KR101821345B1 - Ubiquitin specific protease 47, three-dimensional structure thereof and method of developing a ubiquitin specific protease inhibitor - Google Patents

Abstract

The present invention relates to 47 USP47 (ubiquitin specific protease47) protein derived from Caenorhabditis elegans. The 3-dimensional crystal structure and the active site of the protein are identified and a novel USP47 protein inhibitor is screened Anticancer or anti-viral activity of a cell.

Description

[0001] UBIQUITIN SPECIFIC PROTEASE 47, THREE-DIMENSIONAL STRUCTURE THEREOF AND METHOD OF DEVELOPING A UBIQUITIN SPECIFIC PROTEASE INHIBITOR [0002] This invention relates to a three-dimensional structure of a protein having the ubiquitin-specific protease activity,

The present invention can be effectively used as an inhibitor of a protein having a ubiquitin-specific protease activity and a protein having a ubiquitin-specific protease activity.

Ubiquitin (Ub) is composed of 76 highly conserved amino acids and is present in all eukaryotic cells. Ubiquitin binds to the ε-amino group or rarely the N-terminal amino group of lysine residues (Lysine, Lys, K) of intracellular proteins. The ubiquitin binds to ubiquitin-activating enzyme (E1), ubiquitin-binding enzyme (E2) E3), the covalent bond between ubiquitin and substrate protein occurs sequentially, which is called ubiquitination. E1 enzymes exist in only one species in the body, but the number is the most. E2 exists in several kinds and generally serves to transfer ubiquitin from E1 to E3 or substrate. E3, also called E3 ligase, is the last step enzyme to attach ubiquitin to a substrate, and E3 will mainly determine the specificity of the substrate to be ubiquitinated. That is, the substrate capable of interacting with a given E3 enzyme is specifically determined. Post-transcriptional modification of these proteins changes the fate of substrate proteins such as changes in stability of substrate proteins, interaction with other proteins, and regulation of intracellular location.

Ubiquitin itself has seven lysine residues that are well conserved, so that these lysine residues can also bind other ubiquitin. Thus, the ubiquitination process can have a wide variety of forms depending on how many of the ubiquitin moieties are attached to which moiety. In order to distinguish this, the case where one Ub binds to a substrate protein is referred to as mono-ubiquitination, and the connection of several Ub's as branching is called poly-ubiquitination. In addition, most lysine residues can be ubiquitinated as the substrate protein contains several lysine residues. This is called multi-ubiquitination.

The modification of substrate proteins by ubiquitin plays an important role in regulating various processes in the cell. For example, it involves not only protein metabolism but also chromosome structure regulation, DNA repair, signal transduction, antigen presentation, viral toxicity, stress response and protein migration. These processes are often controlled by the ubiquitin-proteasome system (UPS), in which the poly-ubiquitin-bound protein is degraded by 26S proteasome. The 26S proteasome consists of two parts: a 19S complex with ATPase activity and a 20S proteasome with proteolytic activation.

Substrate protein modification by ubiquitination can occur reversibly, such as by modification by protein phosphorylation / dephosphorylation. That is, it is possible to remove ubiquitin from a substrate protein, which is called deubiquitination, and the enzyme group that activates this reaction is called deubiquitinase (DUB). The deubiquitinization process is still at an earlier stage than the ubiquitin binding process, but it is expected to contribute to the regulation of various physiological functions in the cell, Enzymes also activate the inactive precursor of ubiquitin. Recent deubiquitination studies have revealed the importance of deubiquitinating processes.

It has been reported that about 100 DUBs are present in humans, and the deubiquitinating enzymes are largely UCH (Ub C-terminal hydrolase), USP (Ub-specific processing protease), Otubain (OTU-domain Ub-aldehyde- It is divided into five groups: MJD (Machado-Joseph disease) and JAMM (Jab1 / Pad1 / MPN domain metallo-enzyme) family. Among them, USP is the largest group. In the case of USP, it has an active-domain consisting mainly of 350 amino acids and is a protease (protease). Although the degree of amino acid conservation of these USP active domains is not very high overall, the sites surrounding the active cysteine and histidine residues, namely the cysteine box and the histidine box, are well preserved have. The USP contains many domains that have not yet been shown to be functional in addition to these active domains, and it is presumably involved in substrate recognition, intracellular localization, and protein-protein interaction.

As mentioned above, Ub is not only very diverse in its binding method but also has almost all the functions in the cell, considering that the number of the substrate proteins revealed to date is considerable. Therefore, malfunctions and abnormalities of proteasome and ubiquitinization / deubiquitination systems are directly or indirectly linked to various human diseases, and studies on them are actively under way. In particular, Bertezomib / Velcade, which targets the proteasome in 2003, has been approved as a new drug, and many researchers as well as pharmaceutical companies are spurring on discovering and developing modulators targeting some of these enzymes, particularly E3 and DUB (Cohen & Tcherpakov, Cell 2010 143: 686; Lill and Wertz, Trends in Pharmacological Science 2014 35: 187).

USP47, one of the Ubiquitin-specific proteases (USPs), has not yet been studied. However, up to now, USP47 seems to play an important role in cell division and survival. For example, USP47 has been shown to interact with beta-Trcp, which plays an important role in cell cycle checkpoints, protein synthesis, cell growth and survival. In addition, USP47 siRNA treatment resulted in an increase in the amount of Cdc25A and a marked decrease in cell viability, suggesting that USP47 is closely related to cell survival (Peschiaroli et al ., 2010 Oncogene 29 ): 1384-93). In addition, USP47 has been reported to remove ubiquitin from ubiquitinated DNA polymerase beta (Parsons JL et al., Mol Cell 2011 41 (5) 609-15). Polymerase β is an essential enzyme in DNA base excision repair (BER) repairing damaged DNA base damage. It is known that BER malfunction can lead to premature aging, mutation, and even cancer. Recently, USP47 has also been reported to be involved in axon growth through regulation of caratin p60 with CHIP (C-terminus of HSP70-interacting protein) (Yang et al., 2013 J. Neurosci 33: 12728038).

In addition, USP47 has been reported to be involved in host cell death induced by influenza virus, together with TNFSF13 (APRIL) and TNFSF12 (TWE-PRIL), members of the Tumor necrosis factor ligand superfamily. In addition, knockdown of these proteins has been shown to inhibit at different stages in the proliferation of influenza virus, and USP47 has been shown to be involved in the entry of virus into host cells (Tran. Et al. , Cell Death Dis 2013 4e769).

Although the precise function of USP47 has not been elucidated yet, these reports indicate that USP47 is highly likely as a disease target, and a specific inhibitor that inhibits the activity of USP47 is expected to be an anticancer or antiviral drug. Indeed, US Progenra has already reported compounds that selectively inhibit only USP47 and USP7 based on their potential as anticancer targets, and they have also reported compounds with improved selectivity through further optimization (Weintock J et al ., ACS Med Chem. Lett 2012 3: 789092). For reference, USP7 studies have been conducted for a long time before USP47. USP7 is a typical DUB that regulates protein stability such as p53 and Mdm2. Inhibitors targeting USP7 include cancer, degenerative diseases such as Alzheimer's disease, Parkinson's disease It has been reported that it can be developed as a therapeutic agent for various diseases such as immunity, inflammation and infection.

Thus, the possibility of USP47 as a disease target is high, and development of an inhibitor selectively targeting only USP47 is also very important. To this end, the three-dimensional structure information as well as the production of the protein having the activity of USP47 can be used to understand the mechanism of USP47 and also to develop a substance capable of controlling it. In this way, almost all pharmaceutical companies have already utilized optimization of compounds based on the three-dimensional structure of the target protein, based on information on the structure of complexes of proteins and compounds. Especially, the complex with substrate can obtain much information about the active site, which can be a platform for the search and development of the inhibitor, which is significant. In order to study the three-dimensional structure of proteins, it is essential to design domains capable of securing soluble proteins, to establish a separation purification protocol capable of securing a large amount of high-purity proteins, and to secure crystallization conditions.

In order to meet such a demand, the present invention is to clone a region containing a domain showing the activity of USP47 Ubiquitin specific protease 47 derived from Caenorhabditis elegans ( C. elegans ) The water soluble protein was obtained and crystallized to identify the active site and the three - dimensional crystal structure. In addition, amino acid which contributes mechanismally to the activity was defined by the mutation test of the individual amino acid in the active site. Based on this information, a novel anti-cancer and antiviral agent applied to USP47 The present invention has been completed.

Accordingly, an object of the present invention is to provide a purified purified USP47 mutant protein.

It is another object of the present invention to provide a mutation complex of the USP47 protein and ubiquitin protein isolated and purified from a human.

It is still another object of the present invention to provide a nucleotide sequence encoding the USP47 protein or the mutant complex, and an expression vector containing the same, and a transformant transformed using the same.

Yet another object of the present invention is to identify the active site of the USP47 protein or the mutant complex.

It is yet another object of the present invention to identify the three-dimensional crystal structure of the USP47 protein or the mutant complex.

It is still another object of the present invention to provide a compound having an inhibitory activity by interacting with the USP47 protein using the active site and / or the three-dimensional crystal structure of the USP47 protein or the mutant complex or the purified USP47 protein and the purified ubiquitin A method for screening a compound having an inhibitory activity by interacting with a mutant complex comprising a protein, and a method for verifying the effect on a composition for a target protein for anticancer and antiviral.

The present invention relates to a mutant of USP47 (Ubiquitin specific protease47) protein derived from Caenorhabditis elegans ( C. elegans ) and a complex protein of the USP47 protein mutant and ubiquitin protein, wherein the USP47 protein variant and the activity The present invention relates to a technology for identifying new USP47 protein inhibitors or competitive inhibitors using the same to identify novel three-dimensional crystal structures, and to develop new activity regulating substances exhibiting new anticancer and antiviral effects applied to USP47.

Hereinafter, the present invention will be described more specifically.

The present invention provides a deubiquitinating enzyme, USP47, isolated and purified from C. elegans gene. In the present invention, the amino acid sequence of the protein was firstly developed for mass expression and high purity purification of USP47 protein in an Escherichia coli expression system (Rosetta DE3). As described above, the USP47 protein can be used as a target protein for anticancer and antiviral therapy directly or indirectly as a deubiquitinase, and it can be applied to USP47 specifically among many deubiquitinating enzymes to provide a more selective and efficient new anticancer drug It is very useful as a target protein in the development of antiviral therapeutic agents. The USP47 protein according to the present invention removes part of the C-terminal region of the protein in a range that does not impair the inherent tertiary structure and function of the USP47 protein, And is useful as a novel target protein for the development of novel anticancer and antiviral therapeutic agents based on structural analysis.

The USP47 protein used in the present invention is specifically USP47, which contains the catalytic domain.

As used herein, unless otherwise noted, the term "modified USP47 protein "," USP47 protein variant "or" USP47 variant "refers to the C- terminus of USP47 according to the invention, Means the partially deleted form of the USP47 protein.

