KR101711584B1 - Crystal Structure and Crystallization of modified EPRS protein - Google Patents

Abstract

The present invention relates to a crystal structure of a modified EPRS protein and a method of crystallizing the modified EPRS protein. More particularly, the present invention relates to a modified EPRS protein having a modified bipunctional glutamate / proline tRNA synthetase (EPRS) The present invention relates to a method for mass-expressing an EPRS protein, purifying and crystallizing the EPRS protein, and a crystal structure analyzed thereby.
Accordingly, the present invention provides a crystal structure and a crystallization method of a modified EPRS protein having an amino acid sequence of SEQ ID NO: 1 including a PRS protein domain portion of a human-derived EPRS protein, and can be usefully used for the development of inhibitors using the same .

Description

[0002] Crystal structure and Crystallization of modified EPRS protein [

The present invention relates to a crystal structure of a modified EPRS protein and a method of crystallizing the modified EPRS protein, and more particularly, to a modified EPRS protein having 1000 amino acids at the N-terminal part of EPRS (bifunctional glutamate / proline tRNA synthetase) A method for purifying and crystallizing the same, and a crystal structure analyzed thereby.

Aminoacyl-tRNA synthetase is an enzyme that binds a specific amino acid precisely to the corresponding tRNA, which is essential for protein synthesis. The first step is to consume one ATP and activate the amino acid to aminoacyladenylate. The second step is to activate the amino acid to tRNA .

Aminoacyl tRNA synthetases in mammals have similar roles as aminoacyl tRNA synthetases of prokaryotes, but have additional domains. This additional domain is involved in the formation of various complexes with aminoacyl thiourea synthetase or other regulatory factors. It has recently been shown that this structural complexity is related to the functional diversity of aminoacyl-tRNA synthetases and to various human diseases.

Abnormal expression, mutation, and different formation of aminoacyl tRNA synthetase cause disease in various routes. For example, abnormal cellular accumulation, abnormal molecular interactions, even with normal catalytic activity, interfere with the regulatory network of normal cells, affecting tumor-induced angiogenesis, immune responses, electronic transcription regulation, and signal transduction. Disruption of the cell's proteome and signaling pathway affects the cell's susceptibility to stress related to tumorigenesis or tumorigenesis.

Of the aminoacyl tRNA synthetases, the enzyme (EPRS) that specifically promotes the binding of human glutamate to proline tRNA is specifically present in a single polypeptide chain. The domains catalyzing glutamate are located at the N-terminal region and the domains catalyzing proline are located at the C-terminal, respectively, and these two domains are linked to the WHEP domain. Glutamyl tRNA synthetase belongs to class 1 aminoacyl tRNA synthetase, and proline tRNA synthetase belongs to class 2 aminoacyl tRNA synthetase. The WHEP domain, which links the two domains, consists of 57 amino acid chains repeated three times, mediating protein-protein or protein-RNA interactions.

Most life activities in life are maintained through a medium called a protein made up of a combination of 20 amino acids. The fact that not only the enzymes that catalyze and regulate various in vivo reactions but also the signal transduction systems of various signals are examples of the importance of proteins. Proteins, on the other hand, form a three-dimensional structure or a three-dimensional structure through folding in order to exhibit their unique functions. Deformation of the three-dimensional structure is a major cause of disease development. Identification of the three-dimensional structure of a protein is fundamental to understand the function of the protein accurately, and identification of the three-dimensional structure of the protein whose function is not known becomes an important means of grasping its function. Therefore, developed countries are making national efforts to identify protein structure after the genome project. However, since the three-dimensional structure of a protein depends on the sequence of each amino acid of each polypeptide, it is very difficult to predict the structure of a protein to be produced considering each one.

Recent advances in biotechnology have foreseen a revolution in new drug development. Most human diseases are caused by abnormalities of proteins, and they are being developed into new drugs to inhibit or increase the function of these proteins and turn them into normal functions. Until now, the discovery of new drugs has been conducted through repetition of numerous drug validation experiments for drug candidates. However, in recent years, the structure of disease proteins to identify the binding site of the compound is designed to develop the drug is changing trend.

Thus, the present inventors completed the present invention by crystallizing and analyzing the modified EPRS protein having the amino acid sequence of SEQ ID NO: 1.

Accordingly, an object of the present invention is to provide a protein having the amino acid sequence of SEQ ID NO: 1, wherein the space group is P3 ₁ 21, the unit cell parameter is a = 119.67 Å, b = 119.67 Å, c = 99.36 Å, α = ß = 90 °, γ = 120 ° and providing a crystal of a modified EPRS protein having a dimer structure.

Another object of the present invention is to provide a modified bipunctional glutamate / proline tRNA synthetase (EPRS) represented by the amino acid sequence of SEQ ID NO: 1 by removing (a) 1000 amino acids from the N-terminal part of a human EPRS (Bifunctional Glutamate / Proline synthetase) Expressing and purifying the protein; And (b) crystallizing the protein purified in step (a) with a preservative solution containing sodium citrate / HCl or sodium cacodylate as a precipitant. And to provide a method for crystallizing EPRS protein.

It is another object of the present invention to provide a method for preparing a modified EPRS protein, comprising: (a) reacting the modified EPRS protein with a candidate compound; And (b) selecting a compound that interacts with the modified EPRS protein among the candidate compounds, thereby determining a compound that inhibits the activity of the EPRS protein.

