tyrosyl-tRNA Synthetase Mutant Research Articles

Functional genetic encoding of sulfotyrosine in mammalian cells

Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

RhTyrRS (Y341A), a novel human tyrosyl-tRNA synthetase mutant, stimulates thrombopoiesis through activation of the VEGF-R II/NF-κB pathway

Bacground and purposeTumor chemotherapy and radiotherapy induces hematopoietic cell damage, resulting in thrombocytopenia. Conventional platelet transfusion strategies or drug therapies are used to treat thrombocytopenia. However, these therapies may result in a several side effects, including heightened susceptibility to infectious diseases and the formation of anti-TPO-antibodies. Therefore, a more secure strategy should be explored to overcome and compensate for the shortcomings of conventional strategies. Experimental approachEffects of rhTyrRS(Y341A) on the expression of VCAM-1 on the surface of HUVECs were determined by analysing mRNA expression, promoter activity, protein expression. The molecular mechanisms of the effects of rhTyrRS(Y341A) on the expression of VCAM-1 on the surface of HUVECs were investigated by determining the activation of VEGF-R II/NF-κB pathway. Key resultsOur results provide evidence that rhTyrRS (Y341A) activates NF-κB to upregulate VCAM-1 in a VEGF-R II/NF-κB pathway-dependent, resulting in megakaryocyte adhering to PVECs to induce platelet production. ConclusionsThis study suggested that rhTyrRS (Y341A), a novel human tyrosyl-tRNA synthetase mutation, increased the platelet count under normal conditions. Further more, we confirmed that an NF-κB-mediated mechanism is involved in rhTyrRS (Y341A)-induced thrombopoiesis, which involves its interaction with VEGF-R II.

A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli

We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(()(CUA)), and made an orthogonal tRNA((CUA)) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA(()(CUA)) gene. Although the yields of full-length CaM increased ~3-fold, the ratio of N(3)-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N(3)-Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.

A brief perspective of the determination of crystal structures of site-directed mutants of tyrosyl-tRNA synthetase

Rapid determination of the three-dimensional structures of mutant proteins is so highly embedded within the discipline of protein engineering that it almost seems a routine part of the process of analyzing mutant function. However, in the early days of protein engineering, the time and energy involved in determining mutant structures were substantial compared with today and the dividends unclear. Here is a brief perspective on the determination of the first crystal structures of site-directed mutants of the tyrosyl-tRNA synthetase--an enzyme used to establish many of the early principles of protein engineering.

The nuclear tRNA aminoacylation-dependent pathway may be the principal route used to export tRNA from the nucleus in Saccharomyces cerevisiae

Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNA(Tyr) co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNA(Tyr) export because of a block in nuclear tRNA(Tyr) aminoacylation. tRNA(Tyr) was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.

Utp8p Is an Essential Intranuclear Component of the Nuclear tRNA Export Machinery of Saccharomyces cerevisiae

A yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay based on amber suppression was used to identify proteins that participate in the nuclear tRNA export process in Saccharomyces cerevisiae. One of the proteins identified by this strategy is Utp8p, an essential 80-kDa nucleolar protein that has been implicated in 18 S ribosomal RNA biogenesis. Our characterization indicated that the major function of Utp8p is in nuclear tRNA export. Like the S. cerevisiae Los1p and the mammalian exportin-t, which are proteins known to facilitate nuclear tRNA export, overexpression of Utp8p restored export of tRNAamTyr mutants defective in nuclear export. Furthermore, depletion of Utp8p blocked nuclear export of mature tRNAs derived from both intronless and intron-containing pre-tRNAs but did not affect tRNA and rRNA maturation, nuclear export of mRNA and ribosomes, or nuclear tRNA aminoacylation. Overexpression of Utp8p also alleviated nuclear retention of non-aminoacylated tRNATyr in a tyrosyl-tRNA synthetase mutant strain. Utp8p binds tRNA directly and saturably, indicating that it has a tRNA-binding site. Utp8p does not appear to function as a tRNA export receptor, because it does not shuttle between the nucleus and the cytoplasm. Taken together, the results suggest that Utp8p is an essential intranuclear component of the nuclear tRNA export machinery, which may channel tRNA to the various tRNA export pathways operating in S. cerevisiae.

