Breaking a single hydrogen bond in the mitochondrial tRNAPhe -PheRS complex leads to phenotypic pleiotropy of human disease.

Various pathogenic variants in both mitochondrial tRNAPhe and Phenylalanyl-tRNA synthetase mitochondrial protein coding gene (FARS2) gene encoding for the human mitochondrial PheRS have been identified and associated with neurological and/or muscle-related pathologies. An important Guanine-34 (G34)A anticodon mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) syndrome has been reported in hmit-tRNAPhe . The majority of G34 contacts in available aaRSs-tRNAs complexes specifically use that base as an important tRNA identity element. The network of intermolecular interactions providing its specific recognition also largely conserved. However, their conservation depends also on the invariance of the residues in the anticodon binding domain (ABD) of human mitochondrial Phenylalanyl-tRNA synthetase (hmit-PheRS). A defect in recognition of the anticodon of tRNAPhe may happen not only because of G34A mutation, but also due to mutations in the ABD. Indeed, a pathogenic mutation in FARS2 has been recently reported in a 9-year-old female patient harboring a p.Asp364Gly mutation. Asp364 is hydrogen bonded (HB) to G34 in WT hmit-PheRS. Thus, there are two pathogenic variants disrupting HB between G34 and Asp364: one is associated with G34A mutation, and the other with Asp364Gly mutation. We have measured the rates of tRNAPhe aminoacylation catalyzed by WT hmit-PheRS and mutant enzymes. These data ranked the residues making a HB with G34 according to their contribution to activity and the signal transduction pathway in the hmit-PheRS-tRNAPhe complex. Furthermore, we carried out extensive MD simulations to reveal the interdomain contact topology on the dynamic trajectories of the complex, and gaining insight into the structural and dynamic integrity effects of hmit-PheRS complexed with tRNAPhe . DATABASE: Structural data are available in PDB database under the accession number(s): 3CMQ, 3TUP, 5MGH, 5MGV.

Kinetic and structural changes in HsmtPheRS, induced by pathogenic mutations in human FARS2.

Mutations in the mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three-dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild-type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady-state kinetic measurements of phenylalanine activation and tRNAPhe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease-related mutations in FARS2 gene.

Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids.

Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on. Cytosolic phenylalanyl-tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that "teamwork" of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m-Tyr and l-Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αß)2 architecture only. The fusion protein catalyzes aminoacylation of tRNA(Phe) with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl-tRNAs: Tyr-tRNA(Phe) and m-Tyr-tRNA(Phe) .

Universal pathway for posttransfer editing reactions: insights from the crystal structure of TtPheRS with puromycin.

At the amino acid binding and recognition step, phenylalanyl-tRNA synthetase (PheRS) faces the challenge of discrimination between cognate phenylalanine and closely similar noncognate tyrosine. Resampling of Tyr-tRNA(Phe) to PheRS increasing the number of correctly charged tRNA molecules has recently been revealed. Thus, the very same editing site of PheRS promotes hydrolysis of misacylated tRNA species, associated both with cis- and trans-editing pathways. Here we report the crystal structure of Thermus thermophilus PheRS (TtPheRS) at 2.6 Å resolution, in complex with phenylalanine and antibiotic puromycin mimicking the A76 of tRNA acylated with tyrosine. Starting from the complex structure and using a hybrid quantum mechanics/molecular mechanics approach, we investigate the pathways of editing reaction catalyzed by TtPheRS. We show that both 2' and 3' isomeric esters undergo mutual transformation via the cyclic intermediate orthoester, and the editing site can readily accommodate a model of Tyr-tRNA(Phe) where deacylation occurs from either the 2'- or 3'-OH. The suggested pathway of the hydrolytic reaction at the editing site of PheRS is of sufficient generality to warrant comparison with other class I and class II aminoacyl-tRNA synthetases.

The mechanistic and evolutionary aspects of the 2'- and 3'-OH paradigm in biosynthetic machinery.