The present invention provides USP47 protein variants. Also, the USP47 protein variant of the present invention is a modified USP47 protein comprising an amino acid fragment in which 820 amino acids at the C-terminal side of the amino acid sequence of SEQ ID NO: 1 are deleted. Preferably, a preferred example of the USP47 protein variant of the present invention is a protein having the amino acid sequence of SEQ ID NO: 2, 3 or 4. Hereinafter, the proteins having the amino acid sequences of SEQ ID NOS: 2, 3 and 4 are named "USP47T1", "USP47T2", and "USP47T3", respectively. In the present invention, the protein secondary domain structure and protein domain analysis data obtained from databases such as EXPASY and NCBI are used to remove the C-terminal fragment.

The present invention also provides a complex protein of the USP47 protein variant and a human-derived ubiquitin protein. More preferably, the human ubiquitin protein has the amino acid sequence of SEQ ID NO: 5. Hereinafter, the term "USP47 mutant complex" means a complex protein of the USP47 protein variant and a human-derived ubiquitin protein unless specifically mentioned in the present specification.

The present invention also relates to a USP47 protein variant having the amino acid sequence of SEQ ID NO: 2 to SEQ ID NO: 4 or a coding sequence of the protein variant and a promoter and terminator operably linked to the coding sequence, Lt; / RTI >

The vector may further comprise a conventional selectable marker such as a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, or the like. The present invention also provides a transformant transformed with a vector comprising a nucleic acid molecule encoding the USP47 protein variant. The host used for the transformant may be any host that is a prototype bacterium and has a pBR322 origin of replication and can produce T7 polymerase. For example, Escherichia coli can be used. Also, the present invention includes a step of transforming a host using the vector of the USP47 protein variant to produce a transformant containing the expression vector, and culturing the transformant to produce a USP47 protein variant Lt; RTI ID = 0.0 > USP47 < / RTI > variant.

For the production of the expression vector of the present invention, the coding sequence sequence of the C. elegans USP47 protein variant having the amino acid sequence up to glutamate, which is the 508th amino acid including the amino acid sequence with the deletion of the 818 amino acids at the C-terminus, In order to facilitate the purification of the purified protein, the USP47 protein variant can be produced in the form of a His-tag fusion protein for ease of purification.

The expression vector, the transformant, and the method for producing the USP47 protein variant using the transformant according to the present invention will now be described in detail. However, the following contents are only illustrative examples of the present invention, Is not limited thereto.

First, in order to amplify a gene encoding USP47 protein from genomic DNA of C. elegans, the polymerase chain reaction (PCR) corresponding to the 5'-terminal and 3'-terminal of the gene encoding USP47 protein , Hereinafter referred to as " PCR "). At this time, the primers corresponding to the 5'-terminal and the 3'-end are designed such that restriction enzyme recognition sites identical to the restriction enzyme recognition sites existing in the corresponding vector to be cloned are present, do.

PCR is carried out using the C. elegans genomic DNA as a template and the primer prepared as described above to amplify the USP47 gene. The amplified gene is cleaved with an appropriate restriction enzyme, and the obtained gene fragment is inserted into an appropriate expression vector such as an E. coli vector to prepare an expression vector containing the USP47 mutant gene. Usable restriction enzyme sites are: NdeI, NheI, BamHI, EcoRI, SalI, HindIII, NotI, and XhoI. In the examples of the present invention, NdeI and XhoI restriction enzyme sites were used. As described above, the expression vector includes a conventional selection marker such as a kanamycin resistance gene, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, etc. to facilitate identification of insertion into the host genome . After culturing an appropriate host such as Escherichia coli using the recombinant vector thus prepared, the transformant is cultured under conditions suitable for gene expression. In this case, the culture conditions vary depending on the host used, and the culture can be generally carried out at 15 to 40 ° C for 3 to 15 hours. The conditions are such that the incubation period is shortened at a relatively high temperature and the incubation time is relatively short It can be adjusted by lengthening.

Whether the desired protein is produced can be confirmed by conventional protein identification methods such as SDS-polyacrylamide gel electrophoresis or Western blotting. The transformant transformed with the expression vector containing the USP47 mutant gene is shake cultured in a medium (e.g., ruria medium) containing an antibiotic corresponding to a selection marker such as ampicillin, tetracycline, kanamycin, and chloramphenicol, , And the absorbance at 600 nm is 0.3 to 0.7), IPTG (isopropyl-beta -D-galactopyranoside) is added to induce the expression of the USP47 mutant gene. The culture solution is centrifuged to precipitate the transformant, which is then suspended in a suitable buffer solution and then disrupted. The lysate was centrifuged to separate the supernatant containing the protein, and the resulting USP47 protein was separated and purified using conventional protein separation and purification means such as ion exchange resin, affinity binding resin and gel filtration chromatography. Refine. At this time, the USP47 protein variant can be produced in the form of a histidine-tag fusion protein for ease of purification.

In another aspect, the present invention discloses the three-dimensional structure and active site of the USP47 protein variant or USP47 mutant complex to provide more useful information in screening inhibitory compounds targeting the USP47 protein variant or the complex protein .

In addition, the present invention relates to a three-dimensional crystal structure of the USP47 protein variant or USP47 mutant complex identified as described below and / or to a specific positional characteristic of binding of USP47 to ubiquitin and to the activity of the individual amino acids Role and additionally includes residues that affect activity based on residues and structural changes that result in structural changes that occur when the USP47 protein variant or the complex protein binds to ubiquitin.

The present invention also provides a USP47 protein variant having an amino acid sequence of any one of SEQ ID NOS: 2 to 4 as a target compound; Or a USP47 mutant complex having the amino acid sequence of SEQ ID NO: 5 and the USP47 protein mutant having the amino acid sequence of any one of SEQ ID NOs: 2 to 4, and USP47 mutant complex having the amino acid sequence of SEQ ID NO: 5 is used .

In one embodiment of the present invention, the new drug development method comprises: reacting a USP47 protein variant having the amino acid sequence of SEQ ID NO: 2, or a USP47 mutant complex having the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5 with the candidate compound; And selecting a compound that interacts with the USP47 protein variant or the complex protein among the candidate compounds to screen for a compound that inhibits the activity of the USP47 protein variant or the complex protein.

Preferably, the USP47 protein variant or the complex protein is selected from the group consisting of polyethylene glycol (PEG), and imidazolmalate, ammonium sulfate and 2- [N-morpholino] ethanesulfonic acid (MES) In contact with a reservoir solution comprising at least one precipitant selected from the group consisting of:

In another embodiment, the method for developing a new drug of the present invention uses the X-ray diffraction pattern of the USP47 protein variant and the complex protein of the following Tables 1 and 2 or the three-dimensional crystal structure information of Tables 3 and 4, USP47 protein variant and a novel compound capable of binding to the complex protein in a de-novo design.

In yet another embodiment, the method of the present invention for developing a new drug of the present invention is characterized in that the information on USP47 or an X-ray diffraction pattern of the complex protein shown in the following Tables 1 and 2 or the following three- And screening for a compound capable of binding to the USP47 protein variant or the USP47 mutant complex using virtual screening that matches the three-dimensional crystal structure information of the candidate compound.

Upon selection of a compound that interacts with (binds to) the USP47 protein, information on the three-dimensional crystal structure and / or active site, the structural change site caused by the binding of USP47 and ubiquitin, To thereby more easily and accurately select compounds that inhibit USP47 protein activity. Specifically, the new drug development method includes a finger domain region different from USP2, USP4, USP7, USP8, USP14 or USP21 among the candidate compounds and the USP47 protein variant; The site of the catalyzed tunnel between the thumb and the palm containing the cysteine 97 in the USP47 protein variant; Or whether they interact with each other or not. Also, the new drug development method includes site information targeting target 97 cysteine at the activation site of the USP47 protein variant, which is changed upon binding of the candidate compound, the USP47 variant and the USP47 variant complex and the ubiquitin. Site information targeting a portion of the ubiquitin-binding site of the complex; Or whether they interact with each other or not.

In another aspect, the present invention relates to a composition for screening an anticancer agent and an antiviral agent, which comprises the USP47 protein variant or the USP47 mutant complex as a target protein. Preferably, the virus is an influenza virus.

In another aspect, the present invention relates to a method for screening an anticancer agent or an antiviral therapeutic agent, wherein the USP47 protein variant or the USP47 mutant complex is used as a target protein. Preferably, the virus is an influenza virus.

Among the deubiquitinating enzyme USPs of which the anticancer agent and antiviral agent activity are different from each other, all the deubiquitinating enzymes which essentially require the USP47 protein and the protein having the similar active site, such as USP1, USP2, USP4 , USP7, USP8, USP14, USP15, USP21, and USP44.

The present invention provides a method of crystallizing a modified USP47 protein or a modified USP47 and ubiquitin complex protein. In identifying the three-dimensional structure of the target protein for development of new anticancer and antiviral, it is important that the target protein is soluble and crystalline. Since the USP47 protein according to the present invention is water-soluble, a crystallization step is required to have crystallinity in order to identify its three-dimensional structure. In such a crystallization step, generally, X-ray crystallography is used, and various crystallization methods must be performed as pretreatment steps for using such X-ray crystallization method. In the embodiment of the present invention, the crystallization method such as a sitting-or hanging-drop vapor diffusion method (Jancrik J. et al ., Appl. Cryst., 24, 409-411 Incorporated herein by reference) may be used.

The principle and procedure of crystallization of the droplet mixed steam equilibrium method are as follows. When a small droplet of mother liquor and a much larger reservoir solution coexist in an enclosed space, the migration of water or other volatile material occurs 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 and the like are mixed at various concentrations such as precipitant, protein solution and reservoir solution of such various conditions usually mixed at a ratio of about 1: 1, . 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 droplets differs from the concentration of the reservoir solution, so that the protein does not exist in the crystalline state. However, if the protein is placed in the 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 reservoir solution, the pH of the solution and the temperature of the solution are selected depending on the kind of the protein, It becomes a very important factor.

In the present invention, when the water-soluble USP47 protein or the variant complex is crystallized by using a mixed solution balance method using a reservoir solution, imidazole maleate, ammonium sulfate, polyethylene glycol (PEG) Average molecular weight 1,000 to 20,000) and 2- [N-morpholino] ethane sulfonic acid, and it is preferable to use ammonium sulfate, imidazole maleate and polyethylene glycol together. Preferably, the molecular weight of the polyethylene glycol is 3350 or 4000.

