Figure 4: Critical regulation of ThrRS conformation and activity through the extra binding site of BN.
From: Structural basis for full-spectrum inhibition of translational functions on a tRNA synthetase
(a–d) Binding cavity of ThrRS–BN complex in comparison with those of ThrRSs in complex with L-Thr (PDB: 1EVK), ATP (PDB: 1NYR) and AMP+tRNAThr (PDB: 1QF6). The approximate L-Thr pocket boundary is shown as a red line with bound ligands as sticks. BN binds 4 Å deeper and induces a cavity ~8 Å below the bottom of L-Thr pocket. (e) Extra hydrophobic pocket occupied by BN (orange). Interacting residues are shown as sticks. (f) In addition to human ThrRS full-length (WT) and fragments (N: 1–322; C: 322–723), four indicated mutants were tested to rescue the RRL-based in vitro protein translation inhibited by 250 nM BN. D462L misses one hydrogen bond with the hydroxyl group of L-Thr; F458A misses the stacking interaction with the adenine group of ATP; Y392E causes repulsion to the tRNA backbone phosphate group and L567R fills the space of the fourth subsites. (g–j) Schematic diagrams of the conformational change in ThrRS. The BN-induced upper active site opening (UASO) is denoted, whereas BN is shown as orange lines. Bulky space-filling mutation (L567R) and shorter mutation (L567V) are shown as black lines. (k) Yeast ThrRS (yTHS1) was replaced by human wide-type ThrRS or mutants in supporting yeast growth. Empty vector and human lysyl-tRNA synthetase (LysRS) are used as control. The expression of endogenous yeast ThrRS was switched off by the addition of doxycycline (Dox) to the yeast growth medium. Tenfold serial dilutions of freshly gown yeast cells were spotted onto selective media synthetic complete medium without histodine (SCM-HIS) containing 2% galactose with or without Dox. (l) Schematic map showing BN occupied four sites on ThrRS: L-Thr site, ATP site, tRNA site and an extra site. Each site inhibits the translational activity of ThrRS.
