Computational Insights into the High-Fidelily Catalysis of Aminoacyl-tRNA Synthetases (original) (raw)
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Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases
Methods, 2008
The accuracy of protein synthesis relies on the ability of aminoacyl-tRNA synthetases (aaRSs) to discriminate among true and near cognate substrates. To date, analysis of aaRSs function, including identification of residues of aaRS participating in amino acid and tRNA discrimination, has largely relied on the steady state kinetic pyrophosphate exchange and aminoacylation assays. Pre-steady state kinetic studies investigating a more limited set of aaRS systems have also been undertaken to assess the energetic contributions of individual enzyme-substrate interactions, particularly in the adenylation half reaction. More recently, a renewed interest in the use of rapid kinetics approaches for aaRSs has led to their application to several new aaRS systems, resulting in the identification of mechanistic differences that distinguish the two structurally distinct aaRS classes. Here, we review the techniques for thermodynamic and kinetic analysis of aaRS function. Following a brief survey of methods for the preparation of materials and for steady state kinetic analysis, this review will describe pre-steady state kinetic methods employing rapid quench and stopped-flow fluorescence for analysis of the activation and aminoacyl transfer reactions. Application of these methods to any aaRS system allows the investigator to derive detailed kinetic mechanisms for the activation and aminoacyl transfer reactions, permitting issues of substrate specificity, stereochemical mechanism, and inhibitor interaction to be addressed in a rigorous and quantitative fashion.
Principles of tRNAAla Selection by Alanyl–tRNA Synthetase Based on the Critical G3·U70 Base Pair
ACS Omega, 2019
Throughout evolution, the presence of a single G3•U70 mismatch in the acceptor stem of tRNA Ala is the major determinant for aminoacylation with alanine by alanyl−tRNA synthetase (AlaRS). Recently reported crystal structures of the complexes AlaRS−tRNA Ala / G3•U70 and AlaRS−tRNA Ala /A3•U70 suggest two very different conformations, representing a reactive and a nonreactive state, respectively. On the basis of these structures, it has been proposed that the G3•U70 base pair guides the −CCA end of the tRNA acceptor stem into the active site of AlaRS, thereby enabling aminoacylation. The crystal structures open up the possibility of directly computing the energetics of tRNA specificity by AlaRS. We have carried out molecular dynamics free-energy simulations to quantitatively estimate tRNA discrimination by AlaRS, focusing on the mutations of the single critical base pair G3•U70 to uncover the energetics underlying the accuracy of tRNA selection. The calculations show that the reactive complex is highly selective in favor of the cognate tRNA Ala /G3•U70 over its noncognate analogues (A3•U70/G3•C70/A3•C70). In contrast, the nonreactive complex is predicted to be unselective between tRNA Ala /G3•U70 and tRNA Ala /A3•U70. Utilizing our calculated relative binding free energies, we show how a simple three-step kinetic scheme for aminoacylation, involving both an initial nonspecific binding step and a subsequent transition to a selective reactive complex, accounts for the observed kinetics of the process.
Computational approaches for parameterization of aminoacyl-tRNA synthetase substrates
Biopolymers and Cell, 2018
Aim. To parameterize a modified chained residue and use a newborn topology for molecular dynamics simulation. Method. To deal with the problem, a series of ab initio and semi-empirical methods were combined. The RESP (Restrained ElectroStatic Potential) program, which fits molecular electrostatic potential (MEP) at molecular surfaces using an atom-centered point charge model. All parameters were quantum mechanically calculated and processed with R.E.D.III server. Results. The method of molecular dynamics has potential advantages, like its capability to explore large systems, and disadvantages, like not being feasible to run on fly without a preliminary prepared topologies for identification of each molecule. In an attempt to find a balance between both features speed and accuracy and apply the approach in a computational study, a functional mechanism of prolyl-tRNA synthetase from E.faecalis was investigated. In addition, a well validated protocol of topology preparation for non-canonical structure was developed. Conclusions. Computational approaches like molecular dynamics simulation and molecular docking had now become a strong in silico methods to study biological processes. The major benefits of this methods are expensiveness and speed. It also a strong competitor of quantum modeling approach. However, there is a need to include new structures that are not exist in the GROMACS library is associated with the growing use of different types of modified amino and nucleic acids (DNA derivatives and RNAs) both in fundamental studies in molecular biology, molecular biophy sics, etc., and in applied research related to the search for new drugs. The obtained data well correlate with the experimental data.
