WO2000075182A1 - Crystal of ribosomal recycling factor (rrf) protein and application thereof on the basis of three-dimensional structural data obtained from the crystal - Google Patents

Abstract

A method for designing a compound capable of binding to the active site or the accessory binding site of RRF protein which comprises evaluating with a computer the chemical body of RRF protein on the basis of the structural coordinate obtained from RRF protein crystals; and a method for searching a compound capable of inhibiting the activity of RRF protein on the basis of the activity of inhibiting the binding of RRF protein to ribosome or an activity of inhibiting the behavior of RRF protein on ribosome. These methods are useful in clarifying the three-dimensional structure and function mechanism of RRF, thereby contributing to the development of various bactericides, fungicides, herbicides, etc.

Description

 Description Crystal of ribosome recycling factor (RRF) protein and application based on three-dimensional structural information obtained from the crystal

[Technical field]

 The present invention relates to a crystal of a ribosome recycling factor (RRF). The present invention also relates to a three-dimensional structure of RRF protein obtained by X-ray diffraction of the crystal. Furthermore, the present invention relates to techniques for determining the structure of RRF mutants, homologs, and the like, and for developing next-generation antibacterial agents, antifungal agents, and herbicides by applying the structural information and mechanism of action of RRF proteins.

[Background technology]

 Protein biosynthesis is an essential function of all cell life activities and consists of four stages: “start”, “extension”, “termination”, and “ribosome recycling”. The final step in protein biosynthesis (the fourth step) is to release the messenger—termination RNA, transfer RNA, and ribosome termination complexes, respectively, to reuse the ribosomes for the next “start” step. It ends by dissociation. In Escherichia coli, which is a prokaryotic organism, this “reuse” of ribosomes is based on ribosome recycling factor (RRF) and elongation factor G (EFG) or release factor 3 (Release factory). It is known to be catalyzed by tor3). The process of “reuse” of this ribosome is reviewed by Dr Janosi et al. (1996 Adv. Biophys.

32: 121-201) and a review by Kaji et al. (Biochem, Biophys, Res Communs. 250 1-4, Protein Nucleic Acid Enzyme, Vol. 44, No. 7, 83-84 (1999)).

 It has been suggested that the dissociation of the protein translation termination complex in eukaryotes may be catalyzed by other factors other than RRF, with eukaryotic mRNA being monocistronic and prokaryotic being polycistronic. Nick (Kozak 1987, Mol. Cell.

Biol. 7: 3438-3445; Das et al. 1984, Nucleic Acids Res. 12: 4757-4768; Schoner et al. 1986 Proc. Natl. Acad. Sci. U.S.A. 83: 8506-8510; Sprengel

1985 Nucleic Acids Res. 13: 893-909) Inhibition of dissociation of somal mRNA from mRNA does not affect downstream cistrons. Since the fourth step of dissociation of the protein translation termination complex, which is the final step of protein biosynthesis in eukaryotes, is thought to be different from that of prokaryotes, it is particularly a target for new types of antibiotics. Expected.

 On the other hand, many antibacterial agents have been developed at present, and some of them have very high bactericidal action. However, there are many antibacterial agents obtained in this way whose sites of action remain unknown. Until now, the main method was to use antibacterial agents exhibiting these activities as materials for random screening, and to develop those with even greater utility while establishing a structural activity relationship. This takes a lot of time and effort.

 Therefore, in recent years, a database has been created for the purpose of eliminating this problem and efficiently finding inhibitors. Based on this, a rational drag design method is being studied and developed. An example of this is an inhibitor of the recently marketed anti-HIV drug proteases. HIV protease is crystallized and its three-dimensional structure is known. This structure and the three-dimensional structure of the active site Based on the amino acid sequence, a compound with the highest affinity for this site was selected from compounds known from computers and its inhibitory activity was measured. By forming a co-crystal of the target protein with the active one and measuring the three-dimensional structure, it is possible to predict the compound that binds more, and this is synthesized and its inhibitory activity is measured. Then, a co-crystal of this substance and the target protein is formed again, and an extremely effective substance can be obtained by repeating the above process.

 By the way, there have been many reports of strains that have acquired resistance to conventional antibiotics as described above, and it is urgently necessary to develop new antibiotics that target sites that can directly limit the growth of bacteria. It has been. Accordingly, the present inventors have focused on the fact that the RRF can be a new target of antibacterial agents, and have conducted intensive research, but this idea has recently been spotlighted.

[Disclosure of the Invention]

Regarding RRF, the present inventors have determined several gene sequences for E. coli, not only for prokaryotes, but also for eukaryotes (Japanese Unexamined Patent Publication No. 3-20079). 7, PCT / JP98 / 00734, Japanese Patent Application No. 10-150493). Therefore, the secondary structure can be estimated from the amino acid sequence obtained therefrom. However, in the current state of the art, it has not been possible to identify the actual three-dimensional structure from this secondary structure. In the actual protein, each amino acid residue interacts and, in some cases, undergoes various modifications to form its steric structure. Therefore, if the three-dimensional structure of a protein is known, it is possible to create a substance that can serve as a ligand. In order to create a useful antibiotic in this sense, determination of the three-dimensional structure by crystallization is extremely important. Will have significant significance.

 Accordingly, an object of the present invention is to elucidate the three-dimensional structure of RRF and to contribute to the development of various antibacterial agents, antifungal agents and herbicides.

 [Brief description of drawings]

 FIG. 1 is a photograph showing an XRRF protein crystal.

 FIG. 2 is a photograph showing an X-ray diffraction image of an XRRF protein crystal.

Details of diffraction image: xfl to 1200 of 1200, yf 1 to 1200 of 1200

Diffraction direction: xf to the right, yf up

Data file order:-xf + yf

Maximum pixel value: 65535

Scale limits: min = 1, max = 1200, black indicates high diffraction intensity.

 [FIG. 3] is a photograph of an RRF drawn with a ribbon. As shown in the figure, it consists of two domains, one consisting of three helical forces, and the second domain is a complex of β_sheet and coil helix.

 FIG. 4 is a photograph of an RRF space filling model.

 FIG. 5 is a schematic explanatory view showing a hypothesis about the mechanism of action of RRF.

 FIG. 6 is a graph showing that various inhibitors inhibit release of transfer RNA from the termination complex. Error bars indicate standard deviation.

 [FIG. 7] Lineweaver—Burkf showing inhibition of liposome release in the presence of various concentrations of transferred RNII. This is a graph by lot.

[Fig. 8] shows that RRF was transferred to ribosomes in the presence of paromomycin. It is a graph which shows that binding is inhibited. Error bars indicate standard deviation.

[Explanation of symbols]

 1 ... ribosome, 2 ... transferred RNA, 3 ... messenger RNA, 4 ..- RRF, 5-EFG, 6 ... termination complex.

 Based on the above situation, the present inventors have succeeded in obtaining RRF crystals and identifying the three-dimensional structure for the first time while conducting research on RRF, and as a result of further research, completed the present invention Reached.

 That is, the present invention relates to a crystal of R RF protein, its production method and three-dimensional structure.

 More specifically, a method for designing a compound capable of binding to the active site or an auxiliary binding site of an RRF protein, wherein the chemical entity is evaluated by computer based on the structural coordinates obtained from the RRF protein crystal. The method relates to the method. The present invention also relates to the above method, wherein the RRF protein crystal is any one of a crystal of the RRF protein itself, a crystal of an RRF protein mutant, a crystal of an RRF protein homolog, and a crystal of a co-complex of the RRF protein.

 The present invention also relates to the above method, wherein the RRF protein crystal is a bipyramid system.

Furthermore, the present invention relates to a method in which the RRF protein crystal is a space group. 4,2,2, or have a space group P4 ₃ 2, 2, relates to the aforementioned method.

 The present invention also relates to the above method, wherein the RRF protein crystal has a size of 0.3X0.3X0.5 mm.

 The present invention also relates to the above method, wherein the RRF protein crystal has a unit cell having a size of a = b = 47.3 A and c = 297.6 A.

 Furthermore, the present invention relates to the aforementioned method, wherein the RRF protein crystal is characterized by the structural coordinates according to Table 7.

 The present invention also relates to the above method, wherein the RRF protein crystal is derived from Thermotoga Martinima.

The present invention also relates to the above method, wherein the RRF protein crystal is orthorhombic. Furthermore, the present invention relates to the above method, wherein the RRF protein crystal has a space group P2, 2, 2. Further, the present invention relates to the above method, wherein the RRF protein crystal has a size of 30 3050Χ250Ι1.

 The present invention also relates to the above method, wherein the RRF protein crystal is derived from bacterium X.

 Further, the present invention relates to the above method, wherein the RRF protein crystal is crystallized by a droplet vapor diffusion method.

 The present invention also relates to the present invention, wherein the RRF protein crystal is a heavy atom derivative, and the crystal is any one of a crystal of the RRF protein itself, a crystal of the RRF protein mutant, a crystal of the RRF protein homolog, and a crystal of a co-complex of the RRF protein. The method relates to the method. The present invention also relates to the above method, wherein the heavy atom derivative is formed by reaction with a compound selected from the group consisting of thimerosal, gold thiomalate, peranyl acetate, and lead chloride.

 Further, the present invention relates to the above method, wherein the RRF protein crystal is a heavy atom derivative of platinum or mercury.

 The present invention also relates to the above method, wherein the RRF protein is a monomer.

 Further, the present invention relates to the above method, characterized by amino acid displacement according to Table 5 or Table 6 of RRF protein strength.

 Furthermore, the present invention relates to the above method, wherein the compound characterized by a chemical entity binding to an active site or an auxiliary binding site is an inhibitor of RRF protein.

 In addition, the present invention relates to the method, wherein the inhibitor is a competitive, non-competitive or uncompetitive inhibitor of RRF.

 The present invention also relates to the above method, comprising determining the orientation of the ligand at the active or auxiliary binding site of the RRF protein.

 Furthermore, the present invention relates to the above method, wherein the structural coordinates are the structural coordinates of the RRF protein according to Table 7.

The present invention also relates to the above-mentioned method, wherein the RRF protein is a pocket near the C-terminus located at a bent portion separating two domains of the RRF protein. Furthermore, the present invention relates to the above method, wherein the compound inhibits binding of RRF protein to ribosome or inhibits behavior of RRF protein on liposome.

 The present invention also relates to an RRF protein inhibitor obtained by the method.The present invention also relates to an activity of inhibiting the binding of RRF protein to ribosome or an activity of inhibiting the behavior of RRF protein on ribosome. The present invention relates to a method for searching for a compound that can inhibit the activity of an RRF protein based on the above.

