(a) A schematic of the functional domain arrangement in human KRS. KRS contains an N-terminal extension domain (N-ext), an anticodon-binding domain (ABD), and a catalytic domain (CD). The T52 residue (undergoes phosphorylation) is indicated as a red dot. N-ext and ABD of KRS specifically interact with LR and YH16899. Arrow width indicates intensity of the compound–KRS interaction. (b) Spatial arrangement of the chemically perturbed residues (red) on KRS with tRNALys. (c) Docking positions of the R- (left) and S- (right) YH16899 in the hydrophobic pocket of KRS were identified in NMR experiments. Ten low-energy positions were represented, and the CSP-defined residues are shown in red. (d) A summary of the critical residues around the YH16899 binding site. Specific residues were replaced with alanine, and the effects of the mutations on the binding to YH16899 and LR (in vitro pull-down and co-IP), 67LR stability, and cell invasion were examined (see ). The results of these experiments were calculated based on the relative band intensities (pull-down, IP, and 67LR level) and the relative numbers of migratory cells (invasion assay), and presented as a heat map with the scale from 0 to 10. For the SPR, a –log-transformed value of each KD was obtained and presented on a scale from 0 (KD, 1 × 10−3) to 10 (KD, 1 × 10−10). The location of L142 and F144 in ABD, the most critical residues, is denoted in Fig. 3c.