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

Abstract

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.

Graphical Abstract

Figure 1.

The structure of the EcTrpRS·tRNA^Trp complex. (A) The domain organization of EcTrpRS and HcTrpRS. (B) Cloverleaf models of E. coli tRNA^Trp and human cytoplasmic tRNA^Trp. (C) The cartoon presentation of the homodimeric EcTrpRS in complex with two E. coli tRNA^Trp molecules reveals a symmetric cross-subunit binding mode.

Figure 2.

The recognition of the anticodon triplet of tRNA^Trp by EcTrpRS. (A) The EcTrpRS·tRNA^Trp complex displays structural variations compared to the HcTrpRS·tRNA^Trp complex (PDB ID 2DR2) at their anticodon binding sites. The recognition of C35 and A36 by EcTrpRS (B) and HcTrpRS (C). The recognition of C34 by EcTrpRS (D) and HcTrpRS (E). In silico mutation of C34 to A34 (F) or G34 (G) created spatial conflicts with the residues Tyr270 and Gly271 of EcTrpRS. The nucleotides and amino acids are shown as stick models with transparent spheres.

Figure 3.

Recognition of the tRNA^Trp acceptor arm by EcTrpRS. (A) EcTrpRS interacts with the acceptor stem of tRNA^Trp from the major groove. (B) The charge–charge interactions between EcTrpRS and the acceptor stem of tRNA^Trp. Notably, no base-specific interactions were observed for the minor identity elements A1:U72 and G5:C68 within the acceptor stem. (C) Structural comparison revealed distinct orientations for the major discriminator base G73 of E. coli tRNA^Trp in the EcTrpRS·tRNA^Trp complex (protein in light purple and tRNA^Trp in orange) and A73 of bovine tRNA^Trp in the HcTrpRS·tRNA^Trp complex (PDB ID 2DR2) (protein in green and tRNA^Trp in cyan). (D) The aminoacylation activity of EcTrpRS and its variants. The mutations Q152A, Q155A, and Q152A&E155A resulted in a significant reduction in the charging activity of EcTrpRS for E.coli tRNA^Trp. (E) The discriminator G73 of E.coli tRNA^Trp is recognized by Glu155 of EcTrpRS through two H-bonds. In silico mutation of G73 to A73 results in the loss of H-bonding interactions with Glu155. (F) The 3′ CCA end of bovine tRNA^Trp (cyan) was modeled into the active site cavity of EcTrpRS based on the structure of the HcTrpRS·tRNA^Trp complex. The surface of the active site cavity of EcTrpRS is drawn, with conserved residues between EcTrpRS and HcTrpRS being colored in dark red, similar residues in light red, and non-conserved residues in gray.

Figure 4.

Identification of tirabrutinib and its analogues as selective inhibitors of bacterial TrpRS. (A) The scatterplot for the results of compounds screening against EcTrpRS using the fluorescence-based TSA, with indolmycin serving as a positive control. (B) The thermal melting profile of EcTrpRS in the presence of tirabrutinib and its analogues. Each curve is an average of three measurements. (C) The chemical structures and activities of tirabrutinib and its analogues. (D, E) ITC titrations of N-piperidine ibrutinib and tirabrutinib to EcTrpRS. (F, G) ITC titrations of N-piperidine ibrutinib and tirabrutinib to HcTrpRS.

Figure 5.

The binding modes of N-piperidine ibrutinib and tirabrutinib to EcTrpRS. (A) The overall structure of EcTrpRS in complex with N-piperidine ibrutinib exhibits an “open–closed” asymmetric conformation, wherein the closed subunit bound with the co-purified intermediate product Trp-AMP and the open subunit bound with N-piperidine ibrutinib. The 2F_o–F_c omit map surrounding N-piperidine ibrutinib is shown as mesh and contoured at 1.0σ. (B) The interactions between N-piperidine ibrutinib and EcTrpRS. (C) The interactions of tirabrutinib with EcTrpRS. (D) Structural superimposition of the compound-bound subunit with the Trp-AMP-bound subunit indicates that the upper benzene ring of the compounds occupies the l-Trp binding site within the active site cavity. (E) Structural superimposition of the compound-bound EcTrpRS with the tRNA^Trp-bound HcTrpRS (PDB ID 2DR2) reveals that the pyrrolidine or piperidine moiety of the compounds interferes with the A76 of tRNA^Trp. (F) The ITC titration of l-Trp to EcTrpRS. (G) The ITC titration of l-Trp to EcTrpRS in the presence of 200 μM of N-piperidine ibrutinib. (H) The titration curve of N-piperidine ibrutinib to EcTrpRS in the presence of 100 μM of tRNA^Trp. (I) Thermal melting profiles of EcTrpRS in the presence of N-piperidine ibrutinib as well as its combinations with tRNA^Trp, tRNA^Trp(ΔCCA), or tRNA^Met. (J) Thermal melting profiles of EcTrpRS in the presence of tirabrutinib, its analogues, and their combinations with tRNA^Trp. Each curve represents the average of three measurements.