Aspartate 458 of human glutathione synthetase is important for cooperativity and active site structure

Human glutathione synthetase (hGS) catalyzes the second ATP-dependent step in the biosynthesis of glutathione (GSH) and is negatively cooperative to the γ-glutamyl substrate. The hGS active site is composed of three highly conserved catalytic loops, notably the alanine rich A-loop. Experimental and computational investigations of the impact of mutation of Asp458 are reported, and thus the role of this A-loop residue on hGS structure, activity, negativity cooperativity and stability is defined. Several Asp458 hGS mutants (D458A, D458N and D458R) were constructed using site-directed mutagenesis and their activities determined (10%, 15% and 7% of wild-type hGS, respectively). The Michaelis–Menten constant (K_m) was determined for all three substrates (glycine, GAB and ATP): glycine K_m increased by 30–115-fold, GAB K_m decreased by 8–17-fold, and the ATP K_m was unchanged. All Asp458 mutants display a change in cooperativity from negative cooperativity to non-cooperative. All mutants show similar stability as compared to wild-type hGS, as determined by differential scanning calorimetry. The findings indicate that Asp458 is essential for hGS catalysis and that it impacts the allostery of hGS.     

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.     

Dissociating quaternary structure regulates cell-signaling functions of a secreted human tRNA synthetase

Many tRNA synthetases are homodimers that are catalytically inactive as monomers. An example is the 528-amino acid human tyrosyl-tRNA synthetase, which is made up of an N-terminal catalytic unit (TyrRS(Mini)) and a 164-amino acid C-domain. Although native TyrRS has no known cytokine functions, natural proteolysis of secreted TyrRS releases TyrRS(Mini), which not only has the same aminoacylation activity as native TyrRS but also has strong activity for stimulating migration of polymorphonuclear leukocytes. The migration-stimulating activity is dependent on an ELR tripeptide motif, similar to that in CXC cytokines like IL-8, and also has the familiar bell-shaped concentration dependence seen for CXC cytokines. Here we show that in contrast to IL-8, where the bell-shaped dependence arises from the effects of CXCR1/2 receptor internalization, TyrRS(Mini) does not induce internalization of CXCR1/2. A rationally designed non-associating monomer and a non-dissociating dimer were constructed. With these constructs, the bell-shaped concentration dependence of leukocyte migration was shown to arise from the agonist (for migration) activity of the catalytically inactive monomer and the antagonist activity of the catalytically active dimer. Thus, the dissociating quaternary structure of TyrRS(Mini) regulates two opposing cytokine activities and suggests the possibility of dissociating quaternary structures regulating novel functions of other tRNA synthetases.     

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.     

The crystal structure of phosphinothricin in the active site of glutamine synthetase illuminates the mechanism of enzymatic inhibition

Phosphinothricin is a potent inhibitor of the enzyme glutamine synthetase (GS). The resolution of the native structure of GS from Salmonella typhimurium has been extended to 2.5 A resolution, and the improved model is used to determine the structure of phosphinothricin complexed to GS by difference Fourier methods. The structure suggests a noncovalent, dead-end mechanism of inhibition. Phosphinothricin occupies the glutamate substrate pocket and stabilizes the Glu327 flap in a position which blocks the glutamate entrance to the active site, trapping the inhibitor on the enzyme. One oxygen of the phosphinyl group of phosphinothricin appears to be protonated, because of its proximity to the carboxylate group of Glu327. The other phosphinyl oxygen protrudes into the negatively charged binding pocket for the substrate ammonium, disrupting that pocket. The distribution of charges in the glutamate binding pocket is complementary to those of phosphinothricin. The presence of a second ammonium binding site within the active site is confirmed by its analogue thallous ion, marking the ammonium site and its protein ligands. The inhibition of GS by methionine sulfoximine can be explained by the same mechanism. These models of inhibited GS further illuminate its catalytic mechanism.     