In order to achieve the above object, the invention is represented by the amino acid sequence of SEQ ID NO: 1 by removing the amino acid 1000 of the N-terminal portion of the protein derived from human EPRS (Bifunctional Glutamate / Proline synthetase), space group P3 ₁ 21, a unit cell parameter of a = 119.67 Å, b = 119.67 Å, c = 99.36 Å, α = ß = 90 °, γ = 120 ° and providing a crystal of a modified EPRS protein having a dimer structure do.

In order to accomplish another object of the present invention, the present invention provides a modified EPRS (SEQ ID NO: 1) amino acid sequence represented by amino acid sequence of SEQ ID NO: 1 by (a) removing 1000 amino acids at the N-terminal part of a human EPRS (Bifunctional Glutamate / Proline synthetase) Bifunctional Glutamate / proline tRNA synthetase) protein; And (b) crystallizing the protein purified in step (a) with a preservative solution containing sodium citrate / HCl or sodium cacodylate as a precipitant. A method of crystallizing an EPRS protein is provided.

In order to accomplish another object of the present invention, the present invention provides a method for producing a modified EPRS protein, comprising the steps of: (a) reacting the modified EPRS protein with a candidate compound; And (b) selecting a compound that interacts with the modified EPRS protein among the candidate compounds to determine a compound that inhibits the activity of the EPRS protein.

Hereinafter, the present invention will be described in detail.

In the present invention, 1000 amino acids of the N-terminal portion of the human EPRS (Bifunctional Glutamate / Proline synthetase) protein are removed and represented by the amino acid sequence of SEQ ID NO: 1, and the space group is P3 ₁ 21 and the unit cell parameter is a = 119.67 , b = 119.67 Å, c = 99.36 Å, α = ß = 90 °, γ = 120 ° and provides a crystal of a modified EPRS protein having a dimer structure.

In the present invention, a unit cell parameter is also referred to as a lattice constant, and a unit cell is the smallest repeating unit which is the most easily interpretable constituting a space lattice, and is defined as three crystallographic axes , The lengths (a, b, c) of these three vectors and the angles (?,?,?) Between them.

Space group, also called space group, means the symmetry of the unit cell of crystals. A total of 230 symmetry groups are possible to form a group by combining symmetry elements.

The modified EPRS protein provided in the present invention is a protein containing the PRS domain and is prepared so as to have the amino acid sequence of SEQ ID NO: 1 by removing 1000 amino acids from the N-terminus of the EPRS protein. Protein domain analysis data obtained from Expasy, NCBI, ERGO (http://wit.integratedgenomics.com/Igwit) and the secondary structure of the protein, and the result of comparing the amino acid similarities in various strains, the 1000 amino acids were removed.

The protein of the present invention is characterized by having a dimer structure.

The present invention relates to a method for the production of a modified EPRS (Bifunctional Glutamate / proline tRNA synthetase) protein represented by the amino acid sequence of SEQ ID NO: 1 by removing 1000 amino acids from the N-terminal part of human-derived bifunctional glutamate / proline synthetase (EPRS) And purifying; And (b) crystallizing the protein purified in step (a) with a preservative solution containing sodium citrate / HCl or sodium cacodylate as a precipitant. A method of crystallizing an EPRS protein is provided.

(a) expressing and purifying a modified EPRS (Bifunctional Glutamate / proline tRNA synthetase) protein represented by the amino acid sequence of SEQ ID NO: 1 by removing 1000 amino acids at the N-terminal part of a human EPRS (Bifunctional Glutamate / Proline synthetase) step;

In order to amplify a gene encoding the amino acid sequence of the modified EPRS protein (EPRS_Tr2 of the present invention) (SEQ ID NO: 1), a polymerase chain corresponding to the 5'-terminal and 3'-terminal of the gene reaction, and PCR primers are designed and synthesized. The primer corresponding to the 5 'end of the gene has a restriction enzyme recognition site to facilitate cloning into an E. coli expression vector, and a restriction enzyme recognition site and termination codon are formed in the primer corresponding to the 3' end so that the protein synthesis is artificially terminated I make it. Here, the primer corresponding to the 5 'end is prepared so as to remove the desired number of N-terminal amino acids. The PRS domain gene is amplified by performing PCR using the primer prepared as described above as the template containing the PRS domain in Homo sapiens EPRS DNA.

Expression of the modified EPRS protein of the present invention can be carried out using conventional molecular biology methods known in the art. A gene amplified by PCR is inserted into a vector commonly used in the art, such as pGEM-T, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19 or pET. The expression vector may include a conventional selection mark such as an ampicillin resistance gene, a tetracycline resistance gene, a kanamycin resistance gene, a chloramphenicol resistance gene, or the like. After insertion, suitable host cells such as E. coli JM109, E. coli, BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, Bacillus subtilis, Bacillus subtilis, and Enterobacteria and strains such as Salmonella typhimurium, Serratia marcesensus and various Pseudomonas sp., And the transformed cells are cultured to produce the modified EPRS A large amount of protein can be expressed. The vector used for expression may most preferably be pET, and the host cell may be E. coli.

