Mamkaeva, M. | Search bioRxiv

bioRxiv2026

Local conformational plasticity underlies ligand recognition and 1 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.

biochemistry↗

bioRxiv2025

Antibiotics-induced conformational heterogeneity of a multidrug transporter revealed by single-molecule FRET

Multidrug transporters are membrane proteins that can transport an ensemble of structurally dissimilar compounds and contribute to bacterial multidrug resistance (MDR) by exporting different antibiotics from the cell. However, whether they transport different substrates through a common mechanism or via distinct substrate-dependent mechanisms remains unclear. In this work, we used single-molecule Forster resonance energy transfer (smFRET) to measure time-resolved conformational dynamics of LmrP, a multidrug transporter of the Major Facilitator Superfamily (MFS). We present high-resolution conformational landscapes of LmrP in the presence of different antibiotics. Through multi-parameter Hidden Markov Modeling (mpH2MM), we uncovered transient states and quantified their sub-millisecond interconversion kinetics. We observed antibiotic-dependent heterogeneity in the conformational landscape, both in accessible states and in interconversion rates. Notably, poorly or non-transported antibiotics slow down transition kinetics, pointing to rapid state interconversion as a driver of efficient transport. This suggests that MFS MDR transporters bind and export structurally dissimilar antibiotics by relying on an array of underlying conformational states with ligand-dictated interconversion rates. This work provides novel insights into the mechanism of MDR transporters and advocates for combined structure/dynamics-based drug design when targeting their function.

biophysics↗