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.

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 Kd 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

Temperature-dependence of Glu-AMS binding to GluRS.

<p>Integrated ITC curves of Glu-AMS (90 μM) binding to GluRS (9 μM) at different temperatures; 20°C (circles), 30°C (upside-down triangles), 37°C (squares).</p

Influence of tRNA on GluRS/Glu-AMS binding at 30°C.

<p>n = stoichiometry coefficient (number of moles of Glu-AMS bound per mole of GluRS monomer), ΔH_b = reaction enthalpy, ΔS_b = reaction entropy, ΔG_b = reaction energy (calculated with the formula ΔG_b = -RT Ln <i>K</i>_b, where R = 1.987 cal/mol·K).</p><p>All values and errors in this table were obtained by weighting by inverse variance [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121043#pone.0121043.ref032" target="_blank">32</a>], except for ΔG_b values and errors, obtained by simple average and standard error calculations.</p><p>Raw data and calculated values for each separated ITC runs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121043#pone.0121043.s004" target="_blank">S1 Table</a>.</p><p>Influence of tRNA on GluRS/Glu-AMS binding at 30°C.</p

Temperature-dependance of the GluRS Glu-AMS interaction.

<p>n = stoichiometry coefficient (number of moles of Glu-AMS bound per mole of GluRS monomer), ΔH_b = reaction enthalpy, ΔS_b = reaction entropy, T = temperature, ΔG_b = reaction energy (calculated with the formula ΔG_b = -RT Ln <i>K</i>_b, where R = 1.987 cal/mol·K).</p><p>Injections of Glu-AMS at 90 μM were done in a starting concentration of 9 μM of GluRS.</p><p>Raw data and calculated values for each separated ITC runs are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121043#pone.0121043.s005" target="_blank">S2 Table</a>.</p><p>Temperature-dependance of the GluRS Glu-AMS interaction.</p

Glu-AMS binding to GluRS with/without tRNA.

<p>(a) Integrated ITC curves of Glu-AMS binding to GluRS with/without tRNA. Binding of Glu-AMS: to GluRS alone (circles), to GluRS with saturating concentration of tRNA^Glu in enriched total tRNA from <i>E</i>. <i>coli</i> (upside-down triangles), to GluRS with 11.23 µM tRNA^Phe from brewer’s yeast (squares). A duplicata of each tested condition is shown. The values shown in (b) were calculated from means of two distinct experiments reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121043#pone.0121043.t001" target="_blank">Table 1</a>, weighting by inverse variance [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121043#pone.0121043.ref032" target="_blank">32</a>].</p

Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs.

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs), the enzymes which esterify tRNAs with the cognate specific amino acid, form mainly a different set of proteins than those involved in the cytosolic translation machinery. Many of the mt-aaRSs are of bacterial-type in regard of sequence and modular structural organization. However, the few enzymes investigated so far do have peculiar biochemical and enzymological properties such as decreased solubility, decreased specific activity and enlarged spectra of substrate tRNAs (of same specificity but from various organisms and kingdoms), as compared to bacterial aaRSs. Here the sensitivity of human mitochondrial aspartyl-tRNA synthetase (AspRS) to small substrate analogs (non-hydrolysable adenylates) known as inhibitors of Escherichia coli and Pseudomonas aeruginosa AspRSs is evaluated and compared to the sensitivity of eukaryal cytosolic human and bovine AspRSs. L-aspartol-adenylate (aspartol-AMP) is a competitive inhibitor of aspartylation by mitochondrial as well as cytosolic mammalian AspRSs, with K(i) values in the micromolar range (4-27 microM for human mt- and mammalian cyt-AspRSs). 5'-O-[N-(L-aspartyl)sulfamoyl]adenosine (Asp-AMS) is a 500-fold stronger competitive inhibitor of the mitochondrial enzyme than aspartol-AMP (10nM) and a 35-fold lower competitor of human and bovine cyt-AspRSs (300 nM). The higher sensitivity of human mt-AspRS for both inhibitors as compared to either bacterial or mammalian cytosolic enzymes, is not correlated with clear-cut structural features in the catalytic site as deduced from docking experiments, but may result from dynamic events. In the scope of new antibacterial strategies directed against aaRSs, possible side effects of such drugs on the mitochondrial human aaRSs should thus be considered.journal articleresearch support, non-u.s. gov't2009 May2009 02 28importe