Whether or not the protein is produced can be confirmed by a conventional method such as SDS-polyacrylamide gel electrophoresis or Western blotting method. When Escherichia coli transformed with the expression vector containing the gene of the modified EPRS protein (EPRS_Tr2) was shake cultured in a medium containing an antibiotic corresponding to a selection marker such as ampicillin, tetracycline, kanamycin and chloramphenicol to reach a constant cell concentration , IPTG (isopropyl-D-galactopyranoside) is added to induce the expression of EPRS_Tr2. The culture solution is centrifuged to precipitate E. coli cells, and E. coli is suspended in an appropriate buffer solution and then disrupted. The above crushed material is centrifuged, and the supernatant in which the protein is dissolved is separated, and EPRS_Tr2 is separated and purified by using an ion exchange resin, an affinity binding resin, sizing or the like.

(b) crystallizing the protein purified in step (a) with a preservative solution containing sodium citrate / HCl or sodium cacodylate as a precipitant;

In order to identify the three-dimensional structure of the protein, it is important that the target protein has crystallinity. In the three-dimensional structure determination of target proteins, x-ray crystallography is generally used, and various crystallization methods have to be carried out as a pretreatment step for using this X-ray crystallization method.

The crystallization method is a method using conventional concentration equilibrium. More specifically, it may be a steam equilibrium method or a dialysis method. The steam equilibrium method may be selected from the group consisting of a Sitting Drop Steam Equilibrium Method and a Hanging Drop Steam Equilibrium Method. Most preferably a hanging drop steam equilibration method.

In the present invention, a hanging-drop vapor diffusion method was used as a crystallization method.

The principle and procedure of crystallization of the droplet mixed steam equilibrium method are as follows. When a small drop of mother liquor and a much larger reservoir coexist in an enclosed space, there is a transfer of water or other volatile matter between them. On the other hand, in supersaturated thermodynamically stable state of proteins, precipitation of proteins takes place according to the change of the precipitant, and the crystallization state becomes stable as the precipitation rate progresses slowly. Precipitants that can be used herein are well known and they serve to reduce the solubility of concentrated protein solutions. At this time, in order to reduce the relative adsorption layer around the protein molecules, the protein molecules coalesce to form crystals. Therefore, in the reservoir solution, buffer solution, salt, detergent, etc. are mixed at various concentrations such as precipitant, protein solution and reservoir solution of these various conditions usually mixed at a ratio of about 1: 1 Respectively. The droplets thus obtained are placed on a glass slide coated with silicone and placed on a prepared plate in a state of being turned upside down and sealed. Initially, the concentration of the protein in the droplet differs from the concentration of the preservative solution, so that the protein does not exist in a crystalline state. However, if the protein is placed in a sealed state, the equilibrium is gradually established. At this time, Crystals are formed. In such a mixed-solution steam equilibrium method, the type and proper concentration of the salt, buffer and detergent as well as the precipitant in the preservative solution, the pH of the solution and the temperature of the solution are selected depending on the type of the protein. In some cases, It becomes a very important factor.

The sitting drop steam equilibrium method and the hanging drop steam equilibrium method are based on the above-described principle. The difference lies in the position of placing the sample.

The hanging drop steam equilibration method proceeds as shown below. The hanging drop steam equilibrium method is the most commonly used method for protein crystallization. When a sample mixed with a protein solution and a preservative solution is adhered to the top of a container in a pure equilibrium state with a pure preservative solution, the water content in the sample falls below the equilibrium state of the sample having a lower content than the preservative solution in the vessel . When equilibrium is reached, protein crystals are obtained.

Sitting drop steam equilibration method is shown below. Sitting drop steam equilibrium method has the same basic principle as hanging drop steam equilibrium method. However, there is a difference in the presence of the sample in which the protein solution and preservative solution are mixed in the steam.

Another method is the dialysis method. In the dialysis method, a protein solution is present in a tube composed of a porous membrane, and a reservoir solution (preservative solution) containing a precipitant is present on the outside. Since the porous membrane can move only water molecules having a small size, Water molecules are concentrated through the porous membrane as they move slowly toward the precipitate, resulting in crystals.

In order to prepare a modified EPRS protein crystal represented by the amino acid sequence of SEQ ID NO: 1 of the present invention, sodium citrate / HCl or sodium cacodylate can be used as a precipitant contained in the preservation solution have. More preferably, the two materials can be used together. The concentration of the precipitant in the total preservative solution varies depending on the type of the precipitant to be used, and is preferably in the range of 0.05 to 1.5M. More preferably, the concentration of sodium citrate / HCl is 0.5M to 1.5M and the concentration of sodium cacodylate is 0.05M to 0.15M.

 The preserving solution may contain salts, buffers and detergents conventionally usable in addition to the precipitating agent. In addition, it is preferable that the protein crystallization is performed at pH 6.0 to 7.0. The above range is determined experimentally, and the most excellent crystal can be obtained when the reaction is conducted under the pH of the above range.

The reaction temperature for forming the protein crystals of the present invention is preferably 15 ° C to 25 ° C, more preferably 18 ° C to 22 ° C. The reaction period is preferably from 1 day to 10 days, more preferably from 2 days to 5 days. The reaction temperature and the reaction time range are experimentally found as conditions for obtaining crystals having optimal data for easy X-ray analysis.