Tumor endothelial cell tube formation model for determining anti-angiogenic activity of a tRNA synthetase cytokine

In addition to their key role in protein biosynthesis, aminoacyl-tRNA synthetases have other biological functions that appeared during their long evolutionary development. In mammalian cells, specific members of this family of enzymes are also procytokines that, upon conversion, are active cytokines in pathways for angiogenesis, and thereby connect translation to control of blood vessel development. Here we describe an in vitro assay for tube formation by tumor endothelial cells on a matrigel substrate. In contrast to normal endothelial cells, tumor endothelial cells have strong angiogenic capabilities and the ability to form vessel-like tubes on a solid substrate. In particular, we found that a SV40-immortalized mouse lymphoid endothelial cell line was robust in this assay and yielded data that could be quantified with high precision. Consequently, this specific tube formation model provides an opportunity to discover and analyze potent agents that specifically affect angiogenesis. It has proven effective for studying the angiogenic functions of tRNA synthetase cytokines.     

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.     

tRNA-controlled nuclear import of a human tRNA synthetase

Aminoacyl-tRNA synthetases, essential components of the cytoplasmic translation apparatus, also have nuclear functions that continue to be elucidated. However, little is known about how the distribution between cytoplasmic and nuclear compartments is controlled. Using a combination of methods, here we showed that human tyrosyl-tRNA synthetase (TyrRS) distributes to the nucleus and that the nuclear import of human TyrRS is regulated by its cognate tRNA(Tyr). We identified a hexapeptide motif in the anticodon recognition domain that is critical for nuclear import of the synthetase. Remarkably, this nuclear localization signal (NLS) sequence motif is also important for interacting with tRNA(Tyr). As a consequence, mutational alteration of the hexapeptide simultaneously attenuated aminoacylation and nuclear localization. Because the NLS is sterically blocked when the cognate tRNA is bound to TyrRS, we hypothesized that the nuclear distribution of TyrRS is regulated by tRNA(Tyr). This expectation was confirmed by RNAi knockdown of tRNA(Tyr) expression, which led to robust nuclear import of TyrRS. Further bioinformatics analysis showed that to have nuclear import of TyrRS directly controlled by tRNA(Tyr) in higher organisms, the NLS of lower eukaryotes was abandoned, whereas the new NLS was evolved from an anticodon-binding hexapeptide motif. Thus, higher organisms developed a strategy to make tRNA a regulator of the nuclear trafficking of its cognate synthetase. The design in principle should coordinate nuclear import of a tRNA synthetase with the demands of protein synthesis in the cytoplasm.     

tRNA recognition site of Escherichia coli methionyl-tRNA synthetase

We have previously shown that anticodon bases are essential for specific recognition of tRNA substrates by Escherichia coli methionyl-tRNA synthetase (MetRS) [Schulman, L. H., & Pelka, H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6755-6759] and that the enzyme tightly binds to C34 at the wobble position of E. coli initiator methionine tRNA (tRNAfMet) [Pelka, H., & Schulman, L. H. (1986) Biochemistry 25, 4450-4456]. We have also previously demonstrated that an affinity labeling derivative of tRNAfMet can be quantitatively cross-linked to the tRNA binding site of MetRS [Valenzuela, D., & Schulman, L. H. (1986) Biochemistry 25, 4555-4561]. Here, we have determined the site in MetRS which is cross-linked to the anticodon of tRNAfMet, as well as the location of four additional cross-links. Only a single peptide, containing Lys465, is covalently coupled to C34, indicating that the recognition site for the anticodon is close to this sequence in the three-dimensional structure of MetRS. The D loop at one corner of the tRNA molecule is cross-linked to three peptides, containing Lys402, Lys439, and Lys596. The 5' terminus of the tRNA is cross-linked to Lys640, near the carboxy terminus of the enzyme. Since the 3' end of tRNAfMet is positioned close to the active site in the N-terminal domain [Hountondji, C., Blanquet, S., & Lederer, F. (1985) Biochemistry 24, 1175-1180], this result indicates that the carboxy ends of the two polypeptide chains of native dimeric MetRS are folded back toward the N-terminal domain of each subunit.     

