Stereospecificity control in aminoacyl-tRNA-synthetases: new evidence of d-amino acids activation and editing

Abstract

The homochirality of amino acids is vital for the functioning of the translation apparatus. l-Amino acids predominate in proteins and d-amino acids usually represent diverse regulatory functional physiological roles in both pro- and eukaryotes. Aminoacyl-tRNA-synthetases (aaRSs) ensure activation of proteinogenic or nonproteinogenic amino acids and attach them to cognate or noncognate tRNAs. Although many editing mechanisms by aaRSs have been described, data about the protective role of aaRSs in d-amino acids incorporation remained unknown. Tyrosyl- and alanyl-tRNA-synthetases were represented as distinct members of this enzyme family. To study the potential to bind and edit noncognate substrates, Thermus thermophilus alanyl-tRNA-synthetase (AlaRS) and tyrosyl-tRNA-synthetase were investigated in the context of d-amino acids recognition. Here, we showed that d-alanine was effectively activated by AlaRS and d-Ala-tRNAAla, formed during the erroneous aminoacylation, was edited by AlaRS. On the other hand, it turned out that d-aminoacyl-tRNA-deacylase (DTD), which usually hydrolyzes d-aminoacyl-tRNAs, was inactive against d-Ala-tRNAAla. To support the finding about DTD, computational docking and molecular dynamics simulations were run. Overall, our work illustrates the novel function of the AlaRS editing domain in stereospecificity control during translation together with trans-editing factor DTD. Thus, we propose different evolutionary strategies for the maintenance of chiral selectivity during translation.

Design principles and functional basis of enantioselectivity of alanyl-tRNA synthetase and a chiral proofreader during protein biosynthesis

“…Based on the analysis of the available AlaRS structures in the Protein Data Bank (PDB) ( 22 ), we show that D-alanine is sterically excluded from the active site and hence cannot be charged by AlaRS. Our studies further show that the cis -editing domain of AlaRS is inactive on D-Ala-tRNA Ala thus disproving all the major claims made in the previous work ( 21 ) and clears the serious anomalies ensued on the most fundamental reaction mechanisms that ensure chiral fidelity of the cellular aminoacyl-tRNA pool. Overall, the work experimentally substantiates the design principles governed by Koshland's ‘four-location’ model to explain the size-based disparity in activation of D-amino acids by aaRSs and the universality of L-chiral rejection based chiral proofreading by DTD.…”

“…Recently it has been shown in vitro that AlaRS charges D-alanine on tRNA Ala , and is proofread by the cis -editing domain of AlaRS ( 21 ). In order to test these observations physiologically, we hypothesized that bacterial stain with AlaRS editing defective (AlaRS ED ) gene would be sensitive to excess D-alanine supplementation in growth media, while the wild type bacterial strain would be insensitive to the same.…”

“…The mistranslation of alanine to glycine and serine is effectively prevented by employing both cis- and trans- editing domains including DTD that decouples erroneously attached Gly on tRNA Ala ( 17 , 20 ). Very recently, it was shown that AlaRS also mischarges D-alanine on tRNA Ala apart from glycine and L-serine, which is proofread by the AlaRS cis- editing domain and surprisingly not by DTD ( 21 ). These results deviate from previous works on multiple counts viz.…”

The tRNA identity landscape for aminoacylation and beyond

“…Surprisingly, the high level of d -alanyl-tRNA Ala synthesized by T. thermophilus AlaRS is not edited by a DTD protein but progressively deacylated by post-transfer editing in the synthetic site of AlaRS. This demonstrates the active role of AlaRS in controlling chirality ( 352 ). In summary, chiral proofreading DTD enzymes are a major cellular checkpoint, but other molecules prevent the infiltration of d -amino acids into the translation machinery.…”

Effect of Charge Distribution in a Modified tRNA Substrate on Pre-Reaction Protein-tRNA Complex Geometry

“…The Coulomb cutoff radius of 1.2 nm for the electrostatic and cutoff radius of 1.1 nm for Lennard–Jones interactions were applied. Analysis of the relative position of the substrate, all available water molecules and amino acids of the binding site, was performed with a purpose-written script in Python that integrates step-by-step analysis of all molecules and residues, surrounding an aminoacyl-tRNA fragment during MD simulation . All results of the analyses were performed using Gromacs built-in tools and the last 10 ns were represented in graphs.…”