Isolation and characterization of the mutant form of N-terminal catalytically module of Bos taurus tyrosyl-tRNA synthetase with the replacement of Trp 40 and Trp 283 by alanine

© 2020 V. N. Zayets et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited Structure and Function of Biopolymers ISSN 0233-7657 Biopolymers and Cell. 2020. Vol. 36. N 5. P 329–340 doi: http://dx.doi.org/10.7124/bc.000A37


Introduction
Aminoacyl-tRNA synthetases (ARSases) [EC 6.1.1] are the key enzymes of protein biosynthesis. At the preribosomal stage of translation, ARSases catalyze the activation and covalent attachment of the amino acids to the homologous transfer RNAs, thus carrying out the initial stage of the realization of genetic information into the protein structure [1,2].
Mammalian tyrosyl-tRNA synthetase (TyrRS, EC 6.1.1.1) is one of the most studied eukaryotic ARSases. Under physiological conditions, Bos taurus tyrosyl-tRNA synthetase is an α 2 homodimer with a molecular weight of 2×59.2 kDa. Each monomer consists of two structural parts: the N-terminal catalytic form (mini BtTyrRS, 39 kDa) and the C-terminal EMAP II-like module (20 kDa) [2]. During the isolation of TyrRS from bovine liver, it was shown that along with the full-length main form, the functional proteolytically modified form of tyrosyl-tRNA synthetase with a molecular weight of 39 kDa was released, which retains its enzymatic activity in vitro [3,4].
In addition to the basic tRNA aminoacylation function the mammalian tyrosyl-tRNA synthetases perform also the non-canonical functions: after enzymatic cleavage of tyrosyl-tRNA synthetase by elastase into mini BtTyrRS and C-module, the latter exhibit cytokine properties, thus linking the protein biosynthesis with cell signaling systems [5][6][7]8].
The tests for cytokine activity of the N-terminal catalytic domain of BtTyrRS revealed that mini TyrRS is a chemotaxic factor for neutrophils, activates the migration of endothelial cells and polymorphonuclear leuko-cytes and stimulates angiogenesis in a concentration-dependent manner. The cytokine activity of mini TyrRS has been shown to be mediated by conservative ELR motif in the Rossman fold of the catalytic domain [7,8].
Since the N-terminal catalytic module of the Bos taurus tyrosyl-tRNA synthetase is an interleukin-like cytokine and exhibits proangiogenic properties, it is a promising object for investigation. In the structure of the BtTyrRS catalytic module there are 3 tryptophan residues, which are respectively located in the active center of the enzyme (W40), in the region of dimerization of mini BtTyrRS monomers (W87) and in the binding site of the tRNA anticodon triplet (W283). Such location of tryptophan residues in the functionally important regions of the protein's amino acid sequence makes it very promising to study the properties by fluorescence spectroscopy, especially if there is only one residue in one of three positions in the enzyme structure. Previously, we have cloned the cDNA of the tyrosyl-tRNA synthetase catalytic module in the expression plasmid pET32a and investigated its expression [9]. Subsequently, Trp40 and Trp283 codons were replaced with alanine codons by site-directed mutagenesis in cloned cDNA and only one tryptophan codon was left at the site of dimerization of mini BtTyrRS monomers [10].
The purpose of this work was to determine the optimal expression conditions of mutant mini BtTyrRS and to isolate the recombinant protein for further study of its properties, especially the local conformational changes at the enzyme dimer interface.

Materials and Methods
In order to obtain the mutant form of Bos taurus mini TyrRS we used the bacterial expression system of E. coli cells [11][12][13]. The synthesis of recombinant protein was carried out in the E. coli strain BL21 (DE3) pLysE (Stratagene, USA) transformed with pET30a-39KYRSW87 plasmid.
The pET30a-39KYRSW87 expression construction was created on the basis of the pET-30a(+) vector ("Novagen", USA) and contained a cloned cDNA with the tryptophan-40 and tryptophan-283 codons replaced by alanine codon. A plasmid DNA was isolated using the Gene JET Plasmid Miniprep Kit from "Thermo Scientific". The concentration of plasmid DNA was determined on NanoDrop 2000 spectrophotometer ("Thermo Scientific").
In order to obtain the recombinant plasmid construct pET30a-39KYRSW87, transform it into E.coli cells and express the mutant cDNA of the catalytic module B.taurus tyrosyl-tRNA synthetase, the genetically engineered E. coli DH5α and BL21 (DE3) pLysE strains were used. Competent E. coli cells were obtained according to the Inoue method [14]. All procedures for transformation of plasmid pET30a-39KYRSW87 into competent E. coli cells and analysis of plasmid by 0.7-1 % agarose gel electrophoresis were performed according to [15].
The cultivation of E. coli BL21 (DE3) pLysE cell culture and the induction of expression of recombinant mini BtTyrRS in bacterial culture were performed in Luria-Bertani medium (LB) with 30 μg/ml kanamycin. Transformed with recombinant plasmid pET30a-39KYRSW87 , the competent E. coli BL21 (DE3) pLysE cells were grown on a shaker (BioSun Shaker Incubator ES-20) at 37° C to an optical density of A 600 = = 0.6-0.8 and the target protein synthesis was induced by adding 1M isopropyl-β-D-thiogalactopyranoside (IPTG) up to 1 mM concentration followed by incubation at 37 °C for 4 hours and at 30 °C and 25 °C for 12 hours. The collected biomass from 100 ml of culture was resuspended in 12 ml of cell lysis buffer (50 mM sodium phosphate buffer, pH8.0, 500 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol). Cells lysis was performed using an ultrasonic disintegrator (6 cycles of 20 s, 20 s intervals). The lysate was clarified by centrifugation at 13000 rpm for 30 min at 4 °C.
The supernatant was applied to a Ni-NTA agarose column previously washed with 10 ml of washing buffer (50 mM sodium phosphate buffer, pH 8.0, 500 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol) and with lysis buffer. Recombinant protein was eluted with 5 ml of elution buffer (50 mM sodium phosphate buffer, pH 8.0, 150 mM NaCl, 200 mM imidazole, 5 mM β-mercaptoethanol). All protein containing fractions were combined and dialyzed against 500 ml of dialysis buffer (500 mM sodium phosphate buffer pH 8.0, 150 mM NaCl) for 20 hours at +4 °C. The concentration of purified mini BtTyrRS mutant protein was determined spectrophotometrically on BioMate-5 spectrophotometer using a molar extinction coefficient of 27850 M -1 cm -1 at a wavelength of 280 nm. The optical absorption coefficient of mini BtTyrRS was determined by amino acid analysis of protein using ProtParam server (http://expasy.ch/cgibin/protparam). According to the ProtParam server, the molecular weight of the obtained recombinant mutant mini BtTyrRS is 47364.36 Da and isoelectric point pI = 6.42.
Expression of the recombinant proteins was analyzed by SDS-PAGE [16].
Gels were stained with Coomassie blue R250 dye. The estimation of the amount of recombinant protein in the precipitate and in the supernatant fraction on the electrophoregram was performed densitometrically on a ChemiDocTM XRS + system instrument ("BioRad", USA).
The amino acid sequence of Bos taurus TyrRSs was used from the NCBI Gene database (https://www.ncbi.nlm.nih.gov/protein/) with identification numbers DAA32266.1. The three-dimensional crystal structures were obtained from RCSB PDB archive. Threedimensional coordinates of the protein structural templates were obtained from Protein Data Bank (PDB) (http://www.pdb.org/pdb). Visualization and analysis of protein structure were performed using UCSF Chimera software [17]. The spatial structures of the BtTyrRS dimer and double mutant BtTyrRSW87 were modelled from the crystal structures of HsTyrRS (PDB codes 1N3L:A) as templates using SWISS-MODEL web-server [18]. Highresolution protein structure refinement was done by ModRefiner [19]. The final model of BtTyrRS structure was validated by the MolProbity server [20].
All fluorescence spectra were recorded at 25 o C on a Hitachi Model 850 fluorescence spectrophotometer equipped with thermostated cell holder (Hitachi Ltd., Japan). Fluorescence measurements were performed in a quartz cell with an optical path length of 0.5 cm. The temperature in the quartz cuvette was determined within + 0.2 o C. Both excita-tion and emission slits of 5 nm were used in all fluorescence measurements. The excitation light wavelength was 280 nm or 295 nm, the wavelength interval for the fluorescence spectra was 300-400 nm and the fluorescence registration was performed at the 90 o angle to the beam direction.

Results and Discussion
Previously, we have cloned and sequenced the complete nucleotide sequence of cDNA of the Bos taurus tyrosyl-tRNA synthetase gene [2]. Based on cDNA, an expressive plasmid construct of pET-30a (+)-39KYRS was created with a cloned sequence of the synthetase N-terminal catalytic module. Expressed in strain E. coli BL21 (DE3) recombinant mini BtTyrRS retained the aminoacylating ability inherent in the native aminoacyl-tRNA synthetase.
Based on site-directed mutagenesis, the recombinant plasmid pET-30a (+)-39KYRS was used to create the substitutions of tryptophan residues at positions 40 and 283 by alanine in the cDNA of the synthetase catalytic module [10]. The resulting plasmid pET-30a-39KYRSW87 having only one tryptophan codon in the cloned cDNA was used in this work to obtain one-tryptophan protein for further fluorescence studies of conformational features and intramolecular interactions in protein structure [21,22]. The amino acid alanine was selected for site-directed mutagenesis due to its small hydrophobic radical, which does not affect a secondary protein structure formed by the adjacent amino acid residues in the polypeptide chain.
A considerable amount of recombinant protein is required to investigate the properties of the enzyme by experimental methods. Since the final yield of the target recombinant protein in bacterial systems strongly depends on the culture conditions, the experimentally determined optimal parameters for the expression of mini BtTyrRS in E. coli are required [9,10].
The native mini BtTyrRS cloned in plasmid pET-30a-39KYRS was used to determine the conditions of its optimal expression in the E. coli system [9]. It was shown that the highest level of synthesis of recombinant mini BtTyrRS in E.coli culture was achieved by adding to the culture medium of IPTG at a concentration of 1mM in the logarithmic phase of growth of the culture when it reaches an optical density OD600 = 0.7-0.9 for the induction of protein synthesis and incubation of the culture for 4 hours at 37 o C. It was found that the composition of the culture medium had no significant effect on the expression of the enzyme.
We used these experimentally established optimal expression conditions to express and obtain a mutant single-tryptophan form of mini BtTyrRS in transformed plasmid pET30a-39KYRSW87 E. coli cell culture of strain BL21 (DE3) pLysE.
It is known that the sequence of the cloned genes in the expression vectors of the pET series plays a significant role in both the synthesis of recombinant proteins and obtaining soluble fraction of newly synthesized proteins [13]. Therefore, the replacement of two tryptophan residues with alanine in the mini BtTyrRS structure in our case could be critical and lead to a decrease in enzyme synthesis or its transition into insoluble inclusion bodies. In this regard, we simultaneously expressed in E. coli mutant and native mini BtTyrRS forms of plasmids pET-30a (+) -39KYRS and pET30a-39KYRSW87. Our preliminary electrophoretic data showed that the mutations did not affect the expression of the mutant protein: the number of native and mutant forms of the tyrosyl-tRNA synthetase catalytic module synthesized in bacterial cultures was almost the same. However, our analysis of cell precipitate after clarification of bacterial lysates in the process of protein isolation showed that the majority of both native and mutant forms of the enzyme are in the cytoplasm in insoluble fraction of the inclusion bodies (results not shown).
The temperature of incubation is one of the major factors affecting the transition of recombinant proteins during expression in E. coli to the aggregated state, especially in vectors of the pET series with the extremely strong RNA polymerase promoter of phage T7 [13,23,24]. Therefore, it was decided to analyze the expression of the target protein in E. coli culture of strain BL21 (DE3) pLysE after IPTG induction at a lower incubation temperature of 30 o C and 25 o C. The results of the analysis are shown in Fig. 1 (A, B). The experimental data showed that with decreasing temperature of the bacterial culture growth, the amount of synthesized recombinant protein in the soluble fraction increased in proportion to the temperature decrease. The highest amount of recombinant mini BtTyrRS in soluble cytoplasmic cell fraction was obtained at the incubation temperature of 25 o C. At this temperature, the soluble fraction was about 47 % of the total amount of recombinant protein synthesized, whereas at 37° C it was only 13 %.
The established conditions for optimal expression of the catalytic N-terminal module of the Bos taurus tyrosyl-tRNA synthetase in E. coli in LB medium were taken into account when obtaining a preparative amount of recombinant mutant mini BtTyrRS in E. coli strain BL21 (DE3) pLysE using metal chelating chromatography. After lysis of bacterial cells by sonication and chromatographic purification of lysate on Ni-NTA agarose from 100 ml of the bacterial culture incubated at 25 °C for 8 hours we could get up to 3 mg of homogeneous recombinant protein of the mutant mini BtTyrRS, with the purity according to electrophoresis about 95 % (Fig. 2). Up to 30 mg of purified recombinant enzyme can be obtained from 1 liter of bacterial culture under certain conditions of expression. Taking into account the aggregated protein in the inclusion bodies, the total yield of the synthesized recombinant mini BtTyrRS in transformed plasmid pET30a-39KYRSW87 strain E. coli BL21 (DE3) pLysE was about 75 mg from the 1L LB medium.
