Molecular dynamics simulations show that bound Mg²⁺ contributes to amino acid and aminoacyl adenylate binding specificity in aspartyl-tRNA synthetase through long range electrostatic interactions

Molecular recognition between the aminoacyl-tRNA synthetase enzymes and their cognate amino acid ligands is essential for the faithful translation of the genetic code. In aspartyl-tRNA synthetase (AspRS), the co-substrate ATP binds preferentially with three associated Mg²⁺ cations in an unusual, bent geometry. The Mg²⁺ cations play a structural role and are thought to also participate catalytically in the enzyme reaction. Co-binding of the ATP·Mg₃²⁺ complex was shown recently to increase the Asp/Asn binding free energy difference, indicating that amino acid discrimination is substrate-assisted. Here, we used molecular dynamics free energy simulations and continuum electrostatic calculations to resolve two related questions. First, we showed that if one of the Mg²⁺ cations is removed, the Asp/Asn binding specificity is strongly reduced. Second, we computed the relative stabilities of the three-cation complex and the 2-cation complexes. We found that the 3-cation complex is overwhelmingly favored at ordinary magnesium concentrations, so that the protein is protected against the 2-cation state. In the homologous LysRS, the 3-cation complex was also strongly favored, but the third cation did not affect Lys binding. In tRNA-bound AspRS, the single remaining Mg²⁺ cation strongly favored the Asp-adenylate substrate relative to Asn-adenylate. Thus, in addition to their structural and catalytic roles, the Mg²⁺ cations contribute to specificity in AspRS through long range electrostatic interactions with the Asp side chain in both the pre- and post-adenylation states.