The concentration of the precipitant in the whole reservoir solution can be appropriately adjusted depending on the kind of the precipitant used. Specifically, the concentration of ammonium sulfate in the whole tank solution is preferably 0.05 to 1.5 M, the imidazole maleate is preferably 0.05 to 2 M, and the polyethylene glycol is preferably 5 vol% to 40 vol% (v / v) )%. If the concentration of the precipitant in the reservoir solution is higher than the above range, the protein may precipitate. If the concentration is lower than the above range, crystallization does not occur. In addition, 2- [N-morpholino] ethanesulfonic acid (MES) is related to the stability of the protein mainly in the reservoir solution as a buffer solution. If the concentration is out of the above range, the stability of the protein in the reservoir solution And it is difficult to obtain the desired crystal. The reservoir solution may contain commonly used salts, buffers, detergents and the like in addition to the above-mentioned precipitants.

In addition, the drop mixing steam equilibrium method is preferably performed at a pH of 5.0 to 8.0. The most excellent crystal can be obtained when the reaction is carried out under the pH of the above range. The reaction temperature of the crystal formation reaction by the droplet mixed vapor equilibrium method is preferably 4 to 26 캜, more preferably 16 to 26 캜, more preferably 20 to 24 캜. The reaction period is preferably from 1 day to 20 days, more preferably from 1 week to 2 weeks. The reaction temperature and the reaction time range are experimentally found as conditions under which crystals having optimal data can be easily obtained by x-ray analysis.

X-ray analysis can be used to analyze the structure of the produced crystalline USP47 protein or the complex protein. However, when protein crystals are exposed to high energy of X-rays, their lifespan is shortened and the data intensity is weak, resulting in poor results in structural analysis. In order to avoid such drawbacks, it is preferable to perform USP47 crystals or high-speed nitrogen cooling method in which the complex protein is nitrogen-cooled rapidly before X-ray analysis. For high-speed nitrogen cooling, it is desirable to use ethyleneglycol and glycerol oils in addition to the crystalline conditions for effective protein crystal protection. When ethylene glycol is used, it is preferable to use it at a concentration of 20 to 35% by volume, preferably 25 to 30% by volume. The glycerol is preferably used at a concentration of 15 to 35% by volume, preferably 20 to 25% by volume (v / v)%. It is preferable that the performance temperature of the high-speed nitrogen cooling method is 50 to 200 K, preferably 80 to 120 K. The concentration and temperature range were obtained experimentally as a range where the USP47 protein can withstand the nitrogen stream without affecting its crystallinity and is easy to store in high-speed nitrogen.

The present invention also provides a three-dimensional crystal structure of the USP47 protein or the complex protein crystallized by the above-described method obtained through X-ray crystallization. In one embodiment of the present invention, in order to determine the three-dimensional structure of the crystal, a diffraction pattern is obtained using an X-ray image plate and phase information is obtained by multiwavelength anomalous dispersion (MAD) Can be obtained. Dimensional structure can be obtained by preparing an electron density map from the X-ray diffraction pattern and the phase information of the USP47 protein crystal obtained as described above, and then deriving the atom coordinates from the electron density map.

The method of collecting the diffraction pattern data using the X-ray can be divided into the method using the home source and the synchrotron according to the source of the X-ray. Of these, even a crystal having a crystal size of about 50 탆 can be used by using the radiation, and data can be collected not only at one wavelength but also at a plurality of wavelengths, so that a rapid structure can be obtained. Therefore, in a preferred embodiment of the present invention, diffraction pattern data can be obtained using a synchrotron radiation accelerator. Diffraction pattern data of the thus obtained USP47 protein variant crystal and the USP47 mutant complex are shown in Tables 1 and 2, respectively . The diffraction pattern data of the USP47 protein variant of SEQ ID NO: 2 is shown in Table 1, and the diffraction pattern data of SEQ ID NO: 3 and the complex of ubiquitin is shown in Table 2.

[Table 1]

[Table 2]

The phase information can be obtained by multiple isomorphous replacement, single wavelength anomalous dispersion, and Molecular replacement. In a preferred embodiment of the present invention, the USP47 protein variant and USP47 The phase information for the crystals of the transition complex can be obtained (see Modern X-ray Analysis on Single Crystals, Peter Luger). Specifically, the multi-wavelength analysis method calculates phases using anomalous diffraction through three different wavelengths in a protein crystal having a heavy metal. In order to utilize this multi-wavelength analysis method, the phase must be calculated and the initial model calculation must be performed, and all the programs that can be used for such calculations can be used. In a preferred embodiment of the present invention, SOLVE and RESOLVE (Los Alamos Institute, USA) can be used, and then programs such as CNS (Yale University) and CCP4 (University of Cambridge) can be used for the refinement process and the standardization process.

The refinement step is performed by modifying the electron density from the X-ray data into a structure that best fits the computer monitor with O (Alwyn Jones) and COOT (Paul Emsly) programs. As a result of the refinement step, crystals of the USP47 protein mutant according to the present invention contained two zinc ions and 124 water molecules, and the crystals of the USP47 mutant and the USP47 mutant complex shown in Table 3 below contained two zinc ions And can be represented by an atomic model having a three-dimensional crystal structure as shown in Table 4 below. In the development of a new drug targeting the USP47 protein, the USP47 protein variant may be used singly or in the form of a USP47 mutant and USP47 mutant complex. In particular, It is very useful for development.

Table 3 below shows the crystal data and refinement results of the three-dimensional phase of the USP47 protein variant, and Table 4 shows crystal data and refinement results of the three-dimensional phase of the mutant complex protein containing the USP47 mutant and ubiquitin. Table 3 relates to the crystal data of the USP47 protein variant of SEQ ID NO: 2, and Table 4 relates to the crystal data of the USP47 protein variant and the ubiquitin complex of SEQ ID NO: 3.

[Table 3]

[Table 4]

Table 5 shows the atomic model coordinates of the three dimensional crystal structure of the USP47 protein variant of SEQ ID NO: 2 and Table 6 is the atomic model coordinates of the USP47 mutant of SEQ ID NO: 3 and the three dimensional crystal structure of the ubiquitin complex protein. All of these atomic coordinates were improved using Phenix.

[Table 5]

[Table 6]

After the refinement step, additional analysis steps may be performed to obtain various information from the obtained atomic model. For example, the USP47 variant, USP47 variant and ubiquitin complex model of the three-dimensional space can be graphically displayed, and the distance and space between each atom can be measured by observing the morphology of the active moiety. In the case of the important moiety, And then a modeling procedure for finding a suitable inhibitor can be performed. As a result of the above analysis step, the USP47 protein was structurally located at a site where a well-conserved residue between the thumb and palm domains was present, i.e., located in the catalyzed tunnel, May be the active site of the USP47 protein.

In addition, through mutation experiments, amino acid sequence showing the effect of USP47 protein activity and excellent binding property with ubiquitin was confirmed. For this purpose, one of the amino acid sequences of SEQ ID NO: 2 was selected as a target amino acid, The coding sequence is mutated to encode another amino acid, for example alanine, to prepare a primer. At this time, the 5 'terminal primer and the 3' terminal primer are both used, and the 3 'terminal is modified so as to include the base sequence encoding the amino acid, and the desired recombinant clone is obtained by the PCR method described above. At this time, both the 5'-terminal primer and the 3'-terminal primer are used, and a nucleotide sequence coding for the amino acid to be changed is interposed between these primers. Thus, a desired recombinant clone is obtained by the PCR method described above. The recombinant clone thus obtained is completely digested by the restriction enzyme such as DpnI to obtain the original vector obtained in Escherichia coli. At this time, the reaction time of the restriction enzyme (DpnI) is preferably 30 minutes to 2 hours. Then, only the recombinant clone is introduced into E. coli to obtain a mutant USP47 protein having a desired trait.

Through the mutation experiment conducted in this way, when the 97-position cysteine of the USP47 protein (SEQ ID NO: 2) according to the present invention was substituted with serine (S97), the activity was decreased but the binding force with ubiquitin was greatly increased, It was also found that the substitution of 97 position cysteine with alanine (A97) significantly reduced its activity. The amino acid is an important amino acid that affects the activity of the USP47 protein.

Therefore, the catalytic tunnel between the thumb and finger domains of the USP47 protein variant (SEQ ID NO: 2) according to the present invention is an amino acid in which the cysteine at position 97 and the cysteine at position 97 are substituted with serine, (F215) to 226-position valine (V226) from the finger domain 215 at the time of finger motion, and a beta-strand composed of 11 amino acids and 21 amino acids at position 282 of glutamate (E261) And a more effective USP47 protein inhibitor can be selected by detecting whether or not the protein interacts with at least one site selected from the group consisting of < RTI ID = 0.0 >

In addition, the present invention provides a computer-readable storage medium, such as a floppy diskette, a hard disk, etc., for information on the three-dimensional structure of the USP47 mutant identified above. The three-dimensional structure stored in the medium may include all or a part of the atomic coordinates shown in Tables 5 and 6, or may include information on the amino acid of the active site and a site containing the mutant amino acid residue have. Preferably, the X-ray diffraction pattern data of Table 1 and the three-dimensional crystal structure information of Table 3; Finger domain region information different from USP4, USP7, USP8, USP14 or USP21 among USP47 protein variants; Site information of the catalyzed tunnel between the thumb and the palm including the 97th cysteine of the USP47 protein variant; Or both. Also preferably, the X-ray diffraction pattern data in Table 2 and the three-dimensional crystal structure information in Table 4; USP47 protein variant and USP47 mutant complexes and ubiquitin-binding site of the USP47 protein variant targeting the cysteine 97 and its vicinity; Site information targeting a portion of the ubiquitin-binding site of the complex; Or both.

The present invention also provides a method for developing a new drug, which comprises selecting or designing a compound that interacts with the USP47 mutant or the USP47 mutant complex using the storage medium.

Based on the three-dimensional crystal structure of the USP47 protein variants identified above and the specific positional characteristics of USP47 and ubiquitin binding and the role of individual amino acids affecting activity at the activated site, the present invention provides USP47 protein variants or USP47 A USP47 protein inhibitor that inhibits the activity of the variant complex. A method for screening a protein inhibitor according to the present invention comprises the steps of:

A USP47 protein variant having the amino acid sequence of SEQ ID NO: 2 or a USP47 variant having the amino acid sequence of SEQ ID NO: 3 and SEQ ID NO: 5 and a USP47 mutant complex with the candidate compound; and

Selecting a compound that interacts with the USP47 protein variant or the USP47 variant complex among the candidate compounds.

The USP47 protein variant or USP47 variant complex may be one which has been crystallized by the crystallization method as described above. In selecting a compound that interacts with the USP47 protein variant or USP47 variant complex, compounds that more easily and accurately inhibit USP47 protein activity are selected using information about the three-dimensional crystal structure and / or active site as described above can do. In addition, the screening method of the present invention may be the one using the storage medium as described above.