When X-rays are irradiated to analyze the structure of the crystalline EPRS_Tr2 protein prepared, the lifetime of the protein is shortened due to the high energy of the X-ray, and the data intensity is also weak, resulting in poor results in structural analysis. In order to prevent such drawbacks, it is preferable to perform a high-speed nitrogen cooling method in which protein crystals are nitrogen-cooled at a high speed before X-ray analysis. The solution for rapid cooling consists of sodium malonate, sodium cacodylate trihydrate (pH 6.0) and sodium citrate tribasic dihydrate. The concentration of sodium malonate is 3 to 4 M, the concentration of sodium cacodylate trihydrate (pH 6.0) is 0.05 to 0.2 M, The sodium citrate tribasic dihydrate concentration is preferably 0.5 to 1 M. This concentration represents the concentration of each component in the solution for rapid cooling. Also, it is preferable that the operation temperature of the high-speed nitrogen cooling method is 50 to 200 K, preferably 80 to 120 K. The concentration and the temperature range are obtained experimentally as a range in which the EPRS_Tr2 protein of the present invention is able to withstand the nitrogen stream without affecting its crystallinity and is easy to store in high-speed nitrogen.

In order to accomplish another object of the present invention, the present invention provides a method for producing a modified EPRS protein, comprising: (a) reacting a modified compound of the present invention with a candidate compound; And (b) selecting a compound that interacts with the modified EPRS protein among the candidate compounds to determine a compound that inhibits the activity of the EPRS protein.

(a) reacting a candidate compound with the modified EPRS protein of claim 1;

The step (a) is a step of selecting or designing and reacting the candidate inhibitor substances to inhibit the activity of the EPRS protein.

The candidate compound may be a compound prepared using a conventional compound or a modified EPRS protein crystallization method or structure provided by the present invention. When designing a compound, new compounds can be designed based on the three dimensional crystal structure of the modified EPRS protein of the present invention or the role of the individual amino acids that affect the active site where the protein and proline and adenine bind.

The structure of the modified EPRS protein of the present invention shows detailed information between the enzyme active site and the biological control mechanism. All catalytically important residues at the active site are in a constant position and have an active structure. In the protein of the present invention having the amino acid sequence of SEQ ID NO: 1, histidine 242, threonine 240, 121, glutamic acid 123, arginine 152, arginine 163,278, 152, glutamate 237 residues are included. Histidine 242, threonine 240, 121, glutamic acid 123, arginine 152 residues interact with proline, and arginine 163,278, 152, glutamate 237 residues interact with adenosine (see Example 4-2).

(b) selecting a compound that interacts with the modified EPRS protein among the candidate compounds, thereby determining a compound inhibiting the activity of the EPRS protein;

The step (b) is a step of selecting a compound which interacts with (modified) the modified EPRS protein of the present invention and screening whether the selected compound inhibits the activity of the EPRS protein.

The modified EPRS protein inhibitor of the present invention can be used as a therapeutic agent for an autoimmune disease.

There have been several experiments in the root of Changshan herbal medicine that Halofuginone is effective in the treatment of autoimmune diseases. Recently, the mechanism of its effect has been revealed. Halofuginone inhibits autoimmune disease by activating the amino acid response pathway (AAR). The AAR, which detects nutrients, tells the cells when they need to preserve intracellular resources. For example, when a cell detects that the supply of amino acids needed for protein synthesis is limited, AAR blocks signals that promote inflammation, because tissues in which inflammation occurs require many proteins. Analysis of how halopujinone activates the AAR pathway has shown that halofuginone inhibits EPRS and inhibits the synthesis of proline. Inhibition of EPRS results in the depletion of proline leading to the activation of the AAR response, which alleviates autoimmune disease (Tracy L Keller et al. Nature Chem. Biology 8: 311317, 2012). Therefore, an inhibitor of the protein of the present invention including a PRS domain involved in proline synthesis can be used as a therapeutic agent for an autoimmune disease.

The present invention provides a crystal structure and a crystallization method of a modified EPRS protein having an amino acid sequence of SEQ ID NO: 1 including a PRS protein domain portion of a human-derived EPRS protein, and is effective for the development of an EPRS inhibitor using the same

FIG. 1 shows the amino acid sequence of EPRS (bifunctional glutamate / proline tRNA synthetase), and the red square shows the amino acid sequence of the PRS (proline tRNA synthetase) domain crystallized in the present invention.
FIG. 2 shows the result of amplifying the DNA encoding the EPRS_Tr2 protein of the present invention by PCR followed by extraction by electrophoresis.
FIG. 3 shows SDS-polyacrylamide gel electrophoresis results of cell precipitates obtained by culturing Escherichia coli transformed with the E. coli expression vector pET28a containing the EPRS_Tr2 protein coding gene of the present invention. ( C : Lysis of transformed Escherichia coli without IPTG Induction T : Total lysis of Escherichia coli obtained by induction with IPTG and time of the corresponding concentration S : Supernatant obtained from total sample P : Total Pellets from which samples were obtained).
FIG. 4 is a photograph showing the result of SDS-polyacrylamide gel electrophoresis after purifying EPRS_Tr2 protein of the present invention. ( M : size marker, 45 to 72 lane : protein elution fraction)
5 is a single crystal photograph of the EPRS_Tr2 protein of the present invention.
FIG. 6 is a photograph of a mixed crystal of EPRS_Tr2 protein, ATP, Mg, and proline of the present invention.
Figure 7 is a single crystal X-ray diffraction data image of the EPRS_Tr2 protein of the present invention.
FIG. 8 is a structural schematic diagram of the EPRS_Tr2 protein, which is completed and completed, showing a dimer structure.
9A shows a process in which an aminoacyl tRNA synthetase links an amino acid with a tRNA.
FIG. 9B is a structural schematic diagram showing that the residues of EPRS_Tr2 of the present invention interact with proline and adenosine, and shows a red square state of FIG. 9A.
( Blue : nitrogen element, red : oxygen element, white : carbon element)