Proteins: Structure, Function, and Bioinformatics, 2010
A general approach to genetically encode unnatural amino acids (AA) with diverse chemical, biophysical, and biological properties into prokaryotes and eukaryotes was developed recently. 1-4 Before protein synthesis, each of the 20 standard amino acids (AA) must be attached to their specific tRNA molecule by a specific aminoacyl-tRNA synthetase (AA tRNA-RS). During protein synthesis, mRNA codons are recognized by a specific tRNA anticodon resulting in selective AA incorporation into the elongating protein chain in the ribosome. Aminoacyl-tRNA synthetase/tRNA pairs from archaea have been shown to be orthogonal to the endogenous AA tRNA-RS/AA tRNA pairs in E. coli, which means they do not interfere with any of the host pairs. In vivo incorporation of unnatural AA into proteins was facilitated in response to the amber codon TAG by AA tRNA-RS selectively charging the orthogonal tRNA with a specific unnatural AA. Using this approach, more than 30 unnatural AA have been cotranslationally incorporated into proteins with high fidelity and efficiency in vivo. 1-4 The identity of the AA is determined by the AA tRNA-RS specificity to covalently link a specific AA exclusively to its designated tRNA. This reaction involves several steps: selective binding of the AA and ATP to the synthetase, AA adenylation to activate the AA, selective binding of tRNA, and finally transfer of the adenylated AA to the 3 0 end of the tRNA forming the aminoacyl-tRNA via a covalent ester linkage between AA and tRNA. Experimentally, a directed evolution approach is used to alter the specificity of the orthogonal synthetase enzyme for the target unnatural AA. This is accomplished by randomizing the AA identity of 4-6 positions in the binding pocket. Libraries of enzyme variants comprising on the order of 10 9 mutants are passed through a series of positive and negative selection steps. Repeated rounds of positive and negative selection may result in the isolation of specific enzyme mutants that successfully incorporate target unnatural AA but not endogenous AA.
Protein Engineering Design and Selection, 2006
Seryl-tRNA synthetase (SerRS) charges serine to tRNA Ser following the formation of a seryl adenylate intermediate, but the extent to which other non-cognate amino acids compete with serine to bind to SerRS or for the formation of the activated seryl adenylate intermediate is not known. To examine the mechanism of discrimination against noncognate amino acids, we calculated the relative binding energies of the 20 natural amino acids to SerRS. Starting with the crystal structure of SerRS from Thermus thermophilus with seryl adenylate bound, we used the HierDock and SCREAM (Side-Chain Rotamer Energy Analysis Method) computational methods to predict the binding conformation and binding energy of each of the 20 natural amino acids in the binding site in the best-binding mode and the activating mode. The ordering of the calculated binding energies in the activated mode agrees with kinetic measurements in yeast SerRS that threonine will compete with serine for formation of the activated intermediate while alanine and glycine will not compete significantly. In addition, we predict that asparagine will compete with serine for formation of the activated intermediate. Experiments to check the accuracy of this prediction would be useful in further validating the use of HierDock and SCREAM for designing novel amino acids to incorporate into proteins and for determining mutations in aminoacyl-tRNA synthetase design to facilitate the incorporation of amino acid analogs into proteins. Keywords: aminoacyl-tRNA synthetase/fidelity of protein synthesis/HierDock/SCREAM/seryl-tRNA synthetase Seryl-tRNA synthetase and natural amino acid binding
Journal of Molecular Biology, 2009
Methionyl-tRNA synthetase (MetRS) specifically binds its methionine substrate in an induced fit mechanism, with methionine binding causing large rearrangements. Mutated MetRS able to efficiently aminoacylate the methionine (Met) analog azidonorleucine (Anl) have been identified by saturation mutagenesis combined with in vivo screening procedures. Here, to reveal the structural basis for the altered specificity, the crystal structure of such a mutated MetRS was determined in the apo form as well as complexed with Met or Anl (1.4 to 1.7 Å resolution). The mutations result in both the loss of important contacts with Met, and in the creation of new contacts with Anl, thereby explaining the specificity shift. Surprisingly, the conformation induced by Met binding in wild-type MetRS already occurs in the apo form of the mutant enzyme. Therefore, the mutations cause the enzyme to switch from an induced fit mechanism to a lock and key one, thereby enhancing its catalytic efficiency.
The renaissance of aminoacyl-tRNA synthesis
EMBO reports, 2001
The role of tRNA as the adaptor in protein synthesis has held an enduring fascination for molecular biologists. Over four decades of study, taking in numerous milestones in molecular biology, led to what was widely held to be a fairly complete picture of how tRNAs and amino acids are paired prior to protein synthesis. However, recent developments in genomics and structural biology have revealed an unexpected array of new enzymes, pathways and mechanisms involved in aminoacyl-tRNA synthesis. As a more complete picture of aminoacyl-tRNA synthesis now begins to emerge, the high degree of evolutionary diversity in this universal and essential process is becoming clearer.