glutaminyl-tRNA Synthetase Research Articles

Structural and functional analysis of Glutaminyl-tRNA synthetase (TtGlnRS) from Thermus thermophilus HB8 and its complexes

Aminoacyl-tRNA synthetases (AaRSs) are vital enzymes for translation of proteins in cells. AaRSs catalyse the esterification of a specific amino acid to corresponding tRNAs to form an aminoacyl-tRNA that is used in ribosome-based protein synthesis. We focused on Glutaminyl tRNA synthetase (GlnRS) enzyme from the extreme thermophile Thermus thermophilus for structural studies. Our thermal shift assays show binding of enzyme substrates L-Gln and ATP as well as of various metals including cesium. We resolved crystal structures of apo-GlnRS as well as those in complex with AMP and ATP at 2.8 Å, 2.4 Å and 2.6 Å respectively. The bound cesium was found at the site of magnesium that typically binds to GlnRS. High structural conservation was evident in the Thermus thermophilus GlnRS when compared to those from Escherichia coli GlnRS.

Severe growth deficiency, microcephaly, intellectual disability, and characteristic facial features are due to a homozygous QARS mutation.

Glutaminyl tRNA synthase is highly expressed in the developing fetal human brain. Mutations in the glutaminyl-tRNA synthetase (QARS) gene have been reported in patients with progressive microcephaly, cerebral-cerebellar atrophy, and intractable seizures. We have previously reported a new recessive syndrome of severe linear growth retardation, poor weight gain, microcephaly, characteristic facial features, cutaneous syndactyly of the toes, high myopia, and intellectual disability in two sisters of Ashkenazi-Jewish origin (Eur J Med Genet 2014;57(6):288-92). Homozygosity mapping and whole exome sequencing revealed a homozygous missense (V476I) mutation in the QARS gene, located in the catalytic domain. The patient's fibroblasts demonstrated markedly reduced QARS amino acylation activity in vitro. Furthermore, the same homozygous mutation was found in an unrelated girl of Ashkenazi origin with the same phenotype. The clinical presentation of our patients differs from the original QARS-associated syndrome in the severe postnatal growth failure, absence of epilepsy, and minor MRI findings, thus further expanding the phenotypic spectrum of the glutaminyl-tRNA synthetase deficiency syndromes.

A water-mediated and substrate-assisted aminoacylation mechanism in the discriminating aminoacyl-tRNA synthetase GlnRS and non-discriminating GluRS.

Glutaminyl-tRNA synthetase (GlnRS) catalyzes the aminoacylation of glutamine to the corresponding tRNAGln. However, most bacteria and all archaea lack GlnRS and thus an indirect noncanonical aminoacylation is required. With the assistance of a non-discriminating version of Glutamyl-tRNA synthetases (ND-GluRS) the tRNAGln is misaminoacylated by glutamate. In this study, we have computationally investigated the aminoacylation mechanism in GlnRS and ND-GluRS employing Molecular Dynamics (MD) simulations, Quantum Mechanics (QM) cluster and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations. Our investigations demonstrated the feasibility of a water-mediated, substrate-assisted catalysis pathway with rate limiting steps occurring at energy barriers of 25.0 and 25.4 kcal mol-1 for GlnRS and ND-GluRS, respectively. A conserved lysine residue participates in a second proton transfer to facilitate the departure of the adenosine monophosphate (AMP) group. Thermodynamically stable (-29.9 and -9.3 kcal mol-1 for GlnRS and ND-GluRS) product complexes are obtained only when the AMP group is neutral.

Probing the stereospecificity of tyrosyl- and glutaminyl-tRNA synthetase with molecular dynamics

The stereospecificity of aminoacyl-tRNA synthetases helps exclude d-amino acids from protein synthesis and could perhaps be engineered to allow controlled d-amino acylation of tRNA. We use molecular dynamics simulations to probe the stereospecificity of the class I tyrosyl- and glutaminyl-tRNA synthetases (TyrRS, GlnRS), including wildtype enzymes and three point mutants suggested by three different protein design methods. l/d binding free energy differences are obtained by alchemically and reversibly transforming the ligand from L to D in simulations of the protein–ligand complex. The D81Q mutation in Escherichia coli TyrRS is homologous to the D81R mutant shown earlier to have inverted stereospecificity. D81Q is predicted to lead to a rotated ligand backbone and an increased, not a decreased l-Tyr preference. The E36Q mutation in Methanococcus jannaschii TyrRS has a predicted l/d binding free energy difference ΔΔG of just 0.5±0.9kcal/mol, compared to 3.1±0.8kcal/mol for the wildtype enzyme (favoring l-Tyr). The ligand ammonium position is preserved in the d-Tyr complex, while the carboxylate is shifted. Wildtype GlnRS has a similar preference for l-glutaminyl adenylate; the R260Q mutant has an increased preference, even though Arg260 makes a large contribution to the wildtype ΔΔG value.

