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

本项目旨在确定细菌主要外源性物质输出蛋白AcrB的晶体结构，揭示细菌对外源性物质的识别和外排转运机制。在此之前，还没有确定膜转运蛋白的晶体结构。由于该项目的目的是高风险的，首先我们有一个目标，通过电子显微镜分析确定AcrB的二维结构作为风险对冲。然而，结果是，不需要风险对冲。本实验室的村上等人在开始该项目一年后成功地确定了AcrB的晶体结构。它不仅是外源物质输出的第一结构，也是次级转运的第一结构。这一成果被高度评价为膜转运研究的里程碑。在此基础上，明确揭示了AcrB的识别机理：AcrB结构在底物进入位置为： ...更多信息 分子的一侧向膜的脂质双层区域开放。AcrB通过从脂质双层区域吸收其底物而充当膜真空清洁器。由于外源性物质一般通过简单扩散的方式进入细胞，因此这种机制可以有效地识别外源性物质。我们的第一种结构不含结合底物，因此，它不能回答“结合位点在哪里？".在第一种结构的基础上，估计它是三聚体的中心空腔。一年后，Edward Yu等报道了AcrB的结构，其中底物结合在中心空腔。然而，这一估计与突变研究不一致，在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，Murakami的小组成功地确定了AcrB的底物结合结构。三聚体是不对称的。只有一个原聚体在苯丙氨酸簇区域结合底物，这与中心腔不同。另外两个porotmer分别代表底物挤出步骤和底物进入步骤的站立。换句话说，这一个晶体包含了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全新的机制进行转运，该机制被命名为类似于FoF 1-ATPase的功能旋转结合变化机制。此外，它被揭示，特别广泛的底物识别是通过多位点结合，主要是疏水相互作用实现的。少

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