Local conformational plasticity underlies ligand recognition and proton coupling in MFS multidrug transporters

Polyspecific substrate recognition drives multidrug efflux and antibiotic resistance, yet its molecular basis remains unclear. Here, we use HDX-MS to compare ligand-dependent local dynamics in three multidrug efflux pumps: NorA, QacA and LmrP. In the apo state, all three display high flexibility in specific transmembrane helices, unlike homologous transporters with narrow substrate profiles. Substrate binding remodels these flexible regions, but in a transporter-specific manner, revealing divergent local adaptations within a conserved fold. In LmrP, protonation-mimicking mutation of a conserved acidic residue recapitulates the substrate-induced dynamic changes, supporting a model in which transmembrane helix flexibility couples protonation and substrate binding to the conformational changes required for transport. Together, our study identifies local plasticity beyond what is captured by static high-resolution structures, as an overlooked feature of polyspecific ligand recognition.