Human cytomegalovirus-encoded miR-US4-1 promotes cell apoptosis and benefits discharge of infectious virus particles by targeting QARS.

Human cytomegalovirus (HCMV) can cause congenital diseases and opportunistic infections in immunocompromised individuals. Its functional proteins and microRNAs (miRNAs) facilitate efficient viral propagation by altering host cell behaviour. Identification of functional target genes of miRNAs is an important step in studies on HCMV pathogenesis. In this study, Glutaminyl-tRNA Synthetase (QARS), which could regulate signal transduction pathways for cellular apoptosis, was identified as a direct target of hcmv-miR-US4-1. Apoptosis assay revealed that as silence of QARS by ectopic expression of hcmv-miR-US4-1 and specific small interference RNA of QARS can promote cell apoptosis in HCMV-infected HELF cells. Moreover, viral growth curve assays showed that hcmv-miR-US4-1 benefits the discharge of infectious virus particles. However, silence of hcmv-miR-US4-1 by its specific inhibitor overturned these effects. These results imply that hcmv-miR-US4-1 might have the same effects during HCMV nature infection. In general, hcmv-miR-US4-1 may involve in promoting cell apoptosis and benefiting discharge of infectious virus particles via down-regulation of QARS in HCMV-infected HELF cells.

Development of Glutaminyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis

IntroductionPseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis.ResultsP. aeruginosa glutaminyl‐tRNA synthetase (GlnRS) was overexpressed in E. coli cells, and purified to homogeneity. GlnRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNAGln to produce a stable glutaminyl‐AMP intermediate. When compared with GlnRS from E. coli the amino acid sequence of GlnRS from P. aeruginosa are only 60% conserved. The kinetic parameters for interaction with tRNA were determined and the KM and Vmax were 1.0 μM and 0.23 μM min−1, respectively. The observed kcat was calculated to be 0.15 s−1 resulting in a kcat/KM value of 0.15 s−1μM−1. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa GlnRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Three compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM02E04, BM04B05, and BM04H03 inhibit the activity of P. aeruginosa ArgRS with IC50 values of 1.7, 2.5, and 39.3 μM, respectively. All three of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections.ConclusionGlnRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.Support or Funding InformationResearch was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017) and from the NIH UTPA RISE program grant # 1R25GM100866‐01.

The crystal structure of human GlnRS provides basis for the development of neurological disorders.

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.

Partial suppression of the respiratory defect of qrs1/her2 glutamyl-tRNA amidotransferase mutants by overexpression of the mitochondrial pentatricopeptide Msc6p.

Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.

Dispensability of zinc and the putative zinc-binding domain in bacterial glutamyl-tRNA synthetase.

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNAGln via interaction with GatCAB, whereas its role in tRNAGlu interaction may be a consequence of the presence of pZBD.

In the beginning was the U1A protein: a personal reflection.

In the beginning was the U1A protein: a personal reflection.

Recombinant expression, purification, and crystallization of the glutaminyl-tRNA synthetase from Toxoplasma gondii

Aminoacyl tRNA synthetases play a critical role in protein synthesis by providing precursor transfer-RNA molecules correctly charged with their cognate amino-acids. The essential nature of these enzymes make them attractive targets for designing new drugs against important pathogenic protozoans like Toxoplasma. Because no structural data currently exists for a protozoan glutaminyl-tRNA synthetase (QRS), an understanding of its potential as a drug target and its function in the assembly of the Toxoplasma multi-aminoacyl tRNA (MARS) complex is therefore lacking. Here we describe the optimization of expression and purification conditions that permitted the recovery and crystallization of both domains of the Toxoplasma QRS enzyme from a heterologous Escherichia coli expression system. Expression of full-length QRS was only achieved after the addition of an N-terminal histidine affinity tag and the isolated protein was active on both cellular and in vitro produced Toxoplasma tRNA. Taking advantage of the proteolytic susceptibility of QRS to cleavage into component domains, N-terminal glutathione S-transferase (GST) motif-containing domain fragments were isolated and crystallization conditions discovered. Isolation of the C-terminal catalytic domain was accomplished after subcloning the domain and optimizing expression conditions. Purified catalytic domain survived cryogenic storage and yielded large diffraction-quality crystals over-night after optimization of screening conditions. This work will form the basis of future structural studies into structural–functional relationships of both domains including potential targeted drug-design studies and investigations into the assembly of the Toxoplasma MARS complex.

