JP2006000023A - Human catechol o-methyltransferase crystal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide human catechol O-methyltransferase crystal, and to provide the three-dimensional structure of the crystal. <P>SOLUTION: The human catechol O-methyltransferase crystal comprises human catechol O-methyltransferase, S-adenosylmethionine, magnesium ion, and a ligand, wherein the human catechol O-methyltransferase has a specific amino acid sequence. A detailed three-dimensional structure related to the human catechol O-methyltransferase is obtained by an X-ray crystal analysis of the crystal. Therefore, the crystal is useful for designing the molecule of a human catechol O-methyltransferase inhibitor. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a crystal of human catechol O-methyltransferase, and more particularly, a crystal of human catechol O-methyltransferase comprising four components of human catechol O-methyltransferase, S-adenosyl-L-methionine, magnesium ion and a ligand. About.

  Catechol O-methyltransferase (COMT) plays an important role in metabolism of catecholamines which are neurotransmitters and various compounds having a catechol structure in vivo. For example, COMT is involved in the metabolism of L-dopa, which is used as a therapeutic agent for Parkinson's disease. L-dopa is a precursor of dopamine and is a drug that is metabolized to dopamine in the brain and has an effect, but has a drawback that its blood half-life is very short. Therefore, L-dopa is usually used together with a COMT inhibitor that is a metabolic enzyme inhibitor of L-dopa. COMT is an enzyme that catalyzes the transfer of a methyl group from its coenzyme, S-adenosyl-L-methionine, to a catechol substrate in the presence of magnesium ions. By inhibiting this enzyme, L-DOPA It is known that metabolism to 3-O-methyl-L-dopa is inhibited.

In recent years, intense research has been conducted on mammalian COMT. COMT is known to include a site that accommodates a coenzyme, S-adenosyl-L-methionine, a site that accommodates a ligand that is a substrate for COMT, and a site that accommodates magnesium ions.
Regarding rat COMT, a co-crystal of a quaternary complex of rat COMT, S-adenosyl-L-methionine, magnesium ion and a ligand (for example, a COMT inhibitor such as dinitrocatechol) has been reported (for example, Non-Patent Document 1, 2 and 3) are registered in the protein database as pdb1vid.ent, pdb1h1d.ent, and pdb1jr4.ent, respectively. On the other hand, regarding human COMT, Carola Tilgman et al. Have reported studies on the expression and purification of human catechol O-methyltransferase in E. coli. However, purified human COMT contains impurities that cannot be removed from E. coli. In addition, since a protein similar to human COMT having a different molecular weight is expressed together during protein expression, uniform human COMT cannot be obtained, and crystallization of human COMT has not been successful (for example, see Non-patent Document 4). .

To date, no crystals of human COMT have been reported and therefore the detailed three-dimensional structure of human COMT is not known.
Vidgren J. et al., `` Nature '', 1994, 368, p.354 Bonifacio M. et al., “Mol. Pharmacol.”, 2002, 62, p.795 Lerner C. et al., “Angew. Chem. Intern. Edition”, 2001, 40, p.4040 Tilgmann C. et al., “J. Chromatography”, 1996, 684, p.147-161

  An object of the present invention is to provide human catechol O- consisting of four components of human catechol O-methyltransferase, S-adenosyl-L-methionine, magnesium ion and ligand, which is useful for the molecular design of human catechol O-methyltransferase inhibitors. It is to provide crystals of methyltransferase.

  As a result of intensive research on human catechol O-methyltransferase that can be crystallized, the present inventors have found that recombinant human catechol O-methyltransferase having the amino acid sequence represented by SEQ ID NO: 1 and S-adenosyl- The inventors have found that a quaternary complex of L-methionine, magnesium ion and a ligand can be unexpectedly obtained as extremely good crystals, and has completed the present invention.

  That is, the present invention is a crystal of human catechol O-methyltransferase comprising human catechol O-methyltransferase; S-adenosylmethionine; magnesium ion; and a ligand, wherein the human catechol O-methyltransferase is SEQ ID NO: 1. It relates to a crystal comprising the amino acid sequence represented by

Crystal of the present invention has a space group _P3 2 21, the lattice constants, a is 50.58Å ± 0.3Å, b is 50.58Å ± 0.3Å, c is 167.24Å ± 0.4Å, α has a value equal to 90.0 °, β is 90.0 °, and γ is equal to 120.0 °, and more preferably, a is 50.58 mm, b is 50.58 mm, c is 167.24 mm, α Has a value equal to 90.0 °, β equals 90.0 °, and γ equals 120.0 °.

  In addition, the crystal of the present invention has a pocket that accommodates S-adenosylmethionine, and this pocket is preferably within a range of 5 mm from S-adenosylmethionine, Met42, Asn43, Val44, Leu67, Gly68. , Ala69, Tyr70, Cys71, Gly72, Tyr73, Ser74, Ile91, Glu92, Ile93, Asn94, Cys97, Gly119, Ala120, Ser121, Gln122, Phe141, Asp143, His144 and Trp145.

  Furthermore, the crystal of the present invention has a pocket that accommodates the ligand, which pocket is preferably formed by amino acid residues including Trp40, Met42, Asp143, Trp145, Lys146, Asp171, Asn172, Pro176, Leu200 and Glu201. Is done.