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

EPRS_Tr2 gene expression

<1-1> Amplification of the EPRS_Tr2 gene

(The amino acid sequence of SEQ ID NO: 1) comprising the PRS (proline-tRNA synthetase) domain, among the EPRS proteins, based on the genomic sequence information of EPRS (bifunctional glutamate / proline-tRNA synthetase, Homo sapiens) Was amplified by PCR (polymerase chain reaction) using the following primers (see Fig. 1). The primers are listed in Table 1 below. The PRS domain was named EPRS_Tr2 in the present invention.

SEQ ID NO: directional order Length 3 Forward 5-aaaaa gctagc ggagcaggagaagggcagggg-3 32 mer 4 Reverse 5-aa ctcgag tca gtagctgcgaccaaataaggtgtagtacttg-3 42 mer

The primer having the nucleotide sequence of SEQ ID NO: 3 includes a nucleotide sequence corresponding to the NheI restriction enzyme recognition site (shaded region, gctagc), the primer having the nucleotide sequence of SEQ ID NO: 4 contains the termination codon (underlined portion, tca) , A restriction enzyme XhoI recognition site (shaded region, ctcgag).

The reaction mixture contained 1 μl of homo sapiens EPRS DNA, 2 μl of 10 mM dNTP (final concentration: 0.2 mM), 3 μl of 5 μM primer (final concentration: 0.3 μM), 0.5 μl of HotStarTaq DNA polymerase ), And 10ul PCR buffer (Quiagen) with 80.5ul of distilled water. The reaction solution was incubated at 95 DEG C for 10 minutes; 1 min 30 sec at 94 &lt; 0 &gt;C; 1 min at 58 캜; The reaction at 72 캜 for 1 minute 30 seconds was repeated 28 times. The reaction solution was separated by electrophoresis on 1% agarose gel to obtain about 1,500 base pairs of EPRS_Tr2 gene.

To 16 μl of this eluate was added 2 μl of restriction enzyme reaction buffer 10 times, 1 μl of restriction enzymes NheI and XhoI, and reacted at 37 for 20 minutes. Next, this reaction solution was separated by electrophoresis on 1% agarose gel, and a desired DNA fragment (hereinafter referred to as EPRS_Tr2 Nhe / Xho) was extracted and dissolved in 50 ul of distilled water. A photograph of the electrophoresis result is shown in Fig.

<1-2> EPRS _ Tr2  Generation of expression vector containing gene

Plasmid pET28a purchased from Quiagen was completely digested with restriction enzymes NheI and XhoI and separated on 1% agarose gel (hereinafter pET28a Nhe / Xho). 6 μl of EPRS_Tr2 Nhe / Xho was added to the reaction tube together with 3 μl of the above plasmid vector pET28a Nhe / Xho, and then 2 μl of a 10-fold ligated reaction solution (500 mM Tris-HCl, pH 7.8, 100 mM magnesium chloride, 100 mM DTT, 10 mM ATP ), 10 U of T4 DNA ligase, and distilled water was added thereto so that the total volume became 20 μl, followed by reaction at 37 ° C for 30 minutes.

The reaction solution was transformed into E. coli DH5 competent cells, transformed, and plated on LB-kanamycin medium at a concentration of 50 μl / ml to select E. coli transformants. Plasmids were extracted from these plasmids and confirmed by pET28a-EPRS_Tr2 by restriction enzyme and base sequence analysis, respectively. The nucleotide sequence of the EPRS_Tr2 gene cloned into the recombinant plasmid thus obtained was determined using the Big-Dye Cycle Sequencing System (Applied Biosystems, USA) according to the method of Sanger et al. (Sanger, F., et al. USA). As a result of the analysis by the ABI 377 DNA sequencer, it was confirmed that the nucleotide sequence of EPRS_Tr2 gene was SEQ ID NO: 2.

&Lt; Example 2 >

Purification of proteins

<2-1> Confirmation of EPRS_Tr2 encapsulation and overexpression in E. coli

The expression vector pET28a-EPRS_Tr2 obtained in Example 2 was transformed into Escherichia coli Rosetta2 (DE3) pLysS (Novagen Inc.) as an expression host. The transformed Escherichia coli strain was shake-cultured for 12 hours with shaking culture in Luria medium (LB, 1% pectin, 0.5% yeast extract, 1% sodium chloride) containing kanamycin at 100 ug / ml for 12 hours. 100 ug / ml of kanamycin) and IPTG (isopropyl-ß-D-galactopyranoside) was added at a final concentration of 1 mM when the absorbance of the bacteria reached about 0.6 at 600 nm at 37 ° C. 18 hours after the addition of IPTG, cell suspensions were collected by centrifugation at 5000 rpm for 10 minutes. The collected cell suspensions were suspended in a buffer solution of 20 mM Tris pH 8.0, 100 mM sodium chloride and 5 mM MgCl ₂ , and the cells were disrupted using an ultrasonic disintegrator (Fisher, USA) in an ice bath. After centrifugation at 13000 rpm for 1 hour with a centrifuge, the supernatant was separated.

