tRNAGlu Increases the Affinity of Glutamyl-tRNA Synthetase for Its Inhibitor Glutamyl-Sulfamoyl-Adenosine, an Analogue of the Aminoacylation Reaction Intermediate Glutamyl-AMP: Mechanistic and Evolutionary Implications

Abstract

For tRNA-dependent protein biosynthesis, amino acids are first activated by aminoacyl-tRNA synthetases (aaRSs) yielding the reaction intermediates aminoacyl-AMP (aa-AMP). Stable analogues of aa-AMP, such as aminoacyl-sulfamoyl-adenosines, inhibit their cognate aaRSs. Glutamyl-sulfamoyl-adenosine (Glu-AMS) is the best known inhibitor of Escherichia coli glutamyl-tRNA synthetase (GluRS). Thermodynamic parameters of the interactions between Glu-AMS and E. coli GluRS were measured in the presence and in the absence of tRNA by isothermal titration microcalorimetry. A significant entropic contribution for the interactions between Glu-AMS and GluRS in the absence of tRNA or in the presence of the cognate tRNAGlu or of the non-cognate tRNAPhe is indicated by the negative values of –TΔSb, and by the negative value of ΔCp. On the other hand, the large negative enthalpy is the dominant contribution to ΔGb in the absence of tRNA. The affinity of GluRS for Glu-AMS is not altered in the presence of the non-cognate tRNAPhe, but the dissociation constant K d is decreased 50-fold in the presence of tRNAGlu; this result is consistent with molecular dynamics results indicating the presence of an H-bond between Glu-AMS and the 3’-OH oxygen of the 3’-terminal ribose of tRNAGlu in the Glu-AMS•GluRS•tRNAGlu complex. Glu-AMS being a very close structural analogue of Glu-AMP, its weak binding to free GluRS suggests that the unstable Glu-AMP reaction intermediate binds weakly to GluRS; these results could explain why all the known GluRSs evolved to activate glutamate only in the presence of tRNAGlu, the coupling of glutamate activation to its transfer to tRNA preventing unproductive cleavage of ATP.

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

“…The requirement for tRNA to co-bind may limit the potency of aa-AMS analogues to bind these three aaRSs. In fact, the affinity of Glu-AMS to GluRS decreased by 50-fold without tRNA Glu [ 145 ]. A conceptual triple active site inhibitor with a tRNA-A76 like module plus an AMP and an amino acid-like moiety ( Figure 5 ), as a full mimetic of the aminoacylation state, may give the most potent inhibition.…”

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl‐tRNA synthetase

“…ArgRS is a class-Ic tRNA synthetase that undergoes ATP-PPi exchange with the help of its cognate tRNA [18], like GlnRS [19] and GluRS [20,21]. In Trypanosoma brucei, it showed a good druggability score, and its knockdown led to rapid cell death [22].…”

Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry

“…This approach has been employed to study protein stability in several AARS systems [7,8]. Isothermal titration calorimetry (ITC) has also proven useful for studying the interactions of small molecule ligands with AARSs is [9–11]. In addition to providing binding constants and stoichiometry, ITC is a direct process that does not involve chemical alteration of either the enzyme or the substrate, while providing detailed thermodynamic parameters for a given interaction.…”