The C-terminal domain of the archaeal leucyl-tRNA synthetase prevents misediting of isoleucyl-tRNA(Ile)

In the archaeal leucyl-tRNA synthetase (LeuRS), the C-terminal domain recognizes the long variable arm of tRNA(Leu) for aminoacylation, and the so-called editing domain deacylates incorrectly formed Ile-tRNA(Leu). We previously reported, for Pyrococcus horikoshii LeuRS, that a deletion mutant lacking the C-terminal domain (LeuRS_delta(811-967)) retains normal editing activity, but has severely reduced aminoacylation activity. In this study, we found that LeuRS_delta(811-967), but not the wild-type LeuRS, exhibited surprisingly robust deacylation activity against Ile-tRNA(Ile), correctly formed by isoleucyl-tRNA synthetase ("misediting"). Structural superposition of tRNA(Ile) onto the LeuRS x tRNA(Leu) complex indicated that Ile911, Lys912, and Glu913 of the LeuRS C-terminal domain clash with U20 of tRNA(Ile), which is bulged out as compared to the corresponding nucleotide of tRNA(Leu). The deletion of amino acid residues 911-913 of LeuRS enhanced the Ile-tRNA(Ile) deacylation activity, without affecting the Ile-tRNA(Leu) deacylation activity. These results demonstrate that the clashing between U20 of tRNA(Ile) and residues 911-913 of the LeuRS C-terminal domain is the structural mechanism that prevents misediting. In contrast, the deletion of the C-terminal domains of the isoleucyl- and valyl-tRNA synthetases impaired both the aminoacylation (Ile-tRNA(Ile) and Val-tRNA(Val) formation, respectively) and editing (Val-tRNA(Ile) and Thr-tRNA(Val) deacylation, respectively) activities, and did not cause misediting (Val-tRNA(Val) and Thr-tRNA(Thr) deacylation, respectively) activity. Thus, the requirement of the C-terminal domain for misediting prevention is unique to LeuRS, which does not recognize the anticodon of the cognate tRNA, unlike the common aminoacyl-tRNA synthetases.     

Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles

Leucyl-tRNA synthetase (LeuRS) performs dual essential roles in group I intron RNA splicing as well as protein synthesis within the yeast mitochondria. Deletions of the C terminus differentially impact the two functions of the enzyme in splicing and aminoacylation in vivo. Herein, we determined that a fiveamino acid C-terminal deletion of LeuRS, which does not complement a null strain, can form a ternary complex with the bI4 intron and its maturase splicing partner. However, the complex fails to stimulate splicing activity. The x-ray co-crystal structure of LeuRS showed that a C-terminal extension of about 60 amino acids forms a discrete domain, which is unique among the LeuRSs and interacts with the corner of the L-shaped tRNALeu. Interestingly, deletion of the entire yeast mitochondrial LeuRS C-terminal domain enhanced its aminoacylation and amino acid editing activities. In striking contrast, deletion of the corresponding C-terminal domain of Escherichia coli LeuRS abolished aminoacylation of tRNALeu and also amino acid editing of mischarged tRNA molecules. These results suggest that the role of the leucine-specific C-terminal domain in tRNA recognition for aminoacylation and amino acid editing has adapted differentially and with surprisingly opposite effects. We propose that the secondary role of yeast mitochondrial LeuRS in RNA splicing has impacted the functional evolution of this critical C-terminal domain.     

CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function

The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed. This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase. In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain). This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction. However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products. Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot. These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function.     

Regulation of ex-translational activities is the primary function of the multi-tRNA synthetase complex

During mRNA translation, tRNAs are charged by aminoacyl-tRNA synthetases and subsequently used by ribosomes. A multi-enzyme aminoacyl-tRNA synthetase complex (MSC) has been proposed to increase protein synthesis efficiency by passing charged tRNAs to ribosomes. An alternative function is that the MSC repurposes specific synthetases that are released from the MSC upon cues for functions independent of translation. To explore this, we generated mammalian cells in which arginyl-tRNA synthetase and/or glutaminyl-tRNA synthetase were absent from the MSC. Protein synthesis, under a variety of stress conditions, was unchanged. Most strikingly, levels of charged tRNA^Arg and tRNA^Gln remained unchanged and no ribosome pausing was observed at codons for arginine and glutamine. Thus, increasing or regulating protein synthesis efficiency is not dependent on arginyl-tRNA synthetase and glutaminyl-tRNA synthetase in the MSC. Alternatively, and consistent with previously reported ex-translational roles requiring changes in synthetase cellular localizations, our manipulations of the MSC visibly changed localization.     

Structural basis for the dynamics of human methionyl-tRNA synthetase in multi-tRNA synthetase complexes

In mammals, eight aminoacyl-tRNA synthetases (AARSs) and three AARS-interacting multifunctional proteins (AIMPs) form a multi-tRNA synthetase complex (MSC). MSC components possess extension peptides for MSC assembly and specific functions. Human cytosolic methionyl-tRNA synthetase (MRS) has appended peptides at both termini of the catalytic main body. The N-terminal extension includes a glutathione transferase (GST) domain responsible for interacting with AIMP3, and a long linker peptide between the GST and catalytic domains. Herein, we determined crystal structures of the human MRS catalytic main body, and the complex of the GST domain and AIMP3. The structures reveal human-specific structural details of the MRS, and provide a dynamic model for MRS at the level of domain orientation. A movement of zinc knuckles inserted in the catalytic domain is required for MRS catalytic activity. Depending on the position of the GST domain relative to the catalytic main body, MRS can either block or present its tRNA binding site. Since MRS is part of a huge MSC, we propose a dynamic switching between two possible MRS conformations; a closed conformation in which the catalytic domain is compactly attached to the MSC, and an open conformation with a free catalytic domain dissociated from other MSC components.     

The 2 A crystal structure of leucyl-tRNA synthetase and its complex with a leucyl-adenylate analogue

Leucyl-, isoleucyl- and valyl-tRNA synthetases are closely related large monomeric class I synthetases. Each contains a homologous insertion domain of approximately 200 residues, which is thought to permit them to hydrolyse ('edit') cognate tRNA that has been mischarged with a chemically similar but non-cognate amino acid. We describe the first crystal structure of a leucyl-tRNA synthetase, from the hyperthermophile Thermus thermophilus, at 2.0 A resolution. The overall architecture is similar to that of isoleucyl-tRNA synthetase, except that the putative editing domain is inserted at a different position in the primary structure. This feature is unique to prokaryote-like leucyl-tRNA synthetases, as is the presence of a novel additional flexibly inserted domain. Comparison of native enzyme and complexes with leucine and a leucyl- adenylate analogue shows that binding of the adenosine moiety of leucyl-adenylate causes significant conformational changes in the active site required for amino acid activation and tight binding of the adenylate. These changes are propagated to more distant regions of the enzyme, leading to a significantly more ordered structure ready for the subsequent aminoacylation and/or editing steps.     

Aminoacyl-tRNA synthetases of the multi-tRNA synthetase complex and their role in tumorigenesis

In mammalian cells, 20 aminoacyl-tRNA synthetases (AARS) catalyze the ligation of amino acids to their cognate tRNAs to generate aminoacylated-tRNAs. In higher eukaryotes, 9 of the 20 AARSs, along with 3 auxiliary proteins, join to form the cytoplasmic multi-tRNA synthetase complex (MSC). The complex is absent in prokaryotes, but evolutionary expansion of MSC constituents, primarily by addition of novel interacting domains, facilitates formation of subcomplexes that join to establish the holo-MSC. In some cases, environmental cues direct the release of constituents from the MSC which enables the execution of non-canonical, i.e., “moonlighting”, functions distinct from their essential activities in protein translation. These activities are generally beneficial, but can also be deleterious to the cell. Elucidation of the non-canonical activities of several AARSs residing in the MSC suggest they are potential therapeutic targets for cancer, as well as metabolic and neurologic diseases. Here, we describe the role of MSC-resident AARSs in cancer progression, and the factors that regulate their release from the MSC. Also, we highlight recent developments in therapeutic modalities that target MSC AARSs for cancer prevention and treatment.     

