State Key Laboratory of Molecular Biology and Research Center for Structural Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
Cell Res. 2010 Nov;20(11):1188-200. doi: 10.1038/cr.2010.145. Epub 2010 Oct 26.
Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumor-derived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the "off switch" of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the "on switch" of the hypothetically oncogenic reduction activity of IDH1 by this mutation.
人源细胞质 NADP-依赖的异柠檬酸脱氢酶（IDH1）最近被发现与肿瘤发生有关。值得注意的是，迄今为止鉴定的肿瘤来源的 IDH1 突变主要发生在精氨酸 132 位，而突变 R132H 是最常见的一种。这种突变会损害酶的氧化 IDH 活性，但赋予了新的还原功能，将α-酮戊二酸（αKG）转化为 2-羟基戊二酸。在这里，我们报告了与和不与异柠檬酸（ICT）结合的 R132H 突变 IDH1 的结构。结构数据以及突变和生化数据揭示了一个以前未定义的初始 ICT 结合状态，并证明 IDH 活性需要构象变化到封闭的预过渡状态。Arg132 在催化反应中起多种功能作用；特别是，R132H 突变阻碍了从初始 ICT 结合状态到预过渡状态的构象变化，导致 IDH 活性受损。我们的结果首次描述了存在与初始 ICT 结合状态相对应的中间构象，并且 R132H 突变可以将酶固定在这种构象中，从而阐明了潜在的肿瘤抑制性 IDH 活性的“关闭开关”的分子机制。此外，我们证明了 Tyr139 对于获得的αKG 还原活性的必要性，并提出 Tyr139 可能通过补偿在从 NADPH 向αKG 转移氢阴离子时αKG 的 C2 原子上增加的负电荷来发挥重要作用，这为突变引起的 IDH1 潜在致癌还原活性的“开启开关”机制提供了新的见解。