As described above, the present invention provides anti-cancer and anti relates to a viral target of USP47 protein mutant protein, known as and the USP47 protein variants and ubiquitin mutant complexes, their active sites and the three-dimensional crystal containing structure, according to the invention C The crystals of the USP47 protein variant derived from E. elegans strains have excellent crystallization degree and are easy to analyze by X-ray and have different activation sites from other known deubiquitinating enzymes (USP2, USP4, USP8, USP14, USP21) And a binding site, and shows a three-dimensional structure of crystals similar to those of USP7. Therefore, the USP47 protein variant can be usefully used for the development of other novel activity regulators which are different from other deubiquitinating enzymes (USP2, USP4, USP8, USP14, and USP21). In addition, USP7, which is structurally similar to the USP47 protein variant, may be used as a dual inhibitor in the case of an inhibitor targeting the active site.

1 shows the process of cloning the coding gene of the "USP47 T1" protein of the present invention into the E. coli expression vector pET28a, respectively.
FIG. 2 is a graph showing the results of SDS-polyacrylamide gel electrophoresis of the protein precipitate obtained by culturing Escherichia coli transformed with the E. coli expression vector pET28a containing the "USP47 T1" protein coding gene of the present invention .
3A to 3C are photographs of SDS-polyacrylamide gel electrophoresis after purification of the "USP47 T1" protein obtained in the present invention, wherein 3a is an electrophoresis image using a nickel column and 3b is an ion exchange resin And 3c is an electrophoresis image obtained by purifying the protein using a sizing column.
FIG. 4 is a photograph showing the binding of USP47 protein variant and ubiquitin prepared using SEQ. ID. NO: 3 with a sizing column, showing that the USP47 protein variant and ubiquitin can bind in a water soluble state.
5 shows the isothermal titration calorie of the interaction between ubiquitin and USP47T1 (SEQ ID NO: 2), USP47T2 (SEQ ID NO: 3), or USP47T3 (SEQ ID NO: 4) using isothermal titration calorimetry iTC200, MicroCal .
Figure 6 shows the activity of USP47T1 (SEQ ID NO: 2) on the specificity and efficiency of cutting two ubiquitin chains linked together using lysine (Lys6, Lys11, Lys29, Lys48, Lys63) present in ubiquitin.
Figure 7 is a crystal of a purified USP47 protein variant.
8 is a crystal of the USP47 mutant complex comprising the purified USP47 protein variant and Uibiquitin using SEQ ID NO: 3.
9 shows the skeletal structure of the entire USP 47 as a C alpha ribbon.
Fig. 10 shows the entire skeleton structure of the USP47 protein variant and Uibiquitin complex purified using SEQ ID NO: 3 as a C alpha ribbon.
FIG. 11 shows the activity of the USP47 protein variant after the same spatial positions as FIG. 9 and FIG. 10, showing the major residue changes.

Hereinafter, the present invention will be described in more detail based on examples. However, the following examples are only illustrative of the present invention, and the scope of the present invention is not limited thereto.

≪ Example 1 > Caenorhabditis elegans  Amplification and Expression of the Gene of the USP47 Protein Mutant Derived from

Step 1: Amplification of the USP47 mutant gene

SEQ ID NO. Using the coding base sequence of the C. elegans USP47 protein having the amino acid sequence from the first methionine terminal (N-terminal) to the 508th amino acid glutamate (E), the purified protein can be easily purified , A histidine fusion (His-tag) protein may be bound to the N-terminus. For the synthesis and amplification of the genes, the following primers synthesized using a nucleic acid synthesizer were used for PCR reaction.

Forward primer: 5 'ggggaattccatatggtacgggtcgaggactc 3' (SEQ ID NO: 6)

Reverse primer: 5 'ccgctcgagttattccgcctctcgttcaagtc 3' (SEQ ID NO: 7)

The following primer was used to prepare a (C97S) USP47T2 C97S mutant DNA fragment in which the cysteine at position 97 of USP47 was substituted with serine.

Forward primer: 5 'acccaggcaatgactagttatttgaacagtctt 3' (SEQ ID NO: 8)

Reverse primer: 5 'aagactgttcaaataactagtcattgcctggtt 3' (SEQ ID NO: 9)

USP47T3 C97A mutant DNA fragment (C97A) in which cysteine at position 97 of USP47 was substituted with alanine was prepared using the following primers.

Forward primer: 5 'acccaggcaatgactgcatatttgaacagtctt 3' (SEQ ID NO: 10)

Reverse primer: 5 'aagactgttcaaatatgcagtcattgcctggtt 3' (SEQ ID NO: 11)

The forward primer includes a nucleotide sequence corresponding to the Nde1 restriction enzyme recognition site (SEQ ID NO: 6, 8, and 10), and the reverse primer includes a base corresponding to the XhoI restriction enzyme recognition site (SEQ ID NO: 7, 9, Sequence.

PCR was performed as follows using C. elegans USP47 as a template. 1 μl of each of the above-mentioned C. elegans USP47 full-length cDNA, 8 μl of 2.5 mM dNTP, each 100 pmol forward primer (SEQ ID NOs: 6, 8 and 10) and reverse primers (SEQ ID NOs: 7, 9 and 11), PfuTaq 1 μl of DNA polymerase (5 U / μl, Stratagene, USA) and 10 μl of PCR buffer (Stratagene) were added 79 μl of distilled water to prepare a reaction solution. The reaction solution was incubated at 82 ° C 2 minutes, 94 ° C for 1 minute, 94 ° C for 30 seconds, 56 ° C for 1 minute and 30 seconds, and 72 ° C for 4 minutes and 30 seconds. The reaction solution was separated by electrophoresis on 0.8% agarose gel to elute the gene. To each well of 16 μl of the obtained eluate was added 2 μl of restriction enzyme reaction buffer (10 times) and 1 μl of restriction enzyme Nde1 (NEB (New England Biolabs, USA) and Xho I (NEB, USA) The obtained reaction solution was separated by electrophoresis on 0.8% agarose gel, and the desired USP47 protein mutant coding DNA fragment was dissolved in 50 μl of distilled water solution, which was dissolved in USP47T1 N / X, USP47T2 N / X, USP47T3N / X.

Step 2: Preparation of expression vector containing USP47T1, T2, T3 gene

Plasmid pET-28a (Novagene Inc., USA) expresses 6 histidine residues at the N-terminus. The plasmid pET-28a is treated with restriction enzymes NdeI and XhoI and then subjected to electrophoresis to separate DNA fragments of about 5400 bp size. 28a N / X. 0.5 μg of USP47T1, T2, and T3 N / X were placed in a reaction tube with 0.1 μg of pET-28a N / X and then added to 2 μl of a 10-fold ligation reaction solution (20 mM Hepe-HCl, pH 7.8, 100 mM MgCl ₂ , 100 mM DTT, and 10 mM ATP), 10 U of T4 DNA ligase, and distilled water was added to make the total volume 20 μl, followed by reaction at 16 ° C for 12 hours. This reaction solution was transformed into competent cells of Escherichia coli Rosetta (DE3) (Novagene Inc., USA) and transformed into kanamycin medium containing 1 μg / ml LB (1% Yeast extract and 1% sodium chloride) to select E. coli transformants. The plasmids were extracted from them and the recombinant plasmids pET-28a-USP47T1, pET-28a-USP47T2 and pET-28a-USP47T3 were obtained by restriction enzyme and base sequence analysis.

Plasmids were extracted from these plasmids and pET-28a-USP47T1, pET-28a-USP47T2, and pET-28a-USP47T3 obtained by linking the USP47T1, T2, and T3 DNA fragments prepared in step 1 to the plasmid p28a by restriction enzyme and base sequence analysis Respectively. The process of cloning the coding genes of the USP47T1 protein into the E. coli expression vector pET28a is shown in FIG.

Step 3: Expression of USP47 variant in E. coli

Escherichia coli Rosetta (DE3) (Novagen Inc., USA), which is an expression host, was transformed with the expression vectors pET-28a-USP47T1, T2, and T3 obtained in the above step 2 by a conventional method. The transformed Escherichia coli strain was shake-cultured in LB medium containing 100 μg / ml of kanamycin for 12 hours, and then 1 ml was added to 100 ml of LB medium (containing 100 μg / ml of kanamycin) And cultured at 37 캜 until the absorbance of the culture was about 0.6. Then, the incubation temperature was lowered to 18 캜, and IPTG (isopropyl-β-D-galactopyranoside) was added to a final concentration of 0.6 mM. 1 ml of each of the culture broths before and 15 hours after addition of IPTG was centrifuged at 10,000 g for 2 minutes to collect the cell precipitates. The collected precipitate was subjected to 15% SDS-polyacrylamide gel electrophoresis and analyzed by staining protein (USP47T1) with Coomasie Brilliant Blue (Bio-Rad 161-0400). FIG. 2 shows the result of SDS-polyacrylamide gel electrophoresis of the cell precipitate obtained by culturing Escherichia coli transformed with the E. coli expression vector pET28a containing the coding gene for the obtained protein USP47 T1.

Example 2 Amplification and Expression of Modified Human Uiquitin Gene

SEQ ID NO. 5, the coding sequence sequence of the human Ubiquitin protein having the amino acid sequence from the 1 st methionine terminal (N-terminal) to the 76 amino acid glycine (G) is used and the purified protein is easily purified , A histidine fusion (His-tag) protein may be bound to the N-terminus. For the synthesis and amplification of the gene, a human Ubiquitin T1 DNA fragment was prepared in the same manner as in step 1 of Example 1, except that the following primers synthesized using a nucleic acid synthesizer were used:

Forward primer: 5 'ggggaattccatatgatgcagatcttcgtgaagactctg 3' (SEQ ID NO: 12)

Reverse primer: 5 'ccgctcgagactcccacctctgagacggagcaccaggtgcag 3' (SEQ ID NO: 13)

The forward primer Nde1 restriction enzyme recognition site was used and the reverse XhoI restriction enzyme was used. A pET28a-UbT1 vector containing a UbTl DNA fragment was prepared in the same manner as in step 2 of Example 1. Using the obtained pET28a-UbT1 vector, the protein (pET28a-UbT1) was expressed in the same manner as in step 3 of Example 1.

Example 3 Purification of Modified USP47 Protein

Step 1: Culture and disruption of E. coli cells

In the same manner as in Examples 1 and 2, E. coli cells expressing modified USP47 proteins (USP47T1, T2 and T3) were cultured in a volume of 4 L, and then centrifuged at 3,000 rpm for 15 minutes using a centrifuge, The cell precipitate was collected and suspended in a buffer solution of 20 mM Hepes-hydrochloric acid buffer (pH 7.2), 150 mM sodium chloride and 1 mM TECP [Tris (2-carboxyethyl) phosphine hydrochloride] Dismembrator, Fisher, USA). The resulting solution was centrifuged at 16,000 rpm for 30 minutes using a centrifuge to obtain supernatant.

