JP2006209764A - Specification method for ligand bonding portion of protein and three dimensional structure construction method for protein-ligand complex - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method or the like for specifying a bonding portion of a protein-ligand complex. <P>SOLUTION: In this specification method for the ligand bonding portion of protein, (i) a low molecular compound is disposed around a three dimensional structure of the protein, (ii) water moleculars are further disposed around them, and empirical molecular energy calculation in a water solvent is performed to obtain atomic coordinates of the protein and the low molecular compound, and (iii) the behavior analysis of the low molecular compound inside and around the protein is performed about the obtained atomic coordinates to decide the bonding portion of a ligand. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for constructing a three-dimensional structure of a protein including inductive adaptation and the use thereof. More specifically, the present invention relates to a three-dimensional structure set in which a three-dimensional structure of a reference protein and its atomic coordinates are displaced as a three-dimensional structure of the reference protein. The present invention relates to a method for constructing a three-dimensional structure of a protein comprising preparing a plurality of three-dimensional structure sets of the protein, a method for constructing a three-dimensional structure of a protein-ligand complex using the three-dimensional structure set, a method for specifying a ligand binding site of a protein, and the like.

  The three-dimensional structure of the target protein provided by the method of the present invention is a three-dimensional structure including an induced fit, and is extremely useful for molecular design of medicines and agrochemicals.

  Using information on a protein with a known three-dimensional structure, it is possible to obtain an alignment with a target protein with an unknown three-dimensional structure, and create a three-dimensional structure of the target protein using a computer based on this alignment information. This method is usually called homology modeling. The accuracy of the three-dimensional structure constructed by homology modeling has improved remarkably in recent years, but there are still many problems to be solved.

  When constructing a three-dimensional structure of a receptor protein using this method, it is essential to secure a space for binding a ligand. However, in the conventional three-dimensional structure construction method, the main chain or side chain of the three-dimensional structure constructed in the space where the ligand exists or the binding site is packed and the space is blocked, and the ligand comes into contact with the receptor protein. The problem that it cannot exist in the binding site has occurred.

  In the method for constructing the three-dimensional structure of the protein-ligand complex, if the three-dimensional structure of the target receptor protein has not been experimentally determined, the three-dimensional structure of the receptor protein constructed by the homology modeling method is simply used. The ligand was docked, and the three-dimensional structure of the receptor protein-ligand complex was obtained by optimizing them by molecular force field calculation and molecular dynamics calculation. Also, in the research using the Multiple Copy Simultaneous Search (MCSS) method, the normal vibration mode is not considered in the three-dimensional structure on the receptor protein side, and mainly the vibration of the pico order with respect to the time of the molecule. Long-period thermal fluctuations (hereinafter simply referred to as “thermal fluctuations” or “molecular fluctuations”) have been ignored.

  Furthermore, conventionally, methods for identifying a ligand binding site of a protein by an electrostatic potential that affects a long distance and construction of a three-dimensional structure of a protein-ligand complex based on similar compounds have been carried out. In the absence of similar compounds with low reliability, it was difficult to derive a reliable protein-ligand complex steric structure.

  In view of the above situation, the present invention has been made for the purpose of providing a method for accurately constructing a three-dimensional structure of an arbitrary protein, a method for accurately constructing a three-dimensional structure of a protein-ligand complex, and the like. .

  As a result of diligent studies to achieve the above-mentioned problems, the present inventors have constructed the three-dimensional structure of the receptor protein with reference to the atomic coordinates obtained by displacing the atomic coordinates of the reference protein in the eigenvector direction obtained from the standard vibration analysis method. For example, it is possible to significantly improve the accuracy of the three-dimensional structure of the receptor protein without packing the main chain or side chain of the three-dimensional structure in the space where the ligand exists or the binding site and closing the space. I found it. That is, it has been found that a plurality of receptor protein models can be constructed in consideration of molecular thermal fluctuations based on the normal vibration mode.

  In addition, using the three-dimensional structure of the ligand docked to the receptor protein model thus constructed, the molecular dynamics and molecular dynamics calculations of the Multiple Copy Simultaneous Search (MCSS) method were applied to take into account thermal fluctuations of the molecules. It has been found that it is possible to construct a three-dimensional structure of a protein-ligand complex with high accuracy.

  Furthermore, the present inventors have come to the conclusion that hydrophobic interaction is more important than electrostatic force when considering a phenomenon in an aqueous solution for a protein-ligand complex. Therefore, a solvent is placed around and inside the protein, and the site where the solvent accumulates on the protein or the site where the solvent is difficult to diffuse from the analysis of solvent behavior (solvent diffusion / accumulation) by molecular dynamics matches the ligand binding site. I found.

  The present invention has been accomplished based on these findings.

  That is, in the method of (1) deriving alignment between a reference protein and a target protein and constructing a three-dimensional structure of the target protein based on the alignment and three-dimensional structure information of the reference protein by the method of the present invention, the three-dimensional structure of the reference protein And a method for constructing a three-dimensional structure of a protein including inductive fitting, wherein a plurality of three-dimensional structures of the target protein are created using the three-dimensional structure of which the atomic coordinates are displaced as the three-dimensional structure of the reference protein. .

  According to a preferred embodiment of the present invention, (2) the displacement of the atomic coordinates of the reference protein is performed by a standard vibration analysis method, and (3) the construction of the three-dimensional structure is (i) ) Obtain coordinates from the three-dimensional structure of the reference protein for the Cα atom in the amino acid, optimize the Cα atom coordinate to minimize the objective function, and (ii) change the other coordinates of the main chain to the optimized Cα atom coordinate Optimize the atomic coordinates of the main chain so as to minimize the objective function by adding atoms, and (iii) add other atoms of the side chain to the atomic coordinates of the optimized main chain to minimize the objective function The method according to the above (1) or (2) is provided by performing the optimization as described above.

  According to another aspect of the present invention, (4) (i) a docking operation of a ligand with a plurality of three-dimensional structures of a target protein obtained by the method according to any one of (1) to (3) above and (ii) ) Calculate the empirical molecular energy of one structure of the target protein and the structure of the ligand by the number of the structure of the target protein, and (iii) the target protein side according to the potential energy gradient of each of the multiple structures. (Iv) On the ligand side, move the ligand's atomic coordinates in the direction that averages the calculated potential energy gradients, and (v) the ligand's three-dimensional structure based on the three-dimensional structure of the target protein. Provided is a method for constructing a three-dimensional structure of a protein-ligand complex characterized in that a structure is determined.

  According to a preferred embodiment of the present invention, (5) in the empirical molecular energy calculation, the position of the initial Cα atom coordinate of the target protein is added as an optional Harmonic function, or a potential function that constrains the twist angle of the main chain of the target protein is added The method described in (4) above is provided.

  According to another aspect of the present invention, (6) (i) a low molecular weight compound is arranged around the three-dimensional structure of a protein, (ii) a water molecule is further arranged around them, and an empirical molecule in an aqueous solvent. Perform energy calculation to obtain the atomic coordinates of the protein and low molecular weight compound, and (iii) analyze the behavior of the low molecular weight compound around and inside the protein, and determine the binding site of the ligand. And (7) (i) placing a low-molecular compound around the three-dimensional structure of the protein and the ligand, and (ii) further placing a water molecule around them. Place and perform empirical molecular energy calculations in an aqueous solvent to obtain atomic coordinates of the protein and low molecular weight compounds; (iii) for the obtained atomic coordinates, Performs behavior analysis of the compound, protein - protein and judging the binding site of the ligand complex - How to identify the binding site of the ligand complex is provided.

  According to a preferred embodiment of the present invention, (8) behavior analysis of a low molecular weight compound is performed by cluster analysis for a low molecular weight compound, and the size of the obtained cluster is regarded as the rank of the binding potential site of the ligand, and the binding site is determined. The method according to (6) or (7) above, wherein the determination is performed.

  According to another aspect of the present invention, (9) a ligand is docked to a ligand binding site of a protein identified by the method according to any one of (6) to (8) above, and protein-ligand complex is calculated by empirical molecular energy calculation A method for constructing a three-dimensional structure of a protein-ligand complex is provided.

  According to another aspect of the present invention, (10) the three-dimensional structure of a protein and / or the three-dimensional structure of a protein-ligand complex obtained by the method according to any one of (1) to (5) and (9) above is defined. A computer-readable recording medium characterized in that atomic coordinates to be recorded are recorded, or a database characterized in that the atomic coordinates are included.

  According to another aspect of the present invention, (11) based on the interaction with the three-dimensional structure of a drug candidate molecule using atomic coordinates that define the three-dimensional structure of a protein obtained from the recording medium or database described in (10) above Thus, a drug molecule design method characterized by identifying, searching, evaluating or designing a target drug molecule is provided.

  Hereinafter, the present invention will be described in more detail. In this specification, several terms are used, and have the following meanings unless otherwise specified.

  The “target protein” means an arbitrary protein whose three-dimensional structure has been determined in the present invention, for which a complete three-dimensional structure has not been determined by X-ray crystallography or NMR analysis. This protein includes a protein whose partial structure has been analyzed but a complete three-dimensional structure has not been obtained. In the present invention, it is preferable that the target protein is a receptor protein, an enzyme or the like whose steric structure is unknown. Here, the X-ray crystal analysis includes not only X-rays but also electron beam and neutron beam analysis.

  “Receptor protein” means a protein that is present in a cell and recognizes an exogenous substance or physical stimulus to induce a response in the cell. This receptor protein has the ability to specifically bind a ligand. The “ligand” means a substance having an ability to specifically bind to a protein. Ligand includes not only low molecular weight substances such as medical and agrochemical molecules but also high molecular weight substances such as specific peptides and proteins that interact with antibodies and proteins.

  “Reference protein” means a protein that has been determined in detail by X-ray crystallography, NMR analysis, or the like and whose structure is referred to in order to construct atomic coordinates that define the three-dimensional structure of the target protein. In addition, “alignment” means that amino acid sequence correspondences are established for two or more types of proteins.

  “Atomic coordinates” describe a three-dimensional structure in a three-dimensional space. It is a relative distance in three directions perpendicular to each other with a point in space as the origin, and is a vector quantity consisting of three numbers per atom excluding hydrogen atoms present in the protein.