 Furthermore, the present invention relates to an RRF protein inhibitor obtained by the above method.

 The present invention also relates to a method for determining the three-dimensional structure of the RRF protein, including elucidating the crystal form of the mutant, homolog or co-complex of the RRF protein by molecular replacement.

 The invention also relates to orthorhombic RRF protein crystals.

 The present invention also relates to the RRF protein crystal having a space group 1 ^ 2,2,2. Further, the present invention relates to the RRF protein crystal having a size of 30Χ50Χ250 μm.

 The present invention also relates to the RRF protein crystal, wherein the RRF is derived from bacterium X. The present invention also relates to a bipyramid-based RRF protein crystal.

Furthermore, the present invention relates to a space group? The present invention relates to the RRF protein crystal having 4, 2, 2, or a space group of 4 ₃ 2,2.

 The present invention also relates to the RRF protein crystal having a size of 0.3X0.3X0.5mm.

 The present invention also relates to the aforementioned RRF protein crystal, which has each unit cell of a = b = 47.3A and c = 297.6A.

 The present invention also relates to the aforementioned RRF protein crystals, characterized by the amino acid changes according to Table 5 or Table 6.

Further, the present invention is characterized in that the RRF tank is characterized by structural coordinates according to Table 7. Related to Park crystal.

 The present invention also relates to the RRF protein crystal, which is derived from Thermotoga Multima.

 The present invention also relates to the RRF protein crystal, which has been crystallized by a droplet vapor diffusion method.

 Further, the present invention relates to the RRF protein crystal, which is any one of a crystal of RRF protein itself, a crystal of an RRF protein mutant, a crystal of an RRF protein homologue, and a crystal of a co-complex of the RRF protein.

 The present invention also relates to the RRF protein, wherein the amino acid at the active site is selected from the group consisting of Arg110, Arg129 and Arg132 of SEQ ID NO: 1.

 The invention also provides that the one or more amino acids in the active site or the auxiliary active site are one or more amino acids selected from the group consisting of naturally occurring amino acids, unnatural amino acids, selenocystine and selenomethionine. The RRF protein has been replaced by:

 Further, the present invention relates to the RRF protein, wherein the hydrophilic amino acid and the hydrophobic amino acid in the active site or the auxiliary active site are substituted.

 The present invention also relates to the RRF protein, wherein at least one cysteine amino acid is substituted with an amino acid selected from the group consisting of selenocystine or selenomethionine.

 The present invention also relates to the aforementioned RRF protein, wherein at least one methionine amino acid is substituted by an amino acid selected from the group consisting of selenocystine or selenomethionine.

 Furthermore, the present invention relates to the RRF protein, which is in a crystalline form.

 The present invention also relates to the RRF protein having a specific activity higher or lower than that of the wild-type enzyme.

 The present invention also relates to the RRF protein having an altered substrate specificity. The invention further relates to the use of said RRF protein for measuring the binding interaction between a compound and the RRF protein.

In addition, the present invention provides a method for treating at least 1 The RRF protein of any of the preceding claims, wherein one or more amino acid residues have been substituted, resulting in a change in one or more charge units of surface charge.

 At present, it is presumed that RRF is an ideal target of antibacterial agents, and the three-dimensional structure of RRF disclosed by the present invention is extremely important in industry because it is directly linked to the development of antibacterial agents and the like. . Furthermore, since it is known that the primary structure of RRF of many pathogens is very similar (for example, the RRF of Pseudomonas aeruginosa has 60% homology with that of Escherichia coli), the three-dimensional structure of the RRF according to the present invention is These data make it very easy to understand the three-dimensional structure of the RRF of other pathogens. Therefore, in order to develop a species-specific antibacterial agent, the present invention is applied to the development of a next-generation antibiotic, an antifungal agent and a disinfectant by inhibiting RRF, and particularly to the development of an antibacterial agent by rational derag design. It is extremely useful as an indicator.

 The terms used herein are defined as follows:

 “RRF protein” means an RRF protein that has enzymatic activity under normal conditions.

“Naturally occurring amino acid” means the L-isomer of a naturally occurring amino acid. Naturally occurring amino acids include glycine, alanine, valin, leucine, isoleucine, serine, methionine, threonine, fenylalanine, tyrosine, tryptophan, cystine, proline, histidine, and aspanolaginate. Asparagine, glutamic acid, glutamine, _y -carboxyglutamic acid, arginine, orditin and lysine. Unless otherwise specified, the amino acids in the present specification are in the form of chow.

 “Unnatural amino acid” refers to an amino acid that is not found in nature in a protein. Examples of unnatural amino acids used herein include racemic mixtures of selenocystine and selenomethionine. Further, as non-natural amino acids, there are nor-mouth isine, para-nitrophenylalanine, homophenylalanine, nora-funoleolophenylalanine, and 3-amino-2-benzylpropionic acid. And D or L-form of homoarginine and D-phenylalanine.

“Positively charged amino acid” includes any naturally occurring or unnatural amino acid having a positively charged side chain under normal physiological conditions. Examples of positively charged natural amino acids include arginine, lysine and histidine.

 "Negatively charged amino acid" includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions. Examples of negatively charged natural amino acids include aspartic acid and glutamic acid.

 By "hydrophobic amino acid" is meant any amino acid having an uncharged, non-polar side chain that is relatively insoluble in water. Examples of naturally occurring hydrophobic amino acids are alanine, leucine, isoleucine, valin, proline, phenylalanine

, Tryptophan and methionine.

 "Hydrophilic amino acid" means any amino acid having an uncharged polar side chain that is relatively soluble in water. Examples of naturally occurring hydrophilic amino acids are serine, threonine, tyrosine, asparagine, glutamine and cysteine.

 "Variant" refers to an RRF polypeptide characterized by at least one amino acid substitution in the RRF sequence of wild-type E. coli (ie, a polypeptide that exhibits the biological activity of wild-type RRF). Say. Such variants can be obtained, for example, by expression of cDNA for RRF mutated in its coding sequence by oligonucleotide-specific induction. In addition, RRF mutants can be obtained by general biosynthetic methods according to Noren, CJ, etc. (Science, 224, pl82-188 (1989)) by site-specific incorporation of unnatural amino acids into RRF proteins. be able to.

Selenocystin or selenomethionine is incorporated into wild-type or mutant RRF by expression of a cDNA encoding the RRF in an auxotrophic E. coli strain. In this method, the wild-type or mutant RRF cDNA does not contain the power of natural cysteine or natural methionine (or both) and is grown on a growth medium enriched for selenocystine or selenomethionine (or both). Can be expressed in different hosts. In addition, selenomethionine can be incorporated into wild-type or mutant RRF by the method of inhibiting methionine metabolism (J. B. by Van Dyne GD, 229 ppl05 (1993)). By "change in surface charge" is meant a change in one or more charge units of a variant polypeptide at physiological pH compared to wild-type RRF. This can preferably be obtained by mutation of at least one or more wild-type RRFs into amino acids containing side chains having a different charge from the wild-type side chain at physiological pH in the amino acid. The change in surface charge is determined by measuring the isoelectric point of the polypeptide with the substituted amino acid and comparing this with the isoelectric point of the wild-type RRF molecule.

 “Change in substrate specificity” refers to a change in the substrate of a mutant RRF as compared to a wild-type RRF. Substrate specificity (species specificity) is determined by separating ribosomes, tRNAs, and EF-G from pathogenic bacteria and determining whether they can serve as substrates for RRF and RRF variants of E. coli.

 "Kinetic form" refers to the state of the enzyme in free or unbound form or the state of the enzyme bound to a chemical entity at either its active site or ancillary active site.

 A “competitive” inhibitor is one that inhibits RRF activity by binding to the same kinetic form of the RRF as the substrate of the RRF binds, and thus directly competes with the active site of the RRF.

 "Uncompetitive" inhibitors are inhibitors that inhibit RRF by binding to a different kinetic form of RRF than the substrate binds to.

 A “non-competitive” inhibitor is an inhibitor that binds to either the free or substrate-bound form of RRF.

 By "homolog" is meant a protein having at least 30% homology of the amino acid sequence with RRF or any functional domain of RRF.

 By "co-complex" is meant a RRF or a variant or homologue of an RRF covalently or non-covalently linked to a chemical entity or compound.

 “/ 3-sheet” refers to the conformation of a polypeptide chain that extends into an expanded zigzag conformation. All parallel polypeptide chains extend in the same direction. Polypeptide chains extending antiparallel extend in the opposite direction to the parallel lines.

“Active site” or “active site portion” means any or all of the following sites in the RRF: All parts. The substrate binding site is the site where the ribosome and its complex bind, and the site where degradation of the substrate occurs. The active site is at least near amino acid residues 110, 129 and 132 using SEQ ID NO: 1. It is.

 “Structural coordinates” refers to mathematical coordinates obtained from mathematical expressions relating to the pattern obtained by diffraction of an X-ray monochromatic beam by atoms (dispersion centers) of RRF molecules in a crystalline form. The variance data is used to calculate an electron density map of the repeating unit of the crystal, and the electron density map is used to establish the position of each atom within the unit cell of the crystal.

 "Heavy atom derivative" refers to a chemically modified form of an RRF protein crystal. In its fabrication, in practice, heavy metal atom salts or organometallic compounds (eg, lead chloride, gold thiomaleate, thyromesal or peranil acetate) that can diffuse through crystals and bind to the surface of the protein ). The position (s) of the bound heavy metal atom (s) can be determined by X-ray diffraction analysis of the immersed crystal. This information is then used to create the phase information used to construct the three-dimensional structure of the enzyme. It will be appreciated by those skilled in the art that the set of structural coordinates determined by X-ray crystallography has a standard error. For the purposes of the present invention, they have a root mean square deviation of less than 0.75 A of protein backbone atoms (N, C, C and 0) when superimposed on the structural coordinates listed in Table 7. Any set of structural coordinates of an RRF or RRF homolog or RRF variant should be considered identical.

 A "unit cell" is a basic parallelepiped shaped block. The total volume of the crystal can be constructed by repeated regular stacking of such blocks.

 “Space group” refers to the arrangement of target elements of a crystal.

