Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA
Substrate Binding Stabilizes a Pre-translocation Intermediate in the ATP-binding Cassette Transport Protein MsbA
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DOI：
10.1074/jbc.m113.485714
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发表时间：
2013-07-26
影响因子：
4.8
通讯作者：
van Veen, Hendrik W.
中科院分区：
文献类型：
--
作者：
Doshi, Rupak;van Veen, Hendrik W.
三磷酸腺苷结合盒(ABC)转运蛋白是从原核生物到人类的最大的蛋白质超家族之一。最近细菌和哺乳动物ABC出口蛋白的X射线晶体结构表明，底物运输的一种常见的交替访问机制，这也得到了生物化学的证实。然而，目前的模型还没有解释底物结合和ATP水解之间的耦合，这是依赖于ATP的底物运输的基础。在我们对大肠杆菌同源二聚体多药/脂A ABC出口蛋白MSBA的研究中，我们对嵌入生物膜的转运蛋白的两个报告位置进行了半胱氨酸交联、荧光能量转移和半胱氨酸可及性研究，这两个位置分别位于核苷酸结合域和膜域。我们的结果首次表明，MSBA与底物的结合通过促进核苷酸结合域的二聚化来刺激ATP的最大水解率，这与先前描述的ABC出口体的无核苷酸、面向内和核苷酸结合、面向外的构象明显不同，它与ATP结合。
ATP-binding cassette (ABC) transporters belong to one of the largest protein superfamilies that expands from prokaryotes to man. Recent x-ray crystal structures of bacterial and mammalian ABC exporters suggest a common alternating access mechanism of substrate transport, which has also been biochemically substantiated. However, the current model does not yet explain the coupling between substrate binding and ATP hydrolysis that underlies ATP-dependent substrate transport. In our studies on the homodimeric multidrug/lipid A ABC exporter MsbA from Escherichia coli, we performed cysteine cross-linking, fluorescence energy transfer, and cysteine accessibility studies on two reporter positions, near the nucleotide-binding domains and in the membrane domains, for transporter embedded in a biological membrane. Our results suggest for the first time that substrate binding by MsbA stimulates the maximum rate of ATP hydrolysis by facilitating the dimerization of nucleotide-binding domains in a state, which is markedly distinct from the previously described nucleotide-free, inward-facing and nucleotide-bound, outward-facing conformations of ABC exporters and which binds ATP.