JP2000086696A - Ribosome recycling factor(rrf) protein crystal and application to rational drug design based on three- dimensional structural information obtained from the crystal - Google Patents
Ribosome recycling factor(rrf) protein crystal and application to rational drug design based on three- dimensional structural information obtained from the crystalInfo
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- JP2000086696A JP2000086696A JP25342498A JP25342498A JP2000086696A JP 2000086696 A JP2000086696 A JP 2000086696A JP 25342498 A JP25342498 A JP 25342498A JP 25342498 A JP25342498 A JP 25342498A JP 2000086696 A JP2000086696 A JP 2000086696A
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- 239000013078 crystal Substances 0.000 title claims abstract description 41
- 108090000623 proteins and genes Proteins 0.000 title claims description 16
- 102000004169 proteins and genes Human genes 0.000 title claims description 15
- 108010037379 ribosome releasing factor Proteins 0.000 title abstract description 36
- 238000009510 drug design Methods 0.000 title 1
- 101710102451 Ribosome-recycling factor Proteins 0.000 claims abstract description 31
- 101710150195 Ribosome-recycling factor, chloroplastic Proteins 0.000 claims abstract description 31
- 101710084690 Ribosome-recycling factor, mitochondrial Proteins 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 14
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- 238000009792 diffusion process Methods 0.000 claims abstract description 6
- 239000003112 inhibitor Substances 0.000 claims description 7
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 2
- 229910052753 mercury Inorganic materials 0.000 claims description 2
- 229910052697 platinum Inorganic materials 0.000 claims description 2
- 150000005829 chemical entities Chemical class 0.000 claims 2
- 241000233866 Fungi Species 0.000 claims 1
- 230000002860 competitive effect Effects 0.000 claims 1
- 230000036963 noncompetitive effect Effects 0.000 claims 1
- 230000036967 uncompetitive effect Effects 0.000 claims 1
- 239000004009 herbicide Substances 0.000 abstract description 4
- 239000000126 substance Substances 0.000 abstract description 4
- 239000004599 antimicrobial Substances 0.000 abstract 1
- 244000005700 microbiome Species 0.000 abstract 1
- 239000003242 anti bacterial agent Substances 0.000 description 13
- 230000014616 translation Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 4
- 241000206602 Eukaryota Species 0.000 description 4
- 238000002441 X-ray diffraction Methods 0.000 description 4
- 238000002425 crystallisation Methods 0.000 description 4
- 230000008025 crystallization Effects 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 241000207187 Candidatus Legionella jeonii Species 0.000 description 1
- 102100032566 Carbonic anhydrase-related protein 10 Human genes 0.000 description 1
- 108010010369 HIV Protease Proteins 0.000 description 1
- 101000867836 Homo sapiens Carbonic anhydrase-related protein 10 Proteins 0.000 description 1
- 101000835998 Homo sapiens SRA stem-loop-interacting RNA-binding protein, mitochondrial Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000010562 Peptide Elongation Factor G Human genes 0.000 description 1
- 108010077742 Peptide Elongation Factor G Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 101001062854 Rattus norvegicus Fatty acid-binding protein 5 Proteins 0.000 description 1
- 241000369814 Riccardia aeruginosa Species 0.000 description 1
- 102100025491 SRA stem-loop-interacting RNA-binding protein, mitochondrial Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 229940124411 anti-hiv antiviral agent Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000155 isotopic effect Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000012886 linear function Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 150000002730 mercury Chemical class 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000012887 quadratic function Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、リボソームリサイ
クリング因子（Ribosome recycling factor, 以下RRF)
の結晶に関する。また本発明は、該結晶のＸ線回折によ
り得られるRRFタンパクの立体構造に関する。さらに本
発明は、RRFタンパクの構造情報を応用した次世代抗菌
剤、除草剤を開発する技術に関する。TECHNICAL FIELD The present invention relates to a ribosome recycling factor (RRF).
Related to the crystal. The present invention also relates to a three-dimensional structure of the RRF protein obtained by X-ray diffraction of the crystal. Further, the present invention relates to a technology for developing a next-generation antibacterial agent and a herbicide using the structural information of RRF protein.