“Molecular substitution” means that the structural coordinates (for example, the structural coordinates in Table 7) of another unknown crystal are known in the unit cell of the unknown crystal so as to be optimal for explaining the observed diffraction pattern of the unknown crystal. It refers to a method that includes the step of orienting and positioning a molecule to create a tentative model of an RRF crystal whose structural coordinates are not known. Then the phase is calculated from this model and combined with the observed amplitude, An approximate Fourier composition of a structure whose coordinates are not known is obtained. It can then be applied to the purified material to finally obtain the exact unknown crystal structure. Using the structural coordinates of the RRF according to the present invention, it is possible to determine the structural coordinates of a mutant, homolog, co-complex or different crystal structure of the RRF by using molecular replacement. Crystallization and structural analysis were performed using RRF derived from X bacterium and RRF derived from Thermotoga Mitima, but the same can be performed for other RRFs. In crystallization, not only the RRF protein itself, but also RRF protein mutants, RRF protein homologs, and RRF protein co-complexes can be crystallized and their structures analyzed.

 "Pocket" refers to a dent on the surface of the RRF protein, which binds to a substrate or the like in the expression of RRF activity, in addition to a binding pocket present in the binding site of the RRF protein or an auxiliary binding site. Include other pockets that are not involved in

The present invention provides, for the first time, a crystal of RRF of X bacterium and Thermotoga Maritima RRF and a structure of the RRF determined from the crystal. On the other hand, the crystals of Thermotoga Maritima RRF were formed from ammonium sulfate solution. Crystals have a space group P4 ₃ 2, 2 of the bipyramid type. The unit cell of this crystal has a = b = 47.3A and c = 297.6A.

 Table 7 shows the structural coordinates of the RRF. The crystal backing indicates that the RRF is monomeric.

 Figure 3 shows a ribbon drawing of the Thermotoga Maritima RRF. Helixes A, B, D, E, and F indicate a helix present from the N-terminus to the C-terminus. β-sheets 1, 2, 3, 4, 5, and 6 are / 3-sheet numbers that exist from the か け て end to the C end. As shown in the figure, the RRF is composed of two domains, one is composed of three helices, and the second domain is a complex of β-sheet coil and helix. The active site spans the E and F helices in the figure, and maintains the three-dimensional structure of the domain containing helices B, C, D, / 3-sheets 1, 2, 3, 4, and 5 to maintain activity. Is important.

Figure 4 shows the space filling model of the Thermotoga Maritima RRF, where N and C are Shows the N-terminal and C-terminal, respectively. Gray indicates carbon, red indicates oxygen, purple indicates N atom, numbers indicate amino acid sequence number, and 1 is N-terminal.

 Thus, based on the information on the three-dimensional structure of RRF elucidated by the present inventors, it has become possible for the first time to identify the active site and the auxiliary binding site of the enzyme. In addition to the results of the RRF genetic mutation described below, it is highly likely that the active site portion contains at least the amino acid residues Arg110, Arg129, and Arg132 of SEQ ID NO: 1.

 The present invention enables, for the first time, the use of molecular design techniques to design, select and synthesize chemical entities and compounds with respect to RRF. Chemical entities and compounds include inhibitory compounds that can bind to all or a portion of the active or auxiliary binding site of the RRF. In a possible approach according to the present invention, the structural coordinates of the RRF are used to design compounds that bind to the enzyme and to modify the physical properties (eg, solubility) of the compound in various ways. You. For example, the present invention allows for the design of compounds that act as competitive inhibitors of RRF by binding to all or a portion of the active site of the RRF. The present invention also allows for the design of compounds that act as uncompetitive inhibitors of RRF. These inhibitors can bind to all or a portion of the auxiliary binding site of the RRF already bound to the substrate, and are more potent and more potent than competitive inhibitors, which only bind to the RRF active site. Can be non-specific. Similarly, non-competitive inhibitors that bind to and inhibit RRF, whether or not bound to another chemical entity, can be designed using the structure coordinates obtained according to the present invention.

 A second design approach is to identify RRF crystals with molecules of various chemical entities in order to determine the optimal site for interaction between a candidate RRF inhibitor and RRF. For example, high-resolution X-ray diffraction data collected from solvent-saturated crystals allows the location of each type of solvent molecule to be determined. Small molecules that bind tightly to these sites can then be designed and synthesized, and tested for inhibitor activity (Travis, J., Science, 262, pl374 (1993)).

The present invention also provides for the reaction of a substrate or other compound that binds to the RRF with the RRF. It is useful in the design of improved analogs of RF inhibitors or in the design of new classes of inhibitors based on reaction intermediates of RRF and RRF inhibitor co-complexes. This provides a novel tool for designing RRF inhibitors with both high specificity and high stability.

 Another approach enabled and facilitated by the present invention is the computer screening of chemical entities or compounds that can be wholly or partially bound to the RRF. In this screening, the properties of the fit of such an entity or compound to the binding site can be determined either by shape complementarity or by estimated interaction energies (Meng, EC et al. J. Comp. Chem. , 13, 505-524 (1992)).

 If the RRF can crystallize in more than one crystalline form, the structural coordinates of the RRF, or portions thereof, as provided by the present invention will analyze the structure of other crystalline forms of the RRF Especially important for. The structural coordinates of the RRF, or a portion thereof, may be the structure of the RRF variant, the structure of the RRF co-complex, or the crystalline form of any other protein having an amino acid sequence that is significantly homologous to any functional domain of the RRF. Can also be used to analyze the structure.

 One method that can be used for this purpose is molecular replacement. In this method, the unknown crystal structure may be a crystal form of another form of RRF, an RRF variant or RRF co-complex or any other protein having an amino acid sequence that is significantly homologous to any functional domain of RRF. Can be determined using the structural coordinates of the RRF of the present invention as provided in Table 7. This method provides an accurate structural morphology for an unknown crystal more quickly and efficiently than trying to determine such information from scratch.

Further according to the invention, the RRF variant can be crystallized in a co-complex with a known RRF inhibitor. The crystal structure of such complexes can then be solved by molecular replacement and compared to the crystal structure of wild-type RRF. Thus, potential sites for modification within the various binding sites of the enzyme can be identified. With this information, a means to determine the most effective binding interactions (eg, increased hydrophobic interactions) between the RRF and the chemical entity or compound I will provide a.

 All of the above complexes can be studied using known X-ray diffraction techniques, and can be performed using computer software (eg, X-P0LAR, distributed by Yale University, 1992, Molecular Simulation, Inc). 33A resolution X-ray data can be refined to an R value of about 0.20 or less relative to a control (eg, Blundel & John Son, Protein Crystallography, Academic Press (1976), Methods in Enzymology, Vol. 4, 115, HW Wycoff et. al, Academic Press (1985) This information can therefore be used to optimize RRF inhibitors, and more importantly, to design and synthesize new RRF inhibitors. The structural coordinates of the RRF provided herein also facilitate the identification of related proteins, enzymes or M that are similar in function, structure, or both, to the RRF. Active sites such as serial similar protein, can more accurately estimate the binding sites and the like, a new antimicrobial agent, that connected to herbicides or antifungal agents.

 In designing a compound that binds to or inhibits the RRF of the present invention, two factors generally need to be considered. First, the compound must be able to physically and structurally bind to the RRF. Non-covalent intermolecular interactions that are important for the binding of RRF to its substrate include hydrogen bonding, van der Waals forces, and hydrophobic interactions.

 Second, the compound must be able to assume a conformation that allows it to bind to the RRF. Certain portions of the compound are not directly involved in binding to this RRF, but those portions can still affect the entire conformation of the molecule. This also has a significant effect on efficacy. The prerequisites for such a conformation include the chemical entity or the entire three-dimensional structure and orientation of the compound for all or some of the binding sites (eg, the active or auxiliary binding site of the RRF), or the RRF. Examples include the spacing between functional groups of a compound that contains some chemical entity that interacts directly.

The potential inhibitory or binding effect of a chemical compound on RRF can be analyzed before it is actually synthesized, and using computer modeling techniques. Can be used for testing. If the theoretical structure of a given compound indicates that there is insufficient interaction and binding between that compound and RRF, the synthesis and testing of that compound can be avoided. However, if computer modeling suggests a strong interaction, the molecule is synthesized, and the method of Hirashima and Kaji (Bi ochemi stry, Π, 4037, (1972)) or using oligonucleotides and in vivo Can be tested for its ability to inhibit by screening in Japan (Japanese Patent Application No. 10-158643). By this method, synthesis of ineffective compounds can be avoided.

 Inhibitory compounds of RRF or other binding compounds of RRF can be evaluated computationally and chemical entities or fragments screened for their ability to bind to individual binding pockets or other regions of RRF. It can be designed by means of cleaning and a series of selected steps.

A chemical entity or fragment may be referred to as an RRF, more specifically, an individual binding pocket of a binding site or an auxiliary binding site of an RRF, or another pocket that is not involved in binding to a substrate or the like in expressing RRF activity. One of several ways to screen for their ability to combine with can be used. This process can be started by visual examination of active sites, for example, during computer screening based on the RRF coordinates in Table 7. For example, as shown in the drawing by the ribbon in Fig. 3, the RRF with an “L” shape has a pocket near the C-terminal located at the “L” bend that separates the two domains. However, compounds that bind to this pocket can be potential candidates for RRF inhibitory compounds. The above-mentioned pockets can be easily observed by creating a space filling model using software such as Rasmol based on the RRF coordinates shown in Table 7. This pocket is located between the two domains of RRF, suggesting that it may be involved in the activity of RRF through regulation of the angle between the domains. The selected fragments or chemical entities can then be located in various orientations or can be linked to individual binding pockets of the RRF. Coupling can be accomplished using software such as Quanta and Sybyl, and then using standard molecular mechanics force fields (eg, CHARMM, AMBER). Perform energy minimization and molecular dynamics.

 Specialized computer programs can assist in the process of selecting fragments or chemical entities. Examples of these programs include:

uRID (uoodiora, P. j., A omputat lonal Procedure for Determining tnerget ically Favorable Binding Sites on Biologically Important Mac romolecules ", J. Med. Chem., 28, pp. 849-857 (1985)) Is available from Oxford University, oxford, UK MCSS (Miranker, A and M, Karplus, "Functionality Map of Binding Sites: A Multiple Copy Simultaneous Search Method., Proteins-Structure, Function and Genetics, 11, pp. 29-34 (1991)), which is available from Molecular Simulations, Burlington, MA. AUT0D0CK (Goodsell, DS and ΑJ. Olsen, "Au tomated Docking of of Substrates to Proteins by Simulated Anneal in g", Proteins: Structure, Function and Genetics, 8, pp. 195-202 (1990)) Is available from the Scripps Research Institute, La Jol la, CA. D0CK (Kuntz, ID et al, A Geometric Approach to Macromolecu le-Ligand Interactions ", J. Mol. Biol., 161, pp. 269-288 (1982)), which is the University of Carifornia, San Francisco, CA force, Once the appropriate chemical entity or fragment has been selected, the chemical entity or fragment can be assembled into a single compound or inhibitor. Assembling can be done by visual examination of the interrelationship of the fragments in the 3D image shown on the computer screen with respect to the structural coordinates of the RRF. Next, model building is performed manually using software such as Quanta or Sybyl.

