The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA
The Q-loop disengages from the first intracellular loop during the catalytic cycle of the multidrug ABC transporter BmrA
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DOI：
10.1074/jbc.m503266200
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发表时间：
2005-11-04
影响因子：
4.8
通讯作者：
Jault, JM
中科院分区：
文献类型：
--
作者：
Dalmas, O;Orelle, C;Jault, JM
ATP结合盒是最丰富的转运蛋白家族，包括许多医学相关成员，并聚集了参与多种底物转运的输入者和输出者。虽然三个高分辨率的三维结构已经获得了一个原型的出口商，MsbA，两个受到了很多批评。在这里，BmrA，一个多药细菌转运蛋白结构相关的MsbA的构象变化进行了研究。基于大肠杆菌MsbA的“开放”构象，通过同时引入两个半胱氨酸残基来探测BmrA的三维模型，一个在跨膜结构域的第一胞内环中，另一个在核苷酸结合结构域（NBD）的Q环中。在没有任何效应物的情况下，可以产生分子内二硫键，这阻止了药物转运和ATP酶活性。有趣的是，加入ATP/Mg加钒酸盐强烈阻止了半胱氨酸双突变体中这种键的形成，而当还引入ATP酶失活的E504 Q突变时，单独的ATP/Mg就足够了，这与另外的BmrA模型一致，其中ATP结合位点位于NBD/NBD界面。此外，在ATP/Mg加钒酸盐存在下，当加入间隔大于13埃的同双功能交联剂时，两个半胱氨酸残基之间的交联仍然可以实现。总之，这些结果支持在静息状态下存在与开放MsbA二聚体内发现的类似的BmrA的非聚体构象，并表明在胞内环1和核苷酸结合结构域之间需要大的运动以使多药物ATP结合盒转运蛋白正常发挥功能。
The ATP-binding cassette is the most abundant family of transporters including many medically relevant members and gathers both importers and exporters involved in the transport of a wide variety of substrates. Although three high resolution three-dimensional structures have been obtained for a prototypic exporter, MsbA, two have been subjected to much criticism. Here, conformational changes of BmrA, amultidrug bacterial transporter structurally related to MsbA, have been studied. A three-dimensional model of BmrA, based on the "open" conformation of Escherichia coli MsbA, was probed by simultaneously introducing two cysteine residues, one in the first intracellular loop of the transmembrane domain and the other in the Q-loop of the nucleotide- binding domain (NBD). Intramolecular disulfide bonds could be created in the absence of any effectors, which prevented both drug transport and ATPase activity. Interestingly, addition of ATP/Mg plus vanadate strongly prevented this bond formation in a cysteine double mutant, whereas ATP/Mg alone was sufficient when the ATPase-inactive E504Q mutation was also introduced, in agreement with additional BmrA models where the ATP-binding sites are positioned at the NBD/NBD interface. Furthermore, cross-linking between the two cysteine residues could still be achieved in the presence of ATP/Mg plus vanadate when homobifunctional cross-linkers separated by more than 13 angstrom were added. Altogether, these results give support to the existence, in the resting state, of amonomeric conformation of BmrA similar to that found within the open MsbA dimer and show that a large motion is required between intracellular loop 1 and the nucleotide- binding domain for the proper functioning of a multidrug ATP-binding cassette transporter.