Multidrug efflux pumps present a challenge to the treatment of bacterial infections, making it vitally important to understand their mechanism of action. Here, we investigate the nature of substrate binding within Lactococcus lactis LmrP, a prototypical multidrug transporter of the major facilitator superfamily. We determined the crystal structure of LmrP in a ligand-bound outward-open state and observed an embedded lipid in the binding cavity of LmrP, an observation supported by native mass spectrometry analyses. Molecular dynamics simulations suggest that the anionic lipid stabilizes the observed ligand-bound structure. Mutants engineered to disrupt binding of the embedded lipid display reduced transport of some, but not all, antibiotic substrates. Our results suggest that a lipid within the binding cavity could provide a malleable hydrophobic component that allows adaptation to the presence of different substrates, helping to explain the broad specificity of this protein and possibly other multidrug transporters.

Anion exchanger 1 catalyzes the transmembrane antiport of chloride and bicarbonate ions through a mechanism that has remained unclear. By modeling its inward-facing state and comparing it with the known outward-facing form, Ficici et al. hypothesize that this transporter features an elevator-like mechanism.

Multidrug and toxic-compound extrusion (MATE) proteins comprise an important but largely uncharacterized family of secondary-active transporters. In both eukaryotes and prokaryotes, these transporters protect the cell by catalyzing the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are thus potential pharmacological targets against drug-resistant pathogenic bacteria and tumor cells. The activity of MATEs is powered by transmembrane electrochemical ion gradients, but their molecular mechanism and ion specificity are not understood, in part because high-quality structural information is limited. Here, we use computational methods to study PfMATE, from , whose structure is the best resolved to date. Analysis of available crystallographic data and additional molecular dynamics simulations unequivocally reveal an occupied Na-binding site in the N-lobe of this transporter, which had not been previously recognized. We find this site to be selective against K and broadly conserved among prokaryotic MATEs, including homologs known to be Na-dependent such as NorM-VC, VmrA, and ClbM, for which the location of the Na site had been debated. We note, however, that the chemical makeup of the proposed Na site indicates it is weakly specific against H, explaining why MATEs featuring this Na-binding motif may be solely driven by H in laboratory conditions. We further posit that the concurrent coupling to H and Na gradients observed for some Na-driven MATEs owes to a second H-binding site, within the C-lobe. In summary, our study provides insights into the structural basis for the complex ion dependency of MATE transporters.