Probing interactions of aminoacyl-adenylate with methionyl-tRNA synthetase through site-directed mutagenesis and free energy calculation.

Methionyl-tRNA synthetase (MetRS) is an attractive molecular target for antibiotic discovery. Recently, we have developed several classes of small-molecular inhibitors of MetRS possessing antibacterial activity. In this article, we performed site-directed mutagenesis of aminoacyl-adenylate binding site of MetRS in order to identify crucial amino acid residues for substrate interaction. The umbrella sampling algorithm was used to calculate the binding free energy (ΔG) of these mutated forms with methionyl-adenylate analogue. According to the obtained results, the replacement of Glu24 and Leu293 by alanine leads to the most significant decrease in the binding free energy (ΔG) for adenylate analogue with methionyl-tRNA synthetase indicating increasing of the affinity, which in turn causes the loss of compounds inhibitory activity. Therefore, these amino acid residues can be proposed for further experimental site-directed mutagenesis to confirm binding mode of inhibitors and should be taken into account during chemical optimization to overcome resistance due to mutations.Communicated by Ramaswamy H. Sarma.