  In a preferred embodiment of the present invention, the crystal of the present invention comprises a human catechol O-methyltransferase inhibitor in the pocket containing the ligand, the human catechol O-methyltransferase inhibitor is preferably 3, 5 -Dinitrocatechol.

  In the present invention, the following terms have the following meanings.

  “Ligand” means a low molecular weight compound that binds to a biopolymer. Specific examples of such ligands include, for example, human catechol O-methyltransferase substrate, human catechol O-methyltransferase analog, human catechol O-methyltransferase inhibitor, and preferably human catechol O- It is a methyltransferase inhibitor.

  “Pocket” means a binding region of a compound (eg, cofactor, ligand, etc.) that binds to a biopolymer, and is also referred to as a binding site, a binding region, a binding pocket, or a storage pocket. Specific examples of such pockets include an S-adenosylmethionine accommodation pocket and a ligand accommodation pocket.

The recombinant human catechol O-methyltransferase described in SEQ ID NO: 1 can be prepared as follows.
The design and preparation of primers for amplifying DNA encoding recombinant human catechol O-methyltransferase can be performed according to general methods well known to those skilled in the art. For example, a primer for amplifying a DNA encoding a protein in which the length of the C-terminal site and / or the N-terminal site is changed from a full-length protein is changed in the position to be annealed to the template cDNA, and further 5'-side It can also be designed as a primer containing an appropriate restriction enzyme recognition site on the 3 ′ side. By performing a PCR reaction using such a primer, it is possible to amplify a DNA encoding a recombinant human catechol O-methyltransferase having an arbitrary length. In the present invention, in order to facilitate incorporation into a plasmid, as a primer having a restriction enzyme recognition site added, the following 5′- containing a BamH I on the 5 ′ side and an EcoR I recognition site on the 3 ′ side. Primers and 3′-primers are used.
5'-primer: 5'-tctggatccatgggtacaccagaggag-3 '(SEQ ID NO: 3)
3'-primer: 5'-gaggaattctcagccccttgtagatggccttctc-3 '(SEQ ID NO: 4)
These primers can be subjected to a standard PCR reaction using human liver cDNA as a template and a DNA polymerase (for example, KOD plus). The amplified PCR product is preferably digested with restriction enzymes BamH I and EcoR I and ligated into the desired vector (eg, pGEX-2T). Subsequently, the ligation mixture is used to transform a plasmid amplification host cell (for example, E. coli JM109). Large quantities of recombinant plasmids can be obtained by culturing the host cells in an appropriate medium. The amplified recombinant plasmid is used for transformation of a host cell (for example, E. coli BL21 codon plus (DE3) RP) for expression of the recombinant peptide after purification. Subsequently, the transformed E. coli is cultured in an LB-ampicillin medium in the presence of an appropriate concentration of isopropyl-β-D-thiogalactopyranoside, preferably at room temperature for about 6 to 15 hours. A culture suspension is obtained. After centrifuging this culture suspension, the resulting E. coli pellet is lysed. For example, when pGEX-2T is used as a vector, it is expressed by mixing a resin capable of adsorbing glutathione S-transferase (GST). The adsorbed protein can be adsorbed on the solid phase. Recombinant human catechol O-methyltransferase can then be liberated by adding thrombin at an appropriate concentration and incubating under low temperature conditions (eg about 4 ° C.) for about 12-15 hours. The resulting recombinant human catechol O-methyltransferase can be purified by, for example, anion exchange chromatography, gel filtration chromatography, reverse phase chromatography, or the like.

Crystallization of a quaternary complex composed of human catechol O-methyltransferase of the present invention; S-adenosylmethionine; magnesium ion; and a ligand can be performed as follows.
First, a solution containing recombinant human catechol O-methyltransferase prepared as described above is prepared at an appropriate concentration, preferably about 1.0 to about 10.0 mg / mL, more preferably about 5.0 to about 7. Concentrate to 0 mg / mL. To this concentrated solution, a solution of S-adenosylmethionine (cofactor) and ligand, and optionally a magnesium ion source, preferably a protein: cofactor: magnesium ion: ligand molar ratio of about 1: 2: Add 2: 2 to prepare a protein: cofactor: magnesium ion: ligand complex solution.
Next, a precipitation solution containing a buffer, an ionic strength source, alcohols and the like is prepared. Examples of the buffer used in the precipitation solution include Tris, Hepes, cacodylic acid, citric acid, acetic acid, imidazole, and the like. Preferably, the pH is about 4.0 to about 0.2 in a concentration range of about 0.1 to about 0.2M. A buffer that is in the range of 9.0 is used. Examples of ionic strength sources include calcium chloride, magnesium chloride, sodium citrate, ammonium sulfate, lithium sulfate, magnesium acetate, ammonium acetate, sodium acetate, potassium dihydrogen phosphate, zinc acetate, magnesium formate, sodium formate, sodium phosphate In addition, sodium potassium tartrate is used. As the alcohol, for example, monovalent or polyhydric alcohol, polyethylene glycol, or the like is used.
Subsequently, the droplets of the quaternary complex solution prepared as described above and the droplets of the precipitation solution are mixed, and the obtained mixed droplets are suspended on the wells containing the precipitation solution, hanging drops, It is held by a method such as ing-drop, and is allowed to stand for a long time under a constant temperature range of 4 ° C. to 25 ° C. until the crystal of the quaternary complex grows to a size suitable for X-ray diffraction.
Next, the obtained quaternary complex crystal is immersed in a precipitation solution to which an antifreezing agent is added so as to be about 10 to 15% (V / V) for about 30 seconds to about 1 minute, and 0.5 mm to 0.7 mm. Pick up with a nylon loop of diameter and immerse it directly in liquid nitrogen for quick freezing. Examples of the antifreezing agent used at this time include glycerol, polyethylene glycol, and trehalose. Next, the frozen quaternary complex crystal is sealed in a cryovial and stored in a dewar bin filled with liquid nitrogen until X-ray irradiation.

