An asymmetric structure of bacterial TrpRS supports the half-of-the-sites catalytic mechanism and facilitates antimicrobial screening

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNA(Trp), thereby playing an indispensable role in protein translation. Unlike most class I aminoacyl-tRNA synthetases (AARSs), TrpRS functions as a homodimer. Herein, we captured an 'open-closed' asymmetric structure of Escherichia coli TrpRS (EcTrpRS) with one active site occupied by a copurified intermediate product and the other remaining empty, providing structural evidence for the long-discussed half-of-the-sites reactivity of bacterial TrpRS. In contrast to its human counterpart, bacterial TrpRS may rely on this asymmetric conformation to functionally bind with substrate tRNA. As this asymmetric conformation is probably a dominant form of TrpRS purified from bacterial cells, we performed fragment screening against asymmetric EcTrpRS to support antibacterial discovery. Nineteen fragment hits were identified, and 8 of them were successfully cocrystallized with EcTrpRS. While a fragment named niraparib bound to the L-Trp binding site of the 'open' subunit, the other 7 fragments all bound to an unprecedented pocket at the interface between two TrpRS subunits. Binding of these fragments relies on residues specific to bacterial TrpRS, avoiding undesired interactions with human TrpRS. These findings improve our understanding of the catalytic mechanism of this important enzyme and will also facilitate the discovery of bacterial TrpRS inhibitors with therapeutic potential. [GRAPHICS]

Automated summary

Tryptophanyl-tRNA synthetase (TrpRS) plays a crucial role in protein translation by linking tryptophan to tRNA(Trp). In this study, an 'open-closed' asymmetric structure of bacterial TrpRS was captured, providing evidence for the half-of-the-sites reactivity of bacterial TrpRS. Fragment screening against the asymmetric TrpRS led to the identification of 19 hits, 8 of which were successfully cocrystallized. These findings enhance our understanding of the catalytic mechanism of TrpRS and aid in the discovery of bacterial TrpRS inhibitors with therapeutic potential.