Step 2: Purification by column chromatography

The supernatant obtained in step 1 was dissolved in the above-mentioned buffer solution using a nickel-nitrilotriacetic acid (Ni-NTA) column (GE Healthcare Life Sciences, USA), ion exchange resin ion (GE Healthcare Life Sciences, , And a sizing column (GE Healthcare Life Sciences, USA). The same buffer as above was eluted with an elution gradient of 0-1 M imidazole, followed by SDS-PAGE in Examples 1 and 2 The fractions containing the modified USP47 protein were identified, and the fractions were collected. The modified USP47 protein fraction obtained in the above procedure was injected into an anion exchange resin (Mono Q, GE Healthcare Life Sciences, USA) equilibrated with the same buffer solution except for 20 mM imidazole, and then a 0-1 M sodium chloride concentration gradient And subjected to SDS-PAGE to identify and collect fractions containing the modified USP47 protein of Example 1 and Example 2, respectively. The protein solution was concentrated to about 10 ml, and the supernatant was injected into a gel permeation column (Superdex 200, 26/60, GE Healthcare Life Sciences, USA) pre-equilibrated with the same buffer solution as described above, and eluted with the buffer, . SDS-PAGE was performed to identify and collect fractions containing the modified USP47 proteins of Example 1 and Example 2, respectively. The purified and collected proteins for the modified USP47 protein of Example 1 were named USP47 Truncation T1 (USP47 T1), USP47 Truncation T2 (USP47 T2) and USP47 Truncation T3 (hereinafter "USP47 T3"). Respectively.

3A to 3C show results of SDS-polyacrylamide gel electrophoresis after purifying the USP47 T1 protein obtained above, 3a is an electrophoresis image using a nickel column, and 3b is an ion exchange resin And 3c is a photograph of electrophoresis after purification of the protein using a sizing column.

USP47 Truncation T2 and Ubiquitin Truncation T1 (hereinafter referred to as "UB T1") were combined with each other and then reacted on ice for 4 hours. Then, USP47 Truncation T2: Ubiquitin Truncation T1 ") ≪ / RTI > protein is shown in FIG. From the above results, it was confirmed that USP47 protein and ubiquitin bind in a water-soluble state.

Example 4 Crystallization of USP47 by Drop Mixed Steam Equilibrium

The USP47T1 protein obtained in Example 3 was crystallized by the following mixed-solution steam equilibrium method.

The USP47 T1 protein of the present invention was added to a solution composed of 20 mM Hepes-hydrochloric acid buffer (pH 7.2), 150 mM sodium chloride and 1 mM TECP, and the concentration of the contained protein was adjusted to 10 mg / Respectively. The final protein concentration was determined by the Bradford method.

The results of the screening of the final protein solution and the initial screen solution (Hampton Research Inc., USA) were used to determine the conditions for the determination using the 15% (v / v) PEG 3350, 0.2 M imidazolmalate water tank solution I got the best decision at. Specifically, 1 μl of the USP47T1 protein solution of the present invention and 1 μl of the reservoir solution were brought into contact with the surface of a glass slide coated with silicone, and the slide was placed on a plate containing 0.5 ml of the reservoir solution, Lt; / RTI > After 2 days, seed crystals were formed. After 10 days, the crystal size of USP47T1 crystal was 0.1 X 0.1 X 0.1 mm, and the space group showed monoclinic space group.

The USP47T2: UbT1 protein obtained in Example 3 was crystallized by the following mixed solution vapor equilibrium method. USP47T2: UbT1 protein of the present invention was added to a solution composed of 20 mM Hepes-hydrochloric acid buffer (pH 7.2), 150 mM sodium chloride and 1 mM TECP, and the concentration of the contained protein was adjusted to 15 mg / . The final protein concentration was determined by the Bradford method.

The final protein solution and initial screen solution (Hampton research Inc., USA) were screened for the determination conditions stepwise and found to be 15% (v / v) PEG 4000, 0.15 M ammonium sulfate, 0.1 M 2- [N - morpholino] ethanesulfonic acid (MES) water bath solution. Specifically, 1 μl of the USP47T2: UbT1 protein solution of the present invention and 1 μl of the reservoir solution were contacted with the surface of a glass slide coated with silicone, and the slide was covered with a plate containing 0.5 ml of the reservoir solution Lt; 0 > C. After one day, seed crystals were formed. Three days later, the crystal size of USP47T2: UbT1 crystals was 0.1 X 0.1 X 0.2 mm, and the space group showed an orthorhombic space group.

According to the present invention, crystalline proteins having distinct crystals can be obtained. Photographs of the crystals obtained from the USP47T1 and USP47T2: UbT1 proteins were shown in FIG. 7 and FIG. 8, respectively. 9 shows the skeletal structure of the entire USP 47 as a C alpha ribbon. Fig. 10 shows the entire skeleton structure of USP47 and ubiquitin (Ub) complex purified using SEQ ID NO: 3 as C alpha ribbons. FIG. 11 shows changes in major residues showing the activity of the USP 47 after placing the same spatial positions in FIGS. 9 and 10. FIG.

Example 5 Crystallization of USP47 Protein Performed Further by Nitrogen Fast Cooling

Before X-ray analysis of the obtained USP47T1, USP47T2: UbT1 protein crystals, the following fast Nitrogen cooling method was used to solve the problems that would arise when the USP47 protein crystals obtained from Example 4 were directly exposed to high energy X- Lt; / RTI >

Various cooling solutions such as glycerol, ethylene glycol, sucrose and paratone N were searched at various concentrations, and optimum conditions of the rapid nitrogen cooling method were obtained. Crystals immersed in the cooling solution were recovered using a 0.2-0.4 mm nylon crystal recovery tool (Hampton Research, USA) and immediately poured into a 100 K nitrogen stream.

As a result, when crystals were soaked in a rapid cooling solution containing 25% (v / v) ethylene glycol, 20% (v / v) glycerol and 20% It was found to be most resistant to a liquid nitrogen stream of 100K without any harm.

Example 6 X-ray analysis of USP47 mutant and USP47T2: UbT1 protein crystals through a synchrotron radiation accelerator Data collection and analysis

X-ray analysis data of protein crystals were collected using the USP47, USP47T2: UbT1 crystals obtained from Example 4, using the Accelerator Protein Beamline of the Pohang Accelerator Laboratory (Macromolecular Beam Line PL-5C). The diffraction limits of crystals using the detector of the synchrotron radiation accelerator were 2.6 and 3.0 Å, respectively, and data were processed with DENZO and SCALEPACK (Otwinowski, Z. and Minor, W. Methods Enzymol., 276, 461-472 (1997)). The crystal data collection and refinement results of USP47 mutant and USP47T2: UbT1 protein are shown in Tables 3 and 4, respectively.

[Table 3]

Figures in parentheses for X-ray diffraction data collection are for an outer resolution shell.

Rsym1 = ΣhΣi | Ih, i- <Ih, i> | / ΣhΣiΣh, i (for the intensity I of the measured value i on the reflectivity h) Rcryst2 = Σ | Σ | F observed value |

Rfree3 is the R factor calculated using randomly selected and omitted 5% reflectivity data in the early stages of refinement.

[Table 4]

Figures in parentheses for X-ray diffraction data collection are for an outer resolution shell.

Rsym1 = ΣhΣi | Ih, i- <Ih, i> | / ΣhΣiΣh, i (for the intensity I of the measured value i on the reflectivity h) Rcryst2 = Σ | Σ | F observed value |

Rfree3 is the R factor calculated using randomly selected and omitted 5% reflectivity data in the early stages of refinement.

<Example 7> Analysis of X-ray diffraction data of USP47 protein crystal and structural calculation

Step 1: Identification of the structure of USP47T1

The structure of the USP47 protein obtained from Example 4 was identified through single wavelength analysis (Singlewavelength Anomalous Dispersion). Short wavelength analysis is performed using SOLVE (Terwilliger TC and Berendzen J., Acta Crystallogr. D. Biol. Crystallogr., 55, 849-861 (1999)) program for the initial phase information and for density modification RESOLVE (Terwilliger TC, Acta Crystallogr. D. Biol. Crystallogr., 56, 965-972 (2000)). The diffraction data used was performed with a resolution of 2.6 A.

The CNS program (Brunger, AT. Et al., Acta Cryst. D., 54, 905-921 (1998)) was used in the refinement step. The refinement step was performed using the simulation annealing method of the CNS program. The starting temperature of the simulation was 1500 ° C. The final R and R free factors were 25.4% and 27.4%, respectively. Then, the optimal structure was derived using a 0 program. In order to derive the optimum structure, we performed the refinement step by implementing the electron density from the X-ray data into the computer monitor as 0 program and then correcting it to the most suitable structure.

As a result of the refinement step, the three-dimensional structure of the USP47T1 protein according to the present invention showed a distinctly different finger domain, which is a characteristic structure of the deubiquitinating enzyme compared with USPs, Showed a conservative activation site between the palm domains.

Step 2: USP47T2: Identification of UBT1

For the USP47T2: UbT1 obtained from Example 4 , the structure was identified by performing a molecular replacement (MR), which is a method of finding my structure based on a known structure using a phenix program ( Adams et al, 2010) In the same way, a precise structure was identified.

USP47T2: The three-dimensional structure of the UbT1 protein is different from USP47T1, indicating that ubiquitin is bound between the finger, thumb, and palm and that the conservative activation site between the thumb and palm domains has also changed.

<Example 8> Identification of the active site of USP47 using the affinity of USP47 and ubiquitin using an isothermal calorimetric system (ITC) and a study of mutation of individual amino acid residues around the activated site

The purified USP47 protein and ubiquitin obtained from Example 3 were reconstituted in 20 mM Hepes-HCl buffer (pH 7.2), 150 mM sodium chloride. Calorimetric analysis was performed using an ITC200 system (Malvern Inc. UK). Samples were degassed by vacuum suction for 15 minutes before addition. All experiments were carried out at 20 ° C to 30 ° C with 1 stirring speed of 300 rpm and the heat output was recorded every 10 to 30 seconds. Data was analyzed using the ORIGIN software package (version 7.0) supplied with the instrument. Each set of ITC experiments was repeated two or three times. In the ITC experiment, after obtaining the three-dimensional structure of USP47 protein from Example 7-1, mutations were made to examine the effect of the individual amino acids of the active site of USP47. Mutants of the individual residues according to Example 1-2 were made with cysteine 97 residue as serine (C97S: SEQ ID NOs: 8 and 9) and cysteine 97 residue with alanine (C97A: SEQ ID NOs: 10 and 11). The purified protein of USP47C97S and USP47C97A, an individual residue mutant of USP47, was produced through full-length Example 3-2.

The isothermal calorific value of the interaction between ubiquitin and USP47T1, USP47T2 or USP47T3 was measured and analyzed using ITC, and the result is shown in FIG. The raw data in each panel is shown in the upper figure, and the integrated scan column is displayed in the lower panel. Each titration for USP47T1 is displayed on each panel.