The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells

A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system. The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria. The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E. coli tRNA(Leu) than that previously expressed in E. coli without the N-terminal 21 residues.     

Hierarchical network between the components of the multi-tRNA synthetase complex: implications for complex formation

The macromolecular tRNA synthetase complex consists of nine different enzymes and three non-enzymatic factors. This complex was recently shown to be a novel signalosome, since many of its components are involved in signaling pathways in addition to their catalytic roles in protein synthesis. The structural organization and dynamic relationships of the components of the complex are not well understood. Here we performed a systematic depletion analysis to determine the effects of structural intimacy and the turnover of the components. The results showed that the stability of some components depended on their neighbors. Lysyl-tRNA synthetase was most independent of other components for its stability whereas it was most required for the stability of other components. Arginyl- and methionyl-tRNA synthetases had the opposite characteristics. Thus, the systematic depletion of the components revealed the functional reason for the complex formation and the assembly pattern of these multi-functional enzymes and their associated factors.     

The CP2 domain of leucyl-tRNA synthetase is crucial for amino acid activation and post-transfer editing

Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.     

Expression and characterization of the human mitochondrial leucyl-tRNA synthetase

A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.     

The NH2-terminal extension of rat liver arginyl-tRNA synthetase is responsible for its hydrophobic properties

Rat liver arginyl-tRNA synthetase is found in extracts either as a component (Mr = 72,000) of the multienzyme aminoacyl-tRNA synthetase complex or as a low molecular weight (Mr = 60,000) free protein. The two forms are thought to be identical except for an extra peptide extension at the NH2-terminus of the larger form which is required for its association with the complex, but is unessential for catalytic activity. It has been suggested that interactions among synthetases in the multienzyme complex are mediated by hydrophobic domains on these peptide extensions of the individual proteins. To test this model we have purified to homogeneity the larger form of arginyl-tRNA synthetase and compared its hydrophobicity to that of its low molecular weight counterpart. We show that whereas the smaller protein displays no hydrophobic character, the larger protein demonstrates a high degree of hydrophobicity. No lipid modification was found on the high molecular weight protein indicating that the amino acid sequence itself is responsible for its hydrophobic properties. These findings support the proposed model for synthetase association within the multienzyme complex.     

Structural modelling of the complex of leucyl-tRNA synthetase and mis-aminoacylated tRNA

To assure fidelity of translation, class Ia aminoacyl-tRNA synthetases (aaRSs) edit mis-aminoacylated tRNAs. Mis-attached amino acids and structural water molecules are not included simultaneously in the current crystal structures of the aaRS•tRNA complexes, where the 3′-ends (adenine 76; A76) are bound to the editing sites. A structural model of the completely solvated leucyl-tRNA synthetase complexed with valyl-tRNA^Leu was constructed by exploiting molecular dynamics simulations modified for the present modelling. The results showed that the ribose conformation of A76 is distinct from those observed in the above-mentioned crystal structures, which could be derived from structural constraints in a sandwiched manner induced by the mis-attached valine and tRNA^Leu.     

Vigilin is co-localized with 80S ribosomes and binds to the ribosomal complex through its C-terminal domain

The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.     

Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus

Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.     

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.     

Evidence that arginyl-adenylate is not an intermediate in the arginyl-tRNA synthetase reaction

Arginyl-tRNA synthetase has a reaction mechanism not typical of most aminoacyl-tRNA synthetases. It does not catalyze an amino acid-dependent ATP-PP₁ exchange in the absence of tRNA as do most enzymes of this class. In order to clarify the reaction mechanism by performing experiments with substrate levels of enzyme, we have modified the previous purification procedure. By the method presented, homogeneous enzyme can be prepared in approximately 10% yield. Pulse-labeling experiments indicate that no enzyme-bound arginyl-adenylate is formed in the absence of tRNA. Equilibrium experiments show that no arginyl-adenylate accumulates either in the presence or absence of tRNA^arg. Two mechanisms compatible with these data are suggested.