 Examples of useful programs that can assist one skilled in the art in contacting individual chemical entities or fragments include:

CAVEAT (Bartlett, PA et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologiccal ly Active Molecules", Molle cular Recognition in Chemical and Biological Problems ", Royal Chem. So, 78, pp. 182-196 (1989)), which is the University of Carifornia,

Available from Berkeley, CA. MACCS—3D (3D Database systems such as MDL Information Systems, San Diego, CA; this area is described in Martin, Y. C., "3D Database Searching in Drug Design", J. Med. Chem., 35, pp. 2 145-2154 (1992) H00K (available from Molecular Simulations, Burlington, MA).

 Instead of constructing an RRF inhibitor in a stepwise fashion from one fragment or chemical entity at a time, as described above, the inhibitory compound or other RRF-binding compound may have an active site (or, if necessary, known) from the RRF. (Including some portions of the inhibitors). These methods include:

 LUDI (Bohm, HJ, "The Computer Program LUDI: A New Method for the de novo Design of Enzyme Inhibitors", J. Comp, Aid, Molec, Design, 6 pp. 61-78 (1992), which is Biosym Technologies, Available from San Diego, CA LEGEND (Nishibata, Y. and A. Itai, Tetrahedron, 47, p. 8985 (1991), available from Molecular Simulations, Burlington, MA. LeapFrog ( Tripos Associates, St. Louis, MO Power, etc. Available Ht.

 Other molecular modeling can be used in the present invention. See, for example, Cohen, NC, oMolecular Modeling Software and Methods for Medicinal Chemistry, J. Med. Chem., 33, p883-894 (1990). See also, Navia, M.A. and M.A. Murcko, The Use of Structural Information in Drug Design, Current Opinions in Structural Biology, 2, p. 202-210 (1992).

Once a compound has been designed or selected by the methods described above, the effectiveness with which the compound can bind to the RRF can be tested by computer evaluation and optimized. For example, a compound designed or selected to function as an RRF inhibitor, depending on the active site when binding to a natural substrate Capacity that does not overlap with the capacity occupied should preferably be considered. An effective RRF inhibitor should preferably show a relatively small difference in the energy between its bound and free states (ie, small binding strain forces. Therefore, the most effective RRF inhibitors The RRF inhibitor should be designed with a binding strain of no more than about 10 kcal / mol, preferably no more than about 7 kcal / mol. In their conformation, they can interact with the enzyme, in which case the strain force of the bond is between the energy of the free compound and the average energy of the conformation observed when the inhibitor binds to the enzyme. Makes a difference.

 Compounds designed or selected to bind to RRF, in their bound state, are preferably optimized by the computer to have no repulsive electrostatic interaction with the target enzyme. Such non-complementary (eg, electrostatic) interactions repel, charge-charge, dipole-dipole, and charge-dipole interactions. Specifically, the sum of all electrostatic interactions between the inhibitor and the enzyme when the inhibitor binds to RRF makes a neutral or favorable contribution to the enthalpy of binding.

 Certain computer software is available in the art to evaluate compound strain forces and electrostatic interactions. Examples of programs designed for such uses include Gaussian 92C, MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1992; AMBER, version 4.0 PA Kollman, University of California, San Francisco, 1994; QUANTA / CHARMM Molecular Simulat ions, Inc, San Diego, CA 1994 and the like. These programs can be executed using a general-purpose computer such as Silicon Graphics IRIS 4d / 35 or IBM RISC / 6000 Model550. Other hardware and software are known to those skilled in the art.

Once the RRF binding compound has been optimally selected or designed as described above, substitutions are then made on some of the atoms or side chains of the compound to improve or modify its binding properties. Generally, the first substitution is conservative. That is, the substituent has approximately the same size, shape, hydrophobicity, and charge as the original group. In the field Compounds known to alter the conformation should be avoided. The chemical compounds so substituted are then analyzed for potency compatible with the RRF in a manner similar to the computational methods described above.

 The present invention also allows for variants of RRF and elucidation of their crystal structure. More specifically, the present invention allows the identification of the desired site for mutation by the location of the active site, auxiliary binding site and interface of the RRF based on the crystal structure of the RRF.

 For example, the mutation can be directed to a particular site or combination of only the wild-type RRF site, ie, the active site or the auxiliary binding site. Alternatively, a location on the interface site is selected for mutagenesis. Similarly, only positions on or near the enzyme surface can be replaced, resulting in a change in the surface charge of one or more charged units as compared to the wild-type enzyme. Alternatively, the amino acid residues of the RRF can be selected based on their hydrophilic or hydrophobic characteristics.

 Such variants are characterized by any of several different properties as compared to the wild-type RRF. For example, such a variant may have a change in the surface charge of one or more charged units, or may have increased stability to subunit dissociation. Alternatively, such a mutant may have an altered substrate specificity as compared to the wild-type RRF, or may have a higher or lower specific activity than the wild-type RRF.

The RRF variants prepared according to the present invention can be prepared by a number of methods. For example, a wild-type RRF sequence can be mutated using the present invention at the site identified as desirable for mutation by oligonucleotide-directed mutagenesis or other conventional techniques (eg, deletion, etc.). . Alternatively, variants of RRF can be created by site-specific substitution of a particular amino acid with a non-naturally occurring amino acid. In addition, RRF variants can be made by replacing amino acid residues, ie, specific cysteine or methionine residues, with selenocysteine or selenomethionine. It does not contain either native methionine or cysteine (or both), but does contain wild-type or mutant polypeptides on growth media enriched for selenomethionine or selenocystin (or both). This is achieved by growing a host organism capable of expressing any of the peptides.

 Mutations can be introduced into the DNA sequence encoding the RRF using synthetic oligonucleotides. These oligonucleotides contain nucleotide sequences flanking the desired mutation site. Mutations can be made in the full length DNA sequence of the RRF, or the RRF or the RRF sequence of other organisms, shortened or lengthened (deleted or added).

 In accordance with the present invention, mutated RRF DNA sequences produced by the methods described above or alternative methods known in the art can be expressed using expression vectors. As is well known in the art, expression vectors typically include an element that enables autonomous replication in the host cell independent of the host genome, and one or more phenotypic markers for selection purposes. . The expression vector also encodes a promoter, an operator, a ribosome binding site, a translation initiation signal, and, optionally, a libresor gene and a termination signal, before or after the insert of the DNA sequence surrounding the desired RRF variant coding sequence. Contains regulatory sequences. In some embodiments, if secretion of the produced mutant is desired, a nucleotide coding for a signal sequence can be inserted before the RRF mutant code sequence. For expression under the control of regulatory sequences, the desired DNA sequence must be operably linked to the regulatory sequences. That is, a DNA sequence encoding the RRF variant and maintaining an appropriate reading frame that allows expression of this sequence under the control of a regulatory sequence and production of the desired product encoded by the RRF sequence. Must have an appropriate start signal before the.

A wide variety of well-known and available expression vectors are all useful for expressing the mutated RRF coding sequences of the present invention. These include, for example, various known derivatives of SV40, known bacterial plasmids (eg, plasmids from E. coli including colEl, pCR1, pBR322, pMB9 and derivatives thereof). Plasmids of wider host range (eg, RP4, phage DNA (eg, derivatives of many phage (eg, NM989) and other DNA phages (eg, M13 and filamentous single-stranded DNA phage)), 2 Yeast plasmids such as μplasmid or their derivatives and plasmids and It should consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as vectors obtained from combinations of phage DNAs (eg, phage DNA or plasmids modified to utilize other expression control sequences). Includes turtles. In a preferred embodiment of the present invention, we utilize the E. coli vector.

 In addition, any of a wide variety of expression control sequences that control expression when operably linked to a DNA sequence may be used in these vectors to express a mutated DNA sequence according to the present invention. Such useful expression control sequences include, for example, the early and late promoters of SV40 for animal cells, the lac, trp, TAC or TRC systems, and the λ phage regulatory region of the fd coat protein. Major operator region and promoter region (all for E. coli), 3-phosphoglycerate kinase or other glycolytic enzyme promoter, acid-producing phosphatase promoter (eg Pho5), yeast for yeast The promoter of the mating factor and other sequences known to regulate gene expression in prokaryotic or eukaryotic cells or viruses and combinations thereof. In a preferred embodiment of the present invention, we utilize E. coli expression.

 A wide variety of host species are also useful for producing mutant RRFs according to the present invention. Examples of these hosts include bacteria such as E. coli, Bacillus and Streptomyces, fungi such as yeast, animal cells such as CH0 cells and COS-1 cells, plant cells, and transgenics. Host cells. In a preferred embodiment, the host cell is E. coli.

It is to be understood that not all expression vectors and systems will function in the same manner to express the mutant DNA sequences of the present invention and to produce modified RRFs or RRF variants. Not all hosts work equally well with the same expression system. However, one of skill in the art can select from these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of the present invention. For example, an important consideration in selecting a vector is the ability of the vector to replicate in a given host. Encoded by the vector, such as the number of copies of the vector, the ability to control the number of copies, and the antibiotic marker. The expression of other proteins that must be considered must also be considered.

 Various factors should be taken into account when selecting an expression control sequence. These are, for example, the relative strength of the system, its ability to control, the suitability of the DNA sequences encoding the modified RRFs of the invention, especially with respect to potential secondary structure.

 The host must be compatible with the selected vector, have the toxicity of the modified RRF to the host, have the ability to secrete mature products, have the ability to properly fold the protein, have the fermentation requirements, have the ease of purifying the modified RRF from the host, Should be selected based on safety considerations. Within these parameters, one of skill in the art can select various vector / expression control Z host combinations that can produce useful amounts of mutant RRF.

 Mutant RRF produced in these systems can be purified by a variety of conventional processes and strategies, including those used to purify wild-type RRF and strategies.

 Once the RRF mutation has been made at the desired location (ie, active site or auxiliary binding site), the mutant can be tested for any of several properties of interest.

