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Oxidation alters the architecture of the phenylalanyl-tRNA synthetase editing domain to confer hyperaccuracy.

ABSTRACT: High fidelity during protein synthesis is accomplished by aminoacyl-tRNA synthetases (aaRSs). These enzymes ligate an amino acid to a cognate tRNA and have proofreading and editing capabilities that ensure high fidelity. Phenylalanyl-tRNA synthetase (PheRS) preferentially ligates a phenylalanine to a tRNAPhe over the chemically similar tyrosine, which differs from phenylalanine by a single hydroxyl group. In bacteria that undergo exposure to oxidative stress such as Salmonella enterica serovar Typhimurium, tyrosine isomer levels increase due to phenylalanine oxidation. Several residues are oxidized in PheRS and contribute to hyperactive editing, including against mischarged Tyr-tRNAPhe, despite these oxidized residues not being directly implicated in PheRS activity. Here, we solve a 3.6 Å cryo-electron microscopy structure of oxidized S. Typhimurium PheRS. We find that oxidation results in widespread structural rearrangements in the β-subunit editing domain and enlargement of its editing domain. Oxidization also enlarges the phenylalanyl-adenylate binding pocket but to a lesser extent. Together, these changes likely explain why oxidation leads to hyperaccurate editing and decreased misincorporation of tyrosine. Taken together, these results help increase our understanding of the survival of S. Typhimurium during human infection.

SUBMITTER: Srinivas P

PROVIDER: S-EPMC8599791 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Oxidation of phenylalanyl-tRNA synthetase positively regulates translational quality control.

Project description:Accurate translation of the genetic code is maintained in part by aminoacyl-tRNA synthetases (aaRS) proofreading mechanisms that ensure correct attachment of a cognate amino acid to a transfer RNA (tRNA). During environmental stress, such as oxidative stress, demands on aaRS proofreading are altered by changes in the availability of cytoplasmic amino acids. For example, oxidative stress increases levels of cytotoxic tyrosine isomers, noncognate amino acids normally excluded from translation by the proofreading activity of phenylalanyl-tRNA synthetase (PheRS). Here we show that oxidation of PheRS induces a conformational change, generating a partially unstructured protein. This conformational change does not affect Phe or Tyr activation or the aminoacylation activity of PheRS. However, in vitro and ex vivo analyses reveal that proofreading activity to hydrolyze Tyr-tRNAPhe is increased during oxidative stress, while the cognate Phe-tRNAPhe aminoacylation activity is unchanged. In HPX-, Escherichia coli that lack reactive oxygen-scavenging enzymes and accumulate intracellular H2O2, we found that PheRS proofreading is increased by 11%, thereby providing potential protection against hazardous cytoplasmic m-Tyr accumulation. These findings show that in response to oxidative stress, PheRS proofreading is positively regulated without negative effects on the enzyme's housekeeping activity in translation. Our findings also illustrate that while the loss of quality control and mistranslation may be beneficial under some conditions, increased proofreading provides a mechanism for the cell to appropriately respond to environmental changes during oxidative stress.

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Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase.

Project description:To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA(Phe), indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3'-terminal adenosine, reduced Tyr-tRNA(Phe) deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

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Developmental Angiogenesis Requires the Mitochondrial Phenylalanyl-tRNA Synthetase.

Project description:Background: Mitochondrial aminoacyl-tRNA synthetases (mtARSs) catalyze the binding of specific amino acids to their cognate tRNAs and play an essential role in the synthesis of proteins encoded by mitochondrial DNA. Defects in mtARSs have been linked to human diseases, but their tissue-specific pathophysiology remains elusive. Here we examined the role of mitochondrial phenylalanyl-tRNA synthetase (FARS2) in developmental angiogenesis and its potential contribution to the pathogenesis of cardiovascular disease. Methods: Morpholinos were injected into fertilized zebrafish ova to establish an in vivo fars2 knock-down model. A visualization of the vasculature was achieved by using Tg (fli1: EGFP) y1 transgenic zebrafish. In addition, small interference RNAs (siRNAs) were transferred into human umbilical vein endothelial cells (HUVECs) to establish an in vitro FARS2 knock-down model. Cell motility, proliferation, and tubulogenesis were determined using scratch-wound CCK8, transwell-based migration, and tube formation assays. In addition, mitochondria- and non-mitochondria-related respiration were evaluated using a Seahorse XF24 analyzer and flow cytometry assays. Analyses of the expression levels of transcripts and proteins were performed using qRT-PCR and western blotting, respectively. Results: The knock-down of fars2 hampered the embryonic development in zebrafish and delayed the formation of the vasculature in Tg (fli1: EGFP) y1 transgenic zebrafish. In addition, the siRNA-mediated knock-down of FARS2 impaired angiogenesis in HUVECs as indicated by decreased cell motility and tube formation capacity. The knock-down of FARS2 also produced variable decreases in mitochondrial- and non-mitochondrial respiration in HUVECs and disrupted the regulatory pathways of angiogenesis in both HUVECs and zebrafish. Conclusion: Our current work offers novel insights into angiogenesis defects and cardiovascular diseases induced by FARS2 deficiency.