Expression profile of aminoacyl-tRNA synthetases in dorsal root ganglion neurons after peripheral nerve injury.

Aminoacyl-tRNA synthetases (AminoARSs) are essential enzymes involved in acylating tRNA with amino acids. In addition to the typical functions of AminoARSs, various non-canonical functions have been reported, such as involvement in cellular regulatory processes and signal transduction. Here, to explore the cellular changes in sensory neurons after nerve injury, we evaluated AARS mRNA expression in rat dorsal root ganglia (DRG) neurons using AminoARS-specific primers. Of 20 AminoARSs, we found that expression of lysyl-tRNA synthetase (KARS) and glutaminyl-tRNA synthetase (QARS) was decreased in the DRG injured side. We observed decreased KARS and QARS expression in DRG neuronal cell bodies, but not in satellite cells. Therefore, we suggest the possibility that KARS and QARS may act as signaling molecules to transfer abnormal sensory signals to the spinal dorsal horn after peripheral nerve damage. Therefore, KARS and QARS may represent powerful pharmaceutical targets via control of their non-canonical functions.

Recoding aminoacyl-tRNA synthetases for synthetic biology by rational protein-RNA engineering.

We have taken a rational approach to redesigning the amino acid binding and aminoacyl–tRNA pairing specificities of bacterial glutaminyl–tRNA synthetase. The four-stage engineering incorporates generalizable design principles and improves the pairing efficiency of noncognate glutamate with tRNAGln by over 105-fold compared to the wild-type enzyme. Better optimized designs of the protein–RNA complex include substantial reengineering of the globular core region of the tRNA, demonstrating a role for specific tRNA nucleotides in specifying the identity of the genetically encoded amino acid. Principles emerging from this engineering effort open new prospects for combining rational and genetic selection approaches to design novel aminoacyl–tRNA synthetases that ligate noncanonical amino acids onto tRNAs. This will facilitate reconstruction of the cellular translation apparatus for applications in synthetic biology.

The predatory bacterium Bdellovibrio bacteriovorus aspartyl-tRNA synthetase recognizes tRNAAsn as a substrate.

The predatory bacterium Bdellovibrio bacteriovorus preys on other Gram-negative bacteria and was predicted to be an asparagine auxotroph. However, despite encoding asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase, B. bacteriovorus also contains the amidotransferase GatCAB. Deinococcus radiodurans, and Thermus thermophilus also encode both of these aminoacyl-tRNA synthetases with GatCAB. Both also code for a second aspartyl-tRNA synthetase and use the additional aspartyl-tRNA synthetase with GatCAB to synthesize asparagine on tRNAAsn. Unlike those two bacteria, B. bacteriovorus encodes only one aspartyl-tRNA synthetase. Here we demonstrate the lone B. bacteriovorus aspartyl-tRNA synthetase catalyzes aspartyl-tRNAAsn formation that GatCAB can then amidate to asparaginyl-tRNAAsn. This non-discriminating aspartyl-tRNA synthetase with GatCAB thus provides B. bacteriovorus a second route for Asn-tRNAAsn formation with the asparagine synthesized in a tRNA-dependent manner. Thus, in contrast to a previous prediction, B. bacteriovorus codes for a biosynthetic route for asparagine. Analysis of bacterial genomes suggests a significant number of other bacteria may also code for both routes for Asn-tRNAAsn synthesis with only a limited number encoding a second aspartyl-tRNA synthetase.

