Molecular recognition of substrate intermediate and inhibitors by tryptophanyl-tRNA synthetase from Mycobacterium tuberculosis.

Protein biosynthesis represents a key target for anti-tubercular drug design. Tryptophanyl-tRNA synthetase (TrpRS) is an essential enzyme in Mycobacterium tuberculosis (M.tb) that is involved in translation. Here, we investigate how TrpRS recognizes the substrate-intermediate versus inhibitors and find essential physicochemical features relevant for inhibitor design. We performed systematic molecular dynamics simulations for 24 microseconds across eight TrpRS states, including the apo form, the substrate intermediate, and six indolmycin-based inhibitor-bound states. The substrate-intermediate state was less stable than the inhibitor-bound states, consistent with its transient nature. Inhibitors act by stabilizing the ATP conformation within the TrpRS binding site, whereas compounds that disrupt ATP conformation and its interactions with TrpRS show reduced inhibitory activity. Using data of 28 indolmycin-based inhibitors, we developed simple two-property models (R = 0.82-0.88, p < 0.01) defining the TrpRS inhibitory activity based on electron affinity/solubility and ensemble ligand docking, and further derived a six-feature pharmacophore framework for inhibitor design. TrpRS inhibition was successfully explained by ensemble docking using four TrpRS structures, yielding a correlation coefficient of 0.77 (p < 0.01). These results provide mechanistic insight into TrpRS function and support structure-guided antitubercular drug design.