L-Arginine recognition by yeast arginyl-tRNA synthetase

Abstract

The crystal structure of arginyl-tRNA synthetase (ArgRS) from Saccharomyces cerevisiae, a class I aminoacyl-tRNA synthetase (aaRS), with L-arginine bound to the active site has been solved at 2.75 Å resolution and refined to a crystallographic R-factor of 19.7%. ArgRS is composed predominantly of α-helices and can be divided into five domains, including the class I-specific active site. The N-terminal domain shows striking similarity to some completely unrelated proteins and defines a module which should participate in specific tRNA recognition. The C-terminal domain, which is the putative anticodon-binding module, displays an all-α-helix fold highly similar to that of Escherichia coli methionyl-tRNA synthetase. While ArgRS requires tRNA Arg for the first step of the aminoacylation reaction, the results show that its presence is not a prerequisite for L-arginine binding. All H-bond-forming capability of L-arginine is used by the protein for the specific recognition. The guanidinium group forms two salt bridge interactions with two acidic residues, and one H-bond with a tyrosine residue; these three residues are strictly conserved in all ArgRS sequences. This tyrosine is also conserved in other class I aaRS active sites but plays several functional roles. The ArgRS structure allows the definition of a new framework for sequence alignments and subclass definition in class I aaRSs.

Substrate‐induced conformational changes in Escherichia coli arginyl–tRNA synthetase observed by 19F NMR spectroscopy

“…Five Trp residues (162, 172, 228, 349 and 446) are found in E. coli ArgRS [9,14]. The amino acid sequence of E. coli ArgRS is 28.7% identical to that of yeast homolog enzyme [5]. Five Trp residues of E. coli ArgRS are located in interesting and crucial regions, of which conformations may be altered upon the substrate binding (yeast ArgRS structure; Fig.…”

“…Five Trp residues of E. coli ArgRS are located in interesting and crucial regions, of which conformations may be altered upon the substrate binding (yeast ArgRS structure; Fig. 1) [5,7]. The catalytic core for both amino acid activation and tRNA aminoacylation reactions in ArgRS, which forms the sca¡old for the Rossmann fold, is composed of two halves assembled from three peptides [5,7].…”

“…1) [5,7]. The catalytic core for both amino acid activation and tRNA aminoacylation reactions in ArgRS, which forms the sca¡old for the Rossmann fold, is composed of two halves assembled from three peptides [5,7]. The ¢rst half of the catalytic core contains two peptideŝ Lys143^Lys194 and Trp266^Gly293^while the second half includes residues Thr345^Thr410 in yeast ArgRS [5,7].…”

“…The catalytic core for both amino acid activation and tRNA aminoacylation reactions in ArgRS, which forms the sca¡old for the Rossmann fold, is composed of two halves assembled from three peptides [5,7]. The ¢rst half of the catalytic core contains two peptideŝ Lys143^Lys194 and Trp266^Gly293^while the second half includes residues Thr345^Thr410 in yeast ArgRS [5,7]. Trp162 (yeast Trp192) and Trp228 (yeast Trp266) residues are located at the edge of the ¢rst half of the catalytic core, while Trp349 (yeast Phe383) residue is in the second half.…”

“…Trp446 (yeast Trp475) is in the proximity of a tRNA anticodon stem binding element (a so-called 6 loop from Ser480 to Thr485 in yeast). Trp172 (yeast Gly202) residue is buried in the Ins-1 (yeast Gln195^Ile265) domain that inserts itself into the catalytic core [5,7]. Therefore, it is possible to probe the conformational changes involving these regions in solution with these probes.…”