“Induced fit” means that the protein's conformation is flexible, and when bound to a ligand, such as a pharmaceutical or agrochemical molecule, the conformation of the protein changes to bind better. The “three-dimensional structure including induced fit” means that the three-dimensional structure change of the protein caused by the induction fit can be expressed by, for example, an eigenvector obtained by the standard vibration analysis method. A three-dimensional structure generated by adding eigenvectors.

  The “target protein-ligand complex” refers to a protein-ligand complex that is a target for constructing a three-dimensional structure in the present invention, because the complete three-dimensional structure of the complex has not been elucidated by X-ray crystallography or NMR analysis. means. Of course, it is natural that the protein includes a three-dimensional structure obtained by X-ray crystallography or NMR analysis. This complex includes those in which a partial structure has been analyzed but a complete three-dimensional structure has not been obtained. It means a complex of both ligands bound to a protein.

  The Multiple Copy Simultaneous Search (MCSS) method accepts the three-dimensional structure of the target protein-ligand complex based on the three-dimensional structure of multiple ligands using empirical molecular energy calculation methods, namely molecular mechanics and molecular dynamics calculations. This is a method for obtaining a three-dimensional structure of a body protein. In the present invention, on the contrary, it means a method for obtaining the three-dimensional structure of a target protein-ligand complex from the three-dimensional structure of a plurality of proteins based on the three-dimensional structure of one ligand.

  “Empirical molecular energy calculation” means molecular mechanics calculation and molecular dynamics calculation. Both are molecular energy calculations using empirical potential.

  `` MSAS (Maximum Solvent Accessibility of Sidechain) '' is the maximum solvent contact surface area, the solvent contact surface area of the side chain of each amino acid that constitutes the protein, and the amino acid that does not constitute the protein alone It means the ratio of the side chain to the solvent contact surface area when present. Details of MSAS are described in K. Akahane, Y. Nagano and H. Umeyama, Chem. Pharm. Bull., 1989, 37 (1) 86-92.

The methods I to III described later can be carried out using an appropriate program that executes the method described later using an appropriate computer capable of performing homology modeling.

I. First, a method for constructing a three-dimensional structure including inductive adaptation will be described.

  FIG. 1 is a flowchart showing an example of a method for constructing a three-dimensional structure including an induced fit according to the present invention.

  In step I-10, input the target protein sequence, select the reference protein used to construct the target protein's three-dimensional structure, obtain the atomic coordinates from the reference protein's three-dimensional structure, and minimize the objective function Optimize the coordinates. In step I-20, the optimized vibration analysis method of atomic coordinates is performed. In Step I-30, the atomic coordinates of the reference protein are displaced in the eigenvector direction, and the structure is added to the reference protein to create a set of reference proteins. In step I-40, a set of three-dimensional structures of the target protein is constructed from alignment information and each three-dimensional structure information of the reference protein set by an appropriate homology modeling program such as FAMS. Thus, a three-dimensional structure including an induced fit of the target protein can be constructed with high accuracy. Hereinafter, each step will be described in more detail.

Step I-10: Optimization of Initial Coordinates of Reference Protein First, in the construction of the three-dimensional structure of the target protein, the amino acid sequence of the target protein is input, and the protein to be referenced (reference protein) is selected. Selection of the reference protein is performed using commonly used alignment software known per se. The atomic coordinates of this reference protein are obtained from an appropriate three-dimensional structure database. In this atomic coordinate, there is no hydrogen atom bonded to the nitrogen atom or the like that forms the skeleton of the amino acid, and a hydrogen atom is generated when a hydrogen atom is necessary for the calculation of the normal vibration analysis method in Step I-20. The atomic coordinates are optimized using an objective function composed of the atomic coordinates of the reference protein.

  Here, the amino acid sequence of the target protein to be used may be a sequence derived from any source such as those registered in a database and those analyzed for the first time. As an amino acid sequence database used, for example, “An Internet review: the complete neuroscientist scours the World Wide Web.” Bloom FE, Science 1996; 274 (5290): 1104-9, GCRDb (The G -protein-coupled Receptor Database): http://www.gcrdb.uthscsa.edu/, GPCRDB: http://www.gpcr.org/7tm/, ExPASy: http://www.expasy.ch/cgi- bin / sm-gpcr.pl, ORDB: http://ycmi.med.yale.edu/senselab/ordb/, GeneBank: ftp://ncbi.nlm.nih.gov/genbank/genomes/, PIR: http: //www-nbrf.georgetown.edu/pir/ (National Biomedical Research Foundation (NBRF)), Swiss Plot: http://www.expasy.ch/sprot/sprot-top.html (Swiss Institute of Bioinformatics (SIB) , European Bioinfomatics Institute (EBI)), TrEMBL (URL and administrator are the same as Swiss Plot), TrEMBLNEW (URL and administrator are the same as Swiss Plot), DAD: ftp://ftp.ddbj.nig.ac.jp Human (H. sapiens), Drosophila (D. melanogaster), nematodes (C. elagans), yeasts (S. cerevisiae), Arabidopsis thaliana (A. thaliana) and the like. These databases are merely examples, and any database can be used as long as the amino acid sequence of the protein is registered.

  Further, as a three-dimensional structure database used for obtaining atomic coordinates of a reference protein, for example, PDB (Protein Data Bank): http://www.rcsb.org/pdb/, CCDC (Cambridge Crystallographic Data Center: http: // www.ccdc.cam.au.uk/, SCOP (Structure Classification of Protein): http://scop.mrc-lmb.cam.ac.uk/scop, CATH: http://www.biochem.ucl.ac uk / bsm / cath, etc. These three-dimensional structure databases can be used alone or in combination.In the above-mentioned database, SCOP and CATH are domain units (three-dimensional structure of protein, tertiary It is a three-dimensional structure database divided into structural units).

  For example, FASTA or PSI-BLAST (Position-Specific Iterated BLAST) is preferably used as the alignment software. FASTA is a program for searching a sequence having a high degree of matching with a target sequence from a three-dimensional structure database, and calculating the degree of matching between the final target sequence and a reference protein as an e value. Details of FASTA are described in “Effective protein sequence comparison.” Pearson WR, (1996) Methods Enzymol; 266: 227-58.

  PSI-BLAST is programmed to perform profile alignment. For details on PSI-BLAST, see “Matching a protein sequence against a collection of PSI-BLAST-constructed position-specific score matrices.” Schaffer AA, Wolf YI, Ponting CP, Koonin EV, Aravind L and Altschul SF, Bioinformatics 1999, 12 , 1000-11.

  The method, coordinate system, objective function, etc. for achieving the optimization of the atomic coordinates of the reference protein are not particularly limited, but it is preferable to use, for example, the maximum gradient method, the conjugate gradient method, the Newton-Raphson method, or the like. The maximum gradient method optimizes the objective function of atomic coordinates by using a first-order derivative of the objective function calculated numerically. There are many conjugate gradient methods, but the Fletcher-Reeves method (Fletcher, R., and Reeves, CM (1964) Function Minimization by Conjugate Gradients. Comput J, 7: 149-154) is used as standard. If the objective function is a strict quadratic function of n variables using the first derivative of the objective function, it is guaranteed that the optimization is reached at most by n iterations. The Newton-Raphson method uses the second derivative in addition to the first derivative, and is efficient when the initial structure is close to the optimized structure. Details of these methods are described in Yoshihiro Eguchi, “Physical and Chemical Fundamentals of Protein Engineering (Kyoritsu Shuppan 1991)” and references therein.

  Hereinafter, the structure and coordinates optimized as described above are referred to as the optimized structure and the optimized coordinates, respectively.

Step I-20: Reference vibration analysis method of optimized coordinates Using the optimized coordinates created in Step I-10 above, the atomic coordinates are displaced. The displacement of the atomic coordinates is preferably performed by performing a standard vibration analysis method and obtaining eigenvectors of each eigenvalue. At this time, a coordinate system in which a part of the optimized degrees of freedom is used may be used. In this case, optimization is also achieved for some degrees of freedom.

  Here, the “standard vibration analysis method” means a method in which potential energy is approximated as a quadratic function of displacement, a motion equation is solved exactly, and minute vibrations around the optimized structure are analyzed. “Eigenvalue” means a period of minute vibration. “Eigenvector” means the direction of vibration.

ここでω_kは固有値、U_ikは固有ベクトルであり、δ_ijはクロネッカーのデルタである。T_ijとV_ijはそれぞれ運動エネルギーE_kとポテンシャルエネルギーVに関係し、下記式(3)および(4)の通りである。

ここでq_iは振動の自由度に対応した座標、q_i ⁰は最適化座標、q_iはq_iの時間による微分である。A_jkは集団運動Q_kと個々の原子運動q_jを結ぶ係数であり、下記式(5)の通りである。

ここで、α_kとδ_kは初期条件で定められる。 The eigenvalue equation to be solved by the standard vibration analysis method is the following equation (1) or (2).

Here, ω _k is an eigenvalue, U _ik is an eigenvector, and δ _ij is a Kronecker delta. T _ij and V _ij are related to kinetic energy E _k and potential energy V, respectively, and are expressed by the following equations (3) and (4).

Here q _i is the coordinate corresponding to the degree of freedom of vibrations, q _i ⁰ is optimized coordinates, q _i is the derivative with time of q _i. A _jk is a coefficient connecting the collective motion Q _k and the individual atomic motions q _j , and is given by the following formula (5).

Here, α _k and δ _k are determined by initial conditions.

  Details of the above-described reference vibration analysis method are described in Wilson, E.B., Decius, J.C., and Cross, P.C. 1955. Molecular Vibrations. McGraw-Hill.

Step I-30: Generation of New Reference Protein Using the eigenvalue and eigenvector obtained in Step I-20 above, the position fluctuation of the Cα atom at a certain temperature and a certain eigenvalue is calculated. A position fluctuation equal to the number of eigenvalues is obtained. The temperature factor of the Cα atom of the reference protein is converted into position fluctuation, and the ratio of each Cα atom to the position fluctuation of the standard vibration analysis method is calculated to obtain the average ratio. This average ratio is equal to the number of eigenvalues used, and the eigenvectors belonging to this eigenvalue multiplied by this ratio are added to the atomic coordinates of the reference protein before structure optimization, and the three-dimensional structure consisting of the displaced atomic coordinates, The three-dimensional structure including induced fit is one of the three-dimensional structures of the reference protein. Hereinafter, this is referred to as an induced fit type reference protein, a three-dimensional structure, and coordinates.

