Coding triplets in the tRNA acceptor-TΨC arm and their role in present and past tRNA recognition.

The mechanism and evolution of the recognition scheme between key components of the translation system, that is, tRNAs, synthetases, and elongation factors, are fundamental issues in understanding the translation of genetic information into proteins. Statistical analysis of bacterial tRNA sequences reveals that for six amino acids, a string of 10 nucleotides preceding the tRNA 3' end carries cognate coding triplets to nearly full extent. The triplets conserved in positions 63-67 are implicated in the recognition by the elongation factor EF-Tu, and those conserved in positions 68-72, in the identification of cognate tRNAs, and their derived minihelices by class IIa synthetases. These coding triplets are suggested to have primordial origin, being engaged in aminoacylation of prebiotic tRNAs and in the establishment of the canonical codon set.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

3-Dimensional architecture of the human multi-tRNA synthetase complex.

In mammalian cells, eight cytoplasmic aminoacyl-tRNA synthetases (AARS), and three non-synthetase proteins, reside in a large multi-tRNA synthetase complex (MSC). AARSs have critical roles in interpretation of the genetic code during protein synthesis, and in non-canonical functions unrelated to translation. Nonetheless, the structure and function of the MSC remain unclear. Partial or complete crystal structures of all MSC constituents have been reported; however, the structure of the holo-MSC has not been resolved. We have taken advantage of cross-linking mass spectrometry (XL-MS) and molecular docking to interrogate the three-dimensional architecture of the MSC in human HEK293T cells. The XL-MS approach uniquely provides structural information on flexibly appended domains, characteristic of nearly all MSC constituents. Using the MS-cleavable cross-linker, disuccinimidyl sulfoxide, inter-protein cross-links spanning all MSC constituents were observed, including cross-links between eight protein pairs not previously known to interact. Intra-protein cross-links defined new structural relationships between domains in several constituents. Unexpectedly, an asymmetric AARS distribution was observed featuring a clustering of tRNA anti-codon binding domains on one MSC face. Possibly, the non-uniform localization improves efficiency of delivery of charged tRNA's to an interacting ribosome during translation. In summary, we show a highly compact, 3D structural model of the human holo-MSC.

The Aminoacyl-tRNA Synthetase Complex.

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

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.

Small-angle X-ray solution scattering study of the multi-aminoacyl-tRNA synthetase complex reveals an elongated and multi-armed particle.

In animal cells, nine aminoacyl-tRNA synthetases are associated with the three auxiliary proteins p18, p38, and p43 to form a stable and conserved large multi-aminoacyl-tRNA synthetase complex (MARS), whose molecular mass has been proposed to be between 1.0 and 1.5 MDa. The complex acts as a molecular hub for coordinating protein synthesis and diverse regulatory signal pathways. Electron microscopy studies defined its low resolution molecular envelope as an overall rather compact, asymmetric triangular shape. Here, we have analyzed the composition and homogeneity of the native mammalian MARS isolated from rabbit liver and characterized its overall internal structure, size, and shape at low resolution by hydrodynamic methods and small-angle x-ray scattering in solution. Our data reveal that the MARS exhibits a much more elongated and multi-armed shape than expected from previous reports. The hydrodynamic and structural features of the MARS are large compared with other supramolecular assemblies involved in translation, including ribosome. The large dimensions and non-compact structural organization of MARS favor a large protein surface accessibility for all its components. This may be essential to allow structural rearrangements between the catalytic and cis-acting tRNA binding domains of the synthetases required for binding the bulky tRNA substrates. This non-compact architecture may also contribute to the spatiotemporal controlled release of some of its components, which participate in non-canonical functions after dissociation from the complex.

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain.

Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA(Ile)(2) and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-A resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long alpha-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA(Ile)(2) is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA(Ile)(2) acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29.G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA(Ile)(2) from tRNA(Met) by strategies similar to that used by IleRS, but in distinct manners.

Isolation and characterization of human nuclear and cytosolic multisynthetase complexes and the intracellular distribution of p43/EMAPII.

In this study, the human multienzyme aminoacyl-tRNA synthetase "core" complex has been isolated from the nuclear and cytosolic compartments of human cells and purified to near homogeneity. It is clear from the polypeptide compositions, stoichiometries, and three-dimensional structures that the cytosolic and nuclear particles are very similar to each other and to the particle obtained from rabbit reticulocytes. The most significant difference observed via aminoacylation activity assays and densitometric analysis of electrophoretic band patterns is a lower amount of glutaminyl-tRNA synthetase in the human particles. However, this is not enough to cause major changes in the three-dimensional structures calculated from samples negatively stained with either uranyl acetate or methylamine vanadate. Indeed, the latter samples produce volumes that are highly similar to an initial structure previously calculated from a frozen hydrated sample of the rabbit multisynthetase complex. New structures in this study reveal that the three major structural domains have discrete subsections. This information is an important step toward determination of specific protein interactions and arrangements within the multisynthetase core complex and understanding of the particle's cellular function(s). Finally, gel filtration and immunoblot analysis demonstrate that a major biological role for the cytokine precursor p43 is as an integral part of the multisynthetase complex.

