Probing the ATP Hydrolysis Cycle of the ABC Multidrug Transporter LmrA by Pulsed EPR Spectroscopy
Probing the ATP Hydrolysis Cycle of the ABC Multidrug Transporter LmrA by Pulsed EPR Spectroscopy
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DOI：
10.1021/ja211007t
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发表时间：
2012-04-04
影响因子：
15
通讯作者：
Glaubitz, Clemens
中科院分区：
文献类型：
--
作者：
Hellmich, Ute A.;Lyubenova, Sevdalina;Glaubitz, Clemens
ATP结合盒（ABC）转运蛋白超家族的成员以ATP为代价将各种类型的分子跨膜转运。这需要通过许多催化状态循环。在这里，我们报告整个催化循环的LmrA，同源二聚体多药ABC转运蛋白从L。乳酸菌。使用定点自旋标记和脉冲电子-电子双共振（PELDOR/DEER）光谱，我们已经探测了核苷酸结合结构域和跨膜螺旋6的重新取向，这与药物结合和二聚化界面的一部分特别相关。我们的数据表明，LmrA样品在其载脂蛋白状态下具有非常大的构象空间，这在核苷酸结合时显著减少。需要ATP结合而不是水解来触发这种构象变化，这导致核苷酸结合结构域和跨膜螺旋6的相对固定的取向。这种取向在整个ATP水解循环中保持，直到蛋白质循环回到其apo状态。我们的数据提供了强有力的证据，两个动态和结构不同的状态之间的切换是必要的基板易位。
Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from L. lactis. Using site-directed spin labeling and pulsed electron- electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation.