Proton transport drives allosteric conformational changes in the MmpL3 transporter.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a global health crisis, further complicated by increasing drug resistance. The mycobacterial membrane protein MmpL3, which is essential for transporting cell wall precursors such as trehalose monomycolate (TMM), is a promising therapeutic target. However, the mechanistic link between proton-driven dynamics and substrate transport in MmpL3 is not fully understood. In this study, we use molecular dynamics simulations to investigate how protonation of conserved Asp-Tyr dyads (Asp256-Tyr646 and Asp645-Tyr257) in Mycobacterium smegmatis (Msmg) MmpL3 regulates conformational changes and TMM binding. Protonation at Asp256 induces an open dyad conformation, increases local water accessibility, and is associated with a tendency toward more open configurations of the periplasmic binding pocket. In contrast, protonation at Asp645 stabilizes a closed dyad conformation, reduces water accessibility, and narrows the transmembrane binding pocket. These findings support a proposed sequential protonation model in which protonation-dependent conformational changes are allosterically coupled to substrate translocation.