Deep knot structure for construction of active site and cofactor binding site of tRNA modification enzyme

The tRNA(Gm18) methyltransferase (TrmH) catalyzes the 2'-O methylation of guanosine 18 (Gua18) of tRNA. We solved the crystal structure of Thermus thermophilus TrmH complexed with S-adenosyl-L-methionine at 1.85 A resolution. The catalytic domain contains a deep trefoil knot, which mutational analyses revealed to be crucial for the formation of the catalytic site and the cofactor binding pocket. The tRNA dihydrouridine(D)-arm can be docked onto the dimeric TrmH, so that the tRNA D-stem is clamped by the N- and C-terminal helices from one subunit while the Gua18 is modified by the other subunit. Arg41 from the other subunit enters the catalytic site and forms a hydrogen bond with a bound sulfate ion, an RNA main chain phosphate analog, thus activating its nucleophilic state. Based on Gua18 modeling onto the active site, we propose that once Gua18 binds, the phosphate group activates Arg41, which then deprotonates the 2'-OH group for methylation.     

Definition of the heterocyclic pharmacophore of bacterial methionyl tRNA synthetase inhibitors: potent antibacterially active non-quinolone analogues

Potent inhibitors of bacterial methionyl tRNA synthetase (MRS) have previously been reported. Through SAR of the quinolone moiety, the right hand side pharmacophore for MRS inhibition has now been defined as an NH-C-NH functionality in the context of a bicyclic heteroaromatic system. Potent antibacterial fused-pyrimidone and fused-imidazole analogues have been obtained and enantioselective activity demonstrated. Compound 46 demonstrated very good antibacterial activity against panels of antibiotic-resistant staphylococci and enterococci.     

Functional analysis of hairpin ribozyme active site architecture

The hairpin ribozyme is a small catalytic motif found in plant satellite RNAs where it catalyzes a reversible self-cleavage reaction during processing of replication intermediates. Crystallographic studies of hairpin ribozymes have provided high resolution views of the RNA functional groups that comprise the active site and stimulated biochemical studies that probed the contributions of nucleobase functional groups to catalytic chemistry. The dramatic loss of activity that results from perturbation of active site architecture points to the importance of positioning and orientation in catalytic rate acceleration. The current study focuses on the network of noncovalent interactions that align nucleophilic and leaving group oxygens in the orientation required for the S(N)2-type reaction mechanism and orient the active site nucleobases near the reactive phosphate to facilitate catalytic chemistry. Nucleotide modifications that alter or eliminate individual hydrogen bonding partners had different effects on the activation barrier to catalysis, the stability of ribozyme complexes in the ground state, and the internal equilibrium between cleavage and ligation of bound products. Furthermore, substitution of hydrogen bond donors and acceptors with seemingly equivalent pairs sometimes had very different functional consequences. These biochemical analyses augment high resolution structural information to provide insights into the functional significance of active site architecture.     

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.     

Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site

PRPP (phosphoribosylpyrophosphate) is an important metabolite essential for nucleotide synthesis and PRS (PRPP synthetase) catalyses synthesis of PRPP from R5P (ribose 5-phosphate) and ATP. The enzymatic activity of PRS is regulated by phosphate ions, divalent metal cations and ADP. In the present study we report the crystal structures of recombinant human PRS1 in complexes with SO4(2-) ions alone and with ATP, Cd2+ and SO4(2-) ions respectively. The AMP moiety of ATP binds at the ATP-binding site, and a Cd2+ ion binds at the active site and in a position to interact with the beta- and gamma-phosphates of ATP. A SO4(2-) ion, an analogue of the activator phosphate, was found to bind at both the R5P-binding site and the allosteric site defined previously. In addi-tion, an extra SO4(2-) binds at a site at the dimer interface between the ATP-binding site and the allosteric site. Binding of this SO4(2-) stabilizes the conformation of the flexible loop at the active site, leading to the formation of the active, open conformation which is essential for binding of ATP and initiation of the catalytic reaction. This is the first time that structural stabilization at the active site caused by binding of an activator has been observed. Structural and biochemical data show that mutations of some residues at this site influence the binding of SO4(2-) and affect the enzymatic activity. The results in the present paper suggest that this new SO4(2-)-binding site is a second allosteric site to regulate the enzymatic activity which might also exist in other eukaryotic PRSs (except plant PRSs of class II), but not in bacterial PRSs.     

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.     

Influence of the conserved active site residues of histidyl tRNA synthetase on the mechanism of aminoacylation reaction

The relation between the conservation of active site residues and the molecular mechanism of aminoacylation reaction is an unexplored problem. In the present paper, the influences of the conserved active site residues on the reaction mechanism as well as the electrostatic potential near the reaction center are analyzed for Histidyl tRNA synthetase from Escherichia coli, Thermus thermophilus and Staphylococcus aureus. While the primary structures show both convergence as well as divergence, the secondary level structures of the active sites of the three species show considerable conservation in the respective structural organizations. The conserved active site residues near the reaction center, which have a major role in the reaction mechanism and catalysis, retain their specific position and orientation relative to the substrate in the three species. In order to understand the influence of different conserved and nonconserved residues near the reaction center, two different models are considered. First, a large model of active site with the substrates, Mg²⁺ ions and water is constructed in which the first shell residues (including both conserved as well as nonconserved) near the reaction center are studied. From the large model, a smaller model is constructed for reaction path modeling individually for three species. Validation of the smaller model is carried out by comparing the energy surfaces of large and small models as a function of reaction coordinates. Further, the electrostatic potential near the reaction center for the large and small model are compared. The transition state structures of the activation step of aminoacylation reaction for E. coli, T. thermophilus and S. aureus are calculated using the combined ab-initio/semi-empirical calculation. The similarity of the energy profiles as a function of the relevant reaction coordinate and the orientation of the catalytic residue, Arg259, indicate that the reaction mechanisms are identical which are guided by the strikingly similar structural pattern formed by conserved residues for three species. The energy surfaces have close resemblance in three species and present a clear perspective that how the reaction proceeds with the aid of different conserved residues. The study of electrostatic potential confirms this view. The present study provides an understanding of the relationship between the conservation of residues and the efficient reaction mechanism of aminoacylation reaction.     

Coordination of tRNA synthetase active sites for chemical fidelity

Statistical proteomes that are naturally occurring can result from mechanisms involving aminoacyl-tRNA synthetases (aaRSs) with inactivated hydrolytic editing active sites. In one case, Mycoplasma mobile leucyl-tRNA synthetase (LeuRS) is uniquely missing its entire amino acid editing domain, called CP1, which is otherwise present in all known LeuRSs and also isoleucyl- and valyl-tRNA synthetases. This hydrolytic CP1 domain was fused to a synthetic core composed of a Rossmann ATP-binding fold. The fusion event splits the primary structure of the Rossmann fold into two halves. Hybrid LeuRS chimeras using M. mobile LeuRS as a scaffold were constructed to investigate the evolutionary protein:protein fusion of the CP1 editing domain to the Rossmann fold domain that is ubiquitously found in kinases and dehydrogenases, in addition to class I aaRSs. Significantly, these results determined that the modular construction of aaRSs and their adaptation to accommodate more stringent amino acid specificities included CP1-dependent distal effects on amino acid discrimination in the synthetic core. As increasingly sophisticated protein synthesis machinery evolved, the addition of the CP1 domain increased specificity in the synthetic site, as well as provided a hydrolytic editing site.