Coevolution of Specificity Determinants in Eukaryotic Glutamyl- and Glutaminyl-tRNA Synthetases

The glutaminyl-tRNA synthetase (GlnRS) enzyme, which pairs glutamine with tRNAGln for protein synthesis, evolved by gene duplication in early eukaryotes from a nondiscriminating glutamyl-tRNA synthetase (GluRS) that aminoacylates both tRNAGln and tRNAGlu with glutamate. This ancient GluRS also separately differentiated to exclude tRNAGln as a substrate, and the resulting discriminating GluRS and GlnRS further acquired additional protein domains assisting function in cis (the GlnRS N-terminal Yqey domain) or in trans (the Arc1p protein associating with GluRS). These added domains are absent in contemporary bacterial GlnRS and GluRS. Here, using Saccharomyces cerevisiae enzymes as models, we find that the eukaryote-specific protein domains substantially influence amino acid binding, tRNA binding and aminoacylation efficiency, but they play no role in either specific nucleotide readout or discrimination against noncognate tRNA. Eukaryotic tRNAGln and tRNAGlu recognition determinants are found in equivalent positions and are mutually exclusive to a significant degree, with key nucleotides located adjacent to portions of the protein structure that differentiated during the evolution of archaeal nondiscriminating GluRS to GlnRS. These findings provide important corroboration for the evolutionary model and suggest that the added eukaryotic domains arose in response to distinctive selective pressures associated with the greater complexity of the eukaryotic translational apparatus. We also find that the affinity of GluRS for glutamate is significantly increased when Arc1p is not associated with the enzyme. This is consistent with the lower concentration of intracellular glutamate and the dissociation of the Arc1p:GluRS complex upon the diauxic shift to respiratory conditions.

The phylogenetic utility of acetyltransferase (ARD1) and glutaminyl tRNA synthetase (QtRNA) for reconstructing Cenozoic relationships as exemplified by the large Australian cicada Pauropsalta generic complex

The Pauropsalta generic complex is a large group of cicadas (72 described spp.; >82 undescribed spp.) endemic to Australia. No previous molecular work on deep level relationships within this complex has been conducted, but a recent morphological revision and phylogenetic analysis proposed relationships among the 11 genera. We present here the first comprehensive molecular phylogeny of the complex using five loci (1 mtDNA, 4 nDNA), two of which are from nuclear genes new to cicada systematics. We compare the molecular phylogeny to the morphological phylogeny. We evaluate the phylogenetic informativeness of the new loci to traditional cicada systematics loci to generate a baseline of performance and behavior to aid in gene choice decisions in future systematic and phylogenomic studies. Our maximum likelihood and Bayesian inference phylogenies strongly support the monophyly of most of the newly described genera; however, relationships among genera differ from the morphological phylogeny. A comparison of phylogenetic informativeness among all loci revealed that COI 3rd positions dominate the informativeness profiles relative to all other loci but exhibit some among taxon nucleotide bias. After removing COI 3rd positions, COI 1st positions dominate near the terminals, while the period intron has the most phylogenetic informativeness near the root. Among the nuclear loci, ARD1 and QtRNA have lower phylogenetic informativeness than period intron and elongation factor 1 alpha intron, but the informativeness increases at you move from the tips to the root. The increase in phylogenetic informativeness deeper in the tree suggests these loci may be useful for resolving older relationships.

Glutamyl-tRNAGln amidotransferase is essential for mammalian mitochondrial translation in vivo

Translational accuracy depends on the correct formation of aminoacyl-tRNAs, which, in the majority of cases, are produced by specific aminoacyl-tRNA synthetases that ligate each amino acid to its cognate isoaceptor tRNA. Aminoacylation of tRNAGln, however, is performed by various mechanisms in different systems. Since no mitochondrial glutaminyl-tRNA synthetase has been identified to date in mammalian mitochondria, Gln-tRNAGln has to be formed by an indirect mechanism in the organelle. It has been demonstrated that human mitochondria contain a non-discriminating glutamyl-tRNA synthetase and the heterotrimeric enzyme GatCAB (where Gat is glutamyl-tRNAGln amidotransferase), which are able to catalyse the formation of Gln-tRNAGln invitro. In the present paper we demonstrate that mgatA (mouse GatA) interference in mouse cells produces a strong defect in mitochondrial translation without affecting the stability of the newly synthesized proteins. As a result, interfered cells present an impairment of the oxidative phosphorylation system and a significant increase in ROS (reactive oxygen species) levels. MS analysis of mitochondrial proteins revealed no glutamic acid found in the position of glutamines, strongly suggesting that misaminoacylated Glu-tRNAGln is rejected from the translational apparatus to maintain the fidelity of mitochondrial protein synthesis in mammals.

