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.