The C‑terminal Region of D‑DT Regulates Molecular Recognition for Protein–Ligand Complexes

Systematic analysis of molecular recognition is critical for understanding the biological function of macromolecules. For the immunomodulatory protein D-dopachrome tautomerase (D-DT), the mechanism of protein–ligand interactions is poorly understood. Here, 17 carefully designed protein variants and wild type (WT) D-DT were interrogated with an array of complementary techniques to elucidate the structural basis of ligand recognition. Utilization of a substrate and two selective inhibitors with distinct binding profiles offered previously unseen mechanistic insights into D-DT–ligand interactions. Our results demonstrate that the C-terminal region serves a key role in molecular recognition via regulation of the active site opening, protein–ligand interactions, and conformational flexibility of the pocket’s environment. While our study is the first comprehensive analysis of molecular recognition for D-DT, the findings reported herein promote the understanding of protein functionality and enable the design of new structure-based drug discovery projects.