BACKGROUND: The translation machinery underlies a multitude of biological processes within the cell. The design and implementation of the modern translation apparatus on even the simplest course of action is extremely complex, and involves different RNA and protein factors. According to the "RNA world" idea, the critical link in the translation machinery may be assigned to an adaptor tRNA molecule. Its exceptional functional and structural characteristics are of primary importance in understanding the evolutionary relationships among all these macromolecular components. PRESENTATION OF THE HYPOTHESIS: The 2'-3' hydroxyls of the tRNA A76 constitute chemical groups of critical functional importance, as they are implicated in almost all phases of protein biosynthesis. They contribute to: a) each step of the tRNA aminoacylation reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs); b) the isomerase activity of EF-Tu, involving a mixture of the 2'(3')- aminoacyl tRNA isomers as substrates, thereby producing the required combination of amino acid and tRNA; and c) peptide bond formation at the peptidyl transferase center (PTC) of the ribosome. We hypothesize that specific functions assigned to the 2'-3' hydroxyls during peptide bond formation co-evolved, together with two modes of attack on the aminoacyl-adenylate carbonyl typical for two classes of aaRSs, and alongside the isomerase activity of EF-Tu. Protein components of the translational apparatus are universally recognized as being of ancient origin, possibly replacing RNA-based enzymes that may have existed before the last universal common ancestor (LUCA). We believe that a remnant of these processes is still imprinted on the organization of modern-day translation. TESTING AND IMPLICATIONS OF THE HYPOTHESIS: Earlier publications indicate that it is possible to select ribozymes capable of attaching the aa-AMP moiety to RNA molecules. The scenario described herein would gain general acceptance, if a ribozyme able to activate the amino acid and transfer it onto the terminal ribose of the tRNA, would be found in any life form, or generated in vitro. Interestingly, recent studies have demonstrated the plausibility of using metals, likely abandoned under primordial conditions, as biomimetic catalysts of the aminoacylation reaction.

Anticodon G recognition by tRNA synthetases mimics the tRNA core.

Ancient mechanisms for nucleotide base recognition in the RNA world are candidates for mimicking by early proteins like tRNA synthetases. In the core of the tRNA, conserved G22 interacts with two internal bases in a complex further stabilized by stacking interactions. This particular tRNA format for G recognition is shown here to be adapted by nine different and even nonhomologous anticodon binding domains (ABDs) of tRNA synthetases, in which amino acid side chains mimic all of the tRNA G22 base interactions. We offer the possibility that mimicking this RNA-based mechanism for guanine recognition is perhaps one of the selective pressures for choosing amino acids for the genetic code.

Crystal structure of human mitochondrial PheRS complexed with tRNA(Phe) in the active "open" state.

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 Å. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1-C72 base pair, and the "discriminator" base A73, proposed to contribute to tRNA(Phe) identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS-tRNA(Phe) provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the "closed" to the productive "open" state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions.

Bacterial and eukaryotic phenylalanyl-tRNA synthetases catalyze misaminoacylation of tRNA(Phe) with 3,4-dihydroxy-L-phenylalanine.

Aminoacyl-tRNA synthetases exert control over the accuracy of translation by selective pairing the correct amino acids with their cognate tRNAs, and proofreading the misacylated products. Here we show that three existing, structurally different phenylalanyl-tRNA synthetases-human mitochondrial (HsmtPheRS), human cytoplasmic (HsctPheRS), and eubacterial from Thermus thermophilus (TtPheRS), catalyze mischarging of tRNA(Phe) with an oxidized analog of tyrosine-L-dopa. The lowest level of L-dopa discrimination over the cognate amino acid, exhibited by HsmtPheRS, is comparable to that of tyrosyl-tRNA synthetase. HsmtPheRS and TtPheRS complexes with L-dopa revealed in the active sites an electron density shaping this ligand. HsctPheRS and TtPheRS possessing editing activity are capable of hydrolyzing the exogenous L-dopa-tRNA(Phe) as efficiently as Tyr-tRNA(Phe). However, editing activity of PheRS does not guarantee reduction of the aminoacylation error rate to escape misincorporation of L-dopa into polypeptide chains.

Common peptides study of aminoacyl-tRNA synthetases.

BACKGROUND: Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains. RESULTS: We utilized the Common Peptides (CPs) framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS-class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA) enzyme overlapping binding sites in both families. CONCLUSIONS: The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families.

Idiosyncrasy and identity in the prokaryotic Phe-system: crystal structure of E. coli phenylalanyl-tRNA synthetase complexed with phenylalanine and AMP.