【０００２】[0002]
【従来の技術】蛋白質生合成は、すべての細胞の生命活
動において必要不可欠な機能であり、「開始」、「伸
展」、「終結」及び「リボソームリサイクリング」の四
段階から成り立っている。蛋白質生合成における最終的
なステップ（第4ステップ）は、次の「開始」段階へリ
ボソームを再利用する為に、メッセンジャーRNA、転移R
NA、リボソームからなる終結複合体を各々遊離、解離さ
せることにより終了する。原核生物である大腸菌におい
ては、このリボソームの「再利用」はリボソームリサイ
クリング因子（Ribosome recycling factor, 以下RRF)
とエロンゲーション因子G（elongation factor G, 以下
EFG）により触媒されることが分かっている。このリボ
ソーム「再利用」の過程はJanosi博士らによる総説（19
96 Adv. Biophys. 32:121-201）において紹介されてい
る。真核生物において蛋白質翻訳終結複合体の解離はRR
Fではない他の因子により触媒される可能性が示唆され
ており、真核生物のmRNAはモノシストロニックで原核生
物のそれはポリシストロニックである（Kozak 1987, Mo
l. Cell. Biol. 7:3438-3445; Dasら 1984, Nucleic Ac
ids Res. 12:4757-4768;Schonerら 1986 Proc. Natl. A
cad. Sci. U.S.A. 83:8506-8510; Sprengelら 1985 Nuc
leic Acids Res. 13:893-909）ことから、真核生物にお
いてリボソームのmRNAよりの解離が阻害されても下流の
シストロンを影響することはない。このように真核生物
における蛋白質生合成の最終段階にあたる蛋白質翻訳終
結複合体の解離という第４段階が原核生物のものと異な
ると考えられるので、特に新しい型の抗生物質のターゲ
ットとして期待されている。2. Description of the Related Art Protein biosynthesis is an indispensable function in the life activities of all cells, and is composed of four steps: "start", "extension", "termination" and "ribosome recycling". The final step (fourth step) in protein biosynthesis involves messenger RNA, transfer R, and ribosome recycling to the next “start” step.
The process is terminated by releasing and dissociating a termination complex consisting of NA and ribosome, respectively. In Escherichia coli, which is a prokaryotic organism, this "reuse" of ribosomes is based on Ribosome recycling factor (RRF).
And elongation factor G, below
EFG). This ribosome “reuse” process is reviewed by Dr. Janosi et al. (19
96 Adv. Biophys. 32: 121-201). Dissociation of the protein translation termination complex is RR in eukaryotes
It has been suggested that eukaryotic mRNA may be monocistronic and prokaryotic may be polycistronic (Kozak 1987, Mo
l. Cell. Biol. 7: 3438-3445; Das et al. 1984, Nucleic Ac
ids Res. 12: 4757-4768; Schoner et al. 1986 Proc. Natl. A
cad. Sci. USA 83: 8506-8510; Sprengel et al. 1985 Nuc.
leic Acids Res. 13: 893-909). Therefore, in eukaryotes, even if the dissociation of ribosome from mRNA is inhibited, it does not affect downstream cistron. Thus, the fourth step of dissociation of the protein translation termination complex, which is the last step of protein biosynthesis in eukaryotes, is thought to be different from that of prokaryotes, and is particularly expected as a target for a new type of antibiotic. .
【０００３】一方、現在では数多くの抗菌剤が開発され
ており、この中には例えば非常に高い殺菌作用を示すも
のも存在している。しかし、このようにして得られた抗
菌剤には、その作用部位が不明なままのものが数多く存
在する。これまでは、これらの活性を示す抗菌剤をラン
ダムスクリーニングの材料として用い、構造活性関係を
樹立しつつさらなる効用のあるものを開発していく方法
が中心であったが、これには莫大な時間と労力を要す
る。On the other hand, a large number of antibacterial agents have been developed at present, and some of them have a very high bactericidal action, for example. However, there are many antibacterial agents obtained in this way, the sites of action of which are unknown. Until now, antibacterial agents exhibiting these activities have been used as a material for random screening, and the main method has been to develop structures with further effects while establishing a structure-activity relationship. And labor.
