Dynamics of the Active Sites of Dimeric Seryl <i>t</i>RNA Synthetase from Methanopyrus kandleri

Aminoacyl <i>t</i>RNA synthetases (<i>aa</i>RSs) carry out the first step of protein biosynthesis. Several <i>aa</i>RSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric <i>aa</i>RSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric ^<i>mk</i>SerRS from Methanopyrus kandleri bound with <i>t</i>RNA using an explicit solvent system. Two dimeric states of seryl <i>t</i>RNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound <i>t</i>RNA. The aim is to understand the conformational dynamics of ^<i>mk</i>SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of ^<i>mk</i>SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the reaction center. Synchronously, Arg366 of the β sheet at the base holds the syn oxygen of the attacking carboxylic group so that the attack by the anti oxygen is feasible. This residue also contributes to the reduction of the unfavorable electrostatic potential at the reaction center. Present simulation clearly shows the catalytic role of the residues at reaction center. A precise and stable geometry of hydrogen bonded network develops within the active site, which is essential for the development of an optimum transition state geometry. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bond disappears. The serine binding site is most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn²⁺ and Cys residues. Present simulation further suggests that the helix-turn-helix motif connecting the monomers might have important role in coordinating the functional dynamics of the two active sites. The N-terminal domain is involved in long-range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with <i>t</i>RNA. Overall conformational fluctuation is less in the N terminal compared to the catalytic domain due to the presence of a motif 2 loop, loop <i>f</i>, and serine ordering loop, which change conformation in the later domain during the reaction cycle. The dynamic perspective of the active site of ^<i>mk</i>SerRS with the mobile loop acting as the gate and dynamically silent β sheets performing as the base has similarity with the perception of the active site in various other enzymes