  By analyzing the crystal structure of the quaternary complex consisting of the human catechol O-methyltransferase of the present invention; S-adenosylmethionine; magnesium ion; and a ligand thus obtained, the human catechol O-methyltransferase of A detailed three-dimensional structure can be obtained. Such coordinate data of a three-dimensional structure, particularly coordinate data of a pocket accommodating a ligand, is extremely useful for molecular design of a human catechol O-methyltransferase inhibitor, and a docking study between the ligand-accommodating pocket and an arbitrary compound. By performing the above, it is possible to screen whether or not the compound is suitable as a candidate compound. Such docking studies are well known to those skilled in the art and include various computer software such as ADAM (Pharmaceutical Molecular Design Laboratory), ADAM & EVE (Pharmaceutical Molecular Design Laboratory), DOCK (University of California). San Francisco), FlexX (Tripos) or the like.

  Details on the previously unknown human catechol O-methyltransferase by analyzing quaternary complex crystals of human catechol O-methyltransferase; S-adenosyl-L-methionine; magnesium ion; A three-dimensional structure is obtained. Furthermore, since a detailed three-dimensional structure regarding the S-adenosyl-L-methionine accommodation pocket and the ligand accommodation pocket, which are sites where human catechol O-methyltransferase expresses enzyme activity, is obtained, It is extremely useful for the molecular design of O-methyltransferase inhibitors.

The contents of the present invention will be described in more detail in the following examples, but the present invention is not limited to these contents.
In the following examples, SEQ ID NO: 1 is the sequence of recombinant human catechol O-methyltransferase; SEQ ID NO: 2 is SEQ ID NO: 3 and so as to express the recombinant human catechol O-methyltransferase of SEQ ID NO: 1. 4 shows the DNA sequence amplified with the primer of 4; SEQ ID NO: 3 is the sequence of the 5 ′ primer used to amplify the DNA of SEQ ID NO: 2; SEQ ID NO: 4 is the DNA of SEQ ID NO: 2 The sequence of the 3 ′ primer used to amplify is; SEQ ID NO: 5 is a 5 ′ primer for simply confirming by gel electrophoresis whether or not the DNA shown in SEQ ID NO: 2 has been recombined into a plasmid. Further, SEQ ID NO: 5 is used to confirm whether the DNA shown in SEQ ID NO: 2 has been recombined into a plasmid by the dye terminator method. SEQ ID NO: 6 is the sequence of the 3 ′ primer for easily confirming by gel electrophoresis whether the DNA shown in SEQ ID NO: 2 has been recombined into a plasmid; SEQ ID NO: 7 This is the sequence of the 5 ′ primer in the human catechol O-methyltransferase DNA sequence for confirming whether the DNA shown in SEQ ID NO: 2 has been recombined into a plasmid by the dye terminator method.

Example 1
(Preparation of crystallizable recombinant human catechol O-methyltransferase)
1) Design of crystallizable recombinant human catechol O-methyltransferase of the recombinant human catechol O-methyltransferase of the present invention; S-adenosylmethionine; 3,5-dinitrocatechol; and magnesium ion quaternary complex The recombinant human catechol O-methyltransferase described in SEQ ID NO: 1 was designed as a recombinant human catechol O-methyltransferase used for the crystal. In this recombinant human catechol O-methyltransferase described in SEQ ID NO: 1, a Gly-Ser-residue was added to the N-terminus of the full-length human catechol O-methyltransferase, and 7 amino acids were deleted from the C-terminus. Is.
Based on the DNA sequence of accession number BC011935 registered on NCBI (National Center for Biotechnology Information), which encodes full-length human catechol O-methyltransferase, recombinant human catechol O-methyltransferase described in SEQ ID NO: 1 Two oligonucleotide primers were designed to amplify the encoding DNA sequence. The sequence of the 5 ′ primer is shown in SEQ ID NO: 3, and the sequence of the 3 ′ primer is shown in SEQ ID NO: 4. These primers contain restriction enzyme sites (BamHI on the 5 ′ side and EcoRI on the 3 ′ side) to facilitate insertion of the PCR product into the desired vector.
The insert DNA obtained with these primers is shown in SEQ ID NO: 2. As shown below, this insert DNA is incorporated into a pGEX-2T vector, the protein is expressed as a GST fusion protein, and then treated with thrombin to prepare a recombinant human catechol O-methyltransferase shown in SEQ ID NO: 1. did. The enzyme activity of this recombinant human catechol O-methyltransferase was determined by methods described in the literature (for example, G. Zurcher and M. Da Prada. Et al., “Journal of Neurochemistry”, 1982, 38, p.191-195). Was confirmed to have almost the same activity as that of full-length human catechol O-methyltransferase.

