Coli methionyl-tRNA Synthetase Research Articles

A Comparative Study of All-atom Molecular Dynamics Simulation and Coarse-grained Normal Mode Analysis in Identifying Pre-existing Residue Interaction Networks that Promote Coupled-Domain Dynamics in Escherichia coli Methionyl-tRNA Synthetase

Inter-domain communication plays a key role in the function of modular proteins. Earlier studies have demonstrated that the coupling of domain motions is important in mediating site-to-site communications in modular proteins. In the present study, bioinformatics and molecular simulations were usedto trace “pre-existing” residue-residue interaction networks that mediate coupled-domain dynamics in multi-domain Escherichia coli methionyl-tRNA synthetase (Ec MetRS). In particular, a comparative study was carried out to evaluate the effectiveness of coarse-grained normal mode analysis and all-atom molecular dynamic simulation in predicting pre-existing pathways of inter-domain communications in this enzyme. Integration of dynamic information of residues with their evolutionary features (conserved and coevolved) demonstrated that multiple residue-residue interaction networks exist in Ec MetRS that promote dynamic coupling between the anticodon binding domain and the connective polypeptide I domain, which are > 50Å apart, through correlated motions. Mutation of residues on these pathways have distinct impact on the dynamics and function of this enzyme. Moreover, the present study revealed that the dynamic information obtained from the coarse-grained normal mode analysis is comparable to the atomistic molecular dynamics simulations in predicting the interaction networks that are essential for promoting coupled-domain dynamics in Ec MetRS. KEYWORDS: Domain-domain Communication; Molecular Dynamics; Methionyl-tRNA Synthetase; Normal Mode Analysis; Coupled-domain Dynamics; Course-grained Normal Mode Analysis; Aminoacyl tRNA Synthetases; Statistical Coupling Analysis

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.

A Disease-causing Point Mutation in Human Mitochondrial tRNAMet Results in tRNA Misfolding Leading to Defects in Translational Initiation and Elongation

The mitochondrial tRNA genes are hot spots for mutations that lead to human disease. A single point mutation (T4409C) in the gene for human mitochondrial tRNA(Met) (hmtRNA(Met)) has been found to cause mitochondrial myopathy. This mutation results in the replacement of U8 in hmtRNA(Met) with a C8. The hmtRNA(Met) serves both in translational initiation and elongation in human mitochondria making this tRNA of particular interest in mitochondrial protein synthesis. Here we show that the single 8U-->C mutation leads to a failure of the tRNA to respond conformationally to Mg(2+). This mutation results in a drastic disruption of the structure of the hmtRNA(Met), which significantly reduces its aminoacylation. The small fraction of hmtRNA(Met) that can be aminoacylated is not formylated by the mitochondrial Met-tRNA transformylase preventing its function in initiation, and it is unable to form a stable ternary complex with elongation factor EF-Tu preventing any participation in chain elongation. We have used structural probing and molecular reconstitution experiments to examine the structures formed by the normal and mutated tRNAs. In the presence of Mg(2+), the normal tRNA displays the structural features expected of a tRNA. However, even in the presence of Mg(2+), the mutated tRNA does not form the cloverleaf structure typical of tRNAs. Thus, we believe that this mutation has disrupted a critical Mg(2+)-binding site on the tRNA required for formation of the biologically active structure. This work establishes a foundation for understanding the physiological consequences of the numerous mitochondrial tRNA mutations that result in disease in humans.

A study of communication pathways in methionyl- tRNA synthetase by molecular dynamics simulations and structure network analysis

The enzymes of the family of tRNA synthetases perform their functions with high precision by synchronously recognizing the anticodon region and the aminoacylation region, which are separated by approximately 70 A in space. This precision in function is brought about by establishing good communication paths between the two regions. We have modeled the structure of the complex consisting of Escherichia coli methionyl-tRNA synthetase (MetRS), tRNA, and the activated methionine. Molecular dynamics simulations have been performed on the modeled structure to obtain the equilibrated structure of the complex and the cross-correlations between the residues in MetRS have been evaluated. Furthermore, the network analysis on these simulated structures has been carried out to elucidate the paths of communication between the activation site and the anticodon recognition site. This study has provided the detailed paths of communication, which are consistent with experimental results. Similar studies also have been carried out on the complexes (MetRS + activated methonine) and (MetRS + tRNA) along with ligand-free native enzyme. A comparison of the paths derived from the four simulations clearly has shown that the communication path is strongly correlated and unique to the enzyme complex, which is bound to both the tRNA and the activated methionine. The details of the method of our investigation and the biological implications of the results are presented in this article. The method developed here also could be used to investigate any protein system where the function takes place through long-distance communication.

