The mechanism of lineage-specific tRNA recognition by bacterial tryptophanyl-tRNA synthetase and its implications for inhibitor discovery.

Tryptophanyl-tRNA synthetase (TrpRS) catalyzes the attachment of tryptophan (l-Trp) to tRNATrp, thereby providing the ribosome with a crucial substrate for the decoding of the UGG codon during protein translation. Both bacterial and eukaryotic TrpRSs are unable to efficiently cross-aminoacylate their respective tRNATrp substrates, indicating the evolution of lineage-specific mechanisms for tRNATrp recognition. Herein, we present the first co-crystal structure of bacterial TrpRS from Escherichia coli (EcTrpRS) in complex with its tRNATrp. EcTrpRS demonstrates bacterial-specific interactions with both the anticodon triplet and the acceptor arm of tRNATrp. Particularly, the bacterial-specific residue Glu155 forms hydrogen bonds with the discriminator base G73, thereby stabilizing it in a conformation distinct from that of A73 in the eukaryotic tRNATrp bound to human TrpRS. Through compound screening, we identified tirabrutinib and its analogues as selective inhibitors of bacterial TrpRS. These compounds occupy the l-Trp and tRNATrp CCA end binding sites of bacterial TrpRS, both of which exhibit less conservation compared to the ATP binding site between bacterial and eukaryotic TrpRSs. These findings enhance our understanding of the lineage-specific recognition of tRNATrp by bacterial TrpRS and highlight the CCA end binding site as a promising target for the future development of selective bacterial TrpRS inhibitors as potential antimicrobials.