Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump
Mass spectrometry reveals synergistic effects of nucleotides, lipids, and drugs binding to a multidrug resistance efflux pump
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DOI：
10.1073/pnas.1303888110
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发表时间：
2013-06-11
影响因子：
11.1
通讯作者：
Robinson, Carol V.
中科院分区：
文献类型：
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作者：
Marcoux, Julien;Wang, Sheila C.;Robinson, Carol V.
多药耐药性是成功治疗包括癌症在内的许多人类疾病的严重障碍，其中化疗药物通过膜嵌入泵从靶细胞输出。这些泵中最普遍的是ATP结合盒转运蛋白P-糖蛋白（P-gp），由两个同源的半体组成，每个半体包含一个核苷酸结合结构域和六个跨膜螺旋。跨膜区包裹疏水腔，通过膜中的门户进入，其结合细胞毒性化合物以及脂质和肽。在这里，我们使用质谱（MS）探测完整的P-gp小分子结合复合物的洗涤剂胶束。气相中的活化导致离子的形成，基本上不含去污剂，但保留药物分子以及带电或两性离子脂质。测量脂质结合率和计算表观K-D值表明，多达6个带负电荷的二酰基甘油酯比两性离子脂质更有利地结合。类似的实验证实了心磷脂的结合，并表明免疫抑制剂和抗真菌抗生素环孢菌素A的预先结合增强了心磷脂的后续结合。离子迁移率MS表明，P-gp存在于不同状态之间的平衡，很容易通过配体结合相互转化。总体而言，这些MS结果显示了协同的小分子结合如何导致对多药物外排泵的结合亲和力和构象的协同效应。
Multidrug resistance is a serious barrier to successful treatment of many human diseases, including cancer, wherein chemotherapeutics are exported from target cells by membrane-embedded pumps. The most prevalent of these pumps, the ATP-Binding Cassette transporter P-glycoprotein (P-gp), consists of two homologous halves each comprising one nucleotide-binding domain and six transmembrane helices. The transmembrane region encapsulates a hydrophobic cavity, accessed by portals in the membrane, that binds cytotoxic compounds as well as lipids and peptides. Here we use mass spectrometry (MS) to probe the intact P-gp small molecule-bound complex in a detergent micelle. Activation in the gas phase leads to formation of ions, largely devoid of detergent, yet retaining drug molecules as well as charged or zwitterionic lipids. Measuring the rates of lipid binding and calculating apparent K-D values shows that up to six negatively charged diacylglycerides bind more favorably than zwitterionic lipids. Similar experiments confirm binding of cardiolipins and show that prior binding of the immunosuppressant and antifungal antibiotic cyclosporin A enhances subsequent binding of cardiolipin. Ion mobility MS reveals that P-gp exists in an equilibrium between different states, readily interconverted by ligand binding. Overall these MS results show how concerted small molecule binding leads to synergistic effects on binding affinities and conformations of a multidrug efflux pump.