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Abnormal Proteins Enhance Stress-Induced Cell Death

The effect of abnormal proteins on cell viability was studied using artificially cleaved polypeptides.Escherichia colimethionyl-tRNA synthetase (MetRS) consists of two distinct domains and its activity is essential for cell viability. The polypeptide chain was split by linker insertion and expressed as two fragments. Two pairs of polypeptides, one split within the N-terminal domain and another at the junction of the two domains retained aminoacylation activity. Thein vitroactivities of these split mutants were enhanced by the presence of chaperonin, GroESL. However, cells containing these split polypeptides became sensitive to conditions that induce GroESL. The results of this work suggest that an abnormally generated protein can cause cell death under stressful conditions.

Identification of the metal ligands and characterization of a putative zinc finger in methionyl-tRNA synthetase

A truncated form of the methionyl-tRNA synthetase (delta MTS), which has been cloned, overproduced, and characterized, was used in an attempt to better understand the role of the enzyme-bound zinc in the amino-acylation process. Apo-, Zn(2+)-, Co(2+)-, and 113Cd(2+)-substituted delta MTS proteins were prepared in vivo and purified to homogeneity. Apo-delta MTS was devoid of enzymatic activity in the aminoacylation of tRNA(fMet) and in the methionine-dependent ATP-pyrophosphate exchange reactions. Kinetic constants in both the aminoacylation and ATP-pyrophosphate exchange reactions for the Co(2+)- and 113Cd(2+)-substituted delta MTS proteins were found to be identical with those of the native Zn2+ protein. The low energy absorption spectrum of Co(2+)-substituted delta MTS resembles the d-d transition bands characteristic of tetrahedrally coordinated Co(2+)-substituted proteins. A strong S-->Co2+ charge transfer absorption at 350 nm was clearly evident having a molar absorptivity consistent with four thiolate ligands. The environment of the metal center was further probed by measuring the 113Cd chemical shift of 113Cd(2+)-substituted delta MTS. A single resonance at 759.6 ppm was observed. This chemical shift is consistent with Cd2+ coordinated to four thiolate ligands. The Escherichia coli methionyl-tRNA synthetase contains a potential metal binding sequence Cys-X2-Cys-X9-Cys-X2-Cys in a connecting polypeptide within the nucleotide fold. Titration of a 21-amino acid peptide corresponding to this putative metal binding site, Cys145-Cys161, was shown to bind Co2+ with a Kd of 120 +/- 11 microM. These results demonstrate that the isolated zinc finger binding domain is capable of specifically forming a stoichiometric complex with the divalent cation. Taken together, our studies identify the 4 cysteine residues in the zinc finger-like domain as the metal binding ligands in the E. coli methionyl-tRNA synthetase. The role of the enzyme-bound metal appears to be structural and not directly involved in catalysis.

A novel .alpha.-proton exchange reaction catalyzed by Escherichia coli methionyl-tRNA synthetase

The Escherichia coli truncated methionyl-tRNA synthetase (delta MTS) was shown to catalyze alpha-carbon hydrogen-deuterium exchange of L-selenomethionine, L-methionine, L-ethionine, and L-norleucine in the presence of deuterium oxide. The rate of alpha-proton exchange for L-methionine was shown to be linear with respect to delta MTS concentration. The exchange reaction showed saturation kinetics with apparent Km values of 21 and 4 mM in the absence and presence of saturating adenosine concentrations, respectively. As expected, delta MTS did not catalyze alpha-proton exchange of D-methionine since the enzyme has been shown to be specific for L-amino acids. In the absence of enzyme or in the presence of an equivalent concentration of Zn2+, no hydrogen-deuterium exchange was detected. The exchange reaction was not observed with L-methioninol, an analogue of L-methionine lacking the carboxylate group. These results suggest that the alpha-carboxylate group is a requirement for the delta MTS-catalyzed exchange reaction. The E. coli methionyl-tRNA synthetase (MTS) has previously been shown to be a zinc metalloprotein [Posorske, L. H., Cohn, M., Yanagisawa, N., & Auld, D. S. (1979) Biochim. Biophys. Acta 576, 128]. On the basis of the structural and mechanistic information available on MTS, we propose that the enzyme-bound zinc coordinates the carboxylate of the amino acid, while a base on the enzyme is responsible for exchange of the alpha-proton. The role of the enzyme-bound metal is to render the alpha-proton more acidic through coordination of the carboxylate group.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear overhauser effect studies of the conformations of MG(α, β-Methylene)ATP bound to E. coli isoleucyl-tRNA synthetase