According to the ProtParam program analysis, both native and mutant mini BtTyrRS proteins are stable structures. Their instability indexes are almost identical, 36.2 and 37.15, respectively, indicating that there is no appre- The computational models of the spatial structure of the catalytic modules were constructed on the basis of the X-ray crystallographic data of the N-terminal catalytic module of human tyrosyl-tRNA synthetase [25] and the computational model of the structure of the full-length Bos taurus tyrosyl-tRNA synthetase [26]. The models of spatial structure of native and mutant mini BtTyrRS homodimers with highlighted tryptophan residues in positions 40, 87, 283 in native and in position 87 in mutant forms of the enzyme are shown in Figures 3 and 4, respectively.
Tryptophan 87 in each subunit of mini BtTyrRS is localized at the contact area of protein monomers of the functional mini BtTyrRS. The replacements of two tryptophan residues in mini BtTyrRS by alanine did not resulted in any visible changes of its 3D structure.
Similar data were obtained from the comparison of the spatial structural organization of the contact regions of protein monomers in the environment of the Trp87 residue in native and mutant forms of mini BtTyrRS (Fig. 5). They also did not show any obvious changes after mutagenesis in the structure of the recombinant enzyme.
With the help of UCSF Chimera software the environments of Trp87 residue in the sphere of radius 5 Å in both the native and mutant forms of BtTyrRS were visualized and analyzed ( Table 1). The analysis showed that there are 11 residues in the given region around Trp87: 6 hydrophobic (Tyr79, Ala85, Leu89, Leu90, Thr121, Leu131), two negatively charged residues (Glu88, Glu91), two positively charged ciable effect of substitutions of two tryptophan residues by alanine on the stability of the mini-TyrRS mutant protein.
To evaluate the structure of the mutant form of mini BtTyrRS, we applied the computational modelling method for estimation of the homodimers of native and mutant catalytic modules of Bos taurus TyrRS, microenvironment analysis of tryptophan-87 residue in the residues (Lys127, Arg135) and neutral Pro86. Table 1 represents the distances from the C a atom of Trp87 to the C a atom of the corresponding residue in the native and mutant forms of BtTyrRS. It can be seen that the Trp87 microenvironment is similar in both forms. We observed only minor changes in the solvent accessibility of Trp87.
The fluorescence spectrum of the mutant form of the catalytic modulus of Bos taurus tyrosyl-tRNA synthetase at the excitation wavelengths of 280 nm and 295 nm are shown in Fig. 6. The determined fluorescence cha racteristics of the mutant protein, in particular, the position of the fluorescence maximum, λ m, and the half-width of the fluorescence spectra, ∆λ, are 338 nm and 60 nm, respectively.
According to the three spectral classes model of tryptophan residues in protein structure, the tryptophan residue at position 87 refers to the spectral class II, which is characterized by the emission of indole fluorophore immobilized in the concavity on the surface of the protein, and does not contact with free but only with bound water and other polar groups in the protein structure [21]. The parameters of the fluorescence spectra of tryptophan residues depend on the polarity of their microenvironment, as well as the abi lity of the tryptophan residue to relax during the fluorescence lifetime. It should be keep in mind that the polarity of the tryptophan residue microenvironment is determined not only by its accessibility to the solvent molecules, but also by its own polar protein groups, which are the parts of the microenvironment [21,22].
Previously, we have studied the intrinsic tryptophan fluorescence of native mini TyrRS and analyzed its intramolecular dynamics by fluorescence spectroscopy [27]. The analysis of the localization and microenvironments of three tryptophan residues responsible for the intrinsic fluorescence of mini TyrRS allowed us to characterize their accessibility in the structure of the enzyme dimer and the microenvironment conformational mobility. The characteristics of the tryptophan fluorescence of mutant mini TyrRS with a single tryptophan residue at position 87 indicate the immobilization of the tryptophan residue microenvironment at the dimer interface.
The mutant form of mini-TyrRS with tryptophan-87 residue, which is localized in the  region of dimerization of the enzyme, can be effectively used to investigate the conformational changes of tyrosyl-tRNA synthetase associated with the neurodegenerative disease of Charcot-Marie-Tooth neuropathy [28,29].

Conclusions
In this work it has been found that the replacement of Trp40 and Trp283 residues by alanine in Bos taurus mini-tyrosyl-tRNA synthetase cloned in the expression plasmid pET30a-39KYRSW87 does not affect the synthesis of the mutant form of the enzyme.