For example, mutants can be screened for changes in charge at physiological pH. This is determined by measuring the isoelectric point of the mutant RRF compared to the isoelectric point (pi) of the wild-type parent. The isoelectric point is determined by gel electrophoresis according to the method of Wellner, D. Analyt. Chem. 43. _P 597 (1971). Variants with altered surface charge are RRF polypeptides having a substituted amino acid located on the surface of the enzyme and an altered pi, as provided by the structural information of the present invention.

 In addition, variants can be screened for higher or lower specific activity compared to wild-type RRF. Mutants are measured for activity using the method of Hirashima and Kaji and Atsushi (supra) using oligonucleotides. Mutants can be tested for changes in RRF substrate specificity by measuring the RRF response as described above.

A further object of the present invention includes variants with increased stability. RRF variants with increased stability include those that do not show loss of enzyme activity. Hereinafter, the present invention will be described in more detail with reference to Examples. The examples shown below are for the purpose of detailed explanation only and do not limit other methods.

 [Example]

Example 1: Crystallization of RRF protein of fungus X by the hanging drop vapor ditt'usion technique

 A 5 μl solution containing 4 mg / ml to 8 mg / ml of RRF protein of bacteria X, 50 mM Tris-HCl pH 8.5, sulfate 70-100 mM, polyethylene dalicol 14% to 18% Equilibration was performed in a pool containing a high concentration of crystallization reagent. Equilibration was performed by diffusion of a volatile medium (water or organic solvent) until the vapor pressure of the droplet was equal to the vapor pressure of the pool. If equilibrium is due to water exchange (drops to pools), the volume of the drops will change. As a result, the concentration of all media in the droplet changes. A medium with a higher vapor pressure than water will undergo a conversion from pool to droplets. In this example, the glass container with which the RRF protein solution comes into contact is used after its surface is subjected to a hydrophobic treatment. XRRF crystals were obtained by dialysis against a buffer solution of Tris hydrochloride 100 mM ρΗ8.5 sulfate 150 mM to 200 mM, polyethylene dalicol 28% to 36%. The crystals grew to a size of 30X50X250 im in one to three weeks. Figure 1 shows the results.

Example 2. Three-dimensional structure of RRF by X-ray diffraction analysis

 As a means for determining the three-dimensional structure of the RRF, a multiple isomorphous replacement procedure was used. This is the standard method necessary to obtain diffusion data from isotopic protein crystals due to heavy atoms. From the position of the heavy atom, the difference between the unsubstituted isotope and the isotope was calculated on a Patterson map. The data of the initial protein phase necessary for the calculation of the electron density map for the creation of the protein model was calculated using several types of derivatives.

 X-ray diffraction data of this frozen crystal was collected on BL71 by MaxII synchrotron (Sweden, Lund).

The native crystal diffracted at 2.6 A resolution. Up to 2.9A resolution so far has been used for mosaicity issues of 1.5 and above. This typical times Figure 2 shows the folded image.

 This native data analysis has been completed and Rsym is 1.0. Table 1 shows the statistical data.

 This crystal has a = 98.5A, b = 106.7A, c = 66.7A, and? This asymmetric unit belonging to 2 contains 2 to 4 molecules, with 0.5, 0.33, and 0.5 translations between each molecule. Data were obtained for two derivatives, the platinum derivative diffracted to 4.OA and the mercury derivative to 3.8A.

Statistical examination of native data

Table summarizing diffraction intensity and R-factors by shell size (resolution)

 R value (as a linear function) = 3D SUM (ABS (I-<1>)) / SUM (I)

R value (as a quadratic function) = 3D SUM ((I-<I>) ** 2) I SUM (I ** 2) Chi-square = 3D SUM ((I-<I>) ** 2) I (Error ** 2 * N / (N-1))) All sums were calculated only for values measured more than once.

Shell lower and upper limits Diffraction intensity average first order multiplier second order multiplier Angstrom average error stat. Value Chi-square _ value R-factor R-factor Lower limit

 30.0 7.12 814.6 36.6 18.5 0.709 0.033 0.032

7.12 5.67 227.1 16.7 13.5 1.012 0.084 0.072

5.67 4.95 266.0 19.0 15.3 0.983 0.083 0.075

4.95 4.50 417.3 25.3 18.4 1.052 0.070 0.064

4.50 4.18 394.0 25.5 19.6 1.239 0.087 0.086

4.18 3.93 330.4 24.4 19.7 1. 064 0.093 0.088

3.93 3.74 296.1 24.6 20.9 1.286 0.125 0.219

3.74 3.58 283.8 25.5 22.0 1.275 0.142 0.221

3.58 3.44 207.5 23.2 21.2 1.314 0.170 0.192

3.44 3.32 173.2 22.5 21.1 1.278 0.193 0.298

3.32 3.22 151.8 22.1 20.9 1.414 0.222 0.231

3.22 3.12 130.Ί 21.7 20.8 1.560 0.265 0.280

3.12 3.04 108.3 20.6 19.9 1.552 0.306 0.307

3.04 2.97 92.2 19.9 19.2 1.655 0.334 0.315

2.97 2.90 74.9 19.2 18.7 1. .632 0.411 0.416 All reflections 268.4 23.2 19.3 1.259 0.119 0.102

Example 3. Crystal structure of Thermotoga Maritima RRF

 The RRF cDNA of Thermotoga Maritima was cloned into an expression vector (PET1650) and expressed in E. coli by adding IPTG. As a result, high levels of Thermotog a Maritima RRF accumulated in host cells. The cells were mechanically disrupted and purified by a modification of the method of Hirashima and Kaji (Biochemistry, Π, 4037, (1972)) to obtain Thermotoga Maritima RRF. RRF crystals were grown by vapor diffusion. Mix 5-ΙΟμΙ RRF solution with the same volume of reservoir (00, 1M sodium acetate, (pH 5.5) 2.0M ammonium sulfate, 5mM DTT, 10% glycerol) and mix with 600μ1 of the above reservoir. Equilibrate at 25 ° C and streak this solution for 24 hours to promote crystal formation, and crystals are seen after 15 hours. Three days later, it grew into a 0.3 × 0.3 × 0.5 mm bipyramid type crystal.

 For those skilled in the art, the above-mentioned crystallization conditions can be appropriately modified.

All X-ray data sets (2.55 A resolution) were collected at the beamline BL711 on a Mar345 image plate detector using a MAX II synchrotron. Merging, scaling, indexing and integration of the data were performed using the VDS and Xscale (Kabsch, WJ Appl. Crystallography 26795 (1993)) programs. MAD data (multiware length anomalous dispersior data) was collected using a Mar345 image plate detector at a beamline of BM14 ESRF at wavelengths of 0.9184-0.978 and 0.9788A. This data was processed using Mosflm (Leslie, AGW in Crystallographic computing Oxford Univ. Press (1990)), and scaling and merging were performed in Scala (CCP4). The position of the selenium atom in the RRF was determined by using the shelx program (sheldrick, GM Acta Cryst. A46 P467 (1998)), and using the normalized structure factor. Heavy atom (selenium) parameters were refined using MIphase (CCP4). When I asked the electron density map in both the space group 1 ^ 4,2,2 and Ρ4 ₃ 2,2, space group correct, it has become clear that it is Ρ42,2. Average merit values ranged from 0.66 0.6 to 4.OA. Table 2 shows the crystal data of Thermotoga Maritima RRF. Table 2 Data collection

Maximum resolution shell ^{2 65} -. 2.55 A for the value

 Resolution 30-2.55

 Total number of measurements 79947 (7119)

 Specific catadioptric refraction 11927 (1261)

 Average number of iterations 6.7 (5.6)

R „ _a (%) 0.049 (0.156)

 Data completion (%) 99.8 (99.1)

 I / sigma (I) 26.4 (8.9)

Rmerge = (∑ | I- <I> | / ∑I) where I is the observed intensity,

 <1> is the average intensity of centrally symmetric reflection.

The values in the brackets indicate the values for maximam resolution. Solvent flattering and phase expansion (CCD4_col laborative put iry project ff4, A suite of programr or protein crystallography Daiesburg Laboratory, Warrington, WA4 4. successful polypeptide chains to trace. model building program _{(j ones, A. T et al} Acta. Cryst. A47 PP110 (1991)) refinement position ((CNS) Branger A. T et al Acta Cryst D54 p905 (1998)) and the phase combination of the titles were repeated, and all the side chains were able to be introduced into the model. This model was compared with crystallographic data (native data) to reach the final model using only the model phase.The RRF model according to the present invention R factor for observed data on X and Y axes Root mean square from. The ideal bond lengths and bond angles with 25.3%) of deviation of 0.01 A and 2. OA.

Table 3 shows the results of the statistical processing of the Thermotoga Maritima RRF crystal data. Table 3

Data collection

Data set (wavelength A) Beak (0.9786) Inflection (0.9788) Remote (ϋ.9184) Resolution (A) 2.9 2.9 2.9 Completeness (%) 99 98 98

 Rsym (%) 7.0 7.0 7.2

 Cullis R-centric 0.67 0.56

Cullis R-anomalous 0.60 0.77 0.78

Table 4, _c resolution table 4 showing the Phasing of Thermotoga Maritima RRF

2. Estimation of the active site of RRF

 A series of RRF variants were generated to deduce the location of the active site in the RRF molecule. Mutagenesis was introduced using an error-prone PCR method (Janosi et al EMBO J. 17 1141 (1998)).

 Plasmid having frr (gene encoding RRF) having a lethal mutation was isolated as follows. The pMIX described in EMBO J. 17 1141 (1998) by Janosi et al. Was used. Briefly, pMIX caused various genetic mutations in frr and introduced them into chloramphenicol-resistant plasmid. In this example, E. coli LJ4 (recA_) was used as a host. Escherichia coli is kept alive by the wild-type frr on pPEN (1560) (Janosi et al. EMBO JU 1141 (1998)) because this fungus is inactivated by frr on the chromosome due to frameshifting. . pPEN (1560) has a kanamycin resistance factor and sucrose ill sucrose sensitivity gene 's'.

The E. coli was transformed with pMIX and selected for chloramphenicol resistance as a marker. Since frr is indispensable to bacteria, bacteria having a lethal mutation in pMIX cannot survive without pPEN1560. Therefore, bacteria having both pMIX and PPEN1560 plasmids were searched. By the way, both pMIX and PPEN1560 plasmids do not coexist because they are usually incompatible, but coexist when required (need for antibiotic marker and frr) as described above. To select such Escherichia coli, the transformant is spread on a plate containing CM and sucrose, and then replica-plated on a plate containing CM and KM. To go. If bacteria that grow on the latter and do not grow on the former are selected, bacteria that have lethal frr in pPEN1560 and pMIX can be selected. Since this bacterium has pPEN1560, it cannot grow on plates containing sucrose. Each plasmid was purified from the 153 transformants thus obtained, and used to transform Escherichia coli DH5 (having wild type frr). Since this bacterium has a wild-type frr as described above, it does not require pPEN1560 (kanamycin resistance). Therefore, by selecting Escherichia coli DH5a which is sensitive to kuram ramphenicol and kanamycin, Escherichia coli having lethal mutation and having pMIX can be selected.