  The average ratio is doubled to create an induced fit conformation of the reference protein as well. The eigenvector has a forward / reverse direction, and the eigenvector is similarly displaced in the reverse direction obtained by multiplying the eigenvector by -1. That is, the induced fit type has only four times the number of eigenvalues used. A three-dimensional structure of an induced fit type and a no induced fit type reference protein is set as a reference protein three-dimensional structure set.

ここで、B_iはPDBファイルから得られる原子の温度因子であり、πは円周率、D_iは位置ゆらぎに相当する。本発明ではCα原子に関してのみである。 Here, the relationship between the temperature factor and the position fluctuation is as shown in the following formula (6).

Here, B _i is the temperature factor of the atoms obtained from the PDB file, π corresponds to the circularity, and D _i corresponds to the position fluctuation. In the present invention, only the Cα atom is concerned.

ここでF_i ^vは基準振動解析法から得られるv番目の固有値に対するi番目の原子の位置ゆらぎである。本発明では、Cα原子のみに対して行う。 The ratio of the position fluctuation obtained from the reference vibration method and the position fluctuation converted from the temperature factor of the PDB file is as shown in the following formula (7).

Here, F _i ^v is the position fluctuation of the i-th atom with respect to the v-th eigenvalue obtained from the normal vibration analysis method. In the present invention, it is performed only for Cα atoms.

ここでNは原子数であり、和は原子に対して行う。M^vはv番目の固有値に対する平均の比である。本発明では、Cα原子に対して行う。 The average of the ratio is as shown in the following formula (8).

Here, N is the number of atoms, and the sum is performed on the atoms. M ^v is the ratio of the average to the v th eigenvalue. In the present invention, it is performed on the Cα atom.

ここでC_{i k} ⁰は参照タンパク質の原子座標、V_{i k} ^vはv番目の固有値に属する固有ベクトルの成分をあらわす。 The atomic coordinates of the induced fit type reference protein tertiary structure are as shown in the following formulas (9) and (10).

Here, C _ik ⁰ represents the atomic coordinates of the reference protein, and V _ik ^v represents the component of the eigenvector belonging to the vth eigenvalue.

Step I-40: Modeling of the target protein With reference to the three-dimensional structure set of the reference protein obtained in Step I-30 above, a three-dimensional structure set of the target protein is constructed by an appropriate homology modeling program such as FAMS. The same number of three-dimensional structures of the target protein as the number of three-dimensional structures of the reference protein are constructed. That is, an induced fit and no induced fit target protein conformation that is four times the number of eigenvalues used is constructed, and these are expressed as a target protein conformation set, ie an induction fit ( 3D structure including induced fit).

  Next, each step of FAMS will be described as a suitable example of modeling (construction of a three-dimensional structure). The number of calculations, constants, cut-off values, etc. described in the following Steps I-41 to 43 show examples of parameters that the inventor considers most preferable, and are within the scope of the present invention. It is not limited at all. Details of FAMS are described in Koji Ogata and Hideaki Umeyama, “An automatic homology modeling method consisting of database searches and simulated annealing” Journal of Molecular Graphics and Modeling 18, 258-272, 2000.

Step I-41: Construction and optimization of the initial coordinates of the Cα atom In response to the reference protein set and alignment information from Step I-30, information about amino acid residues inserted and deleted from the reference protein is obtained. In the alignment, select a non-gap region corresponding to three or more amino acids in succession, and in that region, apply the same Cα atom of the target protein as the reference protein in these residue pairs. deep. If the Cα atom is not obtained, coordinates are applied from a database of fragments prepared in advance (see FIG. 2).

  Here, in this specification, the Cα atom means a carbon atom that becomes the center of the skeleton of each amino acid. The Cβ atom means a carbon atom bonded to the side chain side of the Cα atom, and the Cγ atom means a carbon atom bonded to the side chain side of the Cβ atom. The C atom means a carbon atom of a carbonyl group.

ここでU_lenは、配列上隣の残基およびCys残基のペアのCα原子間の距離に関するもので下記式(12)のように設定される。

ここでDi,i+1は残基i と残基i+1のCα間距離である。D_i ^SSはジスルフィド結合を形成するCys残基のペア同士の距離である。K_l とK_SS は定数でありそれぞれ2および5と設定される。 Step I-41 (1): Construction of the Cα atom using the simulated annealing method The Cα atom created in the above step I-41 is optimized using the function composed of the coordinates of the reference protein using the simulated annealing process. It becomes. This objective function is as shown in the following formula (11).

Here, U _len relates to the distance between the Cα atom of the pair of the residue on the sequence and the Cys residue, and is set as shown in the following formula (12).

Here, Di, i + 1 is the distance between Cα of residue i and residue i + 1. D _i ^SS is the distance of the pair between the Cys residues form a disulfide bond. K _l and K _SS are constants and are set to 2 and 5, respectively.

ここで θ_i(rad)は i, i+1, i+2番目の残基Cα原子の角度である。θ₀はPDBのX線構造から(100/180)・π(rad)と設定される。K_aは定数であり1とする。 Uang is a function of the bond angle of the Cα atom and is given by the following formula (13).

Where θ _i (rad) is the angle of the i, i + 1, i + 2nd residue Cα atom. θ ₀ is set to (100/180) · π (rad) from the X-ray structure of the PDB. K _a is a constant and is 1.

ここで||・||が意味する所はノルムであり、M_iは構造を基にしたアライメント上で構造的に等価な位置にあるCα原子間の平均距離である。残基iについてΜ_iの値が求められないとき、Μ_iの値は10と設定される。ここでは、Cα原子の平均座標であり下記式(15)のとおりである。

ここでＸ_jiはj番目の参照タンパク質のi番目の残基のCαの原子座標である。ｗ_jiは、j番目の参照タンパク質のi番目の残基の重みである。この重みは目的タンパク質の大体の形を決定するため重要なパラメータであるが、これはローカルスペースホモロジー（LSH）と呼ばれる着目部位の12Å以内の空間的近傍の局所的な値によって決定している（第３図参照）。LSHと構造がよく保存されている部位（SCRs：Structural Conserved Regions）に存在する残基のペアの比率との相関は第４図に示されているように非常に高い。これは、高いLSH値を持つときは統計的にCα原子の位置が参照タンパク質構造と比べて1.0Å以内にあることを意味する。 Upos is a function related to the position of the Cα atom, and is represented by the following formula (14).

Here, || · || means a norm, and M _i is an average distance between Cα atoms at structurally equivalent positions on the alignment based on the structure. When the value of Micromax _i can not be obtained for residues i, the value of Micromax _i is set to 10. Here, it is an average coordinate of the Cα atom, as shown in the following formula (15).

Here, X _ji is the Cα atomic coordinate of the i-th residue of the j-th reference protein. w _ji is the weight of the i-th residue of the j-th reference protein. This weight is an important parameter for determining the general shape of the target protein, but it is determined by a local value within 12 km of the region of interest called local space homology (LSH) ( (See FIG. 3). As shown in FIG. 4, the correlation between the LSH and the ratio of the residue pair existing in the structurally conserved regions (SCRs) is very high. This means that when having a high LSH value, the position of the Cα atom is statistically within 1.0 cm compared to the reference protein structure.

ここでK_vdwは0. 01(D_i,j < 3.2Å) と0.001(D_i,j > 3.2Å)と設定され6Åをカットオフ値とした。 U _vdw is as shown in the following formula (16).

Here, K _vdw was set to 0.01 (D _{i, j} <3.2 mm) and 0.001 (D _{i, j} > 3.2 mm), and 6 mm was set as a cutoff value.

  Cα atoms are optimized using the simulated annealing method according to equation (11). At this stage of optimization, the Cα atom perturbation is set to be within 1.0 cm. This annealing step is calculated 100 times for all Cα atoms. The parameter corresponding to the temperature was decreased by 0.01 every 25 to 0.5 times, and the parameter was made constant thereafter.

  The two large steps, acquisition of structural information and construction of Cα atoms are repeated 10 times, and the coordinates of the Cα atom having the minimum objective function value are calculated as the optimal solution.

Step I-42: Construction and optimization of main chain atomic coordinates Add other atoms of the main chain to the Cα atomic coordinates of Step I-41 (1), and minimize the objective function by the simulated annealing method To do. First, three-dimensional superposition of Cα atoms is performed, and residues with a Cα interatomic distance of 2.5 cm or less are picked up. The atomic coordinates of the main chain excluding Cα are obtained from the coordinates of the reference protein so as to minimize the distance between the Cα atoms, and are used as a model structure.

  If there is no corresponding residue in the reference protein, the atomic coordinates of the main chain are created from the corresponding 4-residue protein fragment in the database. In this process, the main chain atom of residue i is selected from the residues having the smallest rmsd value between the Cα atoms from the (i−1) th to the (i + 2) th. At that time, for the N-terminal residue, the superposition range of the Cα atom coordinates is from i-th to i + 3-th, and for the C-terminal residue and the previous residue, similarly, from i-3 to i-th And from i-2 to i + 1.

  It is optimized by the simulated annealing method based on the objective function of the main chain atoms.

ここでb_i ⁰は、標準の結合長でありそれぞれの化学結合の種類によって異なる。K_bは定数であり225とする。 The objective function is as shown in the following formula (17).

Here, b _i ⁰ is a standard bond length and differs depending on the type of each chemical bond. K _b is a constant and is 225.

ここでθ_iはi番目の結合角であり、化学結合の種類によって異なる。K_aは定数で45と設定される。 U _ang is a function of the bond angle, as shown in the following formula (19).

Here, θ _i is the i-th bond angle and differs depending on the type of chemical bond. K _a is a constant and set to 45.

ここでε_i,j と r_i,j* は定数で原子の種類によって異なる。
K_nonは定数で0.25とし、カットオフは8Åとする。 U _non-bond is a function of non-bonded interaction and is represented by the following formula (20).

Here, ε _{i, j} and r _{i, j} * are constants and depend on the type of atom.
K _non is a constant of 0.25 and the cutoff is 8mm.

ここで K^SS _CαおよびK^SS _Cβは定数であり7.5である。 U _ss is a function of the disulfide bond generated by the Cys residue and is represented by the following formula (21).