2) Cloning and expression of insert DNA for expressing recombinant human catechol O-methyltransferase Each of the 5 ′ primer described in SEQ ID NO: 3 and the 3 ′ primer described in SEQ ID NO: 4 was diluted with TE buffer to obtain 15 A pmol / μL solution was used. H ₂ O (for PCR, 34.8 μL), 25 mM MgSO ₄ (2.0 μL), 2 mM dNTPs (5.0 μL), 10-fold concentrated DNA polymerase KOD plus buffer (5.0 μL, Toyobo) were mixed, and PCR reaction was performed A working mixture was prepared. Human liver cDNA (Invitrogen) (5.0 μL) was then added to the PCR reaction mixture. Each primer pair (1 μL, 15 pmol) was added to the above mixture, and finally 1.0 μL of KOD plus was added. Thereafter, a PCR reaction was performed. The PCR reaction was conducted at 94 ° C. for 2 minutes, 94 ° C. for 15 seconds, 59 ° C. for 30 seconds, and 68 ° C. for 1 minute for 40 cycles. Subsequently, it was completed at 68 ° C. for 5 minutes and at 4 ° C. for 10 minutes. A 1.0 μL portion of the PCR product was collected and analyzed by electrophoresis after adding 10 × loading buffer (1.2 μL) and H ₂ O (5.8 μL). The PCR product was amplified.
The PCR product was purified with the QIAquick PCR purification kit (Qiagen). The desired insert DNA was eluted with the EB buffer (30 μL) of the same kit. The eluted DNA (1.0 μL) was then diluted with TE buffer (99 μL) to determine the concentration. The insert DNA for expressing the recombinant human catechol O-methyltransferase was 0.112 μg / μL, 260 nm / 280 nm = 1.99.

3) Double digestion of recombinant human catechol O-methyltransferase insert DNA and pGEX-2T vector To recombinant human catechol O-methyltransferase insert DNA (1.5 μg), 10 x EcoR I buffer (New England Biolab) ( 3.0 μL), H ₂ O (11.1 μL), BamH I (1.5 μL, 15 U, 10 u / μL) and EcoR I (1.0 μL, 15 U, 15 U / μL) were added and mixed. The mixed solution was heated at 37 ° C. for 1.5 hours. In addition, 10X loading buffer was added to the solution. The mixed solution was separated by electrophoresis, and the gel portion containing the DNA having the digested fragment was excised and purified using a Qiagen Mini Elute gel extraction kit (Qiagen). The concentration of the digested fragment DNA was 33.1 ng / μL.
pGEX-2T vector DNA (1.5 μg, Amersham) was added to 10 × EcoR I buffer (New England Biolab) (3.0 μL), H ₂ O (21.5 μL), BamH I (1.5 μL, 15 U, 10 u / μL) and EcoR I (1.0 μL, 15 U, 15 U / μL) were added and mixed. The mixed solution was heated at 37 ° C. for 1.5 hours. In addition, 10X loading buffer was added to the solution. The mixed solution was separated by electrophoresis, and the gel portion containing the DNA having the digested fragment was excised and purified using a Qiagen Mini Elute gel extraction kit (Qiagen). The concentration of the digested fragment DNA was 25.0 ng / μL.

4) Ligation and transformation of E. coli JM109 Double digested pGEX-2T vector (2.0 μL, 50 ng, 25 ng / μL) and insert DNA (1.0 μL, 33.1 ng, 33.1 ng / μL) were added to 2 × ligation buffer. Liquid (5.0 μL, PGEM) and H ₂ O (1.7 μL) were added and mixed. Ligase (1.0 μL, 3 U / μL, PGEM) was then added to the mixed solution and the mixture was incubated at 25 ° C. for 1 hour. Next, E. coli JM109 (100 μL) was dissolved at 0 ° C. and the above mixed solution (5 μL) reacted with ligase was added to the JM109 suspension and mixed gently, while avoiding strong vibration (0 ° C., (20 minutes, 42 ° C. for 40 seconds, 0 ° C. for 30 minutes). Next, 450 μL of the SOC solution was added to the solution after heat shock and shaken at 37 ° C. for 1 hour. After shaking, 50 μL and 200 μL of the mixed solution were respectively spread on a plate of LB-ampicillin medium (diameter 9 cm, ampicillin concentration: 100 μg / mL), and static culture was performed at 37 ° C. for 16 hours. As a result, colonies appeared on the plate. The cultured plate was stored at 4 ° C. until use.

