Crystal structures of Saccharomyces cerevisiae tryptophanyl-tRNA synthetase: new insights into the mechanism of tryptophan activation and implications for anti-fungal drug design

Abstract

Specific activation of amino acids by aminoacyl-tRNA synthetases is essential for maintaining translational fidelity. Here, we present crystal structures of Saccharomyces cerevisiae tryptophanyl-tRNA synthetase (sTrpRS) in apo form and in complexes with various ligands. In each complex, there is a sulfate ion bound at the active site which mimics the α- or β-phosphate group of ATP during tryptophan activation. In particular, in one monomer of the sTrpRS–TrpNH2O complex, the sulfate ion appears to capture a snapshot of the α-phosphate of ATP during its movement towards tryptophan. Simulation study of a human TrpRS–Trp–ATP model shows that during the catalytic process the α-phosphate of ATP is driven to an intermediate position equivalent to that of the sulfate ion, then moves further and eventually fluctuates at around 2 Å from the nucleophile. A conserved Arg may interact with the oxygen in the scissile bond at the transition state, indicating its critical role in the nucleophilic substitution. Taken together, eukaryotic TrpRSs may adopt an associative mechanism for tryptophan activation in contrast to a dissociative mechanism proposed for bacterial TrpRSs. In addition, structural analysis of the apo sTrpRS reveals a unique feature of fungal TrpRSs, which could be exploited in rational antifungal drug design.

Re-discovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase inMycobacterium tuberculosis

“…Overall, the SO4 2here engages R201 and R312 in the ATP pocket, and enables multiple interactions to facilitate PF-3845 binding into the ATP pocket of Mtb PheRS. Although SO4 2has been found at the active site of several other aaRS structures, none of them contributes to the binding of a ligand as in the case of PF-3845 (33,34).…”

Unique roles of tryptophanyl-tRNA synthetase in immune control and its therapeutic implications

“…Specific recognition by tryptophanyl tRNA synthetase (WRS) of its substrates, Trp and tRNA, is critical for maintaining fidelity in protein synthesis. WRS belongs to the Class Ic ARS family, containing an RF domain with two highly conserved signature sequences, namely, KMSKS (Lys-Met-Ser-Lys-Ser) and HIGH (His-Ile-Gly-His); the former contributes to amino acid activation and the latter stabilizes both ATP during amino acid activation and the 3′ end of the tRNA for amino acid transfer 2,37,38 . The C-terminal alpha helical domain is the binding site for the tRNA anticodon.…”

“…The C-terminal alpha helical domain is the binding site for the tRNA anticodon. While the WRS from Bacillus stearothermophilus ( bWRS) requires two domains for the catalytic reaction, eukaryotic WRSs, such as those from yeast and archaea, have acquired an extra N-terminal domain, referred to as a eukaryote-specific extension (ESE), of 100 amino acid residues 2,38 . Furthermore, in vertebrates, including humans, WRS has an N-terminal extension of 154 amino acids (N154) that is composed of the ESE and an additional vertebrate-specific extension (VSE), constituting the full-length (FL)-human WRS comprising 471 amino acids 31,39,40 (Fig.…”

Structure of a tryptophanyl-tRNA synthetase containing an iron–sulfur cluster

“…These Tyr and Gln residues are conserved in S. cerevisiae TrpRS as shown in the recent structure (PDB code 3kt0; Zhou et al, 2010). l-Tryptophan recognition in TrpRS is highly conserved among eukaryotes (Zhou et al, 2010) and the interactions observed in TmTrpRS are conserved among prokaryotes (Figs. 4b and 4c).…”