Plasmid was isolated from the Escherichia coli thus obtained, and a Kpnl-Hindlll fragment (0.9 kb, frr) was taken out and subjected to DNA sequencing by a conventional method. Table 5 shows the results.

Table 5

 Gene mutations that inactivate RRF

 Mutation in the primary sequence of the isolated nucleoki ^ ¾ RRF

 "Ko"

 Number of shares

 1 amino acid change frrl46 T (152) C Leui51) Pro

 frrieO T (161) C

 T (19) C Leu (65) Pro

T (99) CT (i 4) C Leu (65) Pro Ser (3; 3) Silent

/ rrlOe! C (123) TG (329) Osamu the A Arg (llO) His Val ( 41) Silent frrll9 C (-91) AC (385 ) T AiH (l29) Cy S promoter

A class frrlld] C (394) G Ax¾ (t32) Gly

 frrl32 1 C (394) T Arg (l32) Cys

 frrlli 1 G (395) A Arg (l32) His

 frrI38 1 G (395) A Arg (132) Gln

 frrl24 T (524) C Leu (l75) Pro

 frrl49 C (447) T T (524) C Lcu (l75) rro Ser (l49) Silent

2 amino acid changes frrl65 G (28) A A (490) C Glu (IO) Lys Thr (l64) Pro

 frr / 13 1 G (28) C G (162) A A (490) C Glu (10) Lys Thi; i64) Pro Leu <154) Silent

B class frrll2 1 T (38) CT (512) C Met (13) Thr Ile (171) Thr-frrll6 T (107) AC (317) A Leu (36) G Thr (106) Lys-frrll] Α (269 ) ϋ G (329) A Asn (90) Ser Arg (UO) His-

3 amino acid changes frrl 1 T (14) CA (103) GT (194) AI! E (5) Thr Ser (35) Gly Lcu (65) Glu

 C class frr l 1 G (88) A T (97) C C (394) TGly (30) Scr Scr (33) Pro Arg (132) Cys-

Short C-terminal frrl27] G (52) TT (4I6) C Glu (18) stop (17 AA long RRF) + mutation bevond stop frrlU A (76) T Lys (26) slop (25 AA long RRF)

 fir 158 C (135) G T r (45) stop (44 ΛΛ long RRF)

 frrl25 C (157) T Gln (53) slop (52 AA long RRF)

 frrl62 T (24) C C (157) T Gln (53) s (op (52 AA long RRF) Asp (8) Silent

 D class frrNO A (196) TT (206) CG (309) Tl, ys (66) stop (65 AA long RRF) +2 mutations beyond stop frrllO C (218) A Ser (73) stop (72 ΛΛ long RRF)

firm CHS) AC (218) A Ser (73) stop (72 AA long RRF) SD-Including starter frrW8 G (364) TA (540) G GIu (122) stop (121 AA long RRF)

Table 5 continued

 frrin 1 A (430) T Lys (144) stop (143 AA long RRF)

 frrl36 1 T (336) C A (430) T Lys (] 44) stop (143 A A long RRF) Asp (112) Silent

 frrl59 I Τ (-46χ: A (508) T Lys (170) stop (169 AA long RRF) + mutation between promoter and SD frr! 42 3 G (514) T Glu (172) stop (171 AA long RRF)

c-end isr force frrllS 2 A (103) G C (157) T Ser (35) Gly Glu (S3) stop (52 AA long RRF)

Nara and frr! 51 1 (All) G G (364) T Asp (4) Gly Glu (122) stop (121 AA long RRF)

 1 Amino acid change frrNO 1 A (61) G C (367) T Lys (21) GIu Glu (123) stop (122 AA long RRF)

 frrl66 1 A (61) G G (I62) A C (367) TLys (2 l) Glu Gln (123) stop (122 ΛΑ long R F) Leu (l 54) Silent

 E class frrl21 1 A (445) G C (469) T Ser 149) Gly Gln (157) stop (156 AA long RRF)

 firl52 I C (467) T C (469) T Scr (156) Phe Gln (157) stop (156 A A long RRF)

 frrl39 1 C (467) TC (469) TC (555) TSer (156) Phe GIn (157) stop (156 AA long RRF) mutation beyond stop

Frame shift frrm IT (5) del Stop at nt 50-52 (16 AA long RRF) Ser (3) to (Cys (16) changed, C-terminal is frrWl 1 G (40) del Stop at nt 50-52 ( 16 AA long RRF) Asp (14) to Cys (16) changed Shortened fr 69] A (70) del Stop at nt 170-172 (56 AA long RRF) Ile (24) to Ser (56) changed

 frrl 1 A (79) del Stop at nt 170-172 (56 AA long RRF) lle (27) to Ser (56) changed frr 1 C (75) del Stop at nt 170-172 (56 AA long RRF) Ser ( 25) to Ser (56) changed frrl26 1 C (101) del A (419) T Stop at nt 170-172 (56 AA long RRF) Ser (35) to Ser (56) changed

 + mutation beyond stop

 F class frrW5 1 T (170) del A (359) TT (492X: Stop at nt 176-178 (58 AA long RRF) Val (57) to Thr (58) changed

 2 mutations beyond stop frrl 4 1 CG (82-83) del Stop at nt 210-212 (69 AA long RRF) Arg (28) to Val (69) changed frrl53 1 AT (I 9-200) C Stop at nt 266 -268 (88 AA long RRF) Ile (67) to Gly (88) changed frrWl 1 A (333) del Slop at nt 338-340 (112 AA long RRF) Asp (l 12) changed

 03 1 C (362) del Stop at nt 16-418 (138 AA long RRF) Ala (121) to Lys (138) changed frri 1 A (346) fcl A (363) GA (50 l) GSlop at nt 416- 418 (138 AA long RRF) Ile (ll6) toLys (138) changed mation in shifted sequence

+ mutation beyond stop frrl 1 A (389) del Stop at nt416- l8 (138 AA long RRF) Asn (I30) to Lys (138) changed fr "i5 1 A (511) dcl Stop at nt 545-547 (181 ΑΑΙοηκ RRF) IIe (168) toOlu (181) changed Nucleotide and amino acid mutations: Mutation sites are indicated by the position numbers of wild-type nucleotides or anoic acids Amino acids are indicated by three letters Abbreviations: AA = amino acid, dd = Deletion

As shown in this table, 61 strains were obtained, which had 53 different genotypes. It was confirmed that the thus isolated plasmid having the lethal gene did not function as frr using LJ4 having a temperature-sensitive RRF (Janosi et al., EMBO J 17 1141 (1998)). None of the resulting plasmids could support LJ4 growth at 42 ° C.

 Next, we describe a method for isolating amino acid mutations that do not affect the function of frr among the structural genes of frr. LJ4 was used as a host for this purpose. This host can be present at 27 ° C due to the plasmid pKH6 with frrl4 as frr on the chromosome does not function as described above.

This Escherichia coli is naturally temperature-sensitive because it lives by frr14 (encoding a temperature-sensitive RRF). Which grow the E. coli 42 ° C at a rate of the growing 4.2x 10- ⁶ Letting and 2 7 ° C the natural reversion rate was obtained. Among these Escherichia coli, those that became temperature-sensitive again when plasmid was replaced with one having tsfrr (pKH6) were selected, and the DNA sequence of the frr portion was determined by a conventional method. In all of the obtained frr, the genetic mutation of frr, Val 117 Asp, was reverted to wild-type palin, but some of them showed mutations at amino acid positions other than position 117. These mutations had no effect on frr function. This mutation is shown in Table 6.

Table 6

 RRF: a mutation that restores the temperature sensitivity of a gene

 Allele phenotypic segregation Nucleotide position Amino acid position description

Amino acids are expressed in three letters. Abbreviations: NA = —, W1-wild cattle type, tS = temperature sensitivity, tr = temperature tolerance

3. Estimation of RRF action mechanism

 From the drawing of the RRF with the ribbon shown in Fig. 2 and the space-filling model shown in Fig. 3, it was found that the RRF had a shape and size similar to the transition RNA (Sel mer, M., Al-Karadaghi, S. , Hirokawa, G., Kaji, A. & Lil jas, A. Science 286, 2 349-2352 (1999)). Therefore, RRF behaves similarly to translocated RNA, suggesting the possibility of expressing the protein translation termination complex dissociation activity. Specifically, the model shown in Fig. 5 is suggested for the mechanism of action of RRF.

 First, RRF4 binds to the aminoacyl site (A site) of the termination complex 6 (a) consisting of transfer RNAs 2a and 2b, messenger RNA 3 and ribosome 1. EFF5 with GTP is bound to RRF4. Transfer RNAs 2a and 2b are bound to the peptidyl site (P site) and the release site (exit site) (E site) of ribosome 1, respectively (b). Next, ribosome-dependent GTP hydrolysis and translocation of the RRF4 bound to the A site to the P site is triggered by EFG5. At the same time, the release of the transfer RNA 2b bound to the E site and the release of the transfer RNA 2a bound to the P site via the transfer to the E site occur, resulting in the transfer of two molecules of transfer RNA. Released (c). Finally, following the release of RRF4 and EFG5 from ribosome 1, ribosome 1 is released from messenger RNA3, and the dissociation of termination complex 6 is terminated (d).

 The following experiment was conducted to verify the hypothesis based on this model.

Example 4. Inhibition of RRF activity by aminoglycosides

Aminoglycosides such as streptomycin, paromomycin, and gentamicin are known to inhibit the binding of translocated RNA to the A site by binding to the A site of the ribosome (Moazed, D. & Noller, HF Nature 327, 389-394 (1987); Fourmy, D., Yoshizawa, S. & Puglisi, JDJ Mol. Biol. 277, 333-345 (1988)); Yoshizawa, S., Fourmy, D. & Puglisi, JD EMBO J.17, 64 37-6448 (1988)). Therefore, according to the model in FIG. 5, the aminoglycosides should also inhibit the binding of RRF to the A site, and if such binding is inhibited, the dissociation process of the termination complex by RRF will be inhibited. Should be. Therefore, whether or not the dissociation process of the termination complex is inhibited in the presence of the above aminodaricosides was examined using the amount of transfer RNA released from liposomes and the amount of liposome released from messenger RNA as indicators. Was.

