Abstract
Chuangxinmycin (CXM) is a promising antimicrobial compound targeting bacterial tryptophanyl-tRNA synthetase (TrpRS), an essential enzyme in protein synthesis. The detailed inhibitory mechanism of CXM, particularly in clinically relevant pathogenic bacteria, is poorly understood. In this study, based on the determination of 10 crystal structures, including Escherichia coli TrpRS (EcTrpRS) and Staphylococcus aureus TrpRS (SaTrpRS) in complex with CXM, ATP, tryptophan, or CXM derivatives, either individually or in combination, as well as the structure of apo-SaTrpRS, we provide key insights into the binding mode of CXM and its species-specific inhibitory mechanisms. Combined with molecular dynamics simulations and binding energy analysis, we demonstrate that CXM binds to EcTrpRS in a manner highly similar to the natural substrate tryptophan. Key residues, including D135 and Y128, play critical roles in CXM recognition and fixation, while conserved hydrophobic residues contribute significantly to binding free energy. This binding pattern is consistent with that observed in Geobacillus stearothermophilus TrpRS (GsTrpRS). However, SaTrpRS exhibits distinct behavior due to structural differences, particularly the orientation of Y126 (corresponding to Y128 in EcTrpRS). This difference results in the selectivity of 3-methylchuangxinmycin (mCXM), a CXM derivative, against SaTrpRS. Furthermore, modeling CXM into the tryptophan-binding site of human cytoplasmic TrpRS (HsTrpRS) reveals the lack of key hydrogen bonds and a salt bridge interaction, which likely underlies CXM's significantly weaker inhibition of HsTrpRS. These findings deepen our understanding of the inhibitory mechanism of CXM and its selectivity toward bacterial TrpRSs, and thus can facilitate the design of next-generation antibiotics targeting bacterial TrpRSs.