【０００４】そこで近年、この問題を排除し能率良く阻
害剤の発見が行われることを目的としてデータベース化
が図られている。それを基本にしてラシヨナルドラグデ
ザイン法が検討され開発されつつある。この例として、
最近上市された抗HIV剤であるプロテアーゼの阻害剤が
挙げられる。HIVのプロテアーゼは結晶化され、その立
体構造が知られている。この構造と活性部位の三次元構
造アミノ酸配列を基にしてコンピューターより既知の化
合物からこの部位に最も親和性の高いものを選び、その
阻害活性が測定されている。活性の出たものと標的蛋白
の共結晶を作り、三次元構造の測定を行うことによりさ
らによりよく結合する化合物を予測できるので、これを
合成しその阻害活性が測定されている。そして再びこの
物質と標的蛋白との共結晶を作り、上記の過程を繰り返
すことにより極めて有効な物質を得ることができる。[0004] In recent years, a database has been created for the purpose of eliminating this problem and efficiently finding inhibitors. Based on this, the Rashionardo rug design method is being studied and developed. As an example of this,
Examples include inhibitors of proteases, which are recently marketed anti-HIV agents. HIV protease is crystallized and its three-dimensional structure is known. Based on this structure and the amino acid sequence of the three-dimensional structure of the active site, a compound having the highest affinity for this site is selected from compounds known from a computer, and its inhibitory activity is measured. By forming a co-crystal of the active protein and the target protein and measuring the three-dimensional structure, it is possible to predict a compound that binds even better, and the compound 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.
【０００５】ところで、上記のような従来の抗生物質へ
の耐性獲得菌株が数多く報告されてきており、細菌の発
育を直接的に制限し得る部位を標的とする、新たな抗生
物質の開発が早急に必要とされている。そこで本発明者
らは前記RRFが抗菌剤の新たなターゲットとなり得るこ
とに着目し鋭意研究を進めてきたが、この着想が近年脚
光を浴びつつある。[0005] By the way, many conventional strains having acquired resistance to antibiotics as described above have been reported, and the development of new antibiotics targeting sites that can directly restrict the growth of bacteria is urgently required. Is needed. Therefore, the present inventors have focused on the fact that the RRF can be a new target of an antibacterial agent, and have conducted intensive research, but this idea has recently been spotlighted.
【０００６】[0006]
【発明が解決しようとする課題】RRFに関して、本発明
者らはこれまでに大腸菌を始め、原核生物のみに留まら
ず真核生物に関するものまで数種の遺伝子配列を決定し
た(特開平3−200797、PCT/JP98/00734、特願平10−1504
93）。従って、そこから得られるアミノ酸配列によりそ
の二次構造までが推定可能ではある。しかしながら、現
在の技術水準においては、この二次構造から実際の立体
構造を同定するまでには至っていない。実際のタンパク
においては各アミノ酸残基が相互的に作用しており、ま
た場合によってはさまざまな修飾を受けてその立体構造
を形成している。従ってタンパクの立体構造が分れば、
そのリガンドとなりうる物質を創製することが可能であ
り、この意味で有用な抗生物質を創製するためには、結
晶化による三次元構造の決定がきわめて重大な意義を有
することとなる。従って本発明の課題は、RRFの立体構
造を解明し、種々の抗菌剤及び除草剤の開発に寄与する
ことにある。With respect to RRF, the present inventors have determined several gene sequences for E. coli and not only for prokaryotes but also for eukaryotes (JP-A-3-200797). , PCT / JP98 / 00734, Japanese Patent Application No. 10-1504
93). Therefore, it is possible to presume the secondary structure from the amino acid sequence obtained therefrom. However, in the current state of the art, the actual three-dimensional structure has not been identified from this secondary structure. In an actual protein, each amino acid residue interacts and, in some cases, undergoes various modifications to form its three-dimensional structure. Therefore, if the three-dimensional structure of the protein is known,
It is possible to create a substance that can serve as the ligand, and in order to create a useful antibiotic in this sense, the determination of the three-dimensional structure by crystallization has extremely important significance. Therefore, 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 and herbicides.
