Efflux pump mechanisms for drug recognition/extrusion

描述（由申请人提供）：多药外排泵通过识别许多结构上不相关的毒性化合物并将其从细胞中主动挤出，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转运蛋白中引起多药识别和挤出的结构和基本机制。该提案的主要目标是大肠杆菌AcrB跨膜外排泵，其在所有已知的多药物转运蛋白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、洗涤剂到简单溶剂。我们已经确定了AcrB的X-射线结构中存在的四种结构不同的代理。这是第一个通过X射线晶体学与各种配体形成复合物的转运蛋白结构。晶体结构表明，三个配体分子同时结合到5000立方埃的极大中心空腔，主要通过疏水性，芳族堆积和货车范德华相互作用。每个配体使用略微不同的AcrB残基子集进行结合。结合的配体分子经常相互作用，稳定结合。随后的研究外排泵结晶突变体AcrB与五个结构不同的配体表明，AcrB由两个不同的结合位点。这五个配体不仅结合到中央腔的各个位置，而且还结合到由C-末端周质结构域形成的深外部凹陷的内衬残基。这些结构还表明AcrB组装成三个相同通道的三聚体，用于挤出药物。三聚体中AcrB的每个亚基形成其自身的多药物转运通道。我们最近收集了AcrB与周质膜融合蛋白AcrA的共晶体的X射线衍射数据。这些数据强烈支持AcrA和AcrB以特定方式相互作用的假设。这两种外排蛋白在周质中形成复合物，并相互协助进行药物转运。具体目标是：1.鉴定AcrB中多药结合的重要残基，2.研究外排泵中多药转运的机制，3.确定AcrAB共晶复合物的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.