Here, K ^SS _C α and K ^SS _C β are constants and are 7.5.

式(22)の＜W_iＸ_i＞は、目的タンパク質および参照タンパク質の間の構造の重ねあわせから求める。
K_posは定数であり0.3である。 U _pos is a function related to the position of the atom, as shown in the following formula (22).

<W _i X _i > in formula (22) is obtained from the superposition of the structure between the target protein and the reference protein.
K _pos is a constant and is 0.3.

ここでφ_i0とψ_i0はRamachandranマップ上での最も近いねじれ角のφ_iおよびψ_iとする。またω_i0は0としてcis-Pro残基の場合のみπ(radian)とする。K_tおよびKωは定数であり、それぞれ10および50とする。 U _tor is a torsion angle of the main chain and is represented by the following formula (24).

Here, φ _i0 and ψ _i0 are the closest twist angles φ _i and ψ _i on the Ramachandran map. In addition, ω _i0 is set to 0 and is set to π (radian) only in the case of a cis-Pro residue. K _t and Kω are constants and are 10 and 50, respectively.

ここでτ_iはN-Cα-Cβ-Cで定められるねじれ角でありK_chi は50とする。 U _chi is related to the chirality of Cα, as shown in the following formula (25).

Here, τ _i is a twist angle determined by N-Cα-Cβ-C, and _Kchi is 50.

U _hydr is related to main-chain hydrogen bonding conserved in homologous proteins, and is defined by the following formula (26).

  Hydrogen bonds are set when the distance between N and O atoms is 2.9 ± 0.5 Å.

When it is determined whether or not there are hydrogen bonds among a plurality of reference proteins, it is determined that there is a hydrogen bond when it is recognized that 75% or more of the reference proteins exist. K _hydr is a constant and is 0.6.

  Next, optimization of the main chain atoms including Cβ is performed by simulated annealing. In this annealing process, the perturbation of the main chain and Cβ atoms is set within 1.0 cm from the initial position. This annealing step is performed 200 times for the main chain and Cβ atoms. The parameter corresponding to the temperature starts from 50 or 25 and is multiplied by 0.5 each time until it reaches 0.01, and then it is set to a constant value.

  In order to sample the configuration of the main chain widely, in the method of the present invention, the above method is preferably performed six times, and the atomic coordinates of the main chain having the minimum objective function value are set as the optimum solution. The parameter corresponding to temperature starts from 50 for the first two times and starts from 25 for the third time.

Step I-43: Construction and optimization of side-chain atomic coordinates Side-chain construction is roughly divided into two stages: “Structure-conserved site side-chain construction” (Step I-43 (1)) and “Overall "Side chain construction" (Step I-43 (2)).

Step I-43 (1): Construction of the side chain of the structure-conserving site For the calculated main chain atom, the twist angle of the side chain is obtained from the homologous protein using the method in the previous study. For details on this method, see "The role of played by environmental residues in side-chain torsional angles within homologous families of proteins: A new method of side chain modeling." Ogata K and Umeyama H, Prot. Struct. Funct. Genet. 1998 , 31, 255-369.

In this method, the proportion of side chains stored in homologous proteins is calculated, and side chains are modeled based on this information. The atomic coordinates of the side chain of the conserved part of the side chain are placed with respect to the fixed main chain atom. For example, if the χ ¹ angle of the arginine residue is conserved in a homologous protein, the coordinates of the Cγ atom can be placed, and if the χ ¹ and χ ² angles are conserved in the Phe residue, all Can place side chain atoms. The process of optimization of simulated annealing using equation (17) was performed only for the main chain and Cβ atoms, and the perturbation of atoms was within 1.0 cm. This stage of annealing of the main chain and Cβ atoms is performed 200 times. The parameter corresponding to the temperature starts from 25 and is increased by 0.5 each time until it becomes 0.01. The unon-bond in formula (17) is performed on the main chain atoms and partially created side chain atoms. At this time, the coordinates of the side chain atoms are preserved throughout the optimization process.

N-O pairs M _i hydrogen bond is the information structure used in the process of optimization. In order to obtain the main chain atom arrangement, the above process is repeated three times, and the coordinates of the minimum main chain atom of the objective function are used as the calculated structure.

Step I-43 (2): Construction of the entire side chain The side chain is constructed under a fixed main chain and Cβ atom. This is done with the research results disclosed in Ogata K and Umeyama H, Prot. Struct. Funct. Genet. 1998, 31, 255-369, and gives an accurate model in a short time. Can do. Next, the main chain structure is optimized by the Monte Carlo method at low temperature, the temperature is set to 0.001, and the objective function U _non-bond of equation (17) is used to calculate for all main chain and side chain atoms. Then, in the process of optimizing the N, Cα, C, and Cβ atoms, the coordinates of the side chains are rearranged so that the twist angle of the side chains is optimized. The perturbation of atoms should be within 0.5mm. The side chain is then deleted and the above side chain construction is repeated. This process is repeated until there is no collision between the 2.4-cm atoms and the twist angle of N-Cα-Cβ-C is in the range of -120 ± 15 °.

Step I-44: Construction of the Final Structure Thus, atomic coordinates that define the three-dimensional structure of any target protein, a no induced fit type and an induced fit type, can be obtained.

II. Next, a method for constructing a three-dimensional structure of a protein-ligand complex, which is another embodiment of the present invention, will be described with reference to the drawings. FIG. 5 is a flowchart showing an example of a method for constructing a target protein-ligand complex, that is, a method for constructing a complex including an induced fit.

  First, in step II-10, the modeled atomic coordinates of the target protein are obtained. The standard vibration mode is obtained by performing the standard vibration analysis method of the optimized reference protein. Then, the atomic coordinates of the target protein obtained mainly in the experiment are displaced in the eigenvector direction to create a set of a plurality of reference proteins. By referring to these coordinates, the three-dimensional structure of the target protein is constructed by homology modeling.

  In Step II-20, an operation for docking the ligand to the obtained three-dimensional structure of the target protein is performed. In Step II-30, empirical molecular energy calculation by MCSS method is performed based on the ligand docked to the target protein set to simulate the three-dimensional structure of the target protein-ligand complex. The three-dimensional structure of the target protein-ligand complex thus obtained is a three-dimensional structure including an inductive fit of the target protein, i.e., periodic thermal motion (molecular fluctuation). Can be used.

  Hereinafter, each step will be described in more detail.

Step II-10: Modeling the target protein The target protein is modeled in the following three steps II-11: Optimization of the initial coordinates of the reference protein, II-12: Normal vibration analysis of the optimized coordinates, II-13: Objective Divided into protein modeling. This step is performed in the same manner as I-10 to I-44. Thus, a three-dimensional structure based on the vibration mode of the reference vibration analysis method, that is, a three-dimensional structure of the target protein including an induced fit can be constructed.

Step II-20: Ligand docking to the target protein Ligand docking is performed on a plurality of three-dimensional structure models of the target protein in consideration of the normal vibration mode. It is docked at a position considered to be a ligand binding site of the target protein. This step is performed using commercially available software that can input and output files in PDB format, such as BIOCES (manufactured by NEC), Cerius2 (manufactured by Accelrys), SYBYL (manufactured by TRIPOS), HyperChem (manufactured by Hypercube), etc. . Generally, docking is performed by rotating and translating a ligand on a display capable of stereo display. Also, docking including a simple energy calculation method may be performed.

  The binding site of the ligand to be used is not particularly limited, and any of the already known binding sites and newly specified binding sites can be used. For a protein with an unknown ligand binding site, the site can also be identified by the method described in III below.

Step II-30: Optimization of the three-dimensional structure of the target protein-ligand complex For the protein-ligand complex structure model obtained in Step II-20, the empirical molecular energy of one structure of the target protein and the structure of the ligand The calculation is performed for the number of target protein structures. At that time, the target protein side moves the atomic coordinates according to the potential energy gradient of each of the multiple structures, and the ligand side averages the calculated potential energy gradients. The ligand coordinate based on a plurality of three-dimensional structures of the target protein is obtained by moving the ligand's atomic coordinates in the converted direction.

  This Step II-30 is performed by, for example, Multiple Copy Simultaneous Search (MCSS) method, and multiple complex structures are optimized simultaneously by empirical molecular energy calculation (molecular force field method) with ligands, and their atomic coordinates are By empirical molecular energy calculation (molecular dynamics method), the structure is relaxed for 10 ps, for example, at a temperature of 300 ° K. Further, the atomic coordinates are optimized by the molecular force field method. Of course, the temperature and time may vary depending on the target system being calculated.

  The MCSS method is proposed by A. Miranker and M. Karplus (Proteins, 1991, 11, 29-34) as a method for optimizing the three-dimensional structure of both a receptor protein and a ligand using a plurality of ligands. As a method, empirical molecular energy calculations of individual ligands and proteins are performed at the same time, and the receptor protein gradient is averaged, so that the receptor protein side moves as a single three-dimensional structure.

  On the other hand, in the method of the present invention, the structure of a ligand based on a plurality of protein structures is obtained using a plurality of molecular structures on the protein side and a single molecular structure on the ligand side. In this empirical molecular energy calculation, protein 1 structure and ligand 1 structure are calculated as many as the number of protein structures, and on the ligand side, the atomic coordinates of the ligand are oriented in the direction of averaging the calculated potential energy gradients. Move. On the other hand, on the target protein side, the atomic coordinates are moved according to the potential energy gradient of each of the plurality of structures, and a ligand structure based on the plurality of three-dimensional structures of the target protein is obtained.

  The above-mentioned empirical molecular energy calculation method is not particularly limited and may be performed by a known method. However, the apricot program developed by the inventors (Yoneda, S., and Umeyama, H., J Chem Phys 1992 97: 6730-6736) is preferred to use the apricot-MCSS program. Empirical potential functions include AMBER type potential functions (SJ Weiner, PA Kollman, DA Case, U. Chandra Singh, C. Ghio, G. Alagona, S. Profeta, Jr., P. Weiner, J. Am. Chem Soc., 1984, 106, 765-784) and para89a Rev A as parameters are preferred. Of course, other experiential potentials can be used.

