol. Based on the observed conformational changes, we propose a mechanism for TMM efflux.