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Studies on the crystal structure of antiporters for organic compounds and the mechanisms

有机化合物反向转运蛋白的晶体结构及其机制研究

基本信息

批准号：
13142205
负责人：
YAMAGUCHI Akihito
金额：
$ 86.66万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2005
项目状态：
已结题

项目摘要

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

本项目旨在测定细菌主要外源物质输出蛋白AcrB的晶体结构，揭示外源物质识别和外排转运机制。在这个项目之前，根本没有确定膜转运蛋白的晶体结构。因为这个项目的目的风险很大，所以一开始我们多了一个目标，就是利用电镜分析确定AcrB的二维结构作为风险对冲。然而，因此，不需要进行风险对冲。 Murakami 等。在该项目启动一年后，我们实验室成功确定了 AcrB 的晶体结构。它不仅是外源物质输出蛋白的第一结构，也是二级转运蛋白的第一结构。这一成果被高度评价为膜转运研究的里程碑。在这种晶体结构的基础上，清楚地揭示了外源生物识别机制：AcrB结构的底物入口位于分子向膜的脂质双层区域开放的一侧。 AcrB 通过从脂质双层区域吸收其底物来充当膜真空清洁器。由于外源物质通常通过简单扩散通过脂质双层区域进入细胞，这种机制可以有效地识别外源物质。我们的第一个结构不包含结合底物，因此，它不能回答“结合位点在哪里？”的问题。在第一个结构的基础上，估计它是三聚体的中心空腔。一年后，Edward Yu 等人。报道了 AcrB 的结构，其中底物结合在中央空腔处。然而，这一估计与突变研究并不相符。中央空腔没有对底物识别重要的氨基酸残基。在该项目的最后一年，村上的研究小组成功确定了AcrB的底物结合结构。三聚体是不对称的。只有一个原聚体在苯丙氨酸簇区域结合底物，该区域不同于中央空腔。另外两个多孔剂分别代表基底挤出步骤和基底进入步骤。换句话说，这一晶体包含了外排运输三个步骤的所有快照。基于这种晶体结构，揭示了底物是通过一种全新的机制进行转运的，这种机制被称为功能性旋转结合变化机制，类似于FoF1-ATPase。此外，研究表明，非凡的广泛底物识别是通过主要具有疏水性相互作用的多位点结合实现的。较少的

项目成果

期刊论文数量（186）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

beta-Lactam resistance modulated by the overexpression o response regulators of two-component signal transduction systems in Escherichia coli

大肠杆菌中双组分信号转导系统的过度表达或反应调节因子调节β-内酰胺抗性

DOI：
发表时间：
2003
期刊：
Journal of Antimicrobial Chemotherapy 52
影响因子：
0
作者：
Hidetada Hirakawa;Kunihiko Nishino;Junko Yamada;Takahiro Hirata;Akihito Yamaguchi
通讯作者：
Akihito Yamaguchi

Roles of To1C-dependent multidrug transporters of Escherichia col in resistance to beta-lactams

大肠杆菌To1C依赖性多药转运蛋白在β-内酰胺耐药中的作用

DOI：
发表时间：
2003
期刊：
Antimicrobial Agents and Chemotherapy 47
影响因子：
0
作者：
Kunihiko Nishino;Junko Yamada;Hidetada Hirakawa;Takahiro Hirata;Akihito Yamaguchi
通讯作者：
Akihito Yamaguchi

Kunihiko NISHINO, Akihito YAMAGUCHI: "EvgA of the two-component signal transduction system modulates production of the YhiUV multidrug transporter in Escherichia coli"Journal of Bacteriology. 184・3(in press). (2002)

Kunihiko NISHINO、Akihito YAMAGUCHI：“双组分信号转导系统的 EvgA 调节大肠杆菌中 YhiUV 多药物转运蛋白的产生”《细菌学杂志》184·3（出版中）。