5) Colony selection by PCR method of GST fusion recombinant human catechol O-methyltransferase The 5 ′ primer described in SEQ ID NO: 5 and the 3 ′ primer described in SEQ ID NO: 6 shown below were each diluted with a TE buffer, A 15 pmol / μL solution was prepared.
5 ′ primer: 5′-gaagctattccccacaaaattgataag-3 ′ (SEQ ID NO: 5)
3 ′ primer: 5′-ctgacggggctgtgtctgctcc-3 ′ (SEQ ID NO: 6)
Next, 5 ′ primer diluted solution (20 μL, 15 pmol / μL), 3 ′ primer diluted solution (20 μL, 15 pmol / μL), H ₂ O (460 μL), 2-fold concentrated AmpliTaq Gold Master Mix ( 500 μL, Applied Biosystems) was mixed and divided into 22 PCR tubes (45 μL × 22 tubes). Twenty-two colonies were selected from the colonies obtained from the LB-ampicillin medium plate obtained in 4), and each was separately added to the above 22 PCR mixed solutions to perform a PCR reaction. The PCR reaction conditions are as shown below. 1) 95 ° C, 5 minutes, 2) 95 ° C, 15 seconds, 3) 61 ° C, 15 seconds, 4) 72 ° C, 1 minute, 5) 1) to 4) repeated 40 times, 6) 72 ° C, 7 7 minutes at 4 ° C. for 10 minutes. Subsequently, 1.0 μL of the PCR product was collected, 2.0 μL of 10 × loading buffer was added, and analyzed by electrophoresis. Colonies into which GST-fused recombinant human catechol O-methyltransferase had been introduced were added to 10 mL LB-ampicillin medium (ampicillin concentration 100 μg / mL) and cultured at 37 ° C. for 15 hours.

6) Extraction and purification of GST-fused recombinant human catechol O-methyltransferase plasmid from Escherichia coli JM109 5) The culture solution of JM109 obtained by transformation of GST-fused recombinant human catechol O-methyltransferase obtained in 5) is one. A portion (100 μL) was used as a glycerol stock, and the remaining culture was centrifuged at 12,000 rpm for 10 minutes to obtain an E. coli pellet. The obtained Escherichia coli pellet was purified by the instruction manual of Qiagen plasmid mini kit (Qiagen) to obtain plasmid DNA encoding GST-fused recombinant human catechol O-methyltransferase. Their concentrations were determined by OD260nm, 311ng / μL, and the ratio of DNA to protein concentration was OD260 / 280 = 1.87. Each plasmid solution was diluted with TE buffer to give a 1 ng / μL solution. The sequence of the obtained plasmid DNA was analyzed by the dye terminator method using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 7 described below. The sequence was confirmed to contain a base.
5'-primer;5'-catcgagatacaccccgactg-3'(SEQ ID NO: 7)

7) Transformation of GST-fused recombinant human catechol O-methyltransferase plasmid DNA into E. coli BL21 CODON PLUS (DE3) RP 1.0 μL (1.0 ng / μL) of purified GST-fused recombinant human catechol O-methyltransferase plasmid DNA The suspension was added to 50 μL of E. coli BL21 CODON PLUS (DE3) RP cell suspension thawed at 0 ° C. and allowed to stand at 0 ° C. for 30 minutes. The mixture was heat shocked at 42 ° C. for 40 seconds without vigorous shaking and cooled at 0 ° C. for 10 minutes. Next, 450 μL of SOC medium was added to the cell suspension and shaken at 37 ° C. for 1 hour. The obtained mixture was plated on a plate of LB-ampicillin medium (diameter: 9 cm, ampicillin concentration: 100 μg / mL), and statically cultured at 37 ° C. for 15 hours.

8) Expression of GST-fused recombinant human catechol O-methyltransferase A plasmid for expressing GST-fused recombinant human catechol O-methyltransferase from E. coli BL21 CODON PLUS (DE3) RP plates after transformation is retained. E. coli colonies were picked up and put into 10 mL of LB-ampicillin medium (ampicillin concentration 100 μg / ml). The above mixture was subjected to shaking culture at 37 ° C. for 4 hours until the OD600nm value became 0.24. A 50 μL portion of the culture was used as a glycerol stock. Further, 1.0 mL was collected as a sample before addition of isopropyl-β-D-thiogalactopyranoside. To the remaining mixed solution after fractionation, 9.0 μL of 0.1 M isopropyl-β-D-thiogalactopyranoside was added and shaken at 20 ° C. for 6 hours. The mixture was then centrifuged at 12,000 rpm for 15 minutes, and the E. coli pellet was recovered and stored frozen at −80 ° C. until use.

9) Thrombin treatment of GST-fused recombinant human catechol O-methyltransferase 8) The cell pellet obtained from 8) was treated with 1.8 mL BugBuster solution (Novagen) per cell pellet for 15 minutes at room temperature with gentle stirring. The obtained lysate was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was recovered. Then, it was previously equilibrated with D-PBS (DULBECCO'S PHOSPHATE BUFFERED SALINE) and resuspended to 50% with D-PBS. 400 μL of glutathione 4B Sepharose (resin bed volume 200 μL) was added to the supernatant solution, and the resulting mixture was shaken at 4 ° C. for 30 minutes. The mixture after shaking was separated into a resin and a filtrate by a filter. The obtained resin was washed 5 times with 500 μL of D-PBS, and 300 μL of thrombin treatment buffer (150 mM NaCl, 50 mM Tris HCl, pH 8.0, 10% glycerol, 2.5 mM CaCl ₂ , 0.5% β-octyl) -D-glucopyranoside) and washed 3 times. Next, the resin for thrombin treatment was added to the resin to make 500 μL, and 5 units of thrombin (Amersham Bioscience) was added. The resin mixture was gently stirred at 4 ° C. for 15 hours, and a portion thereof was analyzed by SDS polyacrylamide gel electrophoresis. As a result, a band corresponding to recombinant human catechol O-methyltransferase was detected by thrombin treatment. The concentration of the protein obtained by filtering a part of the resin mixture was 258 μg / mL. Furthermore, when the molecular weight of the recombinant human catechol O-methyltransferase was measured with an electrospray quadrupole mass spectrometer, the observed value was 23994.97, which is in good agreement with the theoretical value calculated from the peptide described in SEQ ID NO: 1 = 23997.67. I saw.

