Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter.
Conformational dynamics of the nucleotide binding domains and the power stroke of a heterodimeric ABC transporter.
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DOI：
10.7554/elife.02740
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发表时间：
2014-05-16
期刊：
影响因子：
7.7
通讯作者：
Mchaourab HS
中科院分区：
文献类型：
--
作者：
Mishra S;Verhalen B;Stein RA;Wen PC;Tajkhorshid E;Mchaourab HS
多药ATP结合盒（ABC）输出体是普遍存在的ABC转运体，它将细胞毒性分子挤出细胞膜。尽管最近在这些转运体的结构确定方面取得了进展，但将ATP水解的能量转化为底物易位的构象运动仍然不清楚。在这里，我们研究了BmrCD的构象周期，BmrCD是ABC出口商异二聚体家族的代表，具有内在受损的核苷酸结合位点。我们测量了监测核苷酸结合（NBD）和跨膜结构域（TMD）运动的自旋标签对之间的距离。结果揭示了以前未观察到的nbd结构中间体，这些中间体是由催化不相等核苷酸结合位点的不对称配置引起的。TMD的两态转变，从内向到外向的构象，完全由ATP水解驱动。这些发现为ABC出口商的机制差异提供了直接证据。DOI: http://dx.doi.org/10.7554/eLife.02740.001细胞被一层膜包围，这层膜对许多分子起着屏障的作用。这层膜要么阻止分子进入或离开细胞，要么至少减缓它们的运动。然而，重要的是细胞可以清除一些分子，如毒素，营养物质和某些其他分子可以进入细胞。因此，细胞依靠嵌入在膜内的“转运蛋白”来移动这些分子穿过膜。被称为“多药ABC输出体”的转运蛋白几乎存在于所有生物中，它们利用分解三磷酸腺苷（简称ATP）分子所释放的能量将毒素排出细胞。虽然许多转运体的三维形状是已知的，但尚不清楚ATP分子释放的能量如何使转运体将毒素从膜的一边移动到另一边。在这里，Mishra等人研究了一种叫做枯草芽孢杆菌的细菌的ABC输出物的形状在与ATP相互作用时是如何变化的。大多数细菌的ABC输出蛋白是由同一蛋白质的两个副本构成的，但枯草芽孢杆菌的输出蛋白是由两种略有不同的蛋白质构成的，其中一种结合和分解ATP的能力较弱。Mishra等人发现，这两种蛋白质与ATP结合的部分可以采用一系列不同的形状，这是以前从未见过的。此外，当ATP结合时，跨越细胞膜的蛋白质部分面朝细胞内，当ATP被分解时，蛋白质部分面朝细胞外。蛋白质的这种运动将允许细胞内的有毒分子进入出口，然后被推到细胞外。Mishra等人的研究结果表明，并非所有ABC出口商都是通过相同的机制工作的。未来的工作可能会将这种新的认识扩展到人类的多药ABC转运蛋白，它可以从我们的细胞中清除废物和有害分子，并与癌细胞对化疗的耐药性有关。DOI: http://dx.doi.org/10.7554/eLife.02740.002
Multidrug ATP binding cassette (ABC) exporters are ubiquitous ABC transporters that extrude cytotoxic molecules across cell membranes. Despite recent progress in structure determination of these transporters, the conformational motion that transduces the energy of ATP hydrolysis to the work of substrate translocation remains undefined. Here, we have investigated the conformational cycle of BmrCD, a representative of the heterodimer family of ABC exporters that have an intrinsically impaired nucleotide binding site. We measured distances between pairs of spin labels monitoring the movement of the nucleotide binding (NBD) and transmembrane domains (TMD). The results expose previously unobserved structural intermediates of the NBDs arising from asymmetric configuration of catalytically inequivalent nucleotide binding sites. The two-state transition of the TMD, from an inward- to an outward-facing conformation, is driven exclusively by ATP hydrolysis. These findings provide direct evidence of divergence in the mechanism of ABC exporters. DOI: http://dx.doi.org/10.7554/eLife.02740.001 Cells are surrounded by a membrane that acts like a barrier to many molecules. This membrane either stops molecules from entering or exiting the cell, or at least slows their movement. However, it is important that cells can remove some molecules, such as toxins, and that nutrients and certain other molecules can get into cells. As such, cells rely on ‘transporter’ proteins embedded within the membrane to move these molecules through the membrane. Transporters called ‘Multidrug ABC exporters’ are found in almost all living things, and use the energy released by breaking down molecules of adenosine triphosphate (ATP for short) to pump toxins out of cells. Although the three-dimensional shapes of many transporters are known, it is not clear how the energy released from ATP molecules allows the transporter to move a toxin from one side of the membrane to the other. Here, Mishra et al. have looked at how the shape of an ABC exporter from a bacterium called Bacillus subtilis changes as it interacts with ATP. Most bacterial ABC exporters are made from two copies of the same protein, but the B. subtilis exporter is made from two slightly different proteins, one of which is less able to bind to and break down ATP. Mishra et al. found that those parts of the two proteins that bind to ATP can adopt a range of different shapes that had not been seen before. Moreover, the parts of the proteins that extend across the cell membrane face into the cell when the ATP binds, and switch to face out of the cell when the ATP is broken down. This movement of the proteins would allow toxic molecules inside the cell to enter the exporter, and then be pushed to the outside of the cell. The findings of Mishra et al. show that not all ABC exporters work by the same mechanism. Future work could extend this new understanding to multidrug ABC transporters from humans, which remove waste and harmful molecules from our cells and have been implicated in resistance to chemotherapy in cancer cells. DOI: http://dx.doi.org/10.7554/eLife.02740.002