In order to confirm the solubilization and overexpression of the target EPRS_Tr2 protein in the present invention, the transformed Escherichia coli not transformed with IPTG, the transformed Escherichia coli cultured with IPTG, the supernatant obtained by centrifugation at 13000 rpm and the pellet were subjected to SDS- PAGE, and the results are shown in Fig.

3], T is the total sample, S is the supernatant sample, and P is the pellet sample. A band of the target EPRS_Tr2 protein (58 kDa) is seen in the T and S samples (red square in FIG. 3), which means that the EPRS_Tr2 protein has been successfully hydrated and overexpressed.

&Lt; 2-2 > Purification of protein in disrupted Escherichia coli

The supernatant was bound to a pre-equilibrated nickel-nitrilotriacetic acid (Ni-NTA) column (Pharmacia, USA) with a buffer of 20 mM Tris pH 8.0, 100 mM sodium chloride, 5 mM magnesium chloride, After elution with an imidazole concentration gradient of -0.5M, only the EPRS_Tr2 protein portion was collected by SDS-PAGE.

The collected EPRS_Tr2 protein was concentrated to about 2 ml with an ion exchange resin, and then purified by using a gel filtration column (Superdex 200, 26/80, 100 mM sodium chloride, 1% glycerol, 5 mM DTT) equilibrated with 20 mM Tris pH 8.0, Pharmacia), separated by the molecular weight of the protein, confirmed by SDS-PAGE, and the EPRS_Tr2 protein was purified (see FIG. 4).

< Example  3>

Crystallization and cooling of the purified protein

 (EPRS_Tr2) purified in Example 2 by applying a known Hanging-drop vapor diffusion method (Jancarik, J. et al .. J. Appl. Cryst; 24: pp.409-411, The protein crystallized.

EPRS_Tr2 was added to a solution consisting of 1M sodium cacodylate trihydrate (pH 6.0) and 0.85M sodium citrate tribasic dihydrate, and the protein concentration was adjusted to 36 mg / ml to prepare a protein solution. Final protein concentration was determined by the Bradford method (Bradford, M., Anlytical biochemistry ; 72: pp.248-254, 1976).

 The final protein solution and the initial screen solution (Hampton Research Inc, Emerald Biosystems Inc) were used to retrieve the determination conditions step by step. As a result, a pH 6.5 condition consisting of 1.0 M sodium citrate / HCl and 0.1 M sodium cacodylate was found to be the most suitable.

1 μl of the EPRS_Tr2 protein solution and 1 μl of the reservoir solution were brought into contact with the surface of the microbridge and the slide was covered on a plate containing 0.5 ml of the reservoir solution and kept at 20 ° C. After 3 days, crystals were formed and protein monocrystals representing hexagonal space groups were obtained. The obtained crystal was photographed by a camera (SHW-M110S) (FIG. 5).

In order to solve the problem that the EPRS_Tr2 crystal is directly exposed to the X-ray, the EPRS_Tr2 crystal is determined by the high-speed nitrogen cooling method (Garman EF et al .. J. Appl. Cryst. 30, pp.211- 237, 1997).

The optimum condition for the rapid nitrogen cooling method was found to be that the rapid cooling solution consisting of 3.4 M sodium malonate, 0.1 M sodium cacodylate trihydrate (pH 6.0) and 0.85 M sodium citrate tribasic dihydrate had no harmful effect on the EPRS_Tr2 crystal 100 K of liquid nitrogen stream. Also, there was no ice ring (phenomenon in which water freezes when high-speed nitrogen cooling is not completed) often occurred during the experiment.

<Example 4>

Analysis of protein crystal structure

&Lt; 4-1 > diffraction  Data collection

Experiments were carried out by collecting data from a Marcromolecular Beam Line (PL-7A) in POSTECH with the EPRS_Tr2 crystal obtained in Example 3 above. The data limit of the EPRS_Tr2 determination is 2.6, the space group of crystals is the hexagonal space group (P3 ₁ 21), the lattice unit is a = 119.67 Å, b = 119.67 Å, c = 99.36 Å and α = ß = 90 ° and γ = 120 °.

ATP, Mg, proline were mixed with the EPRS_Tr2 protein in order to identify the amino acid residues in which the EPRS_Tr2 protein prepared in the above example interacts with proline and AMP. In order to induce the interaction of the molecules, the solution is dissolved in a solvent such as 1M sodium cacodylate trihydrate (pH 6.0), 0.85M sodium citrate tribasic dihydrate dissolved in EPRS_Tr2 protein solution, To make each concentration 5 mM, and then mixed crystals were produced. The resulting crystals were photographed as shown in Fig. The data limit of the mixed crystal is 2.3 ANGSTROM, and the space group of the mixed crystal is a hexagonal space group (P3 ₁ 21), which is the same as the EPRS_Tr2 single crystal.

A single crystal X-ray diffraction image of the EPRS_Tr2 protein is shown in Fig.

