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WO2001092335A1 - Cristaux de complexes proteiques, coordonnees structurales, et utilisation de ces coordonnees structurales - Google Patents

Cristaux de complexes proteiques, coordonnees structurales, et utilisation de ces coordonnees structurales Download PDF

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Publication number
WO2001092335A1
WO2001092335A1 PCT/JP2001/004519 JP0104519W WO0192335A1 WO 2001092335 A1 WO2001092335 A1 WO 2001092335A1 JP 0104519 W JP0104519 W JP 0104519W WO 0192335 A1 WO0192335 A1 WO 0192335A1
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WIPO (PCT)
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woiv
moiv
structural coordinates
dimensional structural
metabotropic
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PCT/JP2001/004519
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English (en)
Japanese (ja)
Inventor
Hisato Jingami
Naoki Kunishima
Kosuke Morikawa
Takaaki Hiroike
Hiroyuki Toh
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Biomolecular Engineering Research Institute
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Publication of WO2001092335A1 publication Critical patent/WO2001092335A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
    • G01N33/9406—Neurotransmitters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to crystal and three-dimensional structural coordinates of a polypeptide consisting of a glutamate binding portion (hereinafter abbreviated as smG 1 u R) of the extracellular region of metabotropic glutamate receptor (hereinafter abbreviated as mG l R).
  • the present invention also relates to the crystal and three-dimensional structural coordinates of the complex of s mG 1 u R and glutamic acid.
  • the present invention further relates to methods for identifying, searching, evaluating or designing mutants of metabotropic dalbamic acid receptor, glutamate agonists and lupus drugs using these three-dimensional structural coordinates.
  • Glutamate is a type of important neurotransmitter in the central nervous system. Glutamate receptors can be roughly divided into two types, metabotropic dabulin receptors (mG 1 u R) and ionotropic glutamate receptors. By binding to these receptors, dalutabic acid is involved in signal transduction between neurons.
  • G protein coupled receptors G P C R
  • the metabotropic glutamine receptor mG 1 u R
  • Binding of the ligand to these receptors causes the signal to be transmitted to the intracellular part of the receptor, thereby activating the G protein.
  • a signal is transmitted to a specific protein in the cell to increase the concentration of inositol-1,4,5-trisphosphate, causing an increase in intracellular calcium concentration.
  • glutamate receptor subtypes cause a decrease in cyclic AMP concentration.
  • mG 1 u R has 1 to 8 subtypes in humans and rats (mG 1 u R l ⁇ mG l uR 8) is known.
  • GBD glutamic acid binding domain
  • mice cause ataxia and spatial loss recognition.
  • Kano M. et al., Neuron, 18: 779, 1997. Conquet, F. et al., Natures 372: 237-243, 1994.
  • long-term cerebellar long-term depression and hippocampal long-term potentiation are not observed in this mouse, which is assumed to be the basic mechanism of memory and learning (Aiba, A. et al., Cell 79: 365-375, 1994). Kano, M.
  • mG 1 u R 4 gene-disrupted mice exhibit reduced limb coordination motor ability (Pekhletski, R. et al., J. Neuroscience, 16: 6364-6373, 1996).
  • mG 1 uR 7 gene-disrupted mice lack the amygdala-dependent fear response (Masugi, M. et. Al., J. Neuroscience, 19: 955-963, 1999).
  • the efficacy of the mGluR antibody has been reported in the gene epileptogenic rat (Tang et al., Eur. J. Pharmacol., 327: 109-115, 1997).
  • agonists and antagonists of metabotropic glutamic acid receptor are closely related to diseases such as neurological diseases.
  • the glutamate binding domain (GBD) of mGluR is considered to be the most important unit for ligand binding of glutamate receptors and signal transduction. Elucidating the binding of the ligand GBD region of the receptor to the ligand glutamic acid is essential for elucidating the interaction between the ligand and the entire receptor. That is, glutamate receptor variants, mutants of glutamate receptors that can analyze signal transduction mechanisms, and agonists, antagonists that are completely specific to each subtype of glutamate receptors. Designing a gonist is considered to be able to provide a very effective treatment for neurological diseases etc., and is highly desired as a medicine.
  • mGluR is included in GPCRs, but it has not been possible to express these receptors as membrane proteins and conduct structural analysis so far. Also, it has not been possible to produce only the ligand binding region of GPCR as a soluble protein.
  • the present inventors succeeded in producing a large amount of a metabotropic glutamate receptor subtype 1 (mGluR1), a ligand glutamic acid binding region (smGluR 1) as a soluble polypeptide.
  • mGluR1 metabotropic glutamate receptor subtype 1
  • smGluR 1 a ligand glutamic acid binding region
  • This smGluR1 polypeptide was purified, crystals of smGluR1 were produced, and the three-dimensional structural coordinates of smGluRl were revealed for the first time.