Mutations in QARS, Encoding Glutaminyl-tRNA Synthetase, Cause Progressive Microcephaly, Cerebral-Cerebellar Atrophy, and Intractable Seizures

Progressive microcephaly is a heterogeneous condition with causes including mutations in genes encoding regulators of neuronal survival. Here, we report the identification of mutations in QARS (encoding glutaminyl-tRNA synthetase [QARS]) as the causative variants in two unrelated families affected by progressive microcephaly, severe seizures in infancy, atrophy of the cerebral cortex and cerebellar vermis, and mild atrophy of the cerebellar hemispheres. Whole-exome sequencing of individuals from each family independently identified compound-heterozygous mutations in QARS as the only candidate causative variants. QARS was highly expressed in the developing fetal human cerebral cortex in many cell types. The four QARS mutations altered highly conserved amino acids, and the aminoacylation activity of QARS was significantly impaired in mutant cell lines. Variants p.Gly45Val and p.Tyr57His were located in the N-terminal domain required for QARS interaction with proteins in the multisynthetase complex and potentially with glutamine tRNA, and recombinant QARS proteins bearing either substitution showed an over 10-fold reduction in aminoacylation activity. Conversely, variants p.Arg403Trp and p.Arg515Trp, each occurring in a different family, were located in the catalytic core and completely disrupted QARS aminoacylation activity in vitro. Furthermore, p.Arg403Trp and p.Arg515Trp rendered QARS less soluble, and p.Arg403Trp disrupted QARS-RARS (arginyl-tRNA synthetase 1) interaction. In zebrafish, homozygous qars loss of function caused decreased brain and eye size and extensive cell death in the brain. Our results highlight the importance of QARS during brain development and that epilepsy due to impairment of QARS activity is unusually severe in comparison to other aminoacyl-tRNA synthetase disorders.

Differential proteomic studies of the genic male-sterile line and fertile line anthers of upland cotton (Gossypium hirsutum L.)

Pollen development is disturbed in the microspore development stage of the double-recessive nuclear male-sterile line ms5ms6 (Gossypium hirsutum L.). This study aimed to identify differentially expressed anther proteins and their potential roles in pollen development and male sterility. We compared the proteomes of sterile and fertile anthers of the double recessive nuclear male-sterile line ms5ms6. Approximately 1,390 protein spots were detected by two-dimensional differential gel electrophoresis. Proteins with altered accumulation levels in sterile anthers compared with fertile anthers were identified by mass spectrometry and the NCBInr and Viridiplantae EST databases. Down-regulated proteins in the sterile anthers included cytosolic ascorbate peroxidase 1 and glutaminyl-tRNA synthetase (glutamine-tRNA ligase). Several carbohydrate metabolism- and photosynthesis-related enzymes were also present at lower levels in the mutant anthers. By contrast, ATP-dependent RNA helicase eIF4A-13, NADH dehydrogenase subunit 1, enolase, gibberellin 20-oxidase, gibberellin 3-hydroxylase 1, alcohol dehydrogenase 2d, 3-ketoacyl-CoA synthase, and trehalose 6-phosphate synthase were expressed at higher levels in sterile anthers than in fertile anthers. The regulation of upland cotton pollen development involves a complex network of differentially expressed genes. This study provides the foundation for future investigations of gene function in upland cotton pollen development and male sterility.

Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase, Relative to Its Urzyme, Requires Domain Motion Sensed by the D1 Switch, a Remote Dynamic Packing Motif

We previously showed (Li, L., and Carter, C. W., Jr. (2013) J. Biol. Chem. 288, 34736-34745) that increased specificity for tryptophan versus tyrosine by contemporary Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) over that of TrpRS Urzyme results entirely from coupling between the anticodon-binding domain and an insertion into the Rossmann-fold known as Connecting Peptide 1. We show that this effect is closely related to a long range catalytic effect, in which side chain repacking in a region called the D1 Switch, accounts fully for the entire catalytic contribution of the catalytic Mg(2+) ion. We report intrinsic and higher order interaction effects on the specificity ratio, (kcat/Km)Trp/(kcat/Km)Tyr, of 15 combinatorial mutants from a previous study (Weinreb, V., Li, L., and Carter, C. W., Jr. (2012) Structure 20, 128-138) of the catalytic role of the D1 Switch. Unexpectedly, the same four-way interaction both activates catalytic assist by Mg(2+) ion and contributes -4.4 kcal/mol to the free energy of the specificity ratio. A minimum action path computed for the induced-fit and catalytic conformation changes shows that repacking of the four residues precedes a decrease in the volume of the tryptophan-binding pocket. We suggest that previous efforts to alter amino acid specificities of TrpRS and glutaminyl-tRNA synthetase (GlnRS) by mutagenesis without extensive, modular substitution failed because mutations were incompatible with interdomain motions required for catalysis.