ここでUxyzは目的タンパク質におけるCα原子位置に掛ける拘束のポテンシャルエネルギーで、Cαのオリジナル座標値がｘ0、更新された座標値がｘ、Kxyzが原子をどの程度拘束させるかのパラメータである。ここではKxyzとして10.0kcal/mol/Ųを用いたが、一例であるので、式の形を含めて本発明の範囲を限定するものではない。 In molecular dynamics calculations, in addition to the usual energy term, a constraint potential for the Cα atom position is added as a Harmonic function, for example, as shown in the following equation (27), so that the initial three-dimensional structure of the target protein is not greatly broken. Is preferred. This is important in terms of compensating for the roughness of the approximation of the calculation, but the range of the constraint potential may be extended to the entire main chain, and is not limited to this.

Here, Uxyz is a constraint potential energy applied to the position of the Cα atom in the target protein, and is a parameter of how much the original coordinate value of Cα is x0, the updated coordinate value is x, and how much Kxyz constrains the atom. Here it was used 10.0kcal / mol / Å ² as Kxyz, because it is an example, not intended to limit the scope of the invention, including the form of Equation.

Also, instead of the constraint potential for the X, Y, Z coordinates of the Cα atom, it is used for constraint on the torsion angle of the main chain of the target protein shown in formula (24), that is, in the empirical molecular energy calculation, By adding it as a potential function that constrains the twist angle, the initial three-dimensional structure may not be greatly broken.

  Thus, if an induced fit type three-dimensional structure model is used as the target protein, it is possible to obtain atomic coordinates of the target protein-ligand complex in consideration of molecular fluctuations.

  In addition, when the ligand molecule is a protein, a ligand that takes into account the induced fit on the ligand side from multiple three-dimensional structures including the reference vibration mode of the ligand and a single three-dimensional structure of the protein in the same manner as described above. -Construction of a three-dimensional structure of a protein complex is also possible.

III. Next, a method for identifying a protein ligand binding site, which is another embodiment of the present invention, will be described. FIG. 6 is a flowchart showing an example of a method for identifying a ligand binding site of a protein and a method for constructing a three-dimensional structure of a protein-ligand complex by binding a ligand to the obtained binding site.

  In step III-10, the protein / ligand binding site is identified (predicted). In this step, low molecular weight compounds such as nonpolar solvents are generated around and inside the proteins and / or ligands, for example, hydrophobic surfaces, and a number of water molecules are added around them to make the molecules in an apparently aqueous solution appear. Perform dynamics calculations. Based on these results, the binding site of the protein and the ligand is searched from the behavior of a low molecular weight compound on the protein and / or ligand surface, for example, a nonpolar solvent. In Step III-20, referring to the estimated binding site between the protein and the ligand obtained in Step III-10, they are docked to determine the initial atomic coordinates of the three-dimensional structure of the protein-ligand complex. In Step III-30, water molecules are generated around the initial three-dimensional structure of the protein-ligand complex obtained in Step III-20, and apparently the protein- Refine the three-dimensional structure of the ligand complex.

  Hereinafter, each step will be described in more detail.

Step III-10: Identification of Protein Ligand Binding Site Identification of protein and ligand binding site is performed in the following three steps: III-11: Generation of small molecule around protein and / or around ligand, III-12 : Retrieval of behavior of low molecular weight compounds (eg nonpolar solvents) by empirical molecular energy calculation (molecular mechanics, molecular dynamics calculation) in aqueous solvent of proteins and / or ligands, III-13: Low molecular weight compounds (eg It can be divided into the determination of the ligand binding site to the protein and / or the binding site of the ligand to the protein from the behavior of the nonpolar solvent or the like).

Step III-11: Generation of low molecular weight compound around protein and / or ligand First, after generating water molecule around protein and / or ligand, inside of protein, ligand and low molecular weight compound Replaces surrounding water molecules with low molecular weight compounds. In this case, these substitutions may arrange the low molecular weight compound over the entire periphery thereof, or may arrange the low molecular weight compound only around the amino acid or functional group having hydrophobicity or hydrogen bonding ability. Here, when the ligand is a high-molecular substance such as a peptide or protein, a low-molecular compound is generated around the ligand, and the behavior of the low-molecular compound is analyzed by empirical molecular energy calculation as in the case of the protein. . When the ligand is a substance having a low molecular weight, such as a pharmaceutical or agrochemical molecule, it is possible to determine which part is a hydrophobic region, and therefore there is usually no need to specify a binding site. However, when the ligand is a polymer substance, it is necessary to analyze the binding site on the ligand side in the same manner as the binding site on the protein side and specify the binding site of the complex.

  The low molecular compound may be, for example, a nonpolar solvent such as ethane, cyclopentane, or benzene, a hydrogen-bonding solvent such as N-methylacetamide or benzamide, or a medical or agrochemical compound, and is not particularly limited. However, in view of the arbitrary nature of these orientations, compounds having objectivity are preferred. When a nonpolar solvent is used, the binding site of a protein or ligand having a hydrophobic moiety can be specified. In addition, when a compound having an acid amide group, which is a hydrogen bonding ability solvent, is used, a portion capable of hydrogen bonding with an acid amide group, that is, an exposed portion of a β sheet structure or a binding site of a ligand including an oxyanion hole can be specified. it can. Furthermore, when a medicine / pesticidal molecule is used, a moiety to which the medicine / pesticidal molecule can specifically bind can be specified.

  Specifically, for example, when a nonpolar solvent such as benzene is arranged around a protein, water molecules on the surface within 3.5 mm formed by amino acid residues having an MSAS value of 30% or more in the protein. May be substituted with a nonpolar solvent (benzene). In addition, when the nonpolar solvents (benzene) are within 1.5 mm, it is not necessary to replace the water molecules with the nonpolar solvent. All water molecules that have not been replaced by nonpolar solvents are erased once. The above-mentioned criteria for substitution of water molecules with nonpolar solvents are an example when benzene is used, and do not limit the scope of the present invention.

Step III-12: Retrieval of behavior of low molecular weight compounds by empirical molecular energy calculation of protein and / or ligand in water solvent Atom of protein (and / or ligand) and low molecular weight compound created in step III-11 above Using the coordinates, water molecules are generated around them under periodic boundary conditions, and then the three-dimensional structure is optimized by molecular mechanics calculation, which is empirical molecular energy calculation, followed by molecular dynamics calculation. After the molecular dynamics calculation is completed, the water molecules are removed to obtain atomic coordinates of the protein (and / or ligand) and the low molecular compound. For example, when a nonpolar solvent (benzene) is arranged as a low molecular compound, molecular dynamics calculation at a temperature of 300 ° K. and about 10 to 20 ps may be performed. This causes diffusion and accumulation of low molecular weight compounds around and inside the protein. By analyzing the state of diffusion and accumulation, that is, the behavior of the low molecular weight compound by the method of Step III-13 described later, the ligand binding site on the protein side and the protein binding site on the ligand side can be specified.

  The empirical molecular energy calculation method is not particularly limited, but it is preferable to use the apricot program developed by the present inventors. It is preferable to use an AMBER type potential function as the empirical potential function. Of course, other experiential potentials can be used.

Step III-13: Determination of the ligand binding site from the behavior of the low molecular weight compound The distribution of the low molecular weight compound around the protein and / or the ligand obtained in Step III-12 above, for example, the distribution of the nonpolar solvent, was targeted. Cluster analysis is performed, and a site where the ligand is easily docked to the protein is determined from the obtained cluster size.

  Here, cluster analysis is a multivariate analysis method in which a data set given in a multidimensional space is clustered (agglomerated) according to the similarity (or dissimilarity) between individuals. Here, the Euclidean distance between the centroids of nonpolar solvents in a three-dimensional space (coordinate average of 6 carbon atoms in benzene) is calculated, and if there is a nonpolar solvent within the threshold, the distance from nonpolar solvents with a short distance is calculated. It will be clustered. At this time, the clustered set of nonpolar solvents is not the distance from the center of gravity of the cluster, but is checked whether the nonpolar solvents at the shortest distance are within the threshold, unlike the normal cluster analysis. To determine whether to cluster them. In the case of benzene as a non-polar solvent, a threshold value of 6 Å was used, but the value is merely an example and does not limit the scope of the present invention.

  For example, when a nonpolar solvent (benzene) is used, they are classified into several clusters, and it is considered that the larger the cluster, the more likely it is a docking site to a ligand or protein. The clustered nonpolar solvent group can be represented by an ellipsoidal shape, but by solving the eigenvalue problem of coordinates, the length direction of the cluster can be obtained. Several models of protein-ligand complexes are created by docking clusters on both the protein side and the ligand side with reference to the long and short directions of the elliptic sphere. Of course, the complex structure in which protein and ligand overlap is automatically removed. The docked model is fine-tuned for protein and ligand placement with the software described in step II-20.

Step III-20: Ligand docking to protein The low molecular weight compound obtained in Step III-13 above, for example, a cluster of nonpolar solvents (benzene) clustered together, and the protein-ligand complex The initial data of the structure. At this time, low molecular weight compounds such as nonpolar solvent (benzene) data are removed upon docking.

  This step can be performed using commercially available software that can input and output PDB format files. In general, docking is performed by rotating or translating a ligand on a display capable of stereo display. Also, docking including a simple energy calculation method may be performed.

Step III-30: Construction of the three-dimensional structure of the protein-ligand complex The initial atomic coordinate data of the protein-ligand complex obtained in Step III-20 above are generated after generating water molecules around them under periodic boundary conditions. The initial three-dimensional structure is optimized by molecular mechanics calculation, followed by molecular dynamics calculation, and the three-dimensional structure of the protein-ligand complex is obtained by removing water molecules from the coordinate locus of the final step.

  The method of molecular dynamics calculation is not particularly limited, and may be performed, for example, at a temperature of 300 ° K. and about 10 to 20 ps. The program to be used is not particularly limited, but it is preferable to use an AMBER type empirical field with an apricot developed by the inventors. However, the program used and the force field are merely examples, and do not limit the scope of the present invention.

  Thus, taking into account that the formation process of protein-ligand complex is in aqueous solution, the hydrophobic surface of protein and ligand can be obtained by using the accumulation and diffusion of low molecular weight compounds such as non-polar solvent in aqueous solvent. The atomic coordinates of the protein-ligand complex can be obtained more accurately than before by finding them and docking them together.

