We engaged molecular docking and molecular dynamics techniques to study the structural basis of amino acid selectivity by aminoacylation site of LeuRS from T.thermophilus and to describe the binding of pre-transfer substrates. The simulation results showed some differences in the behavior of a number of aminoacylation substrates, amino acids and AMP, which determine the probability of aminoacyladenylate formation. In particular, Tyr43 radical’s shielding could be a mechanism to protect already formed amnoacyladenylates from hydrolysis during pre-transfer editing of erroneously synthesized products.
To simulate the post-transfer editing process in CP1 domain of the LeuRS, the R.E.D.III algorithm of partial charges fitting for aminoacyl-tRNA structures was generated and validated. The protocol of the preparation, run and the analysis of molecular dynamics results was refined based on the results of biochemical experiments for LeuRS systems from T.thermophilus (class I synthetase) and ProRS
from E.faecalis (class II synthetase). The substrate's ability to be bound with the posttransfer editing site was estimated by comparing the interaction energies of the ligand with the amino acids of the site and estimation of the number of intermolecular hydrogen bonds. In the case of R.E.D. III algorithm application the conformation and water accessibility of the ester bond in leucyl-tRNA fragment significantly depends on the residue in 252 position of CP1 domain. In the WT protein the molecule of leucyl-tRNA is weakly interact with Asp347 and rarely with Thr247, a side chain of leucine is exposure out of the binding site. At the same time T252A mutation creates
an additional space, which is sufficient for location of leucine and appropriate orientation of the ester’s bond plane. H-bond interactions with Thr247 and Thr248 increase the probability of nucleophilic attack on the carbonyl carbon of the ligand due to stabilization of the geometry and pulling of the electron cloud density. At the same time Asp347 forms a strong interaction with amino group of leucyl-tRNA with
decrease in the number of degrees of freedom. Similar study was conducted for Ala-tRNAPro from bacterial ProRS E.faecalis to confirm the quality of the simulation results. To run the simulation, a ProRS structure from E.faecalis in complex with tRNA in the editing conformation was modeled, which is significantly different from the existing crystal structures. The trajectory analysis provided an information to determine the pre-reaction coordinates in the editing site. As a result, the 2'-OH group of terminal adenosine (76) of AlatRNAPro has been identified as the most important functional element of this mechanism. The group forms a hydrogen bond with the carbonyl group of the alanine residue, which greatly facilitates the hydrolysis reaction in the presence of two water molecules and a strong hydrogen molecule network between all participants in the reaction. Thus, we demonstrated the mechanism of the post-transfer editing in ProRS and the differences in this process between aminoacyl-tRNA synthetases of class I and II.
A constrained and free molecular dynamics technique have been combined to determine the factor that causes faster hydrolysis of Nva-tRNALeu compared to IletRNALeu in the CP1 domain of bacterial and archaeal LeuRSs. For this purpose, four models were constructed and relaxed via MD simulations of 100 ns. The analysis of MD trajectories showed that Nva-A76 substrate binds more strongly than Ile-A76 and the number of H-bonds and its stability, including Thr247 and Asp347 interactions, is significantly different. On the other hand, our simulation showed another possible cause of the difference in the rate of hydrolysis for Nva- tRNALeuand Ile-tRNALeucin the CP1 domains of LeuRS T.thermophilus and P.horikoshii. The simulation showed that the branched Ile-A76 side chain sterically hinders the approach of the attacking
water molecules, resulting in a significant reduction in the rate of hydrolysis. The molecular dynamics of the LeuRSTt mutant form Asp347Ala, which is critical for the inactivation of the editing function, revealed a number of structural differences in the binding of Nva- and Ile-tRNALeuin the active site of the editing domain, which may affect the selectivity during substrate hydrolysis. In our model,
there are hydrogen bonds between the norvaline’s carbonyl group and 3'-OH group of the A76 tRNALeu and side chain OH group of Thr247. Thus, the obtained results indicated that new quantum-mechanical calculations of the mechanism of posttransferring LeuRSTt editing in ensemble of two water molecules are needed to extend our knowledgebase. Based on the data obtained, we also suggested a mechanism of hydrolysis of Nva-A76 in LeuRSPh, which is similar to the mechanism presented for LeuRSTt, with the only difference in the spatial orientation of the water molecules.
