Efflux pump mechanisms for drug recognition/extrusion

描述(由申请人提供)：多药外排泵通过识别一些结构无关的有毒化合物并将它们主动地从细胞中排出，显著干扰癌症化疗和细菌感染的治疗。我们的长期目标是阐明在这些多药物转运体中引起多重药物识别和挤出的结构和基本机制。这项建议的主要目标是大肠杆菌AcrB跨膜外排泵，它在所有已知的多药物转运体中显示出最广泛的底物特异性，范围从目前使用的大多数抗生素、消毒剂、染料、洗涤剂到简单的溶剂。在四种结构不同的试剂存在下，我们测定了AcrB的X射线结构。这是所有转运蛋白的第一个结构，这些结构已经通过X射线结晶学在与各种配体的络合物中被解决。晶体结构表明，三个配体分子主要通过疏水、芳香堆积和范德华相互作用同时结合到5000立方埃的超大中心空腔上。每个配体使用略有不同的AcrB残基进行结合。结合的配体分子经常相互作用，稳定结合。随后通过使突变的AcrB与五种结构不同的配体结晶来研究外排泵，结果表明AcrB由两个不同的结合部位组成。这五种配体不仅与中央腔的不同位置结合，而且还与C末端周质结构域形成的深外部凹陷内的残基结合。这些结构还表明，AcrB作为三个相同通道的三聚体组装在一起，用于药物的排出。三聚体中AcrB的每个亚基都形成了自己的多药转运通道。我们最近收集了AcrB与周质膜融合蛋白AcrA共晶体的X射线衍射数据。这些数据有力地支持了AcrA和AcrB以特定方式相互作用的假设。这两个外排蛋白在周质中形成一个复合体，并在药物转运过程中相互协助。其具体目的是：1.确定AcrB中多药结合的重要残基；2.检测多药在外排泵中的转运机制；3.确定AcrAB共晶复合体的X射线结构。

DESCRIPTION (provided by applicant): Multidrug efflux pumps interfere significantly with cancer chemotherapy and the treatment of bacterial infections, by recognizing a number of structurally unrelated toxic compounds and actively extruding them from cells. Our long-term goal is to elucidate the structures and fundamental mechanisms that give rise to multiple drug recognition and extrusion in these multidrug transporters. The primary target of this proposal is the Escherichia coli AcrB transmembrane efflux pump, which shows the widest substrate specificity among all known multidrug transporters, ranging from most of the currently used antibiotics, disinfectants, dyes, detergents, to simple solvents. We have determined the x-ray structures of AcrB in the presence of four structurally different agents. These are the first structures of any transporter that have been solved in complex with a variety of ligands by x-ray crystallography. The crystal structures illustrate that three ligand molecules bind simultaneously to the extremely large central cavity of 5000 cubic Angstroms, primarily by hydrophobic, aromatic stacking and van der Waals interactions. Each ligand uses a slightly different subset of AcrB residues for binding. The bound ligand molecules often interact with each other, stabilizing the binding. The subsequent study of the efflux pump by crystallizing a mutant AcrB with five structurally diverse ligands indicates that AcrB consists of two distinct binding sites. These five ligands not only bind to various positions of the central cavity, but also to residues lining the deep external depression formed by the C-terminal periplasmic domain. The structures also suggest that AcrB assembles as a trimer of three identical channels for the extrusion of drugs. Each subunit of AcrB in the trimer forms its own channel for multidrug transport. We recently collected the x-ray diffraction data of a co-crystal of AcrB with a periplasmic membrane fusion protein, AcrA. The data strongly support the hypothesis that AcrA and AcrB interact in a specific manner. These two efflux proteins form a complex in the periplasm, and assist each other for drug transport. The specific aims are to: 1. identify important residues for multidrug binding in AcrB, 2. examine the mechanism of multidrug transport in the efflux pump, 3. determine the x-ray structure of the AcrAB co-crystal complex.