Three-dimensional architecture of the eukaryotic multisynthetase complex determined from negatively stained and cryoelectron micrographs.

This study provides the first description of the three-dimensional architecture of the multienzyme complex of aminoacyl-tRNA synthetases. Reconstructions were calculated from electron microscopic images of negatively stained and frozen hydrated samples using three independent angular assignment methods. In all cases, volumes show an asymmetric triangular arrangement of protein domains around a deep central cavity. The structures have openings or indentations on most sides. Maximum dimensions are ca. 19x16x10 nm. The central cavity is 4 nm in diameter and extends two-thirds of the length of the particle.

Ultrastructure of the eukaryotic aminoacyl-tRNA synthetase complex derived from two dimensional averaging and classification of negatively stained electron microscopic images.

Several aminoacyl-tRNA synthetases in higher eukaryotes are consistently isolated as a multi-enzyme complex for which little structural information is yet known. This study uses computational methods for analysis of electron microscopic images of the particle. A data set of almost 2000 negatively stained images was processed through reference-free alignment and multivariate statistical analysis. Interpretable structural information was evident in five eigenvectors. Hierarchical ascendant classification extracted clusters corresponding to distinct image orientations. The class averages are consistent with rotations around and orthogonal to a central particle axis and provide particle measurements: approximately 25 nm in height, 30 nm at the widest point and 23 nm thick. The results also provide objective evidence in support of the working structural model and demonstrate the feasibility of obtaining the three dimensional structure of the multisynthetase complex by single particle reconstruction methods.

Crystal structure of asparagine synthetase reveals a close evolutionary relationship to class II aminoacyl-tRNA synthetase.

The crystal structure of E. coli asparagine synthetase has been determined by X-ray diffraction analysis at 2.5 A resolution. The overall structure of the enzyme is remarkably similar to that of the catalytic domain of yeast aspartyl-tRNA synthetase despite low sequence similarity. These enzymes have a common reaction mechanism that implies the formation of an aminoacyl-adenylate intermediate. The active site architecture and most of the catalytic residues are also conserved in both enzymes. These proteins have probably evolved from a common ancestor even though their sequence similarities are small. The functional and structural similarities of both enzymes suggest that new enzymatic activities would generally follow the recruitment of a protein catalyzing a similar chemical reaction.

Structural analysis of the high molecular mass aminoacyl-tRNA synthetase complex. Effects of neutral salts and detergents.

The effects of a variety of detergents and neutral salts on the structure of the eukaryotic high molecular mass aminoacyl-tRNA synthetase complex have been directly determined by observing alterations in the composition, sedimentation behavior, and electron microscopic appearance of the rabbit reticulocyte complex. The intact complex is shown to exhibit the enzymatic activities, polypeptide composition, relative stoichiometry, and morphological features that are characteristic of this eukaryotic multienzyme particle. The structure of the high molecular mass aminoacyl-tRNA synthetase complex is seen to be resistant to both ionic and nonionic detergents. However, both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and deoxycholate induce formation of large protein aggregates. In contrast, the chaotropic salts LiCl and NaSCN both selectively remove individual polypeptides from the high molecular mass aminoacyl-tRNA synthetase complex and promote formation of specific particulate subcomplexes which have distinct sizes, polypeptide compositions, and structural features. These data support the view that many of the protein interactions within the high molecular mass amino-acyl-tRNA synthetase complex are hydrophobic in nature. This study also provides direct evidence that the complex contains a core of tightly interacting synthetases onto which the remaining polypeptides are arrayed. The structural alterations observed here may account for the ability of these reagents to markedly inhibit several enzymatic activities within the complex.

The accuracy of aminoacylation--ensuring the fidelity of the genetic code.

The fidelity of protein biosynthesis rests not only on the proper interaction of the messenger RNA codon with the anticodon of the tRNA, but also on the correct attachment of amino acids to their corresponding (cognate) transfer RNA (tRNA) species. This process is catalyzed by the aminoacyl-tRNA synthetases which discriminate with remarkable selectivity amongst many structurally similar tRNAs. The basis for this highly specific recognition of tRNA by these enzymes (also referred to as 'tRNA identity') is currently being elucidated by genetic, biochemical and biophysical techniques. At least two factors are important in determining the accuracy of aminoacylation: a) 'identity elements' in tRNA denote nucleotides in certain positions crucial for protein interactions determining specificity, and b) the occurrence in vivo of competition between synthetases for a particular tRNA which may have ambiguous identity.