Universal pathway for posttransfer editing reactions: Insights from the crystal structure of Tt PheRS with puromycin

Abstract

At the amino acid binding and recognition step, phenylalanyltRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA Phe to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis-and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2′ and 3′ isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA Phe where deacylation occurs from either the 2′-or 3′-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.biosynthesis | aminoacyl-tRNA synthetases | tRNA | puromycin | editing

Loss of protein synthesis quality control in host-restricted organisms

“…For each synthetase, the program calculated area of protein-ligand interactions that were mediated by amino acid side chains of the editing site or the synthetic site. The crystal structure used in this studies were the following: for PheRS (22), ValRS (46), LeuRS (12,47), IleRS (48), and ThrRS (21); the editing domain structures were determined in complex with a near-cognate amino acid bound to AMP, providing accurate information about the ligand-active site interactions. Because for AlaRS the crystal structures were determined in a vacant state, we defined editing-site residues based on previous biochemical studies, but used the structure of a homologous protein, AlaX, bound with near-cognate substrate serine to create an illustration shown in Fig.…”

Evolutionary Limitation and Opportunities for Developing tRNA Synthetase Inhibitors with 5-Binding-Mode Classification

“…The chemical resembles the 3′ end of aminoacylated Phe-tRNA ( Figure 3 G), enters the A site and transfers to the growing chain, causing the formation of a puromycylated nascent chain and premature chain release [ 127 ]. Interestingly, this resemblance of aminoacylated Phe-tRNA also makes puromycin a PheRS editing site binder with a dissociation constant (Kd) of 300 µM, suggesting a way that the mischarged Tyr-tRNA Phe might bind [ 86 ].…”