Release of desacylated transfer RNA from the liposome was examined by the following method. Polysaccharide obtained from tetracycline-treated E. coli Q13 strain (Hirashima, A. & Kaji, AJ Mol. Biol. 65, 43-58 (1972)) (0.6-; 1.8 A) _26. Units) were replaced with 275 μM pure mouth mycin, 0.2 nmole RRF, 0.2 nmole EFG (Kaziro, Y., Inoue-Yokosawa, N. & Kawakita, M. coli. J. Biochem. 72, 853-863 (1972)), and in the presence of 0.37 mM GTP, 550 μl of Buffer R (Tris-CI 10 mM, pH 7. 4. Incubate for 15 minutes at 30 ° C in 8.2 mM magnesium sulfate, 80 mM ammonium chloride and 0.14 mM dithiothritol (DTT). Next, as aminoglycosides, streptomycin, paromomycin or gentamicin were added with an assisting force B of 200 / zM, 100 ^ M and 100% / iM, respectively. Subsequently, the released transfer RNA was separated from the termination complex by centrifugation at 330 G for 40 minutes using Microcon 100 (trade name, manufactured by Millipore). Next, Knocker J (tris-CI 10 mM, pH 7.6, magnesium sulfate 10 mM, ammonium chloride 50 mM, DTT 0.5 mM) was added to the above Microcon 100. The filter was washed once by pouring 550 μl and centrifuging. The combined washing solution and filtrate were centrifuged twice at 1,500 G for 15 minutes using Microc o η30 (Mil 1 ipore, trade name) to obtain 1 It was concentrated to 41. Then, the concentrated transfer RNA was added to 30 μl of buffer solution (Tris_C 150 mM, pH 7.8, magnesium acetate 10 mM, 3_mercaptoethanol 6 mM, ATP 3 mM, phosphoenoenoside). Monorubic acid 5 mM, pyruvate kinase 1 38 μg, aminosyltransferred RNA synthase 33.3 μg (Momose, K. & Kaji, A. Arch. Biochem. Biophys. Ill. 245-252 (1965))), a mixture of ¹⁴ C-amino acids (Amersham, 52 mCi / mg carbon atom) dissolved in 0.15 Ci And aminoacylated. The radioactivity insoluble in cold trichloroacetic acid (4 ° C) obtained in this way corresponds to ¹⁴ C-aminoacyl-transferred RNA, and its amount is determined by a known amount of transfer RNA labeled by the same method. Was calculated based on the radioactivity. As aminoglycosides, those manufactured by Sigma were used. The termination complex was obtained from a natural polysome of E. coli treated with puromycin (Hirashima, A. & Kaji, AJ Biol. Chem. 248, 7580-7587 (1973)). It is known that each ribosome in the isolated polysome is in a stage after the completion of translocation and generally carries two molecules of transfer RNA (Remme, J., Margus, T., Villems, R. & Nierhaus, KH Eur. J. Biochem. 183, 28 g 284 (1989); Stark, H. et al. Cell 88, 19-28 (1977)).

 Fig. 6 shows the results. In FIG. 6, the positive control is the value when RRF, EFG and GTP were added without adding the aminoglycosides, and the negative control was the aminoglycosides, RRF, £ 〇 and 0 Are the values when none of the above was added.

Release of ribosomes from messenger RNA was examined by the following method. The poly Seo one arm _{(0. 5~0. 8 A 2 e} . Units), and 2 7 5 pin Euro puromycin of μΜ, 1 nm ο 1 e purification RRF (Hirashima, A. & Kaji, A. Bioche mistry 11, 4037-4044 (1972)) , 1 nm o 1 e of EFG ^32, the presence of GTP and Aminoguri Koshido such 0. 3 7 mM, and have contact during 2 7 0 1 buffer R, 3 0. Incubated at C for 15 minutes. As for aminoglycosides, streptomycin, paromomycin or gentamicin were added to give 100, 5 and 5 μ 5, respectively. Next, 15 to 30 in buffer J. /. The above buffer solution was incubated on 5 ml of sucrose with a density gradient, and the mixture was incubated at 4 ° C, 75 minutes, 4 ° C using Beckman SW 50.1. Centrifuge. Absorbance at 254 nm

Monitoring was performed with a UA-6 detector to measure the concentration of free 70 S ribosome. Table 7 shows the results. The values in Table 7 are the percentages of the concentration of 70S liposome released in the presence of aminoglycosides and the concentration of free 70S ribosome without aminoglycosides (control). It is expressed in the context. In the control, about 42% of all ribosomes (almost 90% of polysomes) were converted to monosomes by RRF. Table 7 By RRF and EF-G in the presence of various inhibitors

 Release of ribosome from mRNA

Inhibitor concentration based on RRF

 Ribosome release

 (% Of control) 100%

 Noromomycin 5 μΜ 16.5

 Gentamicin 5 μΜ 0

 Streptomycin 100 μΜ 9.9

 Thiostrepton 20 μΜ 0

 Biomycin 50 μΜ 0

 Fusidic acid 200 μΜ 0

 GMPPCP 370 μΜ * 0

From FIG. 6, it can be seen that in the presence of aminoglycosides, the release of transferred RNΑ is inhibited to a value equal to or lower than that of the negative control. Also, from Table 7

However, it can be seen that release of ribosomes is also inhibited in the presence of aminoglycosides. These results strongly suggest that RRF needs to bind to the A site of the ribosome for the expression of RRF activity. It is thought that it can be a strong candidate.

Example 5. Inhibition of RRF activity by thiostrepton and biomycin

Thiostrepton and biomycin are both EFG inhibitors and are known to inhibit the translocation of transfer RNA (Pestka, S.B. iochem. Biophys. Res. Comraun. 40, 667-674 (1970); Rodnina, MV, Savel sbergh, A., Katunin, VI & Wintermeyer, W. Nature 385, 37-41 (1997); Rodn ina, MV et al. Pro Natl. Acad. Sci. USA 96, 9586-9590 (1999)). Thus, according to the model of Figure 5, thiostrepton and biomycin should also inhibit the translocation of the RRF bound to the A site to the P site, and if so, with the translocation. The transfer of RNA from the P and E sites that should occur, and thus the dissociation process of the termination complex by RRF, should also be inhibited. Therefore, whether or not the dissociation process of the termination complex is inhibited in the presence of thiostrepton or biomycin is determined by measuring the amount of transfer RNA released from liposomes and the amount of liposomes released from messenger RNA. I investigated.

 Transfer of RNA from ribosomes Release of RNA was examined in the same manner as in Example 4, except that thiostrepton (Sigma) or biomycin (ICN) was added instead of amidglycosides. Was. Thiostrepton and biomycin were added at 100 μM and 200 / X M, respectively. Figure 6 shows the results.

 Also, the release of ribosomes from messenger RNA can be achieved by adding DMSO to the reaction mixture at the point where thiostrepton or biomycin is added instead of amide glycosides and when thiostrepton is added. Except for the addition, it was examined in the same manner as in Example 4. Thiostrepton and biomycin were added at 20 μM and 50 μM, respectively. Table 7 shows the results.

FIG. 6 shows that in the presence of thiostrepton or biomycin, the release of metastatic RNA was inhibited to a value equivalent to that of the negative control. In addition, Table 7 shows that the release of ribosome is completely inhibited in the presence of thiostrepton or biomycin. These results strongly suggest that the expression of RRF activity requires translocation of the RRF bound to the A site to the P site, and therefore, substances that inhibit RRF translocation are RR It is thought that it can be a strong candidate for F inhibitors. Example 6. Inhibition of termination complex dissociation by GMP PCP and fusidic acid — GMP PCP and fusidic acid are both EFG inhibitors and are terminated by immobilizing EFG on liposomes after translocation of transfer RNA It is known to inhibit the dissociation of RNA, while allowing translocation of transfer RNA only once (Inoue-Yokosawa, N., Ishikawa, C. & Kaziro, YJ Biol. Chem. 249, 4321-4323 (1974); Rodnina, MV, Savelsberg, A., Katunin, VI & Wintermeyer, W. Nature 385, 37-41 (1997); Bodley, J. W., Zieve, FJ, Lin, L. & Zieve. Chem. 245, 5656-5661 (1970); Kuriki, Y., Inoue, N. & Kaziro, Y. Biochim. Biophys. Acta. 224, 487-497 (1970)). Therefore, according to the model in FIG. 5, GMP PCP and fusidic acid allow translocation of the RRF bound to the A site to the P site, so that GMP PCP and fusidic acid are released from the P site and E site that accompany the translocation. Transfer RNA release should not be inhibited. On the other hand, GMP PCP and fusidic acid should inhibit the dissociation of the ribosome from messenger RNA because they inhibit the dissociation of the termination complex. Therefore, it was examined whether or not the release of transfer RNA from ribosomes and the release of liposomes from messenger RNA were inhibited in the presence of GMP PCP or fusidic acid.

 Transfer from ribosome RNA is released by adding GMP PCP (manufactured by Sigma) or fusidic acid (manufactured by ICN) instead of amidoglycosides and without GTP when GMP PCP is added. The procedure was as in Example 4, except that the experiment was performed below. GMP PCP or fusidic acid was added so as to be 37 μM and 200 μM, respectively. Fig. 6 shows the results.

 The release of ribosomes from messenger RNA also depends on the fact that GMP PCP or fusidic acid was added instead of amide glycosides, and that when GMP PCP was added, experiments were performed in the absence of GTP. Except for this, the procedure was the same as in Example 4. GMP PCP or fusidic acid was added so as to be 3700 μM and 200 μM, respectively. Table 7 shows the results.

According to Fig. 6, the release of transfer RNA was equivalent to that of the positive control even when GMP PCΡ or fusidic acid was added, and the release of transfer R R 阻 害 was inhibited. It turns out there is no. On the other hand, Table 7 shows that GMP PCP or fusidic acid completely inhibits ribosome release (Igarashi, K., Ishitsuka, H. & Kaji, A. Biochem. Biophys. Res. Commun. 37, 499-504 (1969); Hirashima, A. & Kaji, AJ Mol. Biol. 65, 43-58 (1972); Ogawa, K. & Kaji, A. Eur. J. Biochem. 58, 411-419 (1975) ); Karimi, R., Pavlov, MY, Buckingham, RH & Ehrenberg, M. Molecular Cell 3, 601-609 (1999)).

