Yeast tRNA(Asp)-aspartyl-tRNA synthetase complex: low resolution crystal structure

Yeast aspartyl-tRNA synthetase, a dimer of molecular weight 125,000, and two molecules of its cognate tRNA (Mr = 24160) cocrystallize in the cubic space group I432 (a = 354 A). The crystal structure was solved to low resolution using neutron and X-ray diffraction data. Neutron single crystal diffraction data were collected in five solvents differing by their D2O content in order to use the contrast variation method to distinguish between the protein and tRNA. The synthetase was first located at 40 A resolution using the 65% D2O neutron data (tRNA matched) tRNA molecules were found at 20 A resolution using both neutron and X-ray data. The resulting model was refined against 10 A resolution X-ray data, using density modification and least-squares refinement of the tRNA positions. The crystal structure solved without a priori phase knowledge, was confirmed later by isomorphous replacement. The molecular model of the complex is in good agreement with results obtained in solution by probing the protected part of the tRNA by chemical reagents.     

Purification and properties of tyrosyl tRNA synthetase of rat liver.

Purification of rat liver tyrosine tRNA synthetase yields two protein fractions A and B and both fractions are required for charging of tyrosine to tRNAtyr. Fraction B catalyzes the activation of tyrosine. Fractions A and B have been purified to near homogeneity and they are composed of single polypeptide chains of 62,000 daltons each. Gel filtration studies suggest a molecular weight of 120,000 for the synthetase.     

Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

Biosynthesis of gramicidin S with the aid of dipeptides by gramicidin S synthetase.

Dipeptides L-phenylalanyl-proline, D-phenylalanyl-proline, prolyl-valine, valyl-lysine, lysyl-leucine and leucyl-phenylalanine, derived from the sequence of gramicidin S, are substrates of the gramicidin S synthetase. When any of these dipeptides are used to replace the two corresponding amino acids in the reaction assay, cyclodecapeptide antibiotic synthesis occurs, and requires the whole multienzyme system. Active esters, like the thiophenyl and p-nitrophenyl esters of D-phenylalanyl-proline are unable to promote gramicidin S biosynthesis with the gramicidin S synthetase system or with the heavy enzyme alone.     

Synthetic analogues of SB-219383. Novel C-glycosyl peptides as inhibitors of tyrosyl tRNA synthetase

Novel inhibitors of bacterial tyrosyl tRNA synthetase have been synthesised in which the cyclic hydroxylamine moiety of SB-219383 is replaced by C-pyranosyl derivatives. Potent and selective inhibition of bacterial tyrosyl tRNA synthetase was obtained.     

Functional and crystal structure analysis of active site adaptations of a potent anti-angiogenic human tRNA synthetase

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.     

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.     

Crystal structure of Staphylococcus aureus tRNA adenosine deaminase TadA in complex with RNA

Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.     

Trypsin inhibition by a peptide hormone: crystal structure of trypsin-vasopressin complex

The large variety of serine protease inhibitors, available from various sources such as tissues, microorganisms, plants, etc., play an important role in regulating the proteolytic enzymes. The analysis of protease-inhibitor complexes helps in understanding the mechanism of action, as well as in designing inhibitors. Vasopressin, an anti-diuretic nonapeptide hormone, is found to be an effective inhibitor of trypsin, with a K(i) value of 5 nM. The crystal structure of the trypsin-vasopressin complex revealed that vasopressin fulfils all the important interactions for an inhibitor, without any break in the scissile peptide bond. The cyclic nature due to a disulfide bridge between Cys1 and Cys6 of vasopressin provides structural rigidity to the peptide hormone. The trypsin-binding site is located at the C terminus, while the neurophysin-binding site is at the N terminus of vasopressin. This study will assist in designing new peptide inhibitors. This study suggests that vasopressin inhibition of trypsin may have unexplored biological implications.     

Inhibitors of bacterial tyrosyl tRNA synthetase: synthesis of carbocyclic analogues of the natural product SB-219383.

Carbocyclic analogues of the microbial metabolite SB-219383 have been synthesised and evaluated as inhibitors of bacterial tyrosyl tRNA synthetase. One compound showed highly potent and selective nanomolar inhibition.     

Crystallization of a tRNA . aminoacyl-tRNA synthetase complex. Characterization and first crystallographic data

A complex formed between the dimeric aspartyl-tRNA synthetase from yeast (Mr congruent to 125,000) and two molecules of its cognate yeast tRNAAsp (Mr = 24,160) was crystallized using ammonium sulfate as the precipitant. The crucial parameter which governs a successful crystallization is the enzyme tRNA stoichiometry. Crystals are only obtained when the starting solution precisely contains two tRNA molecules for one enzyme molecule. It was demonstrated by electrophoresis, biological activity assays, and crystallographic data that the crystals contain the two components in the same two to one stoichiometric ratio. The crystals, of cubic shape with edges up to 0.8 mm, belong to space group 1432. The cell parameter is 354 A and the asymmetric unit contains one particle of complex. The solvent content is about 78%, higher than the values commonly observed. Although particularly soft, the quality of the crystals is suitable for x-ray diffraction studies up to 7-A resolution.     

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.     

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.     

