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

• 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
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13142205/
• 关键词: 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 /

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

本项目旨在确定细菌主要外源生物输出子AcrB的晶体结构，揭示外源生物识别和外排转运的机制。在这个项目之前，根本没有确定膜转运蛋白的晶体结构。由于这个项目的目的是高风险的，我们首先多了一个目标，利用电子显微镜分析作为风险对冲来确定AcrB的二维结构。然而，结果是不需要风险对冲。我们实验室在启动这一项目一年后成功地测定了AcrB的晶体结构。它不仅是异种输出体的第一结构，也是第二转运体的第一结构。这一成果被高度评价为膜转运研究的里程碑。在此晶体结构的基础上，清楚地揭示了异物识别的机制：acrb结构在t…具有进入底物的入口。分子的一侧更多地开放到膜的脂双层区域。AcrB作为膜真空吸尘器，从脂质双层区域吸收其底物。由于外源物质一般通过简单的扩散通过脂类双层进入细胞内，因此该机制能够有效识别外源物质。我们的第一个结构不包含结合底物，因此它不能回答“结合部位在哪里？”这个问题。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。报道称，AcrB的结构中底物结合在中央空腔。然而，这一估计与突变研究不一致；在中央空洞没有对底物识别重要的氨基酸残基。在本项目的最后一年，村上的团队成功地确定了AcrB的底物结合结构。三聚体是不对称的。只有一个原基与苯丙氨酸簇区的底物结合，这与中央空腔不同。另外两个穿孔器分别代表衬底挤出步骤和代表衬底存取步骤。换句话说，这一晶体包含三个流出传输步骤的所有快照。在此晶体结构的基础上，揭示了底物通过一种与FoF1-ATPase类似的全新机制--功能旋转结合改变机制进行转运。此外，还揭示了这种特殊的广泛底物识别是通过以疏水相互作用为主的多位点结合实现的。较少

项目成果

Extramembrane central pore of multidrug exporter AcrB in Escherichia coil plays an important role in drug transport.

大肠杆菌多药输出蛋白AcrB的膜外中心孔在药物转运中发挥重要作用。

• 发表时间: 2004
• 期刊: Journal of Biological Chemistry 279
• 作者: Murakami;S.;N.Tamura;A.Saito;T.Hirata;A.Yamaguchi.
• 通讯作者: A.Yamaguchi.

Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli

• DOI: 10.1093/oxfordjournals.jbchem.a003069
• 发表时间: 2002-01-01
• 期刊: JOURNAL OF BIOCHEMISTRY
• 影响因子: 2.7
• 作者: Fujihira, E;Tamura, N;Yamaguchi, A
• 通讯作者: Yamaguchi, A

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

• 发表时间: 2003
• 期刊: Journal of Antimicrobial Chemotherapy 52
• 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

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

Indole induces the expression of multidrug exporter genes in Echerichia coli

吲哚诱导大肠杆菌多药输出基因的表达

• 发表时间: 2005
• 期刊: Moiecuiar mlcrobiology 55
• 作者: Kamiya;T.;Akahori;T.;Ashikari;M.;Maeshima;M.;Hidetada Hirakawa
• 通讯作者: Hidetada Hirakawa