Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.
Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.
复制标题
使用氢-氘交换质谱法捕获配体和金属结合时人异柠檬酸脱氢酶 1 (IDH1) 的动态构象变化。
DOI：
10.1021/acs.biochem.2c00636
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发表时间：
2023
期刊：
影响因子：
2.9
通讯作者：
Sohl,ChristalD
中科院分区：
文献类型：
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作者：
Sabo,KaitlynA;Albekioni,Elene;Caliger,Danielle;Coleman,NalaniJ;Thornberg,Ella;AvellanedaMatteo,Diego;Komives,ElizabethA;Silletti,Steve;Sohl,ChristalD
人异柠檬酸脱氢酶1（IDH 1）是一种高度保守的代谢酶，催化异柠檬酸和α-酮戊二酸的相互转化。IDH 1的动力学和结构研究揭示了其底物、异柠檬酸盐和NADP+及其催化金属阳离子结合后发生显著构象变化的证据。在这里，我们使用氢-氘交换质谱（HDX-MS）建立一个全面的地图IDH 1经历的动态构象变化后，配体结合。IDH 1被证明非常适合HDX-MS分析，使我们能够捕获底物结合位点和已知调控区域以及更远的局部亚结构域的溶剂可及性的深刻变化，这些亚结构域似乎支持该蛋白质关闭成其活性构象。HDX-MS分析表明，IDH 1主要是在不存在其金属阳离子的情况下与NADP（H）结合纯化的。随后的金属阳离子结合，即使在不存在异柠檬酸盐的情况下，对于驱动大的构象变化是至关重要的。WT IDH 1仅在底物和金属的完全互补存在时折叠成其完全闭合的构象。最后，我们显示的证据支持以前假设的部分开放的构象，形成之前的催化活性状态，我们提出这种构象是由异柠檬酸结合在金属的情况下。
Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen–deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.