A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase.

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNAPro. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNAPro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

Identification of Mycobacterium tuberculosis leucyl-tRNA synthetase (LeuRS) inhibitors among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one.

The increase of antibiotic resistance amongst Mycobacterium tuberculosis strains has become one of the most pressing problems of modern medicine. Therefore, the search of antibiotics against M. tuberculosis with novel mechanisms of action is very important. We have identified inhibitors of M. tuberculosis leucyl-tRNA synthetase (LeuRS) among the derivatives of 5-phenylamino-2H-[1,2,4]triazin-3-one. The most active compounds 5-(5-chloro-2-hydroxy-phenylamino)-6-methyl-2H-[1,2,4]triazin-3-one and 5-(5-chloro-2-hydroxy-phenylamino)-2H-[1,2,4]triazin-3-one inhibit M. tuberculosis LeuRS with IC50 of 7.6 µÐœ and 7.2 µÐœ, respectively. It was established that the inhibitory activity of compounds against pathogenic LeuRS is 10-fold better, than for human enzyme.

Discovery of potent anti-tuberculosis agents targeting leucyl-tRNA synthetase.

Tuberculosis is a serious infectious disease caused by human pathogen bacteria Mycobacterium tuberculosis. Bacterial drug resistance is a very significant medical problem nowadays and development of novel antibiotics with different mechanisms of action is an important goal of modern medical science. Leucyl-tRNA synthetase (LeuRS) has been recently clinically validated as antimicrobial target. Here we report the discovery of small-molecule inhibitors of M. tuberculosis LeuRS. Using receptor-based virtual screening we have identified six inhibitors of M. tuberculosis LeuRS from two different chemical classes. The most active compound 4-{[4-(4-Bromo-phenyl)-thiazol-2-yl]hydrazonomethyl}-2-methoxy-6-nitro-phenol (1) inhibits LeuRS with IC50 of 6µM. A series of derivatives has been synthesized and evaluated in vitro toward M. tuberculosis LeuRS. It was revealed that the most active compound 2,6-Dibromo-4-{[4-(4-nitro-phenyl)-thiazol-2-yl]-hydrazonomethyl}-phenol inhibits LeuRS with IC50 of 2.27µM. All active compounds were tested for antimicrobial effect against M. tuberculosis H37Rv. The compound 1 seems to have the best cell permeability and inhibits growth of pathogenic bacteria with IC50=10.01µM and IC90=13.53µM.

Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain.

Prolyl-tRNA synthetases (ProRSs) are unique among synthetases in that they have diverse architectures, notably the variable presence of a cis-editing domain homologous to the freestanding deacylase proteins YbaK and ProX. Here, we describe crystal structures of two bacterial ProRSs from the pathogen Enterococcus faecalis, which possesses an editing domain, and from Rhodopseudomonas palustris, which does not. We compare the overall structure and binding mode of ATP and prolyl-adenylate with those of the archael/eukaryote-type ProRS from Thermus thermophilus. Although structurally more homologous to YbaK, which preferentially hydrolyzes Cys-tRNA(Pro), the editing domain of E. faecalis ProRS possesses key elements similar to ProX, with which it shares the activity of hydrolyzing Ala-tRNA(Pro). The structures give insight into the complex evolution of ProRSs, the mechanism of editing, and structural differences between prokaryotic- and eukaryotic-type ProRSs that can be exploited for antibiotic design.

The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation.

Leucyl-tRNA synthetase (LeuRS) has a specific post-transfer editing activity directed against mischarged isoleucine and similar noncognate amino acids. We describe the post-transfer-editing and product complexes of Thermus thermophilus LeuRS (LeuRSTT) with tRNA(Leu) at 2.9- to 3.3-A resolution. In the post-transfer-editing configuration, A76 binds in the editing active site exactly as previously found for the adenosine moiety of a small-molecule editing-substrate analog. The 60 C-terminal residues of LeuRSTT, unseen in previous structures, fold into a compact domain flexibly linked to the rest of the molecule and interacting with the G19-C56 tertiary base pair of tRNA(Leu). LeuRS recognition of tRNA(Leu) depends essentially on tRNA shape rather than base-specific interactions. The structures show that considerable domain rotations, notably of the editing domain, accompany the tRNA-3' end dynamics associated successively with aminoacylation, post-transfer editing and product release.

Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion.

The archaeal/eukaryotic tyrosyl-tRNA synthetase (TyrRS)-tRNA(Tyr) pairs do not cross-react with their bacterial counterparts. This 'orthogonal' condition is essential for using the archaeal pair to expand the bacterial genetic code. In this study, the structure of the Methanococcus jannaschii TyrRS-tRNA(Tyr)-L-tyrosine complex, solved at a resolution of 1.95 A, reveals that this archaeal TyrRS strictly recognizes the C1-G72 base pair, whereas the bacterial TyrRS recognizes the G1-C72 in a different manner using different residues. These diverse tRNA recognition modes form the basis for the orthogonality. The common tRNA(Tyr) identity determinants (the discriminator, A73 and the anticodon residues) are also recognized in manners different from those of the bacterial TyrRS. Based on this finding, we created a mutant TyrRS that aminoacylates the amber suppressor tRNA with C34 65 times more efficiently than does the wild-type enzyme.

Structural and mechanistic basis of pre- and posttransfer editing by leucyl-tRNA synthetase.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

Class I tyrosyl-tRNA synthetase has a class II mode of cognate tRNA recognition.

Bacterial tyrosyl-tRNA synthetases (TyrRS) possess a flexibly linked C-terminal domain of approximately 80 residues, which has hitherto been disordered in crystal structures of the enzyme. We have determined the structure of Thermus thermophilus TyrRS at 2.0 A resolution in a crystal form in which the C-terminal domain is ordered, and confirm that the fold is similar to part of the C-terminal domain of ribosomal protein S4. We have also determined the structure at 2.9 A resolution of the complex of T.thermophilus TyrRS with cognate tRNA(tyr)(G Psi A). In this structure, the C-terminal domain binds between the characteristic long variable arm of the tRNA and the anti-codon stem, thus recognizing the unique shape of the tRNA. The anticodon bases have a novel conformation with A-36 stacked on G-34, and both G-34 and Psi-35 are base-specifically recognized. The tRNA binds across the two subunits of the dimeric enzyme and, remarkably, the mode of recognition of the class I TyrRS for its cognate tRNA resembles that of a class II synthetase in being from the major groove side of the acceptor stem.