  • crystals of a complex of glutamate and smG 1 uR 1 were also prepared, and the three-dimensional structural coordinates of the complex of gonore amic acid and smGluR 1 were also clarified for the first time.
  • crystals of a complex of s-MCPG (S-di-methyl 4-carboxyphenylglycine) and smGluR 1 which are antagonists of glutamic acid are prepared, and the complex of s-MCPG and smGluR 1 is prepared. I also clarified the dimensional structural coordinates for the first time at the same time.
  • smGluR the part that binds to glutamate in the extracellular domain of metabotropic glutamate receptors
  • mGluR metabolic glutamate receptor
  • the present invention it is possible to know the detailed mechanism of transmission (stimulatory neurotransmission) of stimulation via glutamate receptor, and based on its three-dimensional structure, a more active ligand or each subtype of It enabled identification, search, evaluation or design of agents capable of specifically identifying receptors. It also made it possible to identify, search, evaluate or design compounds such as antagonists that inhibit the neurotransmission activity of glutamate.
  • smGluRl Identification, search, evaluation or design of selective ligands or easy identification, search, evaluation or design of compounds having selective antagonism has become easy.
  • the present invention provides a crystal of smGluR1 and a crystal of a complex of glutamate and smGluR1 and a crystal of a complex of S-MCPG and smG1 uR1.
  • the present invention relates to the three-dimensional structure of smGluRl, and to glutamic acid and smG 1 used to identify, search, evaluate or design glutamate receptor subtype 1 variants, agonists or antagonists.
  • the three-dimensional structural coordinates of the complex formed by uR1 and the three-dimensional structural coordinates of the complex of s-MCPG and smGluR1 are also summarized.
  • the present invention relates to glutamic acid and metabotropic glutamate receptor subtypes used for identifying, searching, evaluating or designing mutants, agonists or antagonists of glutamic acid receptor-subaver 2-8.
  • the gist is also a three-dimensional structural coordinate of a complex formed by 2-8 (mGluR2-8) glutamic acid binding regions (smGluR2-8).
  • the present invention provides a computer that stores all or part of the above three-dimensional structural coordinates, which is used to identify, search, evaluate or design glutamate receptor variants, agonists or antagonists.
  • the summary also applies to a single use storage medium.
  • the present invention provides all or a part of the above three-dimensional structural coordinates, or a combination thereof as described above for identifying, searching, evaluating or designing mutants, agonists or antagonists of glutamate receptor.
  • the subject matter is also the use of a storage medium.
  • the present invention is characterized by using all or a part of the above three-dimensional structural coordinates, or the above-mentioned storage medium for a complex, and a biological activity equivalent to or superior to that of a natural glutamate receptor.
  • One or more amino acid residues may be substituted, deleted, or inserted, which can be used to analyze the specificity of the gluconate receptor or signal transduction mechanism, such as having or having altered specificity.
  • a method for identifying, searching, evaluating or designing mutants of glutamic acid receptor or chemically modified are also summarized.
  • the present invention is characterized by using a whole or a part of the above three-dimensional structural coordinates, or a computer storage medium as described above, and identifying and searching for an agonist of glucose acid receptor
  • the method of evaluation or design is also the gist.
  • the present invention is characterized by using all or a part of the above three-dimensional structural coordinates, or the above computer storage medium, and identifying, searching, evaluating or identifying an antagonist of glutamic acid receptor.
  • the gist is also how to design.
  • amino acids, peptides and proteins are represented using the abbreviations adopted by the IUPAC-IB Biochemical Nomenclature Committee (CBN) shown below. Also, unless otherwise specified, the sequences of amino acid residues of peptides and proteins are represented from the left end to the right end so as to be from the N-terminus to the C-terminus, and so that the N-terminus is the first.
  • a or A la a galanine residue
  • D or A sp a gzapalagic acid residue
  • E or G lu a glutamic acid residue
  • F or P he a phenyranan residue
  • G or G ly a glycine residue
  • H or His is a histidine residue
  • I or I le isoleucine residue
  • K or L y s lysine residue
  • V or Val valine residue
  • W or Trp tryptophan residue
  • Y or Tyr tyrosine residue
  • C or Cys cysteine residue.
  • Figure 1 is a ribbon diagram of the crystal structure of glutamic acid non-binding smGluRl, viewed from the membrane (estimated) side along the pseudo 2-fold symmetry axis of the molecule (white dots in the center are pseudo) Two-fold axis of symmetry is shown).
  • FIG. 2 is a ribbon diagram of the crystal structure of glutamic acid non-binding smGluR1 and is seen from the entrance side of the ligand of molecule A almost perpendicular to the pseudo 2-fold symmetry axis of the molecule.
  • Figure 3 is a ribbon diagram of the crystal structure of the complex of glutamic acid and s mG 1 uR 1 showing the B from the film (estimated) side along the pseudo 2-fold symmetry axis of the molecule The white point of represents a pseudo two-fold axis of symmetry).
  • FIG. 4 is a ribbon diagram of the crystal structure of a complex of glutamic acid and smGluR1, showing a direction perpendicular to the pseudo 2-fold symmetry axis of the molecule and viewed from the entrance side of the ligand of molecule A.
  • FIG. 5 shows a ribbon diagram of the crystal structures of glutamate (A) and smGluR1 complexes (A) and (B).
  • Fig. 6 is a ribbon diagram of the crystal structure of the complex of s-MCPG and s mG 1 uR 1 and shows a view from the film (estimated) side along the axis of the quasiquasiotic 2-fold symmetry axis. White points indicate pseudo 2-fold symmetry axis).
  • FIG. 7 is a ribbon diagram of the crystal structure of the complex of S-MCPG and smGluR1, showing a direction perpendicular to the pseudo 2-fold symmetry axis of the molecule and viewed from the entrance side of the ligand of molecule A.
  • FIG. 8 shows the equilibrium of the smGluRl dimer (associate).
  • the dimer of smGluRl is considered to be in equilibrium between inactive (R) (left) and active (A) (right).
  • R inactive
  • A active
  • the equilibrium shifts to the right and becomes active
  • LB2-LB2 domain The spacing between the cells is 63A, while that in the active form is as small as 36A, which is thought to transmit the signal into the cell.
  • SmGluRl used in the present invention is derived from mammals, preferably rats, mice, humans, particularly preferably humans.
  • smGluR 1 In the case of smGluR 1 derived from rat, the region from the first Met to the 522nd Ser in the amino acid sequence (Masu, T. et al., Nature, 394, 760-765, 1991) is smGluRl. In the case of s mG 1 uR 1 derived from other mammals, the corresponding region is said, but the start site and the end site of s mG 1 uR 1 are not necessarily strict in the present invention, and the three-dimensional structure of the whole smGluR 1 There is no major impact on the structure.
  • N-terminal and C-terminal shifted by several residues in any direction or N-terminal and / or C-terminal added with several amino acid residues Is also included.
  • Ser from the 33rd Ser to the 522nd Ser of the above amino acid sequence is used.
  • Crystallization is carried out in the case where the protein changes from a solution state to a non-dissolving state by an operation such as adding a precipitant to the target protein solution or reducing the amount of solvent by evaporation etc. It uses the property of precipitating as crystals. Crystallization requires highly purified protein. Further, as conditions for crystallization, physical and chemical factors such as protein concentration, salt concentration, hydrogen ion concentration (pH), type of precipitant to be added, temperature, and the like are involved. Furthermore, there are many crystallization methods such as batch method, dialysis method, vapor diffusion method, etc. as methods of adding precipitant and adjusting solvent volume (Blundell, T. L. and JOhnson, L. N., PROTEIN)
  • Purification methods include chromatography (eg, chromatography, ion exchange, gel filtration, etc.), salting out, centrifugation, electrophoresis, etc., which are commonly performed by those skilled in the art as methods for purifying proteins. Or in combination. It is desirable to use a purification method capable of adjusting the salt concentration and hydrogen ion concentration to conditions closer to those in the living body, for example, gel filtration chromatography.
  • the most commonly used method for clarifying the three-dimensional structure of belay is X-ray crystal structure analysis. That is, the protein is crystallized, and the monochromatized X-ray is applied to the crystal, and the three-dimensional structure of the protein is clarified based on the diffraction pattern of the obtained X-ray (Blundell, TL And J Ohnson, LN, PROTEIN CRYSTALLOGRAPHY, pp. 1-565 (1976), Academic Press, New York). Analyze the crystal structure of smGluR 1 obtained in 1. using X-ray crystal structure analysis technology.
  • the crystal of s mG 1 uR 1 derived from the lato shown in SEQ ID NO: 1 belongs to the tetragonal space group P 4 ⁇ 2i 2 and has 111 ⁇ 10 A as the unit cell in the direction of the a axis and b axis. It has a size of 294 ⁇ 1 OA in the direction of the axis. Furthermore, using the crystal By the method of crystal structure analysis by X-ray diffraction, the three-dimensional structural coordinates (value indicating the spatial positional relationship of each atom) of the metabotropic glutamate receptor according to the present invention are obtained for the first time. The obtained structural coordinates are shown in Table 1 in accordance with the three-dimensional structural coordinate notation of proteins generally used by those skilled in the art.