 These results strongly suggest that RRF may exhibit termination complex dissociation activity by exhibiting behavior similar to that of transfer RNA.Furthermore, the release of transfer RNA and ribosome release are separately inhibited. The fact that can be done supports the model shown in FIG. 5, in which the dissociation of the termination complex proceeds stepwise by EFG and RRF.

Example 7. Inhibition of RRF activity due to excess desacyl transfer RNA

 Example 4 strongly suggested that the expression of RRF activity required RRF to bind to the A site of the ribosome. Therefore, if excess transfer RNA is present, RRF and transfer RNA will compete and bind to the A site of the liposome, which should inhibit dissociation of the termination complex. To confirm this point, the inhibition of dissociation of the termination complex in the presence of various concentrations of transfer RNA was examined.

Polysome (0. 5~ 1 A _26. Units) a, 0 to 1 000 pmo various amount between le RRF, EFG, transfer of various amounts between GTP and 0~ 1 0 nmo 1 e Incubated in the presence of RNA. Then, after performing sucrose density gradient centrifugation (density gradient: 15 to 30%), the amount of free ribosome was measured by measuring the absorbance at 254 nm. Lineweavwe—Burk created based on the measurement results. Figure 7 shows a graph based on the lot. The vertical axis is the reciprocal of the percentage of free ribosome in the presence of various amounts of transfer RNA relative to the amount of free ribosome in the absence of transfer RNA.

In the graph of FIG. 7, the vertical axis intercept regardless of the amount of transfer RNA is constant, and K _m with the increase in the amount of transfer RNA from the larger, transfer RNA is It can be seen that RRF activity is competitively inhibited.

 [009 1]

Example 8. Inhibition of RRF binding to ribosome by paromomycin

Example 4. Based on the results of and example 7, in order to directly verify the paromomycin used in Example 4. inhibit the binding of liposomes RRF, was first subjected to the following experiments, ³⁵ of less than 1 pmo 1 e S-labeled histidine tag RRF to ^{(35 S- H is -RR F)} , in the presence of Paromo Maishishin of 1 0 pmo 1 e washed ribosomes and various concentrations of buffer (g squirrel - CI 5 0mM, p H 7 6. Incubated at 30 ° C for 10 minutes in 10 mM magnesium acetate, 30 mM potassium chloride, 1 mM DTT). After removing free ³⁵ S-His-RRF by microconcentration, the amount of ribosome-bound ³⁵ S-His-RRF was measured by radioactivity. In addition, ³⁵ S—His—RRF was prepared by purifying His—RRF expressed in vitro in the presence of ³⁵ S-labeled methionine using Ni ²⁺ beads. Fig. 8 shows the results. The RRF binding ratio is a value calculated by assuming that the amount of RRF bound to ribosomes in the absence of paromomycin is 100%.

 FIG. 8 shows that the binding rate of RRF decreases depending on the concentration of paromomycin. Therefore, paromomycin is thought to inhibit the activity of RRF by inhibiting the binding of RRF to ribosomes.

Table 8

 RRF structure coordinates

Atom type residue ## XYZ OCC B Atom 1 CB VA then 2 10.355 24.444 73.500 1.00 50.36 Atom 2 CGI VA then 2 11.185 25.300 72.669 1.00 50.36 Atom 3 CG2 VA 2 9.102 25.267 74.125 1.00 50.36 Atom 4 C VAL 2 8.502 23.777 72.304 1.00 83,11 atoms 50 VA then 2 8.267 24.906 71.890 1.00 83.11 atoms 6 N VA then 2. 10.415 23.206 71.242 1.00 83.11 〇Λν

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Claims

The scope of the claims

1. A method for designing a compound that can bind to the active site, an auxiliary binding site, or a pocket of RRF protein. The chemical entity is evaluated by computer based on the structural coordinates obtained from the RRF protein crystal. The method as described above.

 2. The method according to claim 1, wherein the RRF protein crystal is any one of a crystal of the RRF protein itself, a crystal of a mutant RRF protein, a crystal of a homologue of the RRF protein, and a crystal of a co-complex of the RRF protein. .

 3. The method according to claim 1, wherein the RRF protein crystal is a bipyramid system.

4. RRF protein crystals have a space group 1 ^ 4,2,2, or space group P4 ₃ 2, 2, The method according to any one of claims 1 to 3.

 5. The method according to any one of claims 1 to 4, wherein the RRF protein crystal has a size of 0.3X0.3X0.5mm.

 6. The method according to any one of claims 1 to 5, wherein the RRF protein crystal has a unit cell having a size of a = b = 47.3A and c = 297.6A.

 7. The method according to any of claims 1 to 6, wherein the RRF protein crystals are characterized by the structural coordinates according to Table 7.

 8. The method according to any one of claims 1 to 7, wherein the RRF protein crystal is derived from Thermotoga Maritima.

 9. The method according to claim 1, wherein the RRF protein crystal is orthorhombic.

1 0. Is the RRF protein crystal a space group? 10. The method according to any one of claims 1, 2 and 9, comprising 2,2.

 1 1. The method according to any one of claims 1-2 and 9-10, wherein the RRF protein crystal has a size of 30X50 X25 (^: 11).

 12. The method according to any one of claims 1-2 and 9-11, wherein the RRF protein crystal is derived from fungus X.

13. The method according to any one of claims 1 to 12, wherein the RRF protein crystal is crystallized by a droplet vapor diffusion method.

1 4. The RRF protein crystal is a heavy atom derivative, and the crystal is one of a crystal of the RRF protein itself, a crystal of the RRF protein mutant, a crystal of the RRF protein homolog, and a crystal of a co-complex of the RRF protein. A method according to any of the preceding claims.

 15. The method according to any one of claims 1 to 14, wherein the heavy atom derivative is formed by reaction with a compound selected from the group consisting of tyromesal, gold thiomalate, peranyl acetate, and lead chloride.

 1 6. The method according to any one of claims 1, 2 and 9 to 12, wherein the RRF protein crystal is a heavy atom derivative with platinum or mercury.

 17. The method according to any one of claims 1 to 16, wherein the RRF protein is a monomer.

 18. The method according to any of claims 1 to 8, 13 to 15 and 17, wherein the RRF protein is characterized by amino acid changes according to Table 5 or Table 6.

 19. The compound according to any one of claims 1 to 18, characterized in that the compound characterized by an active site, an auxiliary binding site or a chemical entity binding to a pocket is an inhibitor of the RRF protein. The described method.

 20. The method according to any one of claims 1 to 19, wherein the inhibitor is a competitive, non-competitive or uncompetitive inhibitor of RRF.

 21. The method according to any of the preceding claims, comprising determining the orientation of the ligand at the active or auxiliary binding site of the RRF protein.

 22. The method according to any one of claims 1 to 8, 13 to 15, and 17 to 21, wherein the structural coordinates are the structural coordinates of the RRF protein according to Table 7.

 2 3. A method for determining the three-dimensional structure of an RRF protein, comprising elucidating the crystal form of a mutant, homolog or co-complex of the RRF protein by molecular replacement.

 2 4. Orthogonal RRF protein crystals.

 25. The RRF protein crystal according to claim 24, having the space group P2i2,2.

 26. The RRF protein crystal according to claim 24 or 25, having a size of 30X50X250 / im.

27. The RRF tag according to any one of claims 24 to 26, wherein the RRF is derived from bacterium X. Compact crystal.

 2 8. Bipyramid-based RRF protein crystals.

2 9. with space group F ^ A or space group Ρ4 ₃ 2,2, RRF Tan Pak crystal according to claim 28.

 30. The RRF protein crystal according to claim 28 or 29, having a size of 0.3X0.3X0.5mm.

 31. The RRF protein crystal according to any one of claims 28 to 30, having a unit cell having a size of a = b = 47.3A and c = 297.6A.

 32. The RRF protein crystal of any one of claims 28-31, characterized by amino acid changes according to Table 5 or Table 6.

 3 3. An RRF protein crystal according to any of claims 28 to 32, characterized by the structural coordinates according to Table 7.

 3 4. The RRF protein crystal according to 28, wherein the RRF protein crystal is derived from Thermotoga Maritima.

 3 5. The RRF protein crystal according to any one of claims 24 to 34, crystallized by a droplet vapor diffusion method.

 3 6. The claim as claimed in any one of claims 24 to 35, wherein the crystal is any one of a crystal of the RRF protein itself, a crystal of the RRF protein mutant, a crystal of the RRF protein homologue, and a crystal of the RRF protein co-complex. The described RRF protein crystal.

 3 7. An RRF protein wherein the amino acid at the active site is selected from the group consisting of Arg 110, Arg 129 and Arg 132 of SEQ ID NO: 1.

 3 8. One or more amino acids in the active or auxiliary active site are replaced by one or more amino acids selected from the group consisting of naturally occurring amino acids, unnatural amino acids, selenocystine and selenomethionine. 39. The RRF protein of claim 37, wherein

 39. The RRF protein according to claim 37, wherein the hydrophilic amino acid and the hydrophobic amino acid in the active site or the auxiliary active site are substituted.

40. At least one cysteine amino acid has been replaced by an amino acid selected from the group consisting of selenocysteine or selenomethionine. The RRF protein of claim 37.

 41. The RRF protein of claim 37, wherein at least one methionine amino acid is substituted with an amino acid selected from the group consisting of selenocystine or selenomethionine.

 42. The RRF protein according to any of claims 37 to 38, which is in a crystalline form.

43. The RRF protein of claim 37, having a higher or lower specific activity than the wild-type enzyme.

 44. The RRF protein of claim 37, having altered substrate specificity.

 45. Use of the RRF protein according to claim 37 for measuring the binding interaction between a compound and the RRF protein.

 46. The method of claim 37, wherein at least one amino acid residue on or near the surface of the RRF protein has been substituted, resulting in a change in one or more charge units of the surface charge. RRF protein.

 47. The pocket force of RRF protein The method according to any one of claims 1 to 22, which is a pocket near the C-terminus located at a bent portion separating two domains of RRF protein.

 48. The method of any of claims 1-22, wherein the compound inhibits binding of the RRF protein to the ribosome or inhibits the behavior of the RRF protein on the ribosome.

 49. An RRF protein inhibitor obtained by the method according to any one of claims 19 to 23, 47, and 48.

 50. A method for searching for a compound that can inhibit the activity of RRF protein based on the activity of inhibiting the binding of RRF protein to ribosomes or the activity of inhibiting the behavior of RRF protein on ribosomes.

 51. An RRF protein inhibitor obtained by the method according to claim 50.