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

The rapid increase of bacterial multidrug resistance (MDR) is an urgent threat to human health. MDR transporters are membrane proteins that can transport a wide array of structurally dissimilar compounds and contribute to drug resistance by exporting different antibiotics from the cell. Whether such transporters exhibit common or distinct transport mechanisms for different substrates is poorly understood. In this work, we measured the time-resolved conformational dynamics of the model MDR transporter LmrP in its apo and ligand-bound state with single-molecule Forster resonance energy transfer (smFRET). We used multi-parameter Hidden Markov Modeling (mpH2MM) to characterize the underlying conformational states of LmrP in the presence of different antibiotics and to quantify their sub-millisecond interconversion kinetics. We observed substrate-dependent heterogeneity in the conformational landscape, both in terms of accessible states an conversion rates between them, suggesting that MFS MDR transporters can export structurally dissimilar antibiotics by relying on an array of underlying conformational states with substrate-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.