<4-2> Analysis of three-dimensional structure

The structure of the protein crystal was identified by molecular replacement and Zn-MAD (Multi-wavelength anomalous dispersion). The X-ray diffraction data were identified by a three-dimensional structure through a phasing step using structural programs Phenix, CCP4, CNS, and the like.

As a result of analyzing the protein structure, it was confirmed that it is a dimer structure including two monomers (see FIG. 8).

In addition, the electron density structure of crystals obtained by mixing ATP, Mg, proline and EPRS_Tr2 is shown in FIG. 9B. In FIG. 9B, amino acid residues of EPRS_Tr2 interacting with adenine, proline can be identified. The residues interacting with proline are histidine 242, threonine 240, 121, glutamic acid 123, arginine 152, and the residues interacting with the adenosine moiety are arginine 163, 278, 152, glutamate 237.

The present invention provides a crystal structure and a crystallization method of a modified EPRS protein having an amino acid sequence of SEQ ID NO: 1 including a PRS protein domain portion of a human-derived EPRS protein and can be used for the development of EPRS inhibitors using the same, It is highly available.

<110> Medicinal Bioconvergence Research Center <120> Crystal Structure and Crystallization of modified EPRS protein <130> NP12-0075 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 512 <212> PRT <213> Artificial Sequence <220> <223> EPRS_Tr2 protein AA sequence <400> 1 Gly Ala Gly Glu Gly Gly Gly Pro Lys Lys Gln Thr Arg Leu Gly Leu   1 5 10 15 Glu Ala Lys Lys Glu Glu Asn Leu Ala Asp Trp Tyr Ser Gln Val Ile              20 25 30 Thr Lys Ser Glu Met Ile Glu Tyr His Asp Ile Ser Gly Cys Tyr Ile          35 40 45 Leu Arg Pro Trp Ala Tyr Ala Ile Trp Glu Ala Ile Lys Asp Phe Phe      50 55 60 Asp Ala Glu Ile Lys Lys Leu Gly Val Glu Asn Cys Tyr Phe Pro Met  65 70 75 80 Phe Val Ser Gln Ser Ala Leu Glu Lys Glu Lys Thr His Val Ala Asp                  85 90 95 Phe Ala Pro Glu Val Ala Trp Val Thr Arg Ser Gly Lys Thr Glu Leu             100 105 110 Ala Glu Pro Ile Ala Ile Arg Pro Thr Ser Glu Thr Val Met Tyr Pro         115 120 125 Ala Tyr Ala Lys Trp Val Gln Ser His Arg Asp Leu Pro Ile Lys Leu     130 135 140 Asn Gln Trp Cys Asn Val Val Arg Trp Glu Phe Lys His Pro Gln Pro 145 150 155 160 Phe Leu Arg Thr Arg Glu Phe Leu Trp Gln Glu Gly His Ser Ala Phe                 165 170 175 Ala Thr Met Glu Glu Glu Ala Glu Glu Glu Glu Ile Leu Asp Leu             180 185 190 Tyr Ala Gln Val Tyr Glu Glu Leu Ala Ile Pro Val Val Lys Gly         195 200 205 Arg Lys Thr Glu Lys Glu Lys Phe Ala Gly Gly Asp Tyr Thr Thr Thr     210 215 220 Ile Glu Ala Phe Ile Ser Ala Ser Gly Arg Ala Ile Gln Gly Gly Thr 225 230 235 240 Ser His His Leu Gly Gln Asn Phe Ser Lys Met Phe Glu Ile Val Phe                 245 250 255 Glu Asp Pro Lys Ile Pro Gly Glu Lys Gln Phe Ala Tyr Gln Asn Ser             260 265 270 Trp Gly Leu Thr Thr Arg Thr Ile Gly Val Met Thr Met Val His Gly         275 280 285 Asp Asn Met Gly Leu Val Leu Pro Pro Arg Val Ala Cys Val Gln Val     290 295 300 Val Ile Ile Pro Cys Gly Ile Thr Asn Ala Leu Ser Glu Glu Asp Lys 305 310 315 320 Glu Ala Leu Ile Ala Lys Cys Asn Asp Tyr Arg Arg Arg Leu Leu Ser                 325 330 335 Val Asn Ile Arg Val Arg Ala Asp Leu Arg Asp Asn Tyr Ser Pro Gly             340 345 350 Trp Lys Phe Asn His Trp Glu Leu Lys Gly Val Pro Ile Arg Leu Glu         355 360 365 Val Gly Pro Arg Asp Met Lys Ser Cys Gln Phe Val Ala Val Arg Arg     370 375 380 Asp Thr Gly Glu Lys Leu Thr Val Ala Glu Asn Glu Ala Glu Thr Lys 385 390 395 400 Leu Gln Ala Ile Leu Glu Asp Ile Gln Val Thr Leu Phe Thr Arg Ala                 405 410 415 Ser Glu Asp Leu Lys Thr His Met Val Val Ala Asn Thr Met Glu Asp             420 425 430 Phe Gln Lys Ile Leu Asp Ser Gly Lys Ile Val Gln Ile Pro Phe Cys         435 440 445 Gly Glu Ile Asp Cys Glu Asp Trp Ile Lys Lys Thr Thr Ala Arg Asp     450 455 460 Gln Asp Leu Glu Pro Gly Ala Pro Ser Met Gly Ala Lys Ser Leu Cys 465 470 475 480 Ile Pro Phe Lys Pro Leu Cys Glu Leu Gln Pro Gly Ala Lys Cys Val                 485 490 495 Cys Gly Lys Asn Pro Ala Lys Tyr Tyr Thr Leu Phe Gly Arg Ser Tyr             500 505 510 <210> 2 <211> 1536 <212> DNA <213> Artificial Sequence <220> <223> EPRS_Tr2 protein DNA sequence <400> 2 ggagcaggag aagggcaggg gcctaagaaa cagaccaggt tgggtcttga ggcaaaaaaa 60 gaagaaaatc ttgctgattg gtattctcag gtcatcacaa agtcagaaat gattgaatac 120 catgacataa gtggctgtta tattcttcgt ccctgggcct atgccatttg ggaagccatc 180 aaggactttt ttgatgctga gatcaagaaa cttggtgttg aaaactgcta cttccccatg 240 tttgtgtctc aaagtgcatt agagaaagag aagactcatg ttgctgactt tgccccagag 300 gttgcttggg ttacaagatc tggcaaaacc gagctggcag aaccaattgc cattcgtcct 360 actagtgaaa cagtaatgta tcctgcatat gcaaaatggg tacagtcaca cagagacctg 420 cccatcaagc tcaatcagtg gtgcaatgtg gtgcgttggg aattcaagca tcctcagcct 480 ttcctacgta ctcgtgaatt tctttggcag gaagggcaca gtgcttttgc taccatggaa 540 gaggcagcgg aagaggtctt gcagatactt gacttatatg ctcaggtata tgaagaactc 600 ctggcaattc ctgttgttaa aggaagaaag acggaaaagg aaaaatttgc aggaggagac 660 tatacaacta caatagaagc atttatatct gctagtggaa gagctatcca gggaggaaca 720 tcacatcatt tagggcagaa tttttccaaa atgtttgaaa tcgtttttga agatccaaag 780 ataccaggag agaagcaatt tgcctatcaa aactcctggg gcctgacaac tcgaactatt 840 ggtgttatga ccatggttca tggggacaac atgggtttag tattaccacc ccgtgtagca 900 tgtgttcagg tggtgattat tccttgtggc attaccaatg cactttctga agaagacaaa 960 gaagcgctga ttgcaaaatg caatgattat cgaaggcgat tactcagtgt taacatccgc 1020 gttagagctg atttacgaga taattattct ccaggttgga aattcaatca ctgggagctc 1080 aagggagttc ccattagact tgaagttggg ccacgtgata tgaagagctg tcagtttgta 1140 gccgtcagac gagatactgg agaaaagctg acagttgctg aaaatgaggc agagactaaa 1200 cttcaagcta ttttggaaga catccaggtc acccttttca caagggcttc tgaagacctt 1260 aagactcata tggttgtggc taatacaatg gaagactttc agaagatact agattctgga 1320 aagattgttc agattccatt ctgtggggaa attgactgtg aggactggat caaaaagacc 1380 actgccaggg atcaagatct tgaacctggt gctccatcca tgggagctaa aagcctttgc 1440 atccccttca aaccactctg tgaactgcag cctggagcca aatgtgtctg tggcaagaac 1500 cctgccaagt actacacctt atttggtcgc agctac 1536 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward Primer to amplify EPRS_Tr2 DNA <400> 3 aaaaagctag cggagcagga gaagggcagg gg 32 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to amplify EPRS_Tr2 DNA <400> 4 aactcgagtc agtagctgcg accaaataag gtgtagtact tg 42