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Abstract

L'invention concerne des procédés permettant d'identifier, de rechercher, d'évaluer ou d'élaborer un variant du récepteur du glutamate métabolique, présentant la substitution, la délétion, l'insertion ou la modification chimique d'un ou de plusieurs restes d'acide aminé, un agoniste ou un antagoniste de l'acide glutamatique. Ces procédés consistent à utiliser des cristaux d'un polypeptide (smGluR) dans la région se fixant au glutamate dans la région extracellulaire d'un récepteur du glutamate, des cristaux d'un complexe comprenant de l'acide glutamatique et le smGluR, des cristaux d'un complexe comprenant le s-MCPG et le smGluR, et la coordonnée structurale de chaque atome qui est déterminée par des techniques d'analyse de la structure du cristal à l'aide de l'analyse aux rayons X de ces cristaux.
PCT/JP2001/004519 2000-05-31 2001-05-30 Cristaux de complexes proteiques, coordonnees structurales, et utilisation de ces coordonnees structurales WO2001092335A1 (fr)

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JP2000-161615 2000-05-31

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116096517A (zh) * 2020-10-02 2023-05-09 住友电工硬质合金株式会社 立方晶氮化硼烧结体工具

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029449A1 (fr) * 1993-06-04 1994-12-22 The Salk Institute Biotechnology/Industrial Associates, Inc. Recepteurs humains du glutamate metabotropique, acides nucleiques codant ceux-ci et leurs utilisations
WO1997041211A1 (fr) * 1996-04-30 1997-11-06 Vertex Pharmaceuticals Incorporated Molecules comprenant une poche de liaison de type impdh et support code de stockage de donnees representant graphiquement celles-ci

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029449A1 (fr) * 1993-06-04 1994-12-22 The Salk Institute Biotechnology/Industrial Associates, Inc. Recepteurs humains du glutamate metabotropique, acides nucleiques codant ceux-ci et leurs utilisations
WO1997041211A1 (fr) * 1996-04-30 1997-11-06 Vertex Pharmaceuticals Incorporated Molecules comprenant une poche de liaison de type impdh et support code de stockage de donnees representant graphiquement celles-ci

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ASTRID G. CHAPMAN ET AL.: "Anticonvulsant actions of LY 367385 ((+)-2-methyl-4-carboxyphenylglycine) and AIDA ((RS)-1-aminoindan-1.5-dicarboxylic acid)", EUR. J. PHARMACOL., vol. 368, 1999, pages 17 - 24, XP002945095 *
CLAIRE WILSON ET AL.: "Absolute congiguration of (+)-alpha-methyl-4-carboxyphenyglycine (MCPG), a Mwtabotropic glutamate receptor antagonist", ACTA CRYST., vol. C53, 1997, pages 909 - 911, XP002945094 *
ROBERT PELLICCIARI ET AL.: "Synthesis and pharmacological characterization of all sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl) glycine, a novel and selective group II metabotropic", J. MED. CHEM., vol. 39, 1996, pages 2259 - 2269, XP002945096 *
TOMOYUKI OKAMOTO ET AL.: "Expression and purification of the extracelluler ligand binding region of metabotropic glutamate receptor subtype 1", J. BIOL. CHEM., vol. 273, no. 21, 1998, pages 13089 - 13096, XP002945093 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116096517A (zh) * 2020-10-02 2023-05-09 住友电工硬质合金株式会社 立方晶氮化硼烧结体工具

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