Crystal structure of Staphylococcus aureus tyrosyl-tRNA synthetase in complex with a class of potent and specific inhibitors

SB-219383 and its analogues are a class of potent and specific inhibitors of bacterial tyrosyl-tRNA synthetases. Crystal structures of these inhibitors have been solved in complex with the tyrosyl-tRNA synthetase from Staphylococcus aureus, the bacterium that is largely responsible for hospital-acquired infections. The full-length enzyme yielded crystals that diffracted to 2.8 A resolution, but a truncated version of the enzyme allowed the resolution to be extended to 2.2 A. These inhibitors not only occupy the known substrate binding sites in unique ways, but also reveal a butyl binding pocket. It was reported that the Bacillus stearothermophilus TyrRS T51P mutant has much increased catalytic activity. The S. aureus enzyme happens to have a proline at position 51. Therefore, our structures may contribute to the understanding of the catalytic mechanism and provide the structural basis for designing novel antimicrobial agents.     

Catalytic activity of aminoacyl tRNA synthetases and its implications for the origin of life. I. Aminoacyl adenylate formation in tyrosyl tRNA synthetase

The changes in the catalytic activity resulting from amino acid substitutions in the active site region have been theoretically modeled for tyrosyl tRNA synthetase (Tyr-RS). The catalytic activity was calculated as the differential stabilization of the transition state using electrostatic approximation. The results indicate that charged residues His45, His48, Asp78, Asp176, Asp194, Lys225, Lys230, Lys233, Arg265, and Lys268 play essential roles in catalysis of aminoacyl adenylate formation in Tyr-RS, which is in general agreement with previously known experimental data for residues 45, 48, 194, 230, and 233. These catalytic residues have also been used to search for sequence homology patterns among class I aminoacyl RSs of which HIGH and KMSKS conserved sequence motifs are well known. His45 and His48 belong to the HIGH signature sequence of class I aminoacyl tRNA synthetases (aRSs), whereas Arg265 and Lys268 can constitute a part of the KMSKS charge pattern. Lys225, Lys230, and Lys233 may be part of the conservative substitution pattern [HKR]-X(4)-[HKR]-X(2)-[HKR], and Asp194 is part of the new GSDQ motif. This demonstrates that the three dimensional charge distribution near the active site is an essential feature of the catalytic activity of aRS and that the theoretical technique used in this work can be utilized in searches for the catalytically important residues that may provide a clue for a charge residue pattern conserved in evolution. The appearance of patterns I-IV in Arg-, Gln-, Met-, Ile-, Leu-, Trp-, Val-, Glu-, Cys-, and Tyr-RS indicates that all these enzymes could have the same ancestor.     

The crystal structure of yeast mitochondrial ThrRS in complex with the canonical threonine tRNA

In mitochondria of Saccharomyces cerevisiae, a single aminoacyl-tRNA synthetase (aaRS), MST1, aminoacylates two isoacceptor tRNAs, tRNA₁^Thr and tRNA₂^Thr, that harbor anticodon loops of different size and sequence. As a result of this promiscuity, reassignment of the CUN codon box from leucine to threonine is facilitated. However, the mechanism by which a single aaRS binds distinct anticodon loops with high specificity is not well understood. Herein, we present the crystal structure of MST1 in complex with the canonical tRNA₂^Thr and non-hydrolyzable analog of threonyl adenylate. Our structure reveals that the dimeric arrangement of MST1 is essential for binding the 5′-phosphate, the second base pair of the acceptor stem, the first two base pairs of the anticodon stem and the first nucleotide of the variable arm. Further, in contrast to the bacterial ortholog that ‘reads’ the entire anticodon sequence, MST1 recognizes bases in the second and third position and the nucleotide upstream of the anticodon sequence. We speculate that a flexible loop linking strands β4 and β5 may be allosteric regulator that establishes cross-subunit communication between the aminoacylation and tRNA-binding sites. We also propose that structural features of the anticodon-binding domain in MST1 permit binding of the enlarged anticodon loop of tRNA₁^Thr.     

A genomic survey shows that the haloarchaeal type tyrosyl tRNA synthetase is not a synapomorphy of opisthokonts

The haloarchaeal-type tyrosyl tRNA synthetase (tyrRS) have previously been proposed to be a molecular synapomorphy of the opisthokonts. To re-evaluate this we have performed a taxon-wide genomic survey of tyrRS in eukaryotes and prokaryotes. Our phylogenetic trees group eukaryotes with archaea, with all opisthokonts sharing the haloarchaeal-type tyrRS. However, this type of tyrRS is not exclusive to opisthokonts, since it also encoded by two amoebozoans. Whether this is a consequence of lateral gene transfer or lineage sorting remains unsolved, but in any case haloarchaeal-type tyrRS is not a synapomorphy of opisthokonts. This demonstrates that molecular markers should be re-evaluated once a better taxon sampling becomes available.     

tRNA sulfurtransferase: a member of the aminoacyl-tRNA synthetase complex in rat liver

Transfer RNA sulfurtransferase, tRNA methyltransferase, and aminoacyl-tRNA synthetase activity are associated in a complex in rat liver, which is excluded from Sephadex G-200 columns. The complex can also be isolated by subjecting cell supernatants to further centrifugation at 160,000 x g for 18 hours. The resulting pellet contains 70% of the total sulfurtransferase activity, and a 3-fold increase in specific activity is accomplished through pelleting. The data suggest that the enzymes of tRNA metabolism are organized in a large complex in rat liver.