10) Purification and dialysis treatment of recombinant human catechol O-methyltransferase by solid phase extraction The thrombin-treated resin suspension was loaded onto an empty column and the filtrate was recovered. Next, the filtrate obtained by this operation was loaded into GSTrap FF (Amersham Bioscience) with a peristaltic pump, and the filtrate was recovered. The filtrate was dialyzed overnight against 2.0 liters of buffer (10 mM NaCl, 10% Glycerol, 20 mM Tris pH 7.0, 0.5% β-octyl-D-glucopyranoside, 4.0 mM dithiothreitol).

11) Super Q-5PW column chromatography About 10-12 mL of the sample solution after dialysis (equivalent to about 10 mg as the amount of protein) is preliminarily buffered at a flow rate of 0.5 mL / min with buffer A (10 mM NaCl, 10% Glycerol, 20 mM Tris). pH 7.0, 0.5% β-octyl-D-glucopyranoside, 4.0 mM dithiothreitol)) and loaded onto a Super Q-5PW column. The target protein is eluted from this column with a linear gradient at 0.5 mL / min with buffer B (10% glycerol, 50 mM Tris pH 7.0, 4.0 mM dithiothreitol, 0.2% β-octyl-D-glucopyranoside, 700 mM NaCl). ((0% buffer B for 7 minutes (equivalent to about 4 times the volume of Super Q-5PW column volume), 0-20% buffer B for 70 minutes (about 40 times the volume of Super Q-5PW column volume) Equivalent), 2 minutes with 20-100% buffer B (equivalent to about 1 volume of Super Q-5PW column volume)) As a result, the peak obtained by elution of about 50 minutes from the start was analyzed by SDS polyacrylamide gel Analysis by electrophoresis confirmed that the molecular weight of recombinant human catechol O-methyltransferase was given.

12) The fractions obtained by Superdex HR75 prep grade column chromatography 11) were combined and concentrated to about 4 mL with a YM-10 membrane (Millipore). Load 1.0 mL of the sample solution onto a Supedex HR75 prep grade column (Amersham Biosciences) equilibrated with buffer (10% glycerol, 50 mM MES pH 6.5, 1.0 mM dithiothreitol, 2.5 mM MgCl ₂ ) for 2 hours. The elution was performed at a flow rate of 1.0 ml / min. Fractions eluted after 70 minutes were combined and collected as human catechol O-methyltransferase. Moreover, it was confirmed by SDS polyacrylamide gel electrophoresis that the eluted fractions were combined to give the molecular weight of human catechol O-methyltransferase. The obtained fraction of recombinant human catechol O-methyltransferase was analyzed using a Develosil C4 column (Nomura Chemical Co., Ltd.), and it was confirmed that the purity was 95% or more.

Example 2
(Recombinant human catechol O-methyltransferase; S-adenosylmethionine; 3,5-dinitrocatechol; and crystallization of magnesium ion quaternary complex)
10 mL of the fraction containing recombinant human catechol O-methyltransferase prepared in Example 1 was concentrated to about 1.5 mL with a YM-10 membrane (Millipore), and the concentrated solution was further concentrated to a Centricon-10 membrane (Millipore). The final concentration was 5.0 to 4.8 mg / mL. To 60 μL of human catechol O-methyltransferase solution concentrated to the target concentration, 3.0 μL of 60 mM S-adenosylmethionine solution was added and incubated at 0 ° C. for 30 minutes. Subsequently, 6.0 μl of 20 mM 3,5-dinitrocatechol solution was added and incubated at 0 ° C. for 30 minutes in the same manner. Crystallization screening was carried out using Crystal Screen I, II from Hampton Research, set up by the hanging drop method, and incubated at 20 ° C. As a result, yellow wedge-shaped crystals were obtained in about 4 days from the conditions defined by 0.1M Hepes pH 7.5, 2% (v / v) PEG 400, 2.0M (NH ₄ ) ₂ SO ₄ .
The obtained crystal is shown in FIG. These crystals have lattice constants a = 50.584, b = 50.584, c = 167.240 and belong to the space group P3 ₂ 21. They contained one molecule per asymmetric unit (Mw = 23.99 kDa) and had a solvent content of 51.85%. (Vm = 2.57; Matthews BW, “J. Mol. Biol.”, 1968, 33, p.491-497)

  The obtained quaternary complex crystal was immersed in a crystallization buffer containing 10 to 15% glycerol for about 30 to 60 seconds, picked up with a 0.5 to 0.7 mm diameter cryoloop (Hampton Research), and in liquid nitrogen. Quick-frozen by direct pickling.

Example 3
(Analysis of the three-dimensional structure of a quaternary complex crystal composed of recombinant human catechol O-methyltransferase; S-adenosylmethionine; 3,5-dinitrocatechol; and magnesium ion)
1) Crystal data collection The diffraction data of the quaternary complex crystals obtained in Example 2 were collected at the beam line BL32B2 of the SPring-8 large synchrotron radiation facility. The crystals that had been frozen in advance were mounted so as to be immersed in a cooling liquid nitrogen stream at −173 ± 1.0 ° C., and measurement was performed using a Jupiter CCD (Rigaku Corporation) at a wavelength of 1.0 mm. The distance between the CCD detector and the crystal is 150 mm. The data set acquired the image of a total of 100-120 sheets in the range from -60 degrees to 60 degrees at the vibration angle of 1.0 degrees to 1.2 degrees. X-ray exposure time was 40 to 60 seconds / image.

