Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications

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• 作者：Lucía Álvarez ; Ariel Lewis-Ballester ; Adrián Roitberg ; Darío A. Estrin ; Syun-Ru Yeh ; Marcelo A. Marti ; Luciana Capece
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：May 17, 2016
• 年：2016
• 卷：55
• 期：19
• 页码：2785-2793
• 全文大小：504K
• 年卷期：0
• ISSN：1520-4995

文摘

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 loop^C) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop^N) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop^N 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 loop^C, thereby anchoring JK loop^C 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 loop^C 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 loop^C in controlling substrate and product dynamics.