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.

多药外排泵对细菌感染的治疗提出了挑战，因此了解其作用机理至关重要。在这里，我们调查乳酸乳球菌内底物结合的性质LmrP，主要促进者超家族的典型多药转运蛋白。我们确定了LmrP的晶体结构处于配体结合的向外打开状态，并观察到LmrP的结合腔中嵌入的脂质，这一观察得到了天然质谱分析的支持。分子动力学模拟表明，阴离子脂质可稳定观察到的配体结合结构。经过改造以破坏嵌入脂质的结合的突变体显示出某些（但不是全部）抗生素底物的转运减少。我们的结果表明，结合腔内的脂质可以提供可延展的疏水成分，从而可以适应不同底物的存在，从而有助于解释这种蛋白质以及其他多种药物转运蛋白的广泛特异性。