The crystal structure of Phenylalanyl-tRNA synthetase from E. coli (EcPheRS), a class II aminoacyl-tRNA synthetase, complexed with phenylalanine and AMP was determined at 3.05 Å resolution. EcPheRS is a (αß)2 heterotetramer: the αß heterodimer of EcPheRS consists of 11 structural domains. Three of them: the N-terminus, A1 and A2 belong to the α-subunit and B1-B8 domains to the ß subunit. The structure of EcPheRS revealed that architecture of four helix-bundle interface, characteristic of class IIc heterotetrameric aaRSs, is changed: each of the two long helices belonging to CLM transformed into the coil-short helix structural fragments. The N-terminal domain of the α-subunit in EcPheRS forms compact triple helix domain. This observation is contradictory to the structure of the apo form of TtPheRS, where N-terminal domain was not detected in the electron density map. Comparison of EcPheRS structure with TtPheRS has uncovered significant rearrangements of the structural domains involved in tRNA(Phe) binding/translocation. As it follows from modeling experiments, to achieve a tighter fit with anticodon loop of tRNA, a shift of ∼5 Å is required for C-terminal domain B8, and of ∼6 to 7 Å for the whole N terminus. EcPheRSs have emerged as an important target for the incorporation of novel amino acids into genetic code. Further progress in design of novel compounds is anticipated based on the structural data of EcPheRS.

Structure of human cytosolic phenylalanyl-tRNA synthetase: evidence for kingdom-specific design of the active sites and tRNA binding patterns.

The existence of three types of phenylalanyl-tRNA synthetase (PheRS), bacterial (alphabeta)(2), eukaryotic/archaeal cytosolic (alphabeta)(2), and mitochondrial alpha, is a prominent example of structural diversity within the aaRS family. PheRSs have considerably diverged in primary sequences, domain compositions, and subunit organizations. Loss of the anticodon-binding domain B8 in human cytosolic PheRS (hcPheRS) is indicative of variations in the tRNA(Phe) binding and recognition as compared to bacterial PheRSs. We report herein the crystal structure of hcPheRS in complex with phenylalanine at 3.3 A resolution. A novel structural module has been revealed at the N terminus of the alpha subunit. It stretches out into the solvent of approximately 80 A and is made up of three structural domains (DBDs) possessing DNA-binding fold. The dramatic reduction of aminoacylation activity for truncated N terminus variants coupled with structural data and tRNA-docking model testify that DBDs play crucial role in hcPheRS activity.

Structural Aspects of Phenylalanylation and Quality Control in Three Major Forms of Phenylalanyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are a canonical set of enzymes that specifically attach corresponding amino acids to their cognate transfer RNAs in the cytoplasm, mitochondria, and nucleus. The aaRSs display great differences in primary sequence, subunit size, and quaternary structure. Existence of three types of phenylalanyl-tRNA synthetase (PheRS)-bacterial (αß)(2), eukaryotic/archaeal cytosolic (αß)(2), and mitochondrial α-is a prominent example of structural diversity within the aaRSs family. Although archaeal/eukaryotic and bacterial PheRSs share common topology of the core domains and the B3/B4 interface, where editing activity of heterotetrameric PheRSs is localized, the detailed investigation of the three-dimensional structures from three kingdoms revealed significant variations in the local design of their synthetic and editing sites. Moreover, as might be expected from structural data eubacterial, Thermus thermophilus and human cytoplasmic PheRSs acquire different patterns of tRNA(Phe) anticodon recognition.

'RNA walk' a novel approach to study RNA-RNA interactions between a small RNA and its target.

In this study we describe a novel method to investigate the RNA-RNA interactions between a small RNA and its target that we termed 'RNA walk'. The method is based on UV-induced AMT cross-linking in vivo followed by affinity selection of the hybrid molecules and mapping the intermolecular adducts by RT-PCR or real-time PCR. Domains carrying the cross-linked adducts fail to efficiently amplify by PCR compared with non-cross-linked domains. This method was calibrated and used to study the interaction between a special tRNA-like molecule (sRNA-85) that is part of the trypanosome signal recognition particle (SRP) complex and the ribosome. Four contact sites between sRNA-85 and rRNA were identified by 'RNA walk' and were further fine-mapped by primer extension. Two of the contact sites are expected; one contact site mimics the interaction of the mammalian Alu domain of SRP with the ribosome and the other contact sites include a canonical tRNA interaction. The two other cross-linked sites could not be predicted. We propose that 'RNA walk, is a generic method to map target RNA small RNAs interactions in vivo.