【０００７】[0007]
【課題を解決するための手段】本発明者らは上記の現状
を踏まえ、RRFに関し研究を進める中で、RRFの結晶を得
てその立体構造を同定することに初めて成功し、さらに
研究を進めた結果、本発明を完成するに至った。即ち本
発明は、RRFタンパクの結晶及びその製法と立体構造に
関する。より具体的には斜方晶系、特にその空間群P212
12を有するRRFタンパク結晶に関する。さらに本発明
は、該結晶と白金、水銀などとの重原子誘導体に関す
る。また本発明はRRFタンパクの構造情報を使用して、R
RFタンパクの活性部位と結合し得る化合物又はRRFタン
パクを非競合的に又は不競合的に阻害し得る化合物をコ
ンピューター評価して、次世代抗菌剤、除草剤を開発す
る技術に関する。さらに又本発明は、RRFの立体構造に
基づく阻害剤の発見を目的としてデータベース化し、そ
れを基本にしてラシヨナルドラグデザイン法に応用する
ことにも関する。Means for Solving the Problems Based on the above situation, the present inventors have succeeded in obtaining RRF crystals and identifying their three-dimensional structure for the first time while conducting research on RRF. As a result, the present invention has been completed. That is, the present invention relates to a crystal of the RRF protein, a method for producing the same, and a three-dimensional structure. More specifically, orthorhombic, especially its space group P2 1 2
The present invention relates to an RRF protein crystal having 1 2. Further, the present invention relates to a heavy atom derivative of the crystal and platinum, mercury or the like. The present invention also uses the RRF protein structural information,
The present invention relates to a technique for developing a next-generation antibacterial agent and a herbicide by computer evaluation of a compound capable of binding to an active site of an RF protein or a compound capable of non-competitively or non-competitively inhibiting a RRF protein. Furthermore, the present invention relates to the creation of a database for the purpose of discovering inhibitors based on the three-dimensional structure of RRF, and to the application of the database to the lashyonard rag design method based on the database.
【０００８】RRFが理想的な抗菌剤の標的であることが
推定される現在、本発明により解明されたRRFの三次元
構造は、抗菌剤などの開発に直結しているので、産業上
極めて重要である。しかも多くの病原菌のRRFの一次構
造が酷似していることが知られていることから(例えば
緑膿菌のRRFは大腸菌のそれと60%の相同性を有する）、
本発明によるRRFの三次元構造の初期データにより、他
の病原菌のRRFの三次元構造についてもその解明が極め
て容易になる。従って、種特異性の抗菌剤を開発するた
めにも、本発明は、RRF阻害による次世代抗生物質及び
除菌剤開発に、特にラシヨナルドラグデザインにより抗
菌剤を開発する際の一つの指標として極めて有用であ
る。At present, it is presumed that RRF is an ideal target of an antibacterial agent. Since the three-dimensional structure of RRF elucidated by the present invention is directly linked to the development of antibacterial agents and the like, it is extremely important in industry. It is. Moreover, it is known that the primary structure of RRF of many pathogenic bacteria is very similar (for example, R. aeruginosa RRF has 60% homology with that of E. coli)
The initial data of the three-dimensional structure of the RRF according to the present invention makes it very easy to clarify the three-dimensional structure of the RRF of other pathogenic bacteria. Therefore, in order to develop a species-specific antibacterial agent, the present invention is used as an index for developing a next-generation antibiotic and a disinfectant by RRF inhibition, particularly as an index when developing an antibacterial agent by a lashyonard rug design. Extremely useful.
