Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1
Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1
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DOI：
10.1096/fj.12-212746
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发表时间：
2012-11-01
期刊：
影响因子：
4.8
通讯作者：
Eckenhoff, Roderic G.
中科院分区：
文献类型：
--
作者：
Yuki, Koichi;Bu, Weiming;Eckenhoff, Roderic G.
我们之前证明异氟烷靶向淋巴细胞功能相关抗原 1 (LFA-1)，这是白细胞停滞的关键粘附分子。然而，异氟烷如何与完整的胞外域 LFA-1 相互作用并调节其构象和功能仍有待确定。使用可光活化异氟烷（阿齐-异氟烷）进行光标记来探测完整胞外域 LFA-1 上的异氟烷结合位点。通过液相色谱/质谱分析测定加合的残留物。单独进行对接模拟以预测结合位点。在异氟烷结合位点周围引入点突变。通过细胞内粘附分子-1 (ICAM-1) 结合测定和使用流式细胞术对激活敏感抗体进行表位作图来评估异氟烷作用的重要性。使用光标记识别了两个异氟醚结合位点，并通过对接模拟进一步验证：一个位于 ICAM-1 结合域（α I 域）的疏水口袋；另一个位于 ICAM-1 结合域（α I 域）的疏水口袋；另一个位于 beta I 域。 α'1 螺旋的诱变表明，β I 结构域的异氟醚结合位点对于调节 LFA-1 功能和构象非常重要。使用激活敏感抗体进行表位作图表明异氟烷可稳定 LFA-1 的闭合构象。这项研究表明，异氟烷与 ICAM-1 结合位点的 α I 和 β I 结构域变构结合，并且异氟烷结合可稳定 LFA-1 的闭合构象。-Yuki, K.、Bu, W.、Xi, J.、Sen, M.、Shimaoka, M.、Eckenhof, R. G. 异氟烷结合并稳定白细胞功能相关抗原 1 的闭合构象。 FASEB J. 26, 4408-4417 (2012)。 www.fasebj.org
We previously demonstrated that isoflurane targets lymphocyte function-associated antigen-1 (LFA-1), a critical adhesion molecule for leukocyte arrest. However, it remains to be determined how isoflurane interacts with the full ectodomain LFA-1 and modulates its conformation and function. Isoflurane binding sites on the full ectodomain LFA-1 were probed by photolabeling using photoactivatable isoflurane (azi-isoflurane). The adducted residues were determined by liquid chromatography/mass spectrometry analysis. Separately, docking simulations were performed to predict binding sites. Point mutations were introduced around isoflurane binding sites. The significance of isoflurane's effect was assessed in both intracellular adhesion molecule-1 (ICAM-1) binding assays and epitope mapping of activation-sensitive antibodies using flow cytometry. Two isoflurane binding sites were identified using photolabeling and were further validated by the docking simulation: one at the hydrophobic pocket in the ICAM-1 binding domain (the alpha I domain); the other at the beta I domain. Mutagenesis of the alpha'1 helix showed that isoflurane binding sites at the beta I domain were significantly important in modulating LFA-1 function and conformation. Epitope mapping using activation-sensitive antibodies suggested that isoflurane stabilized LFA-1 in the closed conformation. This study suggested that isoflurane binds to both the alpha I and beta I domains allosteric to the ICAM-1 binding site, and that isoflurane binding stabilizes LFA-1 in the closed conformation.-Yuki, K., Bu, W., Xi, J., Sen, M., Shimaoka, M., Eckenhof, R. G. Isoflurane binds and stabilizes a closed conformation of the leukocyte function-associated antigen-1. FASEB J. 26, 4408-4417 (2012). www.fasebj.org