Internuclear distances obtained from transferred nuclear Overhauser effects were used in combination with distance geometry calculations to define the E. coli isoleucyl-tRNA synthetase bound conformation of Mg(alpha, beta-methylene)ATP both in the absence and in the presence of the cognate and noncognate amino acids L-isoleucine and L-valine, respectively. A single nucleotide structure having an anti adenine-ribose glycosidic torsional angle of -114 degrees was found to satisfy the experimental distance constraints. The nearly identical anti glycosidic torsional angles observed in all three complexes demonstrate that the conformation of the adenosine moiety of the enzyme-bound nucleotide is not sensitive to the presence or to the nature of the amino acid bound at the aminoacyladenylate site. In addition, the acceptable range of Mg(alpha, beta-methylene)ATP conformations bound to the E. coli isoleucyl-tRNA synthetase was found to be nearly identical to that previously determined for the E. coli methionyl-tRNA synthetase (Williams and Rosevear (1991) J. Biol. Chem. 266, 2089-2098). Thus, the predicted structural homology between the isoleucyl- and methionyl-tRNA synthetases, both members of the same class of synthetases on the basis of common consensus sequences, is further supported by consensus enzyme-bound nucleotide conformations.

Methionyl-tRNA synthetase fromBacillus stearothermophilus: structural and functional identities with theEscherichia colienzyme

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.

Yeast methionyl-tRNA synthetase: analysis of the N-terminal extension and the putative tRNA anticodon binding region by site-directed mutagenesis

Yeast methionyl-tRNA synthetase has a long N-terminal extension fused to the mononucleotide binding fold that occurs at the N-terminal end of the homologous E coli enzyme. We examined the contribution of this polypeptide region to the activity of the enzyme by creating several internal deletions in MESI which preserve the correct reading frame. The results show that 185 amino acids are dispensable for activity and stability. Removal of the next 5 residues affects the activity of the enzyme. The effect is more pronounced on the tRNA amino-acylation steps than on the adenylate formation step. The K m for ATP and methionine are unaltered, indicating that the global structure of the enzyme is maintained. The K m for tRNA increased slightly by a factor of 3, which indicates that the positioning of the tRNA on the surface of the molecule is not affected. There is, however, a great effect on the V max of the enzyme. Examination of the 3-D structure of the homologous E coli methionyl-tRNA synthetase indicates that the amino acid region preceding the mononucleotide binding fold does not participate directly in the catalytic cleft. It could, however, act at a distance by propagatinga mutational alteration of the catalytic residues. The tRNA Met anticodon binding region of the E coli enzyme has recently been characterized. By mutagenesis of the topologically equivalent region in the yeast enzyme, we could identify residues that alter specifically the aminoacylation of the tRNA. Leu 658 provides a van der Waals contact that is critical for the recognition of the yeast tRNA.

Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity.

Chemical synthesis is described of a 77-nucleotide-long RNA molecule that has the sequence of an Escherichia coli Ado-47-containing tRNA(fMet) species in which the modified nucleosides have been substituted by their unmodified parent nucleosides. The sequence was assembled on a solid-phase, controlled-pore glass support in a stepwise manner with an automated DNA synthesizer. The ribonucleotide building blocks used were fully protected 5'-monomethoxytrityl-2'-silyl-3'-N,N-diisopropylaminophosphoram idites. p-Nitro-phenylethyl groups were used to protect the O6 of guanine residues. The fully deprotected tRNA analogue was characterized by polyacrylamide gel electrophoresis (sizing), terminal nucleotide analysis, sequencing, and total enzyme degradation, all of which indicated that the sequence was correct and contained only 3-5 linkages. The 77-mer was then assayed for amino acid acceptor activity by using E. coli methionyl-tRNA synthetase. The results indicated that the synthetic product, lacking modified bases, is a substrate for the enzyme and has an amino acid acceptance 11% of that of the major native species, tRNA(fMet) containing 7-methylguanosine at position 47.

