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

本项目旨在测定细菌主要外源物质AcrB的晶体结构，揭示其外源物质识别和外排转运的机制。在此项目之前，还没有对膜转运蛋白的晶体结构进行过研究。由于这个项目的目的是高风险的，一开始我们多了一个目标，通过电子显微镜分析来确定AcrB的二维结构，作为风险对冲。然而，结果是不需要进行风险对冲。我们实验室的Murakami等人在开始这个项目一年后，成功地确定了AcrB的晶体结构。它不仅是外源输出体的第一结构，也是次生转运体的第一结构。这一成果被高度评价为膜转运研究的一个里程碑。在此晶体结构的基础上，清楚地揭示了异种生物识别机制：AcrB结构对底物有入口，分子的一侧向膜的脂质双分子层区域开放。AcrB通过从脂质双分子层区域吸收底物而起到膜真空吸尘器的作用。由于外源药物一般通过脂质双分子层区域以简单扩散的方式进入细胞，因此该机制能有效识别外源药物。我们的第一个结构没有包含结合底物，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腔。一年后，Edward Yu等人报道了AcrB的结构，其中底物在中心腔结合。然而，这一估计与突变研究不一致；在中心腔中没有对底物识别重要的氨基酸残基。在这个项目的最后一年，Murakami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。只有一个原聚体在苯丙氨酸簇区与底物结合，这与中心腔不同。另外两个基团分别表示基板挤压步骤和基板接入步骤。换句话说，这一个晶体包含了流出输运的三个步骤的所有快照。在这种晶体结构的基础上，揭示了底物通过一种类似于fof1 - atp酶的功能旋转结合改变机制进行转运。此外，还揭示了多位点结合主要是疏水相互作用，从而实现了非常广泛的底物识别。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