Phosphorylation of ITIM motifs drives the structural transition of indoleamine 2,3-dioxygenase 1 between enzymatic and non-enzymatic states.

Indoleamine 2,3-dioxygenase 1 (IDO1) is the rate-limiting enzyme in tryptophan metabolism that plays a central role in immune regulation across a range of diseases, including cancer. Beyond its enzymatic role, IDO1 has a non-enzymatic function that remains poorly understood. This study explores how phosphorylation of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) modulates IDO1's structural dynamics and functional states. Using molecular dynamics simulations and structural analysis, we show that phosphorylation acts as a molecular switch, inducing conformational changes that regulate heme-binding, remodel specific loop regions, and govern protein-protein interactions with SHP1, SHP2, and SOCS3. Notably, Tyr249 phosphorylation inhibits enzymatic activity by compacting the heme-binding pocket, creating steric hindrance that prevents cofactor binding. In contrast, Tyr111 phosphorylation enhances interactions with SHP1 or SHP2 proteins by embedding their C-terminal regions into the heme-binding pocket, also obstructing heme binding. Furthermore, Tyr249 phosphorylation promotes SOCS3 binding through the formation of a unique loop structure near the phosphorylation site. These findings provide a detailed mechanistic framework for understanding how ITIM phosphorylation orchestrates IDO1's functional transitions, effectively balancing its enzymatic and non-enzymatic functions.