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

Abstract

Tryptophanyl-tRNA synthetase (TrpRS) links tryptophan to tRNATrp, 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.

Graphical Abstract

Crystal structures of Escherichia coli TrpRS reveal an “open-closed” asymmetric conformation which may be important for tRNA binding, and chemical fragments can bind to distinct pockets on the asymmetric TrpRS.

Figure 1.

The ‘open-closed’ asymmetric structure of EcTrpRS. (A) The domain organization of EcTrpRS. (B) The overall structure of homodimeric EcTrpRS in an asymmetric conformation with a molecule of the intermediate product TrpAMP bound at the active site cavity of the closed subunit (sub^Closed). EcTrpRS is presented as a cartoon model and colored according to (A), and TrpAMP is presented as a sphere. (C) Superposition of sub^Open and sub^Closed by aligning their ADs.

Figure 2.

Binding affinities of substrates L-Trp and ATP to EcTrpRS in asymmetric and apo states as measured by isothermal titration calorimetry. (A, B) ITC measurements of L-Trp to ‘open-closed’ asymmetric EcTrpRS and ‘open-open’ apo EcTrpRS showed similar binding affinities, revealing that occupying one active site by the intermediate product TrpAMP does not affect the binding of L-Trp to the other active site. (C, D) ITC measurements of ATP to ‘open-closed’ EcTrpRS and ‘open-open’ EcTrpRS showed that once an active site binds with a TrpAMP, the second active site could not recruit ATP efficiently.

Figure 3.

The binding mode of EctRNA^Trp asks TrpRS to apply the half-of-the-sites reactivity. (A) Crystal structure of the HcTrpRS·tRNA^Trp·Trp complex (PDB code 2DR2). (B, C) Docking of tRNA^Trp to ‘open-closed’ EcTrpRS in two opposite ways·tRNA^Trp·TrpAMP complex. The distance between the anticodon binding site of sub^Closed and acceptor stem binding site of sub^Open of EcTrpRS is shorter than the corresponding distance observed in cocrystal structure of HcTrpRS·tRNA^Trp complex, suggesting that tRNA^Trp cannot functionally bind to EcTrpRS in this way. In the enlarged views of tRNA anticodon binding sites, the tRNA anticodon binding sites are shown as orange (A), pink (B) and skyblue (C) sticks, respectively, while the anticodon nucleotides as white sticks.

Figure 4.

Electrophoretic mobility shifts of EctRNA^Trp(ΔA76) with EcTrpRS in different conformations. tRNA^Trp(ΔA76) was shifted in a does-dependent manner by the ‘open-open’ and ‘open-closed’ EcTrpRSs but not by the ‘closed-closed’ EcTrpRS, suggesting that capturing a substrate EctRNA^Trp by EcTrpRS requires at least one subunit of EcTrpRS to adopt an open conformation, which agrees with the half-of-the-sites reactivity. The gel was dyed with GelRed and then with Coomassie brilliant blue R-250.

Figure 5.

Fragment screening against EcTrpRS using a fluorescence-based thermal shift assay. (A) Schematic illustration of the TSA-based fragment screening process. A fragment was identified as a binder of EcTrpRS if it increased the T_m value of EcTrpRS by > 1.0°C. Fragments were screened in parallel against both the ‘open-open’ EcTrpRS and ‘open-closed’ EcTrpRS. Thirty fragments were identified to bind the ‘open-open’ EcTrpRS, including 19 fragments that could bind the ‘open-closed’ EcTrpRS. (B) The representative thermal melting curves of ‘open-closed’ EcTrpRS in the presence of the eight fragments whose binding modes have later been determined by cocrystal structures with EcTrpRS.

Figure 6.

Binding of seven fragments to the dimeric interface of ‘open-closed’ EcTrpRS. (A) An unprecedented pocket at the dimeric interface of EcTrpRS. This pocket is built by several hydrophobic and hydrophilic residues (Ala89, Gly92, Trp93, Asn96, Asp127, Val130 and Leu131) from both subunits. Binding of fragments chlorzoxazone (B), M1-67 (C), M1-109 (D), M1-158 (E), M2-54 (F), M2-140 (G) and M3-108 (H) to this pocket. Residues from sub^Open and sub^Closed are colored dark blue and light blue, respectively. The hydrogen bonds are shown as black dashes.

Figure 7.

The binding mode and selectivity of niraparib to bacterial TrpRS. (A) Chemical structure of niraparib. (B) The binding mode of niraparib at the active site of EcTrpRS. An annealed omit electron density map of niraparib calculated with Fourier coefficients 2Fo – Fc and contoured at 1.0 σ. (C) The binding mode of L-Trp in EcTrpRS (PDB code 5V0I). (D) Structural explanation for the insensitivity of HcTrpRS to niraparib. In (B–D), the hydrogen bonds are shown as black dashes, and salt bridges as yellow dashes. (E–H) ITC experiments showed that niraparib could potently bind to EcTrpRS (E) and SaTrpRS (F), and this binding was specifically blocked by the high concentration of substrate L-Trp (G). In contrast, niraparib did not bind to HcTrpRS (H).