engleski

Exclusion of the cognate substrate from the leucyl - tRNA synthetase editing pathway

Aminoacyl-tRNA synthetases (aaRS) are enzymes that use ATP to couple tRNAs with their cognate amino acids. Aminoacylated-tRNAs (aa-tRNAs) are substrates for ribosomal protein synthesis, whose fidelity thus relies on the accuracy of aa-tRNA formation. These enzymes exercise amino acid substrate specificity at the level of binding and/or the chemical steps of aminoacylation. Some aaRS cannot discriminate well against near-cognate amino acids in the synthetic pathway alone and thus utilize hydrolytic editing to minimize the error. Leucyl-tRNA synthetase (LeuRS) pairs leucine with tRNALeu. The main threat to the accuracy of leucylation is posed by non- proteinogenic amino acid norvaline. To prevent error in protein synthesis, LeuRS rapidly hydrolyses Nva-tRNALeu in the separate editing domain. Cognate Leu-tRNALeu evades the hydrolytic correction, yet the mechanism by which is excluded is still not clear. We found a 104-fold slower hydrolysis of Leu-tRNALeu as compared with Nva- tRNALeu, while the Kd values describing LeuRS interactions with Leu-tRNALeu and Nva-tRNALeu were similar. This indicates that LeuRS positions Leu- tRNALeu in an unproductive conformation for editing. To address this issue, we employed molecular dynamics (MD) simulations for the wild type LeuRS and the mutant T252A, which hydrolyses Leu-tRNALeu, with the substrate analogues leucyl- and norvalyl-2’-adenosine bound in the editing active site. The simulations suggest that binding of leucine in a conformation defined by N-Cα-Cβ-Cγ torsion angle of approximately ±180°, which is primarily observed in the WT and not in the T252A, results in a decrease in time during which the systems reside in the productive conformation. As norvalines side chain is not branched, its conformation does not influence the system productivity. To ascertain the chemical nature of discrimination and the role of highly conserved T252 we simulated MD for the T252S and T252V mutants. The results indicate that both mutants likely resemble the wild-type enzyme in discrimination against leucine. The kinetic characterization of these mutants is underway.

leucyl -tRNA synthetase ; post-transfer editing ; norvaline

nije evidentirano