【０００９】本発明の実施例においては、X菌のRRF(該R
RFの一次構造については本発明者らによるPCT/JP98/007
34参照）を用いて結晶化し、構造解析を行ったが、その
他のRRFについても同様に実施することができる。また
結晶化に際してはRRFタンパク自体のみならず、RRFタン
パク変異体、RRFタンパクホモログ、RRFタンパク共複合
体を結晶化し、それぞれ構造解析することも可能であ
る。以下、実施例により本発明をより詳細に示す。以下
に示す実施例はあくまでその詳細な解説を目的とするも
のであり、他の方法を制限するものではない。In an embodiment of the present invention, the RRF of X bacterium (the RRF
PCT / JP98 / 007 by the present inventors regarding the primary structure of RF
34), and structural analysis was performed, but other RRFs can be similarly performed. In the crystallization, not only the RRF protein itself, but also the RRF protein mutant, the RRF protein homolog, and the RRF protein co-complex can be crystallized, and the respective structures can be analyzed. Hereinafter, the present invention will be described in more detail with reference to examples. The embodiments described below are for the purpose of detailed explanation only, and do not limit the other methods.
【００１０】[0010]
【実施例】例1 滴状蒸気拡散法（hanging drop vapour
diffusion technique）によるRRFタンパクの結晶化 菌XのRRFタンパク4mg/mlから8mg/ml、トリス塩酸 50mM
pH8.5、硫酸塩 70-100mM、ポリエチレングリコール14％
から18％を含む５μlの溶液を液滴化し、液滴よりも高
い濃度の結晶化試薬を含有する液だまりで平衡化した。
平衡化は揮発性媒体(水又は有機溶媒)の拡散により、液
滴の蒸気圧が液だまりの蒸気圧に等しくなるまで行っ
た。平衡化が水交換(液滴から液だまりへ）によりおき
ると、液滴の容量は変化する。その結果、液滴中の全て
の媒体の濃度は変化する。水よりも高い蒸気圧を有する
媒体には、液だまりから液滴への変換が生起する。本例
においてRRFタンパク溶液が接触するガラス容器は、そ
の表面を疎水化処理して用いられる。トリス塩酸 100mM
pH8.5硫酸塩150mMから200mM、ポリエチレングリコール
28％から36％の緩衝液へ透析してXRRF結晶を得た。結晶
は、１から３週間で 30×50×250μmの大きさに成長し
た。その結果を図１に示す。 EXAMPLE 1 hanging drop vapor diffusion method
Crystallization of RRF protein by diffusion technique) RRF protein of bacteria X 4mg / ml to 8mg / ml, Tris-HCl 50mM
pH 8.5, sulfate 70-100mM, polyethylene glycol 14%
5% of the solution containing from 18% to 18% were dropletized and equilibrated with a pool containing a higher concentration of crystallization reagent than the droplets.
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. As the equilibrium occurs by water exchange (drops to pools), the volume of the drops changes. As a result, the concentration of all media in the droplet changes. For a medium having a higher vapor pressure than water, a conversion from pool to droplets occurs. 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. Tris-HCl 100mM
pH 8.5 sulfate 150 mM to 200 mM, polyethylene glycol
XRRF crystals were obtained by dialysis against 28% to 36% buffer. The crystals grew to a size of 30 × 50 × 250 μm in one to three weeks. The result is shown in FIG.
【００１１】例2 X線回折解析によるRRFの三次元構造 RRF三次元構造決定の手段として、複合同位体置換法（m
ultiple isomorphousreplacement procedure）を用い
た。これは、重原子による同位体タンパク結晶からの拡
散データを得るのに必要な標準的な方法である。重原子
の位置より、未置換のものと同位体との差をパッターソ
ン・マップへ計算した。タンパクモデル作成にあたり電
子密集度図の計算に必要な初期タンパク相のデータは、
数種の誘導体を用いて計算された。この凍結結晶のＸ線
回折のデータは、MaxII synchrotron (Sweden, Lund)
によりBL71へ集めた。そのネイティブな結晶は2.6Åの
解像度で回折した。1.5以上のモザイシティの問題の為
に、現在までの所2.9Åの解像度までを用いた。この典
型的な回折像を図２に示す。このネイティブデータ解析
は終了しており、Rsymが1.0である。この統計データを
表１に示す。この結晶は、a=98.5Å、b=106.7Å、c=66.