IV. Recording medium and database in which atomic coordinates that define the three-dimensional structure of the protein are recorded Predetermined that the computer can use the atomic coordinates that define the three-dimensional structure of the protein or protein-ligand complex obtained by the above method. By storing it in an appropriate recording medium in the above format, a three-dimensional structure database of the target protein can be constructed. The database of the present invention may preferably include alignment information of the reference protein and the target protein together with the atomic coordinates. In addition, the database includes a code number, information on a reference region of a reference protein, information on a target protein, a distance between Cα atoms, and the like as desired.

  In the present invention, the database also means a computer system that writes the atomic coordinates on an appropriate recording medium and performs a search according to a predetermined program. Examples of suitable recording media include magnetic media such as floppy (R) disks, hard disks, and magnetic tapes; optical disks such as CD-ROM, MO, CD-R, and CD-RW, and semiconductor memories. it can.

V. Drug molecular design method A computer running an appropriate program that can design drug molecules such as medical and agrochemicals, and a protein that is the target of the drug molecule obtained by the above method (hereinafter referred to as "target protein") A drug molecule (antagonist or agonist) that interacts with the target protein using all or part of the structure coordinates of the database or recording medium in which they are recorded ) Can be identified, searched, evaluated or designed.

  The identification, search, evaluation or design of the drug molecule is performed based on the presence or absence of the interaction between the three-dimensional structure coordinate obtained by the method of the present invention and the three-dimensional structure coordinate of the drug molecule. In the present specification, identification, search, evaluation, or design of drug molecules may be simply referred to as drug molecular design.

  The computer used for designing the molecule based on the interaction between the three-dimensional structure coordinates of the protein and the three-dimensional structure coordinates of the drug candidate molecule is not particularly limited as long as the computer is adjusted so that an appropriate program can be operated. There is no. There is no particular limitation on the storage medium of the computer. Examples of the program used for molecular design include a computer program Insight II manufactured by Accelrys. In particular, by using a program such as Ludi or DOCK specially created for this purpose alone or in combination, a drug molecule can be identified, searched, evaluated or designed more easily. In addition, docking evaluation between the three-dimensional structure coordinates of the protein and the drug molecule can be performed using software such as BIOCES manufactured by NEC described in Step II-20, for example.

  Here, even if the drug molecule is a known one or a newly synthesized drug molecule having a new chemical structure, any drug molecule can be used as long as the three-dimensional structure can be obtained. It can be used in the inventive method. The three-dimensional coordinate of the drug molecule may be obtained by any method such as X-ray crystallography or modeling. Those whose three-dimensional structural coordinates have been determined may be an appropriate database such as CCDC (Cambridge Crystallographic Data Centre: http://www.ccdc.cam.ac.uk/) or PDB (Protein Data Bank: http: // It can be obtained from www.rcsb.org/pdb/).

  Furthermore, drug molecules can be designed using the three-dimensional structure of the target protein, for example, by the method described in JP-A-2000-178209. In this way, by using the three-dimensional structure coordinates of the protein obtained by the method of the present invention, molecular design by a computer of drug molecules becomes possible. However, the molecular design method of the present invention is not limited to those using these programs and methods.

  Drug molecular design usually has two conceptual stages. The first step is to find the lead compound, and the next step is to optimize the lead compound. Both steps can be performed by methods known per se using the three-dimensional structure coordinates of the target protein. As a result, an optimal candidate molecule for medical and agricultural chemicals can be obtained.

VI. Screening method for candidate molecules for medicines and pesticides obtained by the molecular design method The candidate molecule for medicines and pesticides identified, searched, evaluated or designed by the above method should be obtained by a chemical synthesis method known per se, for example, depending on the nature of the molecule Can do. However, the drug molecule may be a natural compound or a synthetic compound, and may be either a high molecular compound or a low molecular compound. The obtained medicinal and agricultural chemical candidate molecules are further examined by pharmacological or physiological tests in vitro or in vivo by a method known per se, and medicinal and agricultural chemical candidate molecules having a desired activity are selected. By this, what can actually be applied as a medical pesticide can be obtained.

VII. Method for producing pharmaceutical and agrochemical composition Drug molecules such as medical and agrochemical selected by the above screening method, for example, pharmaceutical molecules can be administered to patients with diseases to be treated by themselves, but these active ingredients These can be administered alone or in combination. Further, it is preferable to formulate the substance as a pharmaceutical composition using a pharmacologically acceptable additive for pharmaceutical preparation and the like and administer it. For example, tablets, capsules, granules, fine granules, powders, pills, microcapsules, liposome preparations, troches, sublinguals, liquids, elixirs, emulsions, suspensions, etc. As an injection, a suppository, an ointment, a patch or the like manufactured orally as a sterile aqueous liquid or oily liquid. These are, for example, admixed with the physiologically recognized carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc., in unit dosage forms required for accepted pharmaceutical practice. , And can be produced using methods well known in the art such as filling or tableting. The amount of active ingredient in these pharmaceutical compositions is such that an appropriate volume within the indicated range is obtained.

  When the agrochemical molecule is actually used as an agrochemical, it is mixed with a carrier or diluent, additive, adjuvant, etc. by a known method to form a formulation (composition) usually used as an agrochemical, such as a powder. , Granules, wettable powders, emulsions, aqueous solvents, flowables and the like.

[Example]
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be considered as helping to obtain specific recognition of the present invention and should not limit the scope of the present invention in any way. Absent.

[Example 1]
Construction of the three-dimensional structure of β2 adrenergic receptor According to the method described in detail in I-10 to I-40 of the embodiment of the present invention, a three-dimensional structure including induction conformation of human-derived β2 adrenergic receptor was constructed as follows. FIG. 7 shows a flowchart.

  The three-dimensional structure model was constructed using an NEC workstation (model: Express5800 / 120Rc-2, CPU: Pentium (R) III 933 MHz x 2, OS: Red Hat Linux 6.2J, memory: 1024 Mbytes). The amino acid sequence of the target β2 adrenergic receptor was obtained from ID: QRHUB2 of PIR; http://www-nbrf.georgetown.edu/pir/.

  Alignment by PSI-BLAST (Position-Specific Iterated BLAST) was performed using the amino acid sequence of the β2 adrenergic receptor as the sequence of the target protein. At that time, the motif profile used was 892 total sequences of GCRDb; http://www.gcrdb.uthscsa.edu/. The amino acid sequence of the β2 adrenergic receptor is shown in SEQ ID No. 1.

  Using the structure of the B chain of ID: 1F88 (rhodopsin) of PDB (http://www.rcsb.org/pdb/) as the three-dimensional structure of the protein to be referred to, an alignment for this B chain was obtained. The sequence of B chain of 1F88 (rhodopsin) is shown in SEQ ID No. 2, and the alignment result is shown in FIG. In the crystal lattice of 1F88 (rhodopsin), there is a dimer having almost the same three-dimensional structure consisting of an A chain and a B chain, and the B chain was used as a reference structure. In addition, the coordinates of the A chain and the B chain have large defects, respectively, and are not complete. The 1F88 structure is modeled using the modeling program FAMS detailed in Step I-40, and the constructed three-dimensional structure is β2 adrenaline. The three-dimensional structure of the receptor reference protein was used.

  In the PDB file and FAMS, hydrogen atoms were not added to the appropriate residues. Therefore, hydrogen atoms were generated at the appropriate residues of this reference protein structure, and the initial atomic coordinates serving as the input coordinates for the standard vibration analysis method were obtained.

  As in steps I-10 to I-20, optimization of the initial atomic coordinates obtained with the Cartesian coordinate system, re-optimization with Cartesian coordinate system with some of the potential parameters of SS coupling being zero, dihedral angle coordinates A normal vibration analysis method using a system was performed, and eigenvalues and eigenvectors were obtained.

  In this case, AMBER parm89a Rev A was used as a parameter. The cut-off value for non-bonded interaction is 9.0 内側 for inner side and 10.0 -4 for outer side. The parameter for 1-4 interaction is multiplied by 1/2 of non-bonded interaction, and the dielectric constant is distance-dependent ( 1 / rÅ). The optimization used the Fletcher-Reeves conjugate gradient method. After optimizing the obtained initial atomic coordinates with the Cartesian coordinate system, re-optimize with the Cartesian coordinate system using the same conditions except that the SS bond angle and dihedral angle parameters are set to zero. A standard vibration analysis method using an angular coordinate system was performed to obtain eigenvalues and eigenvectors.

The optimization conditions used were Sumikawa, H., Suzuki, E.-I., Fukuhara, K.-I., Nakajima, Y., Kamiya, K., and Umeyama H. 1998. Dynamics structure of granulocyte colony -Stimulating factor proteins studied by normal mode analysis: Two domain-type motions in low frequency modes. The method described in Chem Pharm Bull 46: 1069-1077 was used. For details of normal vibration analysis using dihedral angle coordinate system, see Noguti, T., and Go, N. 1983. Dynamics of native globular proteins in terms of dihedral angles. J Phys Soc Jpn 52: 3283-3288 and Noguti. , T., and Go, N. 1983. A method of rapid calculation of a second derivative matrix of conformational energy for large molecules. The method described in J Phys Soc Jpn 52: 3685-3690 was used.

As in step I-30, the temperature is set to 300 ° K, the fluctuation of the Cα atom for each eigenvalue of 30 cm ^-1 or less is obtained, and converted from the average temperature factor of the A chain and B chain of PDB ID: 1F88 (rhodopsin) The average ratio for each eigenvalue was obtained. The average ratio was multiplied by the eigenvector belonging to this eigenvalue, and displacement was performed in addition to the atomic coordinates of the reference protein to obtain coordinates defining the three-dimensional structure of the induced fit type reference protein. Similarly, a displacement obtained by multiplying the eigenvector by −1, a displacement obtained by multiplying the eigenvector by the average ratio doubled, and a displacement multiplied by −1 were performed. However, the eigenvector added here is converted from dihedral angle coordinates to Cartesian coordinates. From a single eigenvalue / eigenvector, a three-dimensional structure set of four induced fit type reference proteins can be obtained. The number of eigenvalues below 30 cm ⁻¹ used is 118, and the number of induced fit-type reference proteins obtained is 472. As an example, FIG. 9 shows a temperature factor converted into a fluctuation obtained by multiplying the minimum eigenvalue of 4.47 cm ⁻¹ by M ^v (= 26.4).