The ITC binding affinity study demonstrated that the proteins obtained from USP47C97 and USP47C97A do not bind to ubiquitin, whereas the proteins obtained from USP47C97S demonstrate binding to ubiquitin (Kd = 1.8 μM).

Based on this, it is preferable to use C97S, which is a mutant residue of USP47, as compared with USP47C97 protein, in confirming the binding strength of ubiquitin and USP47 protein. Also, it can be seen that the cysteine 97 residue of USP47 plays a very important role in recognizing and maintaining ubiquitin activity.

Example 9: Selective and quantitative deubiquitinating assay of USP47 using di (di) ubiquitin

(Ubiquitin) chain (linerUb, K6 (ubiquitin)) made in vitro was used in order to confirm whether the purified USP47 obtained from Example 3 showed differentiation to the enzyme activity or to the diu ubiquitin taking the shape of a different form, -diUb, K11-diUb, K29-diUb, K48-DiUb, K63-DiUb boston biochem, USA) were used as substrates. In vitro diu ubiquitin activity was assayed by incubating the substrate with purified USP47 protein in 20 mM Hepes-HCl buffer (pH 7.2), 150 mM sodium chloride buffer for the indicated time at 37 ° C. The reaction was terminated by the addition of the same volume of 2X SDS-PAGE sample buffer. Proteins were digested with 15% SDS-PAGE and blotted with anti-ubiquitin antibody.

