| Project/Area Number |
13142205
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Osaka University |
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Principal Investigator |
YAMAGUCHI Akihito Osaka University, The Institute of Scientific, 産業科学研究所, 教授 (60114336)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Satoshi Osaka University, The Institute of Scientific and Industrial Research, Associate Professor, 産業科学研究所, 助教授 (30300966)
HIRATA Takahiro Osaka University, The Institute of Scientific and Industrial Research, Research Associate, 産業科学研究所, 助手 (90333450)
木村 能章 生物分子工学研究所, 主席研究員
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| Project Period (FY) |
2001 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥135,400,000 (Direct Cost: ¥135,400,000)
Fiscal Year 2005: ¥28,500,000 (Direct Cost: ¥28,500,000)
Fiscal Year 2004: ¥28,900,000 (Direct Cost: ¥28,900,000)
Fiscal Year 2003: ¥25,500,000 (Direct Cost: ¥25,500,000)
Fiscal Year 2002: ¥25,500,000 (Direct Cost: ¥25,500,000)
Fiscal Year 2001: ¥27,000,000 (Direct Cost: ¥27,000,000)
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| Keywords | Xenobiotic exporter / X-ray crystal structure / multidrug resistance / AcrB / multidrug efflux / membrane transport / active transporter / xenobiotic recognition / 異物排出タンパク / 結晶構造 / 多剤排出タンパク / フェニルアラニンクラスタ / 基質結合型結晶 / 部位特異的変異導入 / フェニルアラニンクラスター / アンチポーター / 部位特異変異導入 / Cys走査変異 / NEM / 膜タンパク質 / RND / トランスポーター / 分子構造 |
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| Research Abstract |
This project aimed for determination of the crystal structure of bacterial major xenobiotic exporter AcrB and to reveal the mechanism of xenobiotic recognition and efflux transport. Before this project, no crystal structures of membrane transporters had determined at all. Because the purpose of this project was highly riskful, at first we had one more target to determine two-dimensional structure of AcrB by using electron microscopic analysis as a risk hedge. However, as a result, the risk hedge was not required.
Murakami et al. in our laboratory succeeded to determine the crystal structure of AcrB one year after starting this project. It is not only the first structure of xenobiotic exporters but also the first structure of the secondary transporters. This achievement is highly evaluated as a milestone of the membrane transport study. On the basis of this crystal structure, the xenobiotic recognition mechanism is clearly revealed: The AcrB structure has its entrance for substrates at t
… More
he side of the molecule opened to the lipid bilayer region of the membrane. AcrB acts as a membrane vacuum cleaner by taking up its substrates from the lipid bilayer region. Because xenobiotics generally enter into cells through lipid bilayer region by simple diffusion, this mechanism efficiently recognize xenobiotics.
Our first structure did not contain bound substrate, therefore, it can not answer the question, "where is the binding site?". On the basis of the first structure, it was estimated as the central cavity of the trimer. One years after, Edward Yu et al. reported that the structure of AcrB in which substrates bound at the central cavity. However, this estimation was no consisted with the mutational studies; there is no amino acid residues important for substrate recognition at the central cavity.
In the last year of this project, Murakami's group succeeded to determine the substrate binding structure of AcrB. The trimer is asymmetric. Only one protomer binds substrate at the phenylalanine cluster region, which is different from the central cavity. The other two porotmers represent the substrate extrusion step and the standing for substrate access step, respectively. In other words, this one crystal contains all snapshots for three steps of efflux transport. On the basis of this crystal structure, it was revealed that substrates are transported by the completely new mechanism named as functionally rotating binding change mechanism similar to FoF1-ATPase. In addition, it was revealed that the extraordinary broad substrate recognition is achieved by the multisite binding with mainly hydrophobic interactions. Less
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