2) Processing of diffraction data
The reflection intensity of the image file data set obtained by Mosflm Ver 6.2.1 was integrated. The obtained mtz file is processed sequentially with the program sortmtz, the program scala, and the program truncate (Collaborative Computational Project: (1994) Acta Crystallogr. D50, 760-763; CCP4 suite Ver 4.2.2). Converted to a file. The statistics for each data set are shown below.
Total reflection number = 29525
Number of independent reflections = 9033
Data integrity (overall / outermost shell) = 98.9 / 98.4 (%)
R merge (overall / outermost) = 0.085 / 0.205
Redundancy (overall) = 3.3

3) Determination of the space group For the molecular replacement, the protein coordinates excluding S-adenosylmethionine, magnesium ion, 3,5-dinitrocatechol, and water molecules from pdb1vid.ent in the protein data bank (PDB) are used as a search model. However, amino acid differences due to species differences between human and rat were substituted with alanine. AMoRe (Navaza, J. (1994) AmoRe: Automatic package for molecular replacement, Acta Cryst. D50, 157-163 CCP4 Ver.4.2) As a result of determining the position of rotation and translation, the space group is P3 ₂ I was able to determine 21. Details of the results are shown below.
Resolution range: 20.0 to 3.0 (Å)
Rigid body R factor: 0.429

4) Refinement of the quaternary complex structure The refinement of the structure was performed by CNX2002 (Accelrys Corporation). As a test set, 10% of total reflection was used. QUANTA2000 (Accelrys Co., Ltd.) was used for the main chain and side chain traces and amino acid substitutions, and the model was corrected with reference to the Omit map created by CNX2002. The bulk water mask used the Babynet model. Water molecules are analyzed using the QUANTA2000 Solvation module while interpreting the Fo-Fc difference Fourier map, and ligands such as magnesium ions and S-adenosylmethionine are modeled using the program 2-D sketcher and program Molecular Editor. Modeling by manual fitting was performed to match the electron density of the Fo-Fc difference Fourier map. The final refinement statistic with individual temperature factor refinement was R = 0.254. The statistical quantities that evaluate the quality of the refined structure are shown below.
Final refinement parameter Resolution range: 10.0-2.5Å
Reflection: 9025
R factor: 0.254
R-free factor: 0.283
Number of residues: 212
Number of atoms: 1660
RMS deviation (bond length): 0.010
RMS deviation (bond angle): 1.445
Average temperature factor (protein): 12.94
Average temperature factor (water molecule): 21.15

  The atomic coordinates of the obtained quaternary complex crystals are shown in Table 1 below in accordance with the format of the protein data bank (PDB), which is a standard format for handling biopolymer coordinates.

上記の表において、１〜１３列目はそれぞれ以下：１列目の「ATOM」は原子座標の一つ一つを意味し、「HETATM」は蛋白質以外の化学種を意味する；２列目の数字は原子の通し番号を表す；３列目は各アミノ酸残基、Ｓ−アデノシルメチオニンまたは３，５−ジニトロカテコールにおける位置を表す；４列目は、その原子が属するアミノ酸残基の種類を表し、MGはマグネシウムイオン、SAMはＳ−アデノシルメチオニン、DNCは３，５−ジニトロカテコールを表す；５列目は蛋白質鎖の識別記号を表す；６列目は、そのアミノ酸残基のＮ末端からの番号を表す；７、８および９列目は、それぞれＸ座標、Ｙ座標およびＺ座標（Å単位）を表す；１０列目は占有率を表す；１１列目は等方性温度因子を表す；１２列目はチェインアイデ_イーを表す；１３列目は原子を表す。

In the above table, the 1st to 13th columns are as follows: “ATOM” in the first column means each of the atomic coordinates, and “HETATM” means a chemical species other than protein; The numbers represent the atomic serial numbers; the third column represents the position in each amino acid residue, S-adenosylmethionine or 3,5-dinitrocatechol; the fourth column represents the type of amino acid residue to which the atom belongs. , MG represents magnesium ion, SAM represents S-adenosylmethionine, DNC represents 3,5-dinitrocatechol; the fifth column represents the protein chain identifier; the sixth column represents the N-terminal of the amino acid residue. The seventh, eighth and ninth columns represent the X, Y and Z coordinates (in units of Å), respectively, the tenth column represents the occupancy, and the eleventh column represents the isotropic temperature factor. ; 12 column represents a chain loves _Lee chromatography; 13 Eye represents the atom.