Identification of peptide sequences at the tRNA binding site of Escherichia coli methionyl-tRNA synthetase.

Four different structural regions of Escherichia coli tRNAfMet have been covalently coupled to E. coli methionyl-tRNA synthetase (MetRS) by using a tRNA derivative carrying a lysine-reactive cross-linker. We have previously shown that this cross-linking occurs at the tRNA binding site of the enzyme and involves reaction of only a small number of the potentially available lysine residues in the protein [Schulman, L. H., Valenzuela, D., & Pelka, H. (1981) Biochemistry 20, 6018-6023; Valenzuela, D., Leon, O., & Schulman, L. H. (1984) Biochem. Biophys. Res. Commun. 119, 677-684]. In this work, four of the cross-linked peptides have been identified. The tRNA-protein cross-linked complex was digested with trypsin, and the peptides attached to the tRNA were separated from the bulk of the tryptic peptides by anion-exchange chromatography. The tRNA-bound peptides were released by cleavage of the disulfide bond of the cross-linker and separated by reverse-phase high-pressure liquid chromatography, yielding five major peaks. Amino acid analysis indicated that four of these peaks contained single peptides. Sequence analysis showed that the peptides were cross-linked to tRNAfMet through lysine residues 402, 439, 465, and 640 in the primary sequence of MetRS. Binding of the tRNA therefore involves interactions with the carboxyl-terminal half of MetRS, while X-ray crystallographic data have shown the ATP binding site to be located in the N-terminal domain of the protein [Zelwer, C., Risler, J. L., & Brunie, S. (1982) J. Mol. Biol. 155, 63-81].(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus.

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.

Modification of the amino acid acceptor stem of E. coli tRNA fMet by ligation of chemically synthesized ribooligonucleotides

The single-stranged region of the amino acid acceptor stem corresponding to the 3'-end of E. coli tRNA Met f was replaced by ligation of chemically synthesized ribooligonucleotides, in order to change the length of the single-stranded CCA terminus. The chemically synthesized ribooligomers, CCA, ACCA, AACCA and CAACCA, were ligated to nuclease-treated E. coli tRNA Met f, which lacked the ACCA sequence at the 3'-end. The methionine acceptor activities of these modified tRNAs were examined using E. coli methionyl-tRNA synthetase. Ligation of the chemically synthesized pentamer (AACCA) to the acceptor terminus restored the methionine acceptor activity, whereas ligation of the hexamer (CAACCA) or trimer (CCA) to the acceptor terminus did not Modification of the acceptor terminus had no effect on the formylation of accepted methionine.

Preparation and analysis of l-[ 35S]methionine labeled transfer ribonucleic acids from rabbit liver

The two methionine-specific tRNA species found in rabbit liver cytoplasm can be individually labeled with radioactive methionine under the proper conditions. Crude rabbit liver tRNA aminoacylated with radio-active methionine using the homologous rabbit methionyl-tRNA synthetase, and then deacylated using the Escherichia coli methionyl-tRNA synthetase, yields specifically labeled Met-tRNA m Met . This is the species of methionine-specific tRNA which transfers its methionine residue into internal positions in growing polypeptide chains. Transfer RNA f Met can be specifically aminoacylated using the E. coli methionyl-tRNA synthetase in the absence of a formyl donor to yield Met-tRNA f Met , the initiator tRNA in eukaryotes. In the presence of Leucovorin, a formyl group donor, the use of the E. coli methionyl-tRNA synthetase and the E. coli transformylase yields N-formyl-Met-tRNA f Met . This last tRNA species is not found naturally in the cytoplasm of eukaryotes, but it often can serve as a useful substrate in in vitro studies on the initiation of protein synthesis in eukaryotes. These labeling methods appear to be of general use for the methionine-specific tRNAs found in most eukaryotes.