Fig. 6 shows the activity and specificity of USP47T1 (SEQ ID NO: 2) for the cleavage of two ubiquitin chains linked by lysine (Lys6, Lys11, Lys29, Lys48 and Lys63) present in ubiquitin. In the case of liner diUb in the pair of diubiquitin, USP47T1 did not cleave mono ubiquitin and K11-diUb, K48-DiUb, and K6-DiUb cleaved mono ubiquitin during 90 min reaction time. . In the case of K6-diUb, K29-diUb and K63-diUb, only half of the K6-diUb was cleaved into mono ubiquitin during the 90 min reaction time. We found that the USP47T1 protein possessed ubiquitin recognition and cleavage activity.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> UBIQUITIN SPECIFIC PROTEASE 47, THREE-DIMENSIONAL STRUCTURE          THEREOF AND METHOD OF DEVELOPING A UBIQUITIN SPECIFIC PROTEASE          INHIBITOR <130> DPP20151847 <160> 13 <170> Kopatentin 1.71 <210> 1 <211> 1326 <212> PRT <213> Caenorhabditis elegans USP47 full length (T0) <400> 1 Met Val Arg Val Glu Asp Ser Asn Gly Ala Asp Thr Asp Thr Asn Pro   1 5 10 15 Glu Gly Ser Ser Gly Tyr Val Arg Leu Ala Gly Asn Gly Leu Pro              20 25 30 Gly Gly Glu GIn Gln Gln Ala Ser Ser Gly Val Ala Ala Ala Pro          35 40 45 Ser Val Asp Ser Ser Pro Gly Arg Ser Val Pro Leu Ala Ile Ala Ser      50 55 60 Ser Ser Ser Pro Ala Asn Val Ala Asn Asn Gln Tyr Ala Ile Pro Val  65 70 75 80 Asp Glu Asn Gly His Arg Tyr Val Gly Leu Val Asn Gln Ala Met Thr                  85 90 95 Cys Tyr Leu Asn Ser Leu Val Gln Ser Leu Tyr Met Thr Pro Glu Phe             100 105 110 Arg Asn Ala Met Tyr Asp Trp Glu Tyr Val Gln Gln Pro Ala His Ile         115 120 125 Lys Glu Gln Arg Lys Lys Ala Glu Gln Ser Ile Pro Cys Gln Leu Gln     130 135 140 Lys Leu Phe Leu Leu Leu Gln Thr Ser Glu Asn Asp Ser Leu Glu Thr 145 150 155 160 Lys Asp Leu Thr Gln Ser Phe Gly Trp Thr Ser Asn Glu Ala Tyr Asp                 165 170 175 Gln His Asp Val Gln Glu Leu Cys Arg Leu Met Phe Asp Ala Leu Glu             180 185 190 His Lys Trp Lys Gly Thr Glu His Glu Lys Leu Ile Gln Asp Leu Tyr         195 200 205 Arg Gly Thr Met Glu Asp Phe Val Ala Cys Leu Lys Cys Gly Arg Glu     210 215 220 Ser Val Lys Thr Asp Tyr Phe Leu Asp Leu Pro Leu Ala Val Lys Pro 225 230 235 240 Phe Gly Ala Ile His Ala Tyr Lys Ser Val Glu Glu Ala Leu Thr Ala                 245 250 255 Phe Val Gln Pro Glu Leu Leu Asp Gly Ser Asn Gln Tyr Met Cys Glu             260 265 270 Asn Cys Lys Ser Lys Gln Asp Ala His Lys Gly Leu Arg Ile Thr Gln         275 280 285 Phe Pro Tyr Leu Leu Thr Ile Gln Leu Lys Arg Phe Asp Phe Asp Tyr     290 295 300 Asn Thr Met His Arg Ile Lys Leu Asn Asp Lys Met Thr Phe Pro Asp 305 310 315 320 Val Leu Asp Leu Asn Asp Tyr Val Asn Lys Glu Lys Arg Ser Thr Thr                 325 330 335 Ser Ser Ala Trp Gln Gln Ile Gly Lys Asn Lys Ser Glu Asn Glu Glu             340 345 350 Asp Asp Met Glu Leu Gly Ser Pro Asn Pro Lys Arg Cys Thr Pro Gly         355 360 365 Val Gln Ser Pro Asn Arg Tyr Gln Gly Ser Glu Asn Val Cys Val Gly     370 375 380 Gln Pro Ile Asp His Ala Ala Val Asp Asp Ile Val Lys Thr Ser Gly 385 390 395 400 Asp Asn Val Tyr Glu Leu Phe Ser Val Met Val His Ser Gly Asn Ala                 405 410 415 Ala Gly Gly His Tyr Phe Ala Tyr Ile Lys Asn Leu Asp Gln Asp Arg             420 425 430 Trp Tyr Val Phe Asn Asp Thr Arg Val Asp Phe Ala Thr Pro Leu Glu         435 440 445 Ile Glu Lys Ser Phe Gly Gly His Pro Ser Gly Trp Asn Gln Ser Asn     450 455 460 Thr Asn Ala Tyr Met Leu Met Tyr Arg Arg Ile Asp Pro Lys Arg Asn 465 470 475 480 Ala Arg Phe Ile Leu Ser Asn Gln Leu Pro Gln His Ile Lys Asp Ser                 485 490 495 Gln Glu Lys Trp Lys Arg Leu Glu Arg Glu Ala Glu Asp Glu Arg Leu             500 505 510 Arg Lys Leu Ser Leu Ile Gln Val Tyr Val Thr Ile Asn Tyr Pro Phe         515 520 525 Pro Ser Val Val Thr Leu Pro Asp Lys Lys Gln Leu Asp Leu Thr Pro     530 535 540 Gln Lys Tyr Gln Ile Ala Glu Asp Phe Gly Glu Tyr Lys Thr Glu Ile 545 550 555 560 Ser Arg Glu Met Pro Ile Lys Asn Val Phe Asn His Ala Phe Glu Phe                 565 570 575 Phe Asn Glu Arg Ala Arg Ala Tyr Asn Leu Pro Phe Thr Lys Asn Ser             580 585 590 Ala Arg Leu Ile Tyr Val Glu His Gly Ser Leu Met Met Asp Phe Lys         595 600 605 Ser Lys Ala Asp Met Asp Lys Lys Leu Arg Ser Val Phe Asn Val Asn     610 615 620 His Gly Glu Pro Gly Ser Met Tyr Gly Val His Phe Ile Leu Asp Val 625 630 635 640 Arg Ile Ala Ser Ser Phe Phe Pro Val Asp Ile Lys Asn Lys Ile Thr                 645 650 655 Ile Lys Val Gln Arg Val Asp Ile Gly Lys Lys Thr Thr Ala Asn Glu             660 665 670 Leu Ile Val Val Val Asp Ala Asn Glu Lys Met Ile Lys Val Lys Ser         675 680 685 Trp Ile Gly Gly Gln Phe Arg Asp Asp Ile His Gly Val Leu Asn Ala     690 695 700 Arg Val Val Leu Glu Val Ala Ser Ser Arg Cys Glu Phe Met Met Ile 705 710 715 720 Asp Val Ser His Asn Ala Thr Glu Phe Gly Ser Leu Ile Gln Gln Tyr                 725 730 735 Val Gly His Thr Thr Pro Thr Leu Tyr Tyr Asp Gly Gly Leu Asn Thr             740 745 750 Met Val Thr Lys Glu Ser Gln Glu Ala Thr Leu Ala Asp Arg Lys Leu         755 760 765 Pro Phe Asp Lys Ser Val Met Tyr His Ile Leu Asp Arg Lys Cys Phe     770 775 780 Ser Thr Met Val Lys Val Arg Leu Pro Ser Gln Glu Glu Ile Glu Lys 785 790 795 800 Ala Ala Ser Thr Lys Asn Ala Tyr Gln Gly Pro Thr Trp Ala Glu Thr                 805 810 815 Ile Ala Ile Met Lys Glu Glu Asp Arg Leu Trp Asn Glu Pro Arg Ala             820 825 830 Ala Val Glu Val Met Ser Thr Val Ser Arg Thr Asp Thr Thr Asp His         835 840 845 Ala Leu Val Ala Asp Thr Asp Asp Glu Pro Ile Pro Ser Gly Arg Gly     850 855 860 Ser Thr Ala Ser Met Arg Ser Val Ser Met Asp Asp Ile Asp Ala Asp 865 870 875 880 Ile Gly Ile Ser Gly Ser Leu Cys Asn Asn Thr Pro Gln Met Ser Pro                 885 890 895 Cys Val Ser Glu Gly Asp Asp Ala Asp Glu Ser Gln Leu Asp Gly Lys             900 905 910 Ser Gln Leu Met Ser Asp Tyr Met Gln Lys Thr Ser Pro Asp Phe Tyr         915 920 925 Tyr Asn Arg Asp Pro Gln Asn His Leu Asn Lys Asn Leu Lys Ile Ala     930 935 940 Leu Gly Asp Glu Thr Pro Ser Glu Ser Ser Ser Ser Gly 945 950 955 960 Gln Ser Thr Leu Val Ser Ser Ser Ser Gln Ala Leu Ser Ser Met                 965 970 975 Thr Arg Ser Asp Glu Ala Val Asp Gly Lys Ile Val Thr Val Phe Ser             980 985 990 His Glu Asn Phe His Lys Leu Asp Val Asp Ser Arg Met Arg Val Leu         995 1000 1005 Glu Phe Lys Lys Trp Val Ala Glu Gln Leu Glu Met Asp Lys Asp Gln    1010 1015 1020 Phe Val Ile Val Lys His Ala Ser Asp Asp Gly Ser Asp Ser Gly Tyr 1025 1030 1035 1040 Glu Ala Asn Phe Met Asp Asp Glu Thr Val Ser Gly Ala Phe Gln Ser                1045 1050 1055 Cys Phe Ile Ser Ile Lys Leu Arg Ala Pro Leu Lys Ser Asp Glu Lys            1060 1065 1070 Met Met Gln Ile Leu Phe Asp Ile Leu Glu Asn Leu Arg Glu Asn        1075 1080 1085 Trp Lys Pro Leu Phe Glu Leu Pro Val Ser Gln Ser Thr Val Ile Gly    1090 1095 1100 Asp Val Leu Leu Gln Cys Leu Arg Met Tyr Lys Glu Val Tyr Gly Glu 1105 1110 1115 1120 Glu Leu Thr Pro Lys Met Val Arg Leu Arg Glu Leu Gly Gly Ser Gly                1125 1130 1135 Val Gly Thr Gly Arg Ala Val Leu Asn Pro Asn Asp Thr Leu Glu Lys            1140 1145 1150 Arg Ser Tyr Asn Trp Cys Ser His Leu Tyr Leu Gln Ile Ile Thr Asp        1155 1160 1165 Glu Ala Met Ile Gly Lys Pro Gly Glu Pro Ile Met Val Arg Arg Phe    1170 1175 1180 Arg Pro Ser Thr Val Glu Val Asn Pro Thr His Glu Val Leu Val Asp 1185 1190 1195 1200 Ala Asn Ala Glu Asn Pro Val Val Ser Phe Val Glu Ala Leu Ser Lys                1205 1210 1215 Ile Ser Gly Ile Pro Val Glu Arg Leu Ala Ile Thr Asp Leu Lys Glu            1220 1225 1230 Phe Asn Trp Gln Lys Trp Pro Tyr Leu Lys Ser Arg Leu Asp Met Leu        1235 1240 1245 Glu Asn Lys Val Asn Phe Thr Lys Asp Leu Gln Val Thr Tyr Pro Leu    1250 1255 1260 Pro Arg Glu Phe Leu Asp Lys Val Gly Ser Arg Val Leu Tyr Tyr Lys 1265 1270 1275 1280 Asp Ser Asp Glu Glu Ala Lys Val Leu Ser Glu Asp Glu Arg Lys Gln                1285 1290 1295 Ile Lys Ile Lys Glu Asn Gly Gln Ser Ala Asn Ala Asn Arg Arg Lys            1300 1305 1310 Glu Arg Pro Leu Arg Ile Gln Met Ser Ser Val Cys Glu Ala        1315 1320 1325 <210> 2 <211> 508 <212> PRT <213> Catalytic domain of USP47 T1 <400> 2 Met Val Arg Val Glu Asp Ser Asn Gly Ala Asp Thr Asp Thr Asn Pro   1 5 10 15 Glu Gly Ser Ser Gly Tyr Val Arg Leu Ala Gly Asn Gly Leu Pro              20 25 30 Gly Gly Glu GIn Gln Gln Ala Ser Ser Gly Val Ala Ala Ala Pro          35 40 45 Ser Val Asp Ser Ser Pro Gly Arg Ser Val Pro Leu Ala Ile Ala Ser      50 55 60 Ser Ser Ser Pro Ala Asn Val Ala Asn Asn Gln Tyr Ala Ile Pro Val  65 70 75 80 Asp Glu Asn Gly His Arg Tyr Val Gly Leu Val Asn Gln Ala Met Thr                  85 90 95 Cys Tyr Leu Asn Ser Leu Val Gln Ser Leu Tyr Met Thr Pro Glu Phe             100 105 110 Arg Asn Ala Met Tyr Asp Trp Glu Tyr Val Gln Gln Pro Ala His Ile         115 120 125 Lys Glu Gln Arg Lys Lys Ala Glu Gln Ser Ile Pro Cys Gln Leu Gln     130 135 140 Lys Leu Phe Leu Leu Leu Gln Thr Ser Glu Asn Asp Ser Leu Glu Thr 145 150 155 160 Lys Asp Leu Thr Gln Ser Phe Gly Trp Thr Ser Asn Glu Ala Tyr Asp                 165 170 175 Gln His Asp Val Gln Glu Leu Cys Arg Leu Met Phe Asp Ala Leu Glu             180 185 190 His Lys Trp Lys Gly Thr Glu His Glu Lys Leu Ile Gln Asp Leu Tyr         195 200 205 Arg Gly Thr Met Glu Asp Phe Val Ala Cys Leu Lys Cys Gly Arg Glu     210 215 220 Ser Val Lys Thr Asp Tyr Phe Leu Asp Leu Pro Leu Ala Val Lys Pro 225 230 235 240 Phe Gly Ala Ile His Ala Tyr Lys Ser Val Glu Glu Ala Leu Thr Ala                 245 250 255 Phe Val Gln Pro Glu Leu Leu Asp Gly Ser Asn Gln Tyr Met Cys Glu             260 265 270 Asn Cys Lys Ser Lys Gln Asp Ala His Lys Gly Leu Arg Ile Thr Gln         275 280 285 Phe Pro Tyr Leu Leu Thr Ile Gln Leu Lys Arg Phe Asp Phe Asp Tyr     290 295 300 Asn Thr Met His Arg Ile Lys Leu Asn Asp Lys Met Thr Phe Pro Asp 305 310 315 320 Val Leu Asp Leu Asn Asp Tyr Val Asn Lys Glu Lys Arg Ser Thr Thr                 325 330 335 Ser Ser Ala Trp Gln Gln Ile Gly Lys Asn Lys Ser Glu Asn Glu Glu             340 345 350 Asp Asp Met Glu Leu Gly Ser Pro Asn Pro Lys Arg Cys Thr Pro Gly         355 360 365 Val Gln Ser Pro Asn Arg Tyr Gln Gly Ser Glu Asn Val Cys Val Gly     370 375 380 Gln Pro Ile Asp His Ala Ala Val Asp Asp Ile Val Lys Thr Ser Gly 385 390 395 400 Asp Asn Val Tyr Glu Leu Phe Ser Val Met Val His Ser Gly Asn Ala                 405 410 415 Ala Gly Gly His Tyr Phe Ala Tyr Ile Lys Asn Leu Asp Gln Asp Arg             420 425 430 Trp Tyr Val Phe Asn Asp Thr Arg Val Asp Phe Ala Thr Pro Leu Glu         435 440 445 Ile Glu Lys Ser Phe Gly Gly His Pro Ser Gly Trp Asn Gln Ser Asn     450 455 460 Thr Asn Ala Tyr Met Leu Met Tyr Arg Arg Ile Asp Pro Lys Arg Asn 465 470 475 480 Ala Arg Phe Ile Leu Ser Asn Gln Leu Pro Gln His Ile Lys Asp Ser                 485 490 495 Gln Glu Lys Trp Lys Arg Leu Glu Arg Glu Ala Glu             500 505 <210> 3 <211> 508 <212> PRT <213> Catalytic domain of USP47 T2 <400> 3 Met Val Arg Val Glu Asp Ser Asn Gly Ala Asp Thr Asp Thr Asn Pro   1 5 10 15 Glu Gly Ser Ser Gly Tyr Val Arg Leu Ala Gly Asn Gly Leu Pro              20 25 30 Gly Gly Glu GIn Gln Gln Ala Ser Ser Gly Val Ala Ala Ala Pro          35 40 45 Ser Val Asp Ser Ser Pro Gly Arg Ser Val Pro Leu Ala Ile Ala Ser      50 55 60 Ser Ser Ser Pro Ala Asn Val Ala Asn Asn Gln Tyr Ala Ile Pro Val  65 70 75 80 Asp Glu Asn Gly His Arg Tyr Val Gly Leu Val Asn Gln Ala Met Thr                  85 90 95 Ser Tyr Leu Asn Ser Leu Val Gln Ser Leu Tyr Met Thr Pro Glu Phe             100 105 110 Arg Asn Ala Met Tyr Asp Trp Glu Tyr Val Gln Gln Pro Ala His Ile         115 120 125 Lys Glu Gln Arg Lys Lys Ala Glu Gln Ser Ile Pro Cys Gln Leu Gln     130 135 140 Lys Leu Phe Leu Leu Leu Gln Thr Ser Glu Asn Asp Ser Leu Glu Thr 145 150 155 160 Lys Asp Leu Thr Gln Ser Phe Gly Trp Thr Ser Asn Glu Ala Tyr Asp                 165 170 175 Gln His Asp Val Gln Glu Leu Cys Arg Leu Met Phe Asp Ala Leu Glu             180 185 190 His Lys Trp Lys Gly Thr Glu His Glu Lys Leu Ile Gln Asp Leu Tyr         195 200 205 Arg Gly Thr Met Glu Asp Phe Val Ala Cys Leu Lys Cys Gly Arg Glu     210 215 220 Ser Val Lys Thr Asp Tyr Phe Leu Asp Leu Pro Leu Ala Val Lys Pro 225 230 235 240 Phe Gly Ala Ile His Ala Tyr Lys Ser Val Glu Glu Ala Leu Thr Ala                 245 250 255 Phe Val Gln Pro Glu Leu Leu Asp Gly Ser Asn Gln Tyr Met Cys Glu             260 265 270 Asn Cys Lys Ser Lys Gln Asp Ala His Lys Gly Leu Arg Ile Thr Gln         275 280 285 Phe Pro Tyr Leu Leu Thr Ile Gln Leu Lys Arg Phe Asp Phe Asp Tyr     290 295 300 Asn Thr Met His Arg Ile Lys Leu Asn Asp Lys Met Thr Phe Pro Asp 305 310 315 320 Val Leu Asp Leu Asn Asp Tyr Val Asn Lys Glu Lys Arg Ser Thr Thr                 325 330 335 Ser Ser Ala Trp Gln Gln Ile Gly Lys Asn Lys Ser Glu Asn Glu Glu             340 345 350 Asp Asp Met Glu Leu Gly Ser Pro Asn Pro Lys Arg Cys Thr Pro Gly         355 360 365 Val Gln Ser Pro Asn Arg Tyr Gln Gly Ser Glu Asn Val Cys Val Gly     370 375 380 Gln Pro Ile Asp His Ala Ala Val Asp Asp Ile Val Lys Thr Ser Gly 385 390 395 400 Asp Asn Val Tyr Glu Leu Phe Ser Val Met Val His Ser Gly Asn Ala                 405 410 415 Ala Gly Gly His Tyr Phe Ala Tyr Ile Lys Asn Leu Asp Gln Asp Arg             420 425 430 Trp Tyr Val Phe Asn Asp Thr Arg Val Asp Phe Ala Thr Pro Leu Glu         435 440 445 Ile Glu Lys Ser Phe Gly Gly His Pro Ser Gly Trp Asn Gln Ser Asn     450 455 460 Thr Asn Ala Tyr Met Leu Met Tyr Arg Arg Ile Asp Pro Lys Arg Asn 465 470 475 480 Ala Arg Phe Ile Leu Ser Asn Gln Leu Pro Gln His Ile Lys Asp Ser                 485 490 495 Gln Glu Lys Trp Lys Arg Leu Glu Arg Glu Ala Glu             500 505 <210> 4 <211> 508 <212> PRT <213> Catalytic domain of USP47 T3 <400> 4 Met Val Arg Val Glu Asp Ser Asn Gly Ala Asp Thr Asp Thr Asn Pro   1 5 10 15 Glu Gly Ser Ser Gly Tyr Val Arg Leu Ala Gly Asn Gly Leu Pro              20 25 30 Gly Gly Glu GIn Gln Gln Ala Ser Ser Gly Val Ala Ala Ala Pro          35 40 45 Ser Val Asp Ser Ser Pro Gly Arg Ser Val Pro Leu Ala Ile Ala Ser      50 55 60 Ser Ser Ser Pro Ala Asn Val Ala Asn Asn Gln Tyr Ala Ile Pro Val  65 70 75 80 Asp Glu Asn Gly His Arg Tyr Val Gly Leu Val Asn Gln Ala Met Thr                  85 90 95 Ala Tyr Leu Asn Ser Leu Val Gln Ser Leu Tyr Met Thr Pro Glu Phe             100 105 110 Arg Asn Ala Met Tyr Asp Trp Glu Tyr Val Gln Gln Pro Ala His Ile         115 120 125 Lys Glu Gln Arg Lys Lys Ala Glu Gln Ser Ile Pro Cys Gln Leu Gln     130 135 140 Lys Leu Phe Leu Leu Leu Gln Thr Ser Glu Asn Asp Ser Leu Glu Thr 145 150 155 160 Lys Asp Leu Thr Gln Ser Phe Gly Trp Thr Ser Asn Glu Ala Tyr Asp                 165 170 175 Gln His Asp Val Gln Glu Leu Cys Arg Leu Met Phe Asp Ala Leu Glu             180 185 190 His Lys Trp Lys Gly Thr Glu His Glu Lys Leu Ile Gln Asp Leu Tyr         195 200 205 Arg Gly Thr Met Glu Asp Phe Val Ala Cys Leu Lys Cys Gly Arg Glu     210 215 220 Ser Val Lys Thr Asp Tyr Phe Leu Asp Leu Pro Leu Ala Val Lys Pro 225 230 235 240 Phe Gly Ala Ile His Ala Tyr Lys Ser Val Glu Glu Ala Leu Thr Ala                 245 250 255 Phe Val Gln Pro Glu Leu Leu Asp Gly Ser Asn Gln Tyr Met Cys Glu             260 265 270 Asn Cys Lys Ser Lys Gln Asp Ala His Lys Gly Leu Arg Ile Thr Gln         275 280 285 Phe Pro Tyr Leu Leu Thr Ile Gln Leu Lys Arg Phe Asp Phe Asp Tyr     290 295 300 Asn Thr Met His Arg Ile Lys Leu Asn Asp Lys Met Thr Phe Pro Asp 305 310 315 320 Val Leu Asp Leu Asn Asp Tyr Val Asn Lys Glu Lys Arg Ser Thr Thr                 325 330 335 Ser Ser Ala Trp Gln Gln Ile Gly Lys Asn Lys Ser Glu Asn Glu Glu             340 345 350 Asp Asp Met Glu Leu Gly Ser Pro Asn Pro Lys Arg Cys Thr Pro Gly         355 360 365 Val Gln Ser Pro Asn Arg Tyr Gln Gly Ser Glu Asn Val Cys Val Gly     370 375 380 Gln Pro Ile Asp His Ala Ala Val Asp Asp Ile Val Lys Thr Ser Gly 385 390 395 400 Asp Asn Val Tyr Glu Leu Phe Ser Val Met Val His Ser Gly Asn Ala                 405 410 415 Ala Gly Gly His Tyr Phe Ala Tyr Ile Lys Asn Leu Asp Gln Asp Arg             420 425 430 Trp Tyr Val Phe Asn Asp Thr Arg Val Asp Phe Ala Thr Pro Leu Glu         435 440 445 Ile Glu Lys Ser Phe Gly Gly His Pro Ser Gly Trp Asn Gln Ser Asn     450 455 460 Thr Asn Ala Tyr Met Leu Met Tyr Arg Arg Ile Asp Pro Lys Arg Asn 465 470 475 480 Ala Arg Phe Ile Leu Ser Asn Gln Leu Pro Gln His Ile Lys Asp Ser                 485 490 495 Gln Glu Lys Trp Lys Arg Leu Glu Arg Glu Ala Glu             500 505 <210> 5 <211> 76 <212> PRT <213> Human ubiquitin T1 <400> 5 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu   1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp              20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys          35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu      50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly  65 70 75 <210> 6 <211> 32 <212> DNA &Lt; 213 > USP47 T1 < <400> 6 ggggaattcc atatggtacg ggtcgaggac tc 32 <210> 7 <211> 32 <212> DNA <213> USP47 T1 xho1 3` <400> 7 ccgctcgagt tattccgcct ctcgttcaag tc 32 <210> 8 <211> 33 <212> DNA <213> USP47 C97S 5 ' <400> 8 acccaggcaa tgactagtta tttgaacagt ctt 33 <210> 9 <211> 33 <212> DNA <213> USP47 C97S 3` <400> 9 aagactgttc aaataactag tcattgcctg gtt 33 <210> 10 <211> 33 <212> DNA <213> USP47 C97A 5` <400> 10 acccaggcaa tgactgcata tttgaacagt ctt 33 <210> 11 <211> 33 <212> DNA <213> USP47 C97A 3` <400> 11 aagactgttc aaatatgcag tcattgcctg gtt 33 <210> 12 <211> 39 <212> DNA <213> UiquitinT1 nde1 5 ` <400> 12 ggggaattcc atatgatgca gatcttcgtg aagactctg 39 <210> 13 <211> 42 <212> DNA <213> UiquitinT1 xho1 3` <400> 13 ccgctcgaga ctcccacctc tgagacggag caccaggtgc ag 42