5) Characterization of ligand-accommodating pocket FIG. 2 contains the ligand of the human catechol O-methyltransferase of the present invention; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex crystals. The structure near the pocket is shown.
The ligand-accommodating pocket of human catechol O-methyltransferase accommodates a ligand and magnesium ion, and the magnesium ion present in the active center interacts with the side chain of the three amino acid residues Asp143, Asn172, and Glu201. Furthermore, it was confirmed that the two hydroxyl groups of 3,5-dinitrocatechol also form a coordination interaction. Furthermore, it is considered that the side chain of Glu201 forms a hydrogen bond with one hydroxyl oxygen atom of 3,5-dinitrocatechol and stabilizes the complex. It was confirmed that the indole ring, which is the side chain of Trp40, caused an edge-to-face CH-π interaction with the benzene ring of 3,5-dinitrocatechol. In addition, the carbon atom at the γ-position of Pro176 formed van der Waals interaction with the benzene ring of 3,5-dinitrocatechol. Further, the Nz atom in the side chain of Lys 146 is directed toward the hydroxyl oxygen atom on the S-adenosylmethionine side of 3,5-dinitrocatechol, which is considered to stabilize the complex electrostatically.
In the vicinity of the ligand-accommodating pocket, it was revealed that the side chain of Arg203 forms a hydrogen bond with Glu36 and is not oriented to the 3,5-dinitrocatechol side. Comparison of human catechol O-methyltransferase crystals of the present invention with the three-dimensional structure of known rat catechol O-methyltransferase; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex crystals As a result, in rat catechol O-methyltransferase, the amino acid residue corresponding to Arg203 is hydrophobic methionine, and the side chain of this methionine residue protrudes in the direction of the ligand, In human catechol-O-methyltransferase, as shown in FIG. 2, the side chain of Arg203 is directed in the opposite direction to the ligand to form a hydrogen bond with Glu36, and human catechol-O-methyltransferase is composed of rat rat catechol- O-methyl transfer It was found to have a broad ligand receiving pocket compared to. Such knowledge is revealed for the first time by analyzing the crystal of the quaternary complex of the present invention.

6) Characterization of S-adenosylmethionine-accommodating pocket Figure 3 shows the human catechol O-methyltransferase of the present invention; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex crystals The structure near the pocket accommodating S-adenosylmethionine was shown.
The pocket containing S-adenosylmethionine was characterized as follows. That is, when QUANTA (manufactured by Accelrys) was used to cut out amino acid residues located within a range of 5 mm from S-adenosylmethionine, the following amino acid residues were: , Tyr70, Cys71, Gly72, Tyr73, Ser74, Ile91, Glu92, Ile93, Asn94, Cys97, Gly119, Ala120, Ser121, Gln122, Phe141, Asp143, His144 and Trp145 were identified.
Trp145 was stabilized by causing an edge-to-face interaction with the adenine ring of S-adenosylmethionine. Similarly, His144 formed an edge-to-face interaction with the adenine ring of S-adenosylmethionine. The side chain of Ile93 was located on the adenine ring of S-adenosylmethionine and made van der Waals interaction. Glu92 formed hydrogen bonds with two hydroxyl groups on the ribose ring of S-adenosylmethionine.

  Since the quaternary complex crystal of human catechol O-methyltransferase of the present invention; S-adenosyl-L-methionine; magnesium ion; and ligand provides a detailed three-dimensional structure for human catechol O-methyltransferase, the crystal Is extremely useful for the molecular design of human catechol O-methyltransferase inhibitors.

FIG. 1 shows photographs of crystals of human catechol O-methyltransferase of the present invention; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex. FIG. 2 shows the three-dimensional structure in the vicinity of the pocket accommodating the ligand among human catechol O-methyltransferase; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex. The black ball and stick model shows S-adenosylmethionine. Magnesium ions are shown as gray spheres. The gray ball and stick model shows 3,5-dinitrocatechol. FIG. 3 shows the three-dimensional structure near the pocket that accommodates S-adenoylmethionine among human catechol O-methyltransferase; S-adenosylmethionine; magnesium ion; and 3,5-dinitrocatechol quaternary complex. . The black ball and stick model shows S-adenosylmethionine. Magnesium ions are shown as gray spheres. The gray ball and stick model shows 3,5-dinitrocatechol.

Claims (5)

A crystal of human catechol O-methyltransferase comprising human catechol O-methyltransferase; S-adenosylmethionine; magnesium ion;
The human catechol O-methyltransferase comprises the amino acid sequence represented by SEQ ID NO: 1,
The crystal has a space group P3 ₂ 21, and the lattice constants are 50.58Å ± 0.3Å, b is 50.58Å ± 0.3Å, c is 167.24Å ± 0.4Å, and α is 90 A crystal having a value equal to 0.0 °, β equal to 90.0 °, and γ equal to 120.0 °. The crystal according to claim 1, wherein the ligand is 3,5-dinitrocatechol. The crystal according to claim 2, having the atomic coordinates described in Table 1. The human catechol O-methyltransferase has a pocket that accommodates S-adenosylmethionine, and the pocket falls within the range of 5 mm from S-adenosylmethionine, Met42, Asn43, Val44, Leu67, Gly68, Ala69 , Tyr70, Cys71, Gly72, Tyr73, Ser74, Ile91, Glu92, Ile93, Asn94, Cys97, Gly119, Ala120, Ser121, Gln122, Phe141, Asp143, His144, and Trp145. The crystal | crystallization as described in any one of -3. The human catechol O-methyltransferase has a pocket accommodating a ligand, and the pocket is formed by amino acid residues including Trp40, Met42, Asp143, Trp145, Lys146, Asp171, Asn172, Pro176, Leu200, and Glu201. The crystal according to any one of claims 1 to 4.

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