As in step I-40 above, the 3D structure of the target protein β2 adrenergic receptor was modeled by FAMS from the no induced fit reference protein 3D structure and the induced fit reference protein 3D structure set. . The three-dimensional structure of the target protein and the three-dimensional structure of the reference protein are in a one-to-one relationship, and 472 induced fit target protein three-dimensional structures and one non-induced fit obtained from the conventional method (no induced) fit) type target protein tertiary structure was obtained. As an example, FIG. 10 shows a no induced fit constructed from the no induced fit reference protein obtained above.
The 3D structure of the induced fit target protein constructed from the induced fit type reference protein 3D structure obtained by multiplying the 3D structure of the target protein and the eigenvector of the lowest eigenvalue by ± 2 × M ^v (± 2 × 26.4) A part of the structure is shown. In the figure, the central structure is a non-inductive compatible target protein.

[Example 2]
Construction of the three-dimensional structure of a complex of trypsin and trypsin inhibitor alone In this example, β-Trypsin (trypsin) derived from bovine pancreas and X-ray crystallographic analysis of the receptor, ligand, and receptor-ligand complex are known. Using the trypsin inhibitor (BPTI) system, the method for constructing the three-dimensional structure of the protein-ligand complex of the present invention was verified. Here, trypsin is a receptor protein (target protein) and BPTI is a ligand.

The amino acid sequence of triprin used is shown in SEQ ID No. 3, and the amino acid sequence of trypsin inhibitor (BPTI) is shown in SEQ ID No. 4. Since the amino acid number of trypsin is described by the amino acid sequence number of chymotrypsinogen (a precursor of chymotrypsin), it becomes 223 residues from amino acid numbers 16 to 245 as shown below (FIG. 17). In the middle, amino acid numbers 35, 36, 68, 128, 131, 188, 205, 206, 207, 208 are missing, and 184, 188, 221 are duplicated (indicated by 184A, 188A, 221A).

Ile Val Gly Gly Tyr Thr Cys Gly Ala Asn Thr Val Pro Tyr Gln Val
16 20 25 30
Ser Leu Asn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu Ile Asn Ser
34 37 40 45
Gln Trp Val Val Ser Ala Ala His Cys Tyr Lys Ser Gly Ile Gln Val
50 55 60 65
Arg Leu Gly Glu Asp Asn Ile Asn Val Val Glu Gly Asn Glu Gln Phe
67 69 70 75 80
Ile Ser Ala Ser Lys Ser Ile Val His Pro Ser Tyr Asn Ser Asn Thr
85 90 95
Leu Asn Asn Asp Ile Met Leu Ile Lys Leu Lys Ser Ala Ala Ser Leu
100 105 110
Asn Ser Arg Val Ala Ser Ile Ser Leu Pro Thr Ser Cys Ala Ser Ala
115 120 125 127 130 132
Gly Thr Gln Cys Leu Ile Ser Gly Trp Gly Asn Thr Lys Ser Ser Gly
135 140 145
Thr Ser Tyr Pro Asp Val Leu Lys Cys Leu Lys Ala Pro Ile Leu Ser
150 155 160
Asp Ser Ser Cys Lys Ser Ala Tyr Pro Gly Gln Ile Thr Ser Asn Met
165 170 175 180
Phe Cys Ala Gly Tyr Leu Glu Gly Gly Lys Asp Ser Cys Gln Gly Asp
183 184A184 185 187 188A188 190
Ser Gly Gly Pro Val Val Cys Ser Gly Lys Leu Gln Gly Ile Val Ser
195 200 204 209 210
Trp Gly Ser Gly Cys Ala Gln Lys Asn Lys Pro Gly Val Tyr Thr Lys
215 217 219 220 221A221 225 230
Val Cys Asn Tyr Val Ser Trp Ile Lys Gln Thr Ile Ala Ser Asn
235 240 245

  According to the method described in detail in steps II-10 to II-30, a three-dimensional model of trypsin-BPTI complex was constructed by the following procedure, and the position of the complex active site was compared with the X-ray crystallographic data. .

  The three-dimensional structure model of the receptor protein-ligand complex was constructed using a DEL personal computer (model: Dimension XPS B866, CPU: Pentinum III 864 MHz, OS: RedHat Linux 6.2J, memory: 512 Mbytes). Coordinates of X-ray crystallographic analysis of trypsin and BPTI alone, as well as that of trypsin-BPTI complex, from Protein Data Bank (PDB); http://www.rcsb.org/pdb/, respectively, 1TLD (trypsin alone), 4PTI (BPTI) and 2PTC (trypsin-BPTI complex) were obtained and used.

  The 3D coordinate system of trypsin and BPTI was superimposed on the coordinate system of 1TLD and 4PTI to the coordinate system of 2PTC with a least square foot so that the results of the trypsin-BPTI complex could be easily considered. The three-dimensional coordinates of trypsin and BPTI were optimized for the initial coordinates of each single atom after generating a hydrogen atom in the heteroatom. Next, trypsin performed a standard vibration analysis in a system that does not contain BPTI, and obtained a vibration vector for each wavelength.

Among them, BPTI three-dimensional structure is docked to five trypsin three-dimensional structures consisting of long-period vibration vectors, and MCSS calculation is performed by the apricot-MCSS program. The three-dimensional structure of trypsin-BPTI complex Was refined. The breakdown of the MCSS calculation is as follows: First, optimize the three-dimensional structure of the trypsin-BPTI complex in 1000 steps by molecular dynamics calculation, and then trypsin--by molecular dynamics calculation at 300 ° K and 10 ps with 1 fs as one step. The steric structure of the BPTI complex was relaxed. The molecular dynamics calculations was added constraint Kxyz = 10.0kcal / mol / Å ² for Cα atoms shown in Formula (27) so as not collapse significantly three-dimensional structure of complex. And about the three-dimensional structure after 10 ps, the coordinate data of the trypsin-BPTI complex were obtained in the PDB format.

  FIG. 11 shows the trypsin structure of the trypsin-BPTI complex system after MCSS calculation. Looking at the atomic coordinates of trypsin, there were parts where both the main chain and side chain were greatly dispersed, and parts where they were not scattered much. Among them, the trypsin active site, His57, Asp102, Gly193-Asp194-Ser195 (oxyanion hole) part on the trypsin side was in good agreement with the main chain and side chain. By utilizing this fact, a site on the side of the receptor protein important for the ligand binding site can be found. It is very helpful in designing new ligands.

  The initial three-dimensional structure of trypsin-BPTI complex before MCSS calculation is shown in FIG. 12, and the three-dimensional structure of trypsin-BPTI complex after MCSS calculation is shown in FIG. 13, together with the three-dimensional structure of the X-ray crystallographic analysis of the complex. . In these figures, His57, Asp102, and oxyanion holes (Gly193-Asp194-Ser195) corresponding to the active site of trypsin-BPTI complex are extracted and displayed on the BPTI side, with Lys15 alone. The line displayed in black is the three-dimensional structure of X-ray crystallographic analysis of trypsin-BPTI complex, and the line displayed in gray is the initial three-dimensional structure of the complex model assembled according to the present invention (FIG. 12). This is a refined result (FIG. 13).

  The active sites of trypsin, His57, Asp102, and oxyanion holes, are the initial three-dimensional structure before MCSS calculation (Fig. 12) and the refined three-dimensional structure after MCSS calculation (Fig. 13). It matches well including. The Lys15 main chain of BPTI also agrees well before and after the MCSS calculation because the carbonyl oxygen is connected to the Gly193 of the oxyanion hole and the Ser195 peptide NH group by two hydrogen bonds. On the other hand, the direction of the Lys15 side chain of BPTI is not in the active pocket of trypsin before MCSS calculation, but by entering the active pocket by refining the three-dimensional structure by MCSS calculation, the X-ray crystal of trypsin-BPTI complex Matches well with analysis.

  The purpose of this is to use a plurality of model three-dimensional structures including the normal vibration mode of the target protein and to simulate the initial three-dimensional structure of the target protein-ligand complex obtained by docking with them by MCSS calculation. It is useful for constructing the three-dimensional structure of a protein-ligand complex.

[Example 3]
Identification of trypsin and trypsin inhibitor binding sites According to the method detailed in steps III-10 to III-30 above, trypsin and BPTI binding sites were identified by the following procedure, and these sites were identified as X-rays of the complex. It was compared with crystal analysis data. In this example, a system of β-Trypsin (trypsin) and trypsin inhibitor (BPTI) derived from bovine pancreas, which is known for protein-ligand complex X-ray crystallography, was used. Here, trypsin is a receptor protein (target protein) and BPTI is a ligand. Since BPTI is also a protein, not only the protein side but also the binding site on the ligand side was specified. The trypsin and trypsin inhibitor (BPTI) amino acid sequences used are as shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

  The three-dimensional structure coordinates of the trypsin-BPTI complex were obtained as 2PTC from Protein Data Bank (PDB); http://www.rcsb.org/pdb/. The three-dimensional structure of the X-ray crystallographic analysis of 2PTC trypsin-BPTI complex is shown in FIG.

  A personal computer manufactured by DEL (model: Dimension XPS B866, CPU: Pentinum III 864 MHz, OS: RedHat Linux 6.2J, memory: 512 Mbytes) was used to search for protein and ligand binding sites.

The three-dimensional coordinates of trypsin and BPTI were handled separately, and after generating a hydrogen atom as a heteroatom, an aqueous solvent was generated around it. Next, water molecules within 3.5 mm of the surface formed by amino acid residues with MSAS of 30% or more in trypsin and BPTI were replaced with benzene molecules. At that time, when the benzenes were within 1.5 mm, the water molecules were not replaced with benzene. When the substitution with benzene was completed, the water molecule was erased once. Three-dimensional structure coordinates of trypsin and BPTI containing benzene molecules generated periodic boxes filled with water molecules around them, and then empirical molecular energy calculations were performed using the apricot program under the periodic boundary conditions of water molecules. The breakdown of these energy calculations is first to optimize the structure by molecular dynamics calculation in 1,000 steps, and then to search for the behavior of benzene molecules by molecular dynamics calculation at 300 ° K and 10 ps with 1 fs as one step. In the molecular dynamics calculation, a constraint condition of Uxyz = 10.0 kcal / mol / Å ² according to Eq. (27) was added to the Cα atom of all amino acid residues so that the three-dimensional structure of the protein was not greatly damaged.