Claims (7)

1, the space group is represented by P3 ₁ 21, the unit cell parameter is represented by a = 119.67 Å, b = ₁ , _2, 3, 4, 119.67 Å, c = 99.36 Å, α = ß = 90 °, γ = 120 °, and a dimer structure.
(a) expressing and purifying a modified EPRS (Bifunctional Glutamate / proline tRNA synthetase) protein represented by the amino acid sequence of SEQ ID NO: 1 by removing 1000 amino acids at the N-terminal part of a human EPRS (Bifunctional Glutamate / Proline synthetase) step; And
(b) crystallizing the protein purified in step (a) with a preservative solution containing sodium citrate / HCl or sodium cacodylate as a precipitant. Crystallization method.
[3] The method of claim 2, wherein the crystallization method of step (b) is a droplet mixed steam equilibrium method. The method according to claim 2, wherein the concentration of sodium citrate / HCl is 0.5M to 1.5M, and the concentration of sodium cacodylate is 0.05M to 0.15M. &Lt; / RTI &gt;
The crystallization method according to claim 2, wherein the protein crystallization in step (b) is performed at a pH of 6.0 to 7.0.
The crystallization method according to claim 2, wherein the protein crystallization in step (b) is performed at a temperature of 15 to 25 ° C.
(a) reacting a candidate compound with the modified EPRS protein crystal of claim 1; And
(b) selecting a compound that interacts with the modified EPRS protein crystal among the candidate compounds to determine a compound that inhibits the activity of the modified EPRS protein
Lt; RTI ID = 0.0 &gt; EPRS &lt; / RTI &gt; protein activity inhibitor.

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