Claims (32)

A USP47 protein variant comprising the amino acid sequence of SEQ ID NO: 2. The USP47 protein variant of claim 1, wherein the cysteine located at position 97 is further substituted with serine, based on the amino acid sequence of SEQ ID NO: 2.
The USP47 protein variant according to claim 1, wherein the cysteine located at position 97 is further substituted with alanine, based on the amino acid sequence of SEQ ID NO: 2.
4. A USP47 mutant complex comprising a USP47 protein variant according to any one of claims 1 to 3 and an ubiquitin protein consisting of the amino acid sequence of SEQ ID NO:
4. A nucleic acid encoding a USP47 protein variant according to any one of claims 1 to 3.
A vector comprising a nucleic acid according to claim 5.
7. The vector of claim 6, wherein said vector comprises a promoter operably linked to said nucleic acid sequence and a terminator.
The vector according to claim 6, which is capable of expressing in E. coli.
A transformant transformed with a vector according to claim 6.
Transforming a host with a vector according to claim 6 to produce a transformant; And culturing the produced transformant to produce a protein.
A method for crystallizing a protein variant comprising contacting a protein variant according to any one of claims 1 to 3 with a precipitant solution to carry out a droplet mixed steam equilibrium method.
12. The method of claim 11, wherein the precipitant is at least one selected from the group consisting of ammonium sulfate, polyethylene glycol (PEG), imidazolmalate, and 2- [N-morpholino] ethanesulfonic acid Lt; / RTI &gt;
13. The method of claim 12,
Wherein the ammonium sulfate concentration ranges from 0.1 to 0.3 M; Wherein the polyethylene glycol (PEG) ratio is in the range of 15 to 25 vol% (v / v); Wherein the imidazolmalate concentration ranges from 0.1 to 0.5 M; Or the 2- [N-morpholino] ethanesulfonic acid (MES) concentration is in the range of 0.1 to 0.2 M.
14. The method of claim 13,
Wherein the polyethylene glycol (PEG) has a molecular weight of 3350 or 4000.
The crystallization method according to claim 11, wherein the drop mixing steam equilibrium method is carried out under the reaction conditions of a reaction pH of 5.0 to 8.0, a reaction temperature of 16 to 26 캜 and a reaction period of 1 to 20 days.
The crystallization method according to claim 11, further comprising performing a rapid nitrogen cooling method after performing the droplet mixed steam equilibrium method.
The crystallization method according to claim 16, wherein the high-speed nitrogen cooling method is performed in a temperature range of 50 to 200 K.
The crystals of the USP47 protein variant of claim 1 having x-ray diffraction pattern data of Table 1:
[Table 1]

.
Crystals of the USP47 mutant complex comprising the USP47 variant and the ubiquitin protein of claim 2 having x-ray diffraction pattern data of Table 2:
[Table 2]

.
The crystals of the USP47 protein variant of claim 1 having the three-dimensional crystal structure of Table 3:
[Table 3]

.
Crystals of the USP47 mutant complex protein comprising the USP47 variant and the ubiquitin protein of claim 2 having the three-dimensional crystal structure of Table 4:
[Table 4]

.
The crystals of the USP47 protein variant of claim 1 having the atomic coordinates of Table 5.
A crystal of the USP47 mutant complex comprising the USP47 protein variant and the ubiquitin protein of claim 2, having the atomic coordinates of Table 6.
A USP47 protein variant according to any one of claims 1 to 3 or a USP47 mutant complex comprising the USP47 protein variant and the ubiquitin protein consisting of the amino acid sequence of SEQ ID NO: A composition for screening a therapeutic agent.
25. The composition of claim 24, wherein the virus is an influenza virus.
A USP47 protein variant according to any one of claims 1 to 3; Or a variant complex comprising the USP47 protein variant and the ubiquitin comprising the amino acid sequence of SEQ ID NO: 5 is used.
27. The method of claim 26,
The USP47 protein variant; Or a variant complex comprising the USP47 protein variant and the ubiquitin comprising the amino acid sequence of SEQ ID NO: 5 with a candidate compound; And
Selecting a compound that interacts with the USP47 protein variant or the mutant complex among the candidate compounds to screen for a compound that inhibits the activity of the USP47 protein variant or the mutant complex.
27. The drug screening method according to claim 26, wherein the USP47 protein variant has the x-ray diffraction pattern data of Table 1 or the three-dimensional crystal structure of Table 3:
[Table 1]

[Table 3]

.
27. The drug screening method according to claim 26, wherein the mutant complex comprising the USP47 protein variant and the ubiquitin has the x-ray diffraction pattern data of Table 2 or the three-dimensional crystal structure of Table 4:
[Table 2]

[Table 4]

.


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