Exit strategies for charged tRNA from GluRS.

For several class I aminoacyl-tRNA synthetases (aaRSs), the rate-determining step in aminoacylation is the dissociation of charged tRNA from the enzyme. In this study, the following factors affecting the release of the charged tRNA from aaRSs are computationally explored: the protonation states of amino acids and substrates present in the active site, and the presence and the absence of AMP and elongation factor Tu. Through molecular modeling, internal pK(a) calculations, and molecular dynamics simulations, distinct, mechanistically relevant post-transfer states with charged tRNA bound to glutamyl-tRNA synthetase from Thermus thermophilus (Glu-tRNA(Glu)) are considered. The behavior of these nonequilibrium states is characterized as a function of time using dynamical network analysis, local energetics, and changes in free energies to estimate transitions that occur during the release of the tRNA. The hundreds of nanoseconds of simulation time reveal system characteristics that are consistent with recent experimental studies. Energetic and network results support the previously proposed mechanism in which the transfer of amino acid to tRNA is accompanied by the protonation of AMP to H-AMP. Subsequent migration of proton to water reduces the stability of the complex and loosens the interface both in the presence and in the absence of AMP. The subsequent undocking of AMP or tRNA then proceeds along thermodynamically competitive pathways. Release of the tRNA acceptor stem is further accelerated by the deprotonation of the alpha-ammonium group on the charging amino acid. The proposed general base is Glu41, a residue binding the alpha-ammonium group that is conserved in both structure and sequence across nearly all class I aaRSs. This universal handle is predicted through pK(a) calculations to be part of a proton relay system for destabilizing the bound charging amino acid following aminoacylation. Addition of elongation factor Tu to the aaRS.tRNA complex stimulates the dissociation of the tRNA core and the tRNA acceptor stem. Copyright (c) 2010. Published by Elsevier Ltd.