A comparative electron paramagnetic resonance study of the nucleotide-binding domains' catalytic cycle in the assembled maltose ATP-binding cassette importer
A comparative electron paramagnetic resonance study of the nucleotide-binding domains' catalytic cycle in the assembled maltose ATP-binding cassette importer
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DOI：
10.1529/biophysj.108.132456
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发表时间：
2008-09-15
影响因子：
3.4
通讯作者：
Schneider, Erwin
中科院分区：
文献类型：
--
作者：
Grote, Mathias;Bordignon, Enrica;Schneider, Erwin
我们提出了一个定量分析的核苷酸结合亚基，MalK（2），麦芽糖ATP结合盒进口商MalFGK（2）在运输周期中的构象变化。所选残基之间发生的距离变化进行了监测，通过定点自旋标记电子顺磁共振光谱和定点化学交联在全转运。我们认为S83 C和A85 C来自MalK的保守Q环，V117 C位于MalK的外表面。此外，研究中还包括两种天然半胱氨酸（C350、C360）。在ATP结合时，检测到天然位点之间的小重排，并且位置117之间没有距离变化。相反，位置85在ATP结合状态和钒酸盐捕获的中间体中更靠近在一起，并在ATP水解后向脱辅基状态移动。位置83之间的距离显示在ATP结合时略微减小，并且在ATP水解后进一步减小。从交联实验的结果与这些发现是一致的。将数据与来自分离的MalK（2）和MalFGK（2）-E复合物的计算机自旋标记的X射线结构进行比较。我们的研究结果与MalK（2）在催化循环过程中关闭和重新打开的略微修改的“镊子样”模型一致，并显示了MalK和跨膜亚基MalG之间不可预见的潜在相互作用。
We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK(2), of the maltose ATP-binding cassette importer MalFGK(2) during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. OnATPbinding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK(2) and the MalFGK(2)-E complex. Our results are consistent with a slightly modified "tweezers-like'' model of closure and reopening of MalK(2) during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.