  At the end of these empirical molecular energy calculations, water molecules in the periodic box are erased for both trypsin and BPTI, and the atomic coordinates of trypsin and benzene and the atomic coordinates of BPTI and benzene after the molecular dynamics calculation in 10 ps are displayed in PDB format. Obtained. From these, cluster analysis was performed with a threshold of 6% for the distribution of benzene excluding trypsin and BPTI. Of the 94 and 40 benzene molecules placed around trypsin and BPTI, the largest clusters were 29 and 11, respectively. The distribution of benzene molecules around trypsin and BPTI is shown in FIGS. 15 and 16 together with trypsin and BPTI.

  These figures are viewed from the same direction as FIG. In the figure, the black hexagon is the largest benzene cluster.

  From FIG. 14 to FIG. 16, it can be seen that the largest benzene clusters around trypsin and BPTI are well matched in direction. In other words, by placing benzene molecules around the hydrophobic residues of proteins, performing molecular dynamics calculations in aqueous solvents, and searching for large benzene cluster distributions by cluster analysis, binding site candidates for protein ligands are selected. You can see that it can be identified. Moreover, it is thought that the initial configuration of the protein-ligand complex can be roughly predicted by docking the protein and the ligand so that these clusters overlap each other on the graphics. By adjusting the initial configuration manually or with molecular design software, it becomes one of the promising candidates for the configuration of the protein-ligand complex.

  As described above, the method of the present invention is a method capable of accurately constructing a protein structure that is closer to the true, in particular, the vicinity that binds to a ligand, as compared with the conventional method. Therefore, the method of the present invention is extremely useful for designing medical and agrochemical molecules.

  That is, the method for constructing a three-dimensional structure including inductive fitting according to the present invention uses a plurality of coordinate data obtained from a reference vibration analysis based on a model three-dimensional structure of a target protein, and an average model three-dimensional structure considering molecular vibration. Can be constructed with high accuracy. In particular, when predicting the three-dimensional structure of a target protein-ligand complex, an important induced fit can be included in the three-dimensional structure of the target protein-ligand complex. The three-dimensional structure of multiple receptor proteins was averaged over time by simulating the three-dimensional structure of the protein-ligand complex using the Multiple Copy Simultaneous Search (MCSS) method, which optimizes the structure of one receptor with that of a single ligand. A three-dimensional structure of the complex is obtained.

  In addition, the method for constructing the three-dimensional structure of the protein-ligand complex of the present invention examines the variation of the atomic coordinates on the receptor side in the target protein-ligand complex model after MCSS calculation, and the site important for activity is atomic coordinates. It is possible to design a new ligand by utilizing the fact that the variation in the size is relatively small and the variation in the other sites is large, and can be effectively used in the molecular design of medicines and agrochemicals.

  Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

  This application is based on a Japanese patent application (Japanese Patent Application No. 2001-011783) filed on January 19, 2001, the contents of which are incorporated herein by reference. The contents of documents cited in this specification are also incorporated herein by reference.

It is a flowchart which shows an example of the protein three-dimensional structure construction method including the induction | guidance | derivation fitting of this invention. It is a figure which shows the construction method of the C (alpha) atom coordinate of step I-41. The matching part of the alignment is obtained from the reference protein, and the non-matching part is obtained from the database with the smallest rmsd of the superposition of the two residues that overlap each of the N and C ends. It is a figure which shows local space homology (LSH). The calculations for T residues in the figure take into account the shaded (gray) residues. In the lower alignment in the figure, the part enclosed by a square is a residue pair to be considered, and the ratio where the mark is * is LSH. In this case, the LSH is 56.2%. It is a figure which shows the relationship between LSH and the ratio which exists in a structure preservation | save part (SCRs). LSH is calculated from the superposition of Cα atoms of the target protein and the reference protein, and the ratio in SCRs is the number of residues in SCRs relative to the total number of residues in the target protein. It is a flowchart which shows an example of the three-dimensional structure construction method of the protein-ligand complex of this invention. It is a flowchart which shows an example of the identification method of the ligand binding site of this invention, and the three-dimensional structure construction method of the protein-ligand complex using the binding site specified by this method. It is a flowchart which shows an example of the Example of the three-dimensional structure construction method of protein including the induction | guidance | derivation fitting of this invention. It is a figure which shows alignment of QRHUB2 ((beta) 2 adrenergic receptor) obtained by using 1F88 (rhodopsin) as a reference protein. In the figure, the numbers on the right side of QRHUB2 and 1F88 are the number of amino acids targeted for alignment in the amino acid sequence of each protein. The upper sequence shows QRHUB2 (β2 adrenergic receptor), and the lower sequence shows 1F88 (rhodopsin). The amino acid sequence of each protein is indicated by a one-letter code. Is a diagram showing the M ^v (= 26.4) multiplied by fluctuation and converted to temperature factors of the lowest eigenvalues 4.47cm ^-1. The solid line is the fluctuation of the Cα atom converted from the average temperature factor of the A chain and B chain of PDB ID: 1F88, and the dotted line is the fluctuation of the Cα atom position of 4.47 cm ^-1 obtained from the normal vibration analysis method M ^v (= 26.4) It is doubled. Printout of a display showing part of the conformation of an induced fit target protein constructed from the target protein and an induced fit reference protein multiplied by ± 2 x M ^v (± 2 x 26.4) It is a photograph of. The central structure is the no induced fit target protein. It is the photograph of the printout of the display which shows the trypsin of the trypsin-BPTI composite system after MCSS calculation. It is a photograph of the printout of the display which shows the initial three-dimensional structure of the trypsin-BPTI complex before MCSS calculation. In this figure, His57, Asp102, and oxyanion holes (Gly193-Asp194-Ser195) corresponding to the active site of trypsin-BPTI complex are extracted and displayed on Lys15 alone on the BPTI side. In the figure, the black line represents the three-dimensional structure of the trypsin-BPTI complex in the X-ray crystal analysis, and the gray line represents the initial three-dimensional structure of the complex model assembled. It is a photograph of the printout of the display which shows the three-dimensional structure of the trypsin-BPTI complex after MCSS calculation. In this figure, His57, Asp102, and oxyanion holes (Gly193-Asp194-Ser195) corresponding to the active site of trypsin-BPTI complex are extracted and displayed on Lys15 alone on the BPTI side. In the figure, the black line is the three-dimensional structure of the X-ray crystallographic analysis of the trypsin-BPTI complex, and the gray line is the refined three-dimensional structure of the assembled complex model. . It is a photograph of the printout of the display which shows the three-dimensional structure coordinate of the X ray crystal analysis of a trypsin-BPTI complex. FIG. 6 is a photograph of a display printout showing the distribution of benzene molecules around trypsin. In the figure, the black hexagon is the largest benzene cluster. A display printout showing the distribution of benzene molecules around BPTI. In the figure, the black hexagon is the largest benzene cluster. It is the figure which described the amino acid number of trypsin with the amino acid sequence number of chymotrypsinogen (precursor of chymotrypsin).

Claims (5)

A method for identifying a ligand binding site of a protein using a computer,
The computer includes at least a CPU and storage means.
The storage means stores at least three-dimensional structure information describing atomic coordinates of the three-dimensional structure of the protein, low molecular compound, and water molecule,
Executed in the CPU,
(i) arranging the three-dimensional structure information of the low-molecular compound around the three-dimensional structure information of the protein;
(ii) The three-dimensional structure information of the water molecules is further arranged around them, and empirical molecular energy calculation in an aqueous solvent is performed to obtain atomic coordinates of the protein and the low molecular compound,
(iii) For the obtained atomic coordinates, the behavior of the low-molecular compound around and inside the protein is analyzed, and the binding site of the ligand is determined.
A method for identifying a ligand binding site of a protein, comprising each step. A method for identifying a ligand binding site of a protein using a computer,
The computer includes at least a CPU and storage means.
The storage means stores at least three-dimensional structure information describing atomic coordinates of the three-dimensional structure of the protein, the ligand, the low molecular compound, and the water molecule,
Executed in the CPU,
(i) arranging the three-dimensional structure information of the low-molecular compound around the three-dimensional structure information of the protein and the ligand;
(ii) The three-dimensional structure information of the water molecules is further arranged around them, and empirical molecular energy calculation in an aqueous solvent is performed to obtain atomic coordinates of the protein and the low molecular compound,
(iii) With respect to the obtained atomic coordinates, the behavior analysis of the low molecular compound around and inside the protein and the ligand is performed, and the binding site of the protein-ligand complex is determined.
A method for identifying a binding site of a protein-ligand complex, comprising each step.   The behavior analysis of the low molecular weight compound executed in the CPU is performed by cluster analysis for the low molecular weight compound, and the size of the obtained cluster is set as the rank of the binding potential site of the ligand, and the binding site is determined. The method according to claim 1, wherein the method is determined. A method for constructing a three-dimensional structure of a protein-ligand complex using a computer,
The computer includes at least a CPU and storage means.
The storage means stores at least three-dimensional structure information describing atomic coordinates of the three-dimensional structure of the protein, the ligand, the low molecular compound, and the water molecule,
Executed in the CPU,
The structure information of the ligand is docked to the ligand binding site of the structure information of the protein identified by the method according to any one of claims 1 to 3, and the protein-ligand structure is calculated by empirical molecular energy calculation. A protein-ligand complex conformation construction step for obtaining a conformation of the complex;
A method for constructing a three-dimensional structure of a protein-ligand complex, comprising: The protein-ligand complex three-dimensional structure construction step executed in the CPU includes:
(i) The empirical molecular energy calculation of the one-dimensional structure information of the protein and the three-dimensional structure information of the ligand is performed by the number of the three-dimensional structure information of the protein,
(ii) The protein side moves the atomic coordinates according to the potential energy gradient of each of the three-dimensional structure information of the protein,
(iii) On the ligand side, move the atomic coordinates of the above-mentioned ligand three-dimensional structure information in the direction in which a plurality of calculated potential energy gradients are averaged,
(iv) obtaining the three-dimensional structure information of the ligand based on the plurality of three-dimensional structure information of the protein;
The method for constructing a three-dimensional structure of a protein-ligand complex according to claim 4, comprising each step.

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