Large-scale movement of functional domains facilitates aminoacylation by human mitochondrial phenylalanyl-tRNA synthetase.

Structural studies suggest rearrangement of the RNA-binding and catalytic domains of human mitochondrial PheRS (mtPheRS) is required for aminoacylation. Crosslinking the catalytic and RNA-binding domains resulted in a "closed" form of mtPheRS that still catalyzed ATP-dependent Phe activation, but was no longer able to transfer Phe to tRNA and complete the aminoacylation reaction. SAXS experiments indicated the presence of both the closed and open forms of mtPheRS in solution. Together, these results indicate that conformational flexibility of the two functional modules in mtPheRS is essential for its phenylalanylation activity. This is consistent with the evolution of the aminoacyl-tRNA synthetases as modular enzymes consisting of separate domains that display independent activities.

Intra-protein compensatory mutations analysis highlights the tRNA recognition regions in aminoacyl-tRNA synthetases.

The aminoacyl-tRNA synthetases (aaRSs) covalently attach amino acids to their corresponding nucleic acid adapter molecules, tRNAs. The interactions in the tRNA-aaRSs complexes are mostly non-specific, and largely electrostatic. Tracing a way of aaRS-tRNA mutual adaptation throughout evolution offers a clearer view of understanding how aaRS-tRNA systems preserve patterns of tRNA recognition and binding. In this study, we used the compensatory mutations analysis to explore adaptation of aaRSs in respond to random mutations that can occur in the tRNA-recognition area. We showed that the frequency of compensatory mutations among residues that belong to the recognition region is 1.75-fold higher than that of the exposed residues. The highest frequencies of compensatory mutations are observed for pairs of charged residues, wherein one residue is located within the tRNA-recognition area, while the second is placed outside of the area, and contributes to the formation of the aaRS electrostatic landscape. Given charged residues are compensated by buried charge residues in more than 60% of the analyzed mutations. The cytoplasmatic and mitochondrial aaRSs preserve similar patterns of compensatory mutations in the tRNA recognition areas. Moreover, we found that mitochondrial aaRSs demonstrate a significant increase in the frequency of compensatory mutations in the area. Our findings shed light on the physical nature of compensatory mutations in aaRSs, thereby keeping unchanged tRNA-recognition patterns.

Eukaryotic cytosolic and mitochondrial phenylalanyl-tRNA synthetases catalyze the charging of tRNA with the meta-tyrosine.

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROS-damaged amino acids are incorporated into proteins remains unclear. We provide herein evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases (HsmtPheRS and HsctPheRS, respectively) catalyze direct attachment of m-Tyr to tRNA(Phe), thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal complexes of mitochondrial and bacterial PheRSs with m-Tyr reveal the net of highly specific interactions within the synthetic and editing sites.

Crystallization and X-ray analysis of human cytoplasmic phenylalanyl-tRNA synthetase.

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.

The tRNA-induced conformational activation of human mitochondrial phenylalanyl-tRNA synthetase.

All class II aminoacyl-tRNA synthetases (aaRSs) are known to be active as functional homodimers, homotetramers, or heterotetramers. However, multimeric organization is not a prerequisite for phenylalanylation activity, as monomeric mitochondrial phenylalanyl-tRNA synthetase (PheRS) is also active. We herein report the structure, at 2.2 A resolution, of a human monomeric mitPheRS complexed with Phe-AMP. The smallest known aaRS, which is, in fact, 1/5 of a cytoplasmic analog, is a chimera of the catalytic module of the alpha and anticodon binding domain (ABD) of the bacterial beta subunit of (alphabeta)2 PheRS. We demonstrate that the ABD located at the C terminus of mitPheRS overlaps with the acceptor stem of phenylalanine transfer RNA (tRNAPhe) if the substrate is positioned in a manner similar to that seen in the binary Thermus thermophilus complex. Thus, formation of the PheRS-tRNAPhe complex in human mitochondria must be accompanied by considerable rearrangement (hinge-type rotation through approximately 160 degrees) of the ABD upon tRNA binding.

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea.

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.3%). All cysteine-rich organisms belong to the methanogenic Archaea, which generates special cysteine clusters associated with primitive metabolic reactions. Our findings suggest that cysteine plays an important role in early forms of life.

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids.

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.