Download Exclusion of the cognate substrate from the leucyl-tRNA

Exclusion of the cognate substrate from the leucyl-tRNA synthetase editing
pathway
Isključenje pripadnog supstrata iz korektivnog mehanizma leucil-tRNAsintetaze
Igor Živković, Morana Dulić, Nevena Cvetešić, Branimir Bertoša and Ita Gruić Sovulj
Department of Chemistry, Faculty of Science, University of Zagreb, Croatia
Email: [email protected]
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 [1].
Leucyl-tRNA synthetase (LeuRS) pairs leucine with tRNALeu. The main threat to the
accuracy of leucylation is posed by non-proteinogenic amino acid norvaline [2]. 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 NvatRNALeu 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
[3]. 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.
References:
[1] J.J. Perona and I. Gruic-Sovulj, Top Curr Chem 344 (2013) 1-41.
[2] N. Cvetesic, A. Palencia, I. Halasz, S. Cusack and I. Gruic-Sovulj, EMBO J 33 (2014) 1639-1653.
[3] M. Boero, J. Phys. Chem. B 115 (2011) 12276-12286.