7Åを有し、P21212に属している。この非対称のユニッ
トは２から４分子を含んでおり、各分子間には0.5、0.3
3、0.5のトランスレーションが存在する。２つの誘導体
のデータが得られ、プラチナの誘導体は4.0Åに回折
し、水銀の誘導体は3.8Åに回折した。 Example 2 Three-dimensional structure of RRF by X-ray diffraction analysis As a means for determining the three-dimensional structure of RRF, a compound isotope replacement method (m
ultiple isomorphous replacement procedure). This is the standard method required to obtain diffusion data from isotopic protein crystals due to heavy atoms. From the position of the heavy atom, the difference between the unsubstituted one and the isotope was calculated on a Patterson map. The initial protein phase data required for calculating the electron density map in creating the protein model is:
Calculated using several derivatives. X-ray diffraction data of this frozen crystal was obtained from MaxII synchrotron (Sweden, Lund)
To BL71. The native crystal diffracted at a resolution of 2.6 mm. Due to the mosaicity problem of 1.5 or more, we used up to 2.9Å resolution so far. This typical diffraction image is shown in FIG. This native data analysis has been completed, and Rsym is 1.0. The statistical data is shown in Table 1. This crystal has a = 98.5Å, b = 106.7Å, c = 66.
7Å and belongs to P2 1 2 1 2 This asymmetric unit contains 2 to 4 molecules, with 0.5, 0.3 between each molecule.
There are 3, 0.5 translations. Data were obtained for two derivatives, the platinum derivative diffracted to 4.0 ° and the mercury derivative to 3.8 °.
【００１２】ネイティブデータの統計学的検討 回折強度の要約とR−因子をシェルの大きさ(解像度)に
より示した表 Rの値(一次関数として)=3D SUM ( ABS(I - <I>)) / SUM
(I) Rの値(二次関数として)=3D SUM ( (I - <I>) ** 2) / S
UM (I ** 2) カイ自乗=3D SUM ( (I - <I>) ** 2) / (エラー** 2 *
N / (N-1) ) ) 全ての和の計算には二度以上測定した値についてのみ行
った。Statistical examination of native data Table R showing summary of diffraction intensity and R-factor by shell size (resolution) (as a linear function) = 3D SUM (ABS (I-<I>) ) / SUM
(I) The value of R (as a quadratic function) = 3D SUM ((I-<I>) ** 2) / S
UM (I ** 2) Chi square = 3D SUM ((I-<I>) ** 2) / (Error ** 2 *
N / (N-1))) All sums were calculated only for values measured twice or more.
【００１３】[0013]
【表１】 [Table 1]
【図１】XRRFタンパク結晶を示す写真図である。FIG. 1 is a photograph showing an XRRF protein crystal.
【図２】XRRFタンパク結晶のX線回折像を示す写真図で
ある。 回折像の詳細 : xf1 to 1200 of 1200, yf 1 to 1200 o
f 1200 回折像の方向 : xf to the right, yf up データのファイル順 : -xf + yf 最大ピクセル値: 65535 スケールの限界:最小=1、最大=1200、黒は回折強度の高
い価を示す。FIG. 2 is a photograph showing an X-ray diffraction image of an XRRF protein crystal. Details of diffraction image: xf1 to 1200 of 1200, yf 1 to 1200 o
f 1200 Direction of diffraction image: xf to the right, yf up Data file order: -xf + yf Maximum pixel value: 65535 Scale limit: minimum = 1, maximum = 1200, black indicates high value of diffraction intensity.
───────────────────────────────────────────────────── フロントページの続き Ｆターム(参考） 4B064 AG01 BG09 BH20 CA01 CE06 CE15 DA01 DA11 4C084 AA07 AA17 BA33 BA44 CA04 DC50 ZB352 4H045 AA10 AA30 BA52 CA10 EA05 EA29 GA10 GA24 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B064 AG01 BG09 BH20 CA01 CE06 CE15 DA01 DA11 4C084 AA07 AA17 BA33 BA44 CA04 DC50 ZB352 4H045 AA10 AA30 BA52 CA10 EA05 EA29 GA10 GA24
Claims (11)
求項1に記載のRRFタンパク結晶。2. The RRF protein crystal according to claim 1, wherein the crystal is crystallized by a droplet vapor diffusion method.
