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

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.