Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.
Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.
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DOI：
10.1021/acs.biochem.6b00077
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发表时间：
2016-05-17
期刊：
影响因子：
2.9
通讯作者：
Capece L
中科院分区：
文献类型：
--
作者：
Álvarez L;Lewis-Ballester A;Roitberg A;Estrin DA;Yeh SR;Marti MA;Capece L
人吲哚胺2，3-双加氧酶催化色氨酸氧化裂解为N-甲酰犬尿氨酸，这是犬尿氨酸途径中的起始和限速步骤。此外，这种酶已被确定为癌症治疗的可能靶点。连接J和K螺旋的20个氨基酸的蛋白质片段(JK环)在已报道的HIDO晶体结构中没有被解析。以前的研究表明，这个环在底物结合时进行结构重排。在这项工作中，我们应用复制交换分子动力学模拟和定点突变实验相结合的方法来表征该蛋白质区域的结构和动力学。我们的模拟结果表明，JK环可以分为两个区域：第一个区域(JK LoopC)显示出特定的、明确的构象，并且位于衬底的氢键距离内；第二个区域(JK LoopN)是高度无序的，并且暴露在溶剂中。JK loopN特殊的柔性性质表明它可能是翻译后修饰的目标和/或蛋白质-蛋白质相互作用的介体。相反，在高度保守的JK loopC的“GTGG”基序中，观察到底物与Thr379之间的氢键作用，从而将JK loopC锚定在封闭的构象中，这为催化提供了合适的底物结合模式。定点突变实验证实了该残基的关键作用，突出了JK loopC构象在调节酶活性中的重要性。此外，无底物形式的部分和完全开放构象的存在表明JK loopC在控制底物和产物动力学方面发挥了作用。
Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loopC) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loopN) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loopN suggests that it may function as a target for post-translational modifications and/or a mediator for protein–protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved “GTGG” motif of JK loopC, thereby anchoring JK loopC in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loopC conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loopC in controlling substrate and product dynamics.