記載のRRFタンパク結晶。3. The RRF protein crystal according to claim 1, which has a space group P2 1 2 1 2.
項1〜3のいずれかに記載のRRFタンパク結晶。4. The RRF protein crystal according to claim 1, which has a size of 30 × 50 × 250 μm.
のいずれかに記載のRRFタンパク結晶。5. The method of claim 1, wherein the RRF is derived from fungus X.
The RRF protein crystal according to any one of the above.
パク変異体の結晶、RRFタンパクホモログの結晶及びRRF
タンパクの共複合体の結晶のいずれかである、請求項１
に記載のRRFタンパク結晶。6. The crystal of the RRF protein itself, the crystal of an RRF protein mutant, the crystal of an RRF protein homolog, and the RRF.
2. The protein of any one of the crystals of the co-complex of the protein.
The RRF protein crystal according to the above.
金又は水銀による重原子誘導体。7. A heavy atom derivative of the RRF protein crystal according to claim 1 with platinum or mercury.
物を設計するに際し、その化学的実体をコンピューター
評価するための、請求項１に記載のRRFタンパク結晶か
ら得られる構造情報の使用。8. Use of the structural information obtained from the RRF protein crystal according to claim 1 for designing a compound capable of binding to an active site of the RRF protein by computer evaluation of its chemical entity.
徴付けられる化合物が、RRFタンパクの阻害物質であ
る、請求項8に記載の構造情報の使用。9. Use of structural information according to claim 8, wherein the compound characterized by a chemical entity that binds to the active site is an inhibitor of the RRF protein.
又は不競合的阻害物質である、請求項9に記載の構造情
報の使用。10. Use of structural information according to claim 9, wherein said inhibitor is a competitive, non-competitive or uncompetitive inhibitor of RRF.
得られる、RRFタンパクの阻害物質。An inhibitor of an RRF protein obtained by using the structural information according to claim 9.
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|---|---|---|---|
| JP25342498A JP2000086696A (en) | 1998-09-08 | 1998-09-08 | Ribosome recycling factor(rrf) protein crystal and application to rational drug design based on three- dimensional structural information obtained from the crystal |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP25342498A JP2000086696A (en) | 1998-09-08 | 1998-09-08 | Ribosome recycling factor(rrf) protein crystal and application to rational drug design based on three- dimensional structural information obtained from the crystal |
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|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000075182A1 (en) * | 1999-06-04 | 2000-12-14 | Akira Kaji | Crystal of ribosomal recycling factor (rrf) protein and application thereof on the basis of three-dimensional structural data obtained from the crystal |
| KR20020004089A (en) * | 2000-07-01 | 2002-01-16 | 오현숙 | Three-Dimensional Structure And Crystallization Method of Ribosome Recycling Factor |
| WO2007066787A1 (en) | 2005-12-05 | 2007-06-14 | National University Corporation Nagoya University | Method of optimizing multiple parameters by hybrid ga, method of analyzing data by pattern matching, method of estimating substance based on radiation diffractometric data and program, recording medium and various devices related thereto |
-
1998
- 1998-09-08 JP JP25342498A patent/JP2000086696A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000075182A1 (en) * | 1999-06-04 | 2000-12-14 | Akira Kaji | Crystal of ribosomal recycling factor (rrf) protein and application thereof on the basis of three-dimensional structural data obtained from the crystal |
| KR20020004089A (en) * | 2000-07-01 | 2002-01-16 | 오현숙 | Three-Dimensional Structure And Crystallization Method of Ribosome Recycling Factor |
| WO2007066787A1 (en) | 2005-12-05 | 2007-06-14 | National University Corporation Nagoya University | Method of optimizing multiple parameters by hybrid ga, method of analyzing data by pattern matching, method of estimating substance based on radiation diffractometric data and program, recording medium and various devices related thereto |
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