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Project description:Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA(Ala) and hydrolysis of mis-acylated Ser- and Gly-tRNA(Ala) at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned >35 A from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.

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Unique protein architecture of alanyl-tRNA synthetase for aminoacylation, editing, and dimerization.

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Project description:Human cytosolic phenylalanyl-tRNA synthetase (hcPheRS) is responsible for the covalent attachment of phenylalanine to its cognate tRNA(Phe). Significant differences between the amino-acid sequences of eukaryotic and prokaryotic PheRSs indicate that the domain composition of hcPheRS differs from that of the Thermus thermophilus analogue. As a consequence of the absence of the anticodon-recognizing B8 domain, the binding mode of tRNA(Phe) to hcPheRS is expected to differ from that in prokaryotes. Recombinant hcPheRS protein was purified to homogeneity and crystallized. The crystals used for structure determination diffracted to 3.3 A resolution and belonged to space group C2, with unit-cell parameters a = 362.9, b = 213.6, c = 212.7 A, beta = 125.2 degrees . The structure of hcPheRS was determined by the molecular-replacement method in combination with phase information from multiwavelength anomalous dispersion.

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The role of a novel auxiliary pocket in bacterial phenylalanyl-tRNA synthetase druggability.

Project description:The antimicrobial activity of phenyl-thiazolylurea-sulfonamides against Staphylococcus aureus PheRS are dependent upon phenylalanine levels in the extracellular fluids. Inhibitor efficacy in animal models of infection is substantially diminished by dietary phenylalanine intake, thereby reducing the perceived clinical utility of this inhibitor class. The search for novel antibacterial compounds against Gram-negative pathogens led to a re-evaluation of this phenomenon, which is shown here to be unique to S. aureus. Inhibition of macromolecular syntheses and characterization of novel resistance mutations in Escherichia coli demonstrate that antimicrobial activity of phenyl-thiazolylurea-sulfonamides is mediated by PheRS inhibition, validating this enzyme as a viable drug discovery target for Gram-negative pathogens. A search for novel inhibitors of PheRS yielded three novel chemical starting points. NMR studies were used to confirm direct target engagement for phenylalanine-competitive hits. The crystallographic structure of Pseudomonas aeruginosa PheRS defined the binding modes of these hits and revealed an auxiliary hydrophobic pocket that is positioned adjacent to the phenylalanine binding site. Three viable inhibitor-resistant mutants were mapped to this pocket, suggesting that this region is a potential liability for drug discovery.

| S-EPMC4118124 | biostudies-literature

Strictly conserved lysine of prolyl-tRNA Synthetase editing domain facilitates binding and positioning of misacylated tRNA(Pro.).

Project description:To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNA(Pro). This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2'-OH of misacylated tRNA(Pro) and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNA(Pro) deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNA(Pro) hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNA(Pro) and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNA(Pro) hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site.

| S-EPMC3986007 | biostudies-literature

Purification, crystallization and preliminary X-ray characterization of a human mitochondrial phenylalanyl-tRNA synthetase.

Project description:Human monomeric mitochondrial phenylalanyl-tRNA synthetase (mitPheRS) is an enzyme that catalyzes the charging of tRNA with the cognate amino acid phenylalanine. Human mitPheRS is a chimera of the bacterial alpha-subunit of PheRS and the B8 domain of its beta-subunit. Together, the alpha-subunit and the 'RNP-domain' (B8 domain) at the C-terminus form the minimal structural set to construct an enzyme with phenylalanylation activity. The recombinant human mitPheRS was purified to homogeneity and crystallized in complex with phenylalanine and ATP. The crystals diffracted to 2.2 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 55, b = 90, c = 96 A.

| S-EPMC2376306 | biostudies-literature

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