Molecular recognition in proteins: Simulation analysis of substrate binding by a tyrosyl-tRNA synthetase mutant

Alchemical molecular dynamics simulations are performed to determine the difference in the free energy of binding of the tyrosine substrate between the wild type of tyrosyl-tRNA synthetase (TyrRS) from Bacillus stearothermophilus and the mutant Tyr169 → Phe. The results are of general interest because the Tyr169 hydroxyl group interacts with the ammonium group of the substrate in a manner corresponding to that found in other amino acid binding proteins (e.g. the Asp receptor of the chemotactic bacterium Salmonella typhimurium and class I major histocompatibility complex molecules). The calculated free-energy change due to the Tyr169 → Phe mutation is 3·4 kcal/mol (the statistical error is ± 0·5 kcal/mol) in satisfactory agreement with the experimental value of 3(±0·5) kcal/mol. By use of thermodynamic integration, the contribution of the different terms to the free energy change are estimated. The path dependence of such a decomposition is discussed and it is suggested that the alchemical choice is of primary interest for understanding the interactions involved. There are large protein contributions to the alchemical free energy difference of the bound and free enzyme that cancel in the overall result. Due to this cancellation, the essential interactions contributing to the free-energy change are those between the OH group of Tyr169 and water in the free enzyme and those between the OH group of Tyr169 and the ammonium group of the substrate in the bound system. The results thus support simple models based on a balance of hydrogen bonding interactions.

Kinetic and thermodynamic properties of wild-type and engineered mutants of tyrosyl-tRNA synthetase analyzed by pyrophosphate-exchange kinetics

The first step of the reaction catalyzed by the aminoacyl-tRNA synthetases is the formation of enzyme-bound aminoacyl adenylate. The steady-state kinetics of this step has conventionally been studied by measuring the rate of isotopic exchange between pyrophosphate and ATP. A simple kinetic analysis of the pyrophosphate-exchange reaction catalyzed by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus is given in which all the observed rate and binding constants can be assigned to identifiable physical processes under a variety of limiting conditions. The free energies of binding to the enzyme of tyrosine, ATP, and the transition state for tyrosyl adenylate formation can be measured in relatively straightforward experiments. The excellent agreement between parameters measured in these experiments and those from earlier pre-steady-state kinetics confirms that the intermediates isolated in the presteady state are kinetically competent. The dissociation constant of ATP from the unligated enzyme, a constant that has previously been experimentally inaccessible, has been measured for wild-type and several mutant enzymes. The changes in enthalpy and entropy of activation on mutation have been measured by a rapid procedure for mutants that have altered contacts with tyrosine and ATP. Those mutants that have large changes of enthalpy and entropy of binding are likely to have structural changes and so warrant further examination by protein crystallography.

Linear free energy relationships in enzyme binding interactions studied by protein engineering

Experiments on mutants of tyrosyl-tRNA synthetase have shown that there can be linear free energy relationships (LFERs) between changes in activation free energies and changes in binding energies when groups are deleted that bind to non-reacting parts of the substrate (Fersht et al., 1986, 1987). It has now been proposed (Straub and Karplus, 1990) that such LFERs can occur for the mutation of hydrogen bonding groups only for the limiting examples of Brønsted beta of 0, 1 or infinity, and that fractional values of beta are not permissible. The reasoning behind this is that the energy of a hydrogen bond is not linear with distance and the (false) premise that an LFER requires that there is a linear relationship between bond energy and distance. We show from a simple model how LFERs can arise for binding interactions and how they can give fractional values of beta, in accord with experimental evidence. An LFER occurs between binding and catalysis when a set of interactions exists in which each member contributes to the binding energy of the transition state the same fraction of the binding energy it contributes to the products (both relative to the ground state).

Crystal structures of two factitious mutants of tyrosyl-tRNA synthetase

Crystal structures of two factitious mutants of tyrosyl-tRNA synthetase

Improved mapping of the tyrS locus in Escherichia coli.

A tyrosyl-tRNA synthetase mutant of Escherichia coli was isolated and the tyrS gene assigned a map position between man and pdxH at 36.0 min on the chromosome. The tyrS mutant grew badly on broth as did previously described tyrS mutants. This sensitivity to broth was suppressed by tyrR mutations. F-prime factors were found to complement the tyrS mutation.