Detailed mapping of drug binding and translocation sites in the AcrB pump protein

 描述（由申请人提供）：人类细菌病原体正在以惊人的速度变得多重耐药，这是一个严重的全球健康问题。虽然药物特定的药代动力学和药效学知识被用来管理正确剂量的抗生素，正在作出巨大的努力，寻找新的抗菌化合物和细胞靶点，并获得更深入的了解耐药性的机制。一种这样的机制涉及多药耐药外排泵的表达升高，以对抗穿过细菌包膜的药物流入，从而赋予对多种抗生素的抗性。在所有革兰氏阴性人类细菌病原体中一致保守的此类药物外排泵的主要类别属于耐药-结瘤-分裂（RND）家族。大肠杆菌的组成型表达的RND泵复合物包含AcrA、AcrB和TolC蛋白，其在组装时桥接内膜和外膜。来自不同细菌物种的所有三种蛋白质的三维结构已经得到解决。虽然这使我们更接近了解外排机制，但仍有很多东西需要了解药物是如何被捕获和排出的。当与质子从周质到细胞质的运动耦合时，AcrB（内膜的实际泵蛋白）具有与结构上不同的抗生素和抑制剂结合并将它们转运到外膜中的通道蛋白TolC的显著能力。AcrA是一种锚定在内膜上的周质脂蛋白，它完成了三联外排泵的组装。该应用程序的两个目标将整合结构和建模数据与遗传，分子动力学模拟和生化数据，以创建AcrB中药物结合和易位位点的详细地图，并揭示其结构-功能可塑性。鉴于AcrB样多药外排泵在人类病原体中的保守性和积极作用，该应用的结果将产生广泛而重要的影响。

 DESCRIPTION (provided by applicant): The alarming rate at which human bacterial pathogens are becoming multidrug resistant is a grave global health concern. While antibiotic-specific pharmacokinetic and pharmacodynamic knowledge is being used to administer correct dosage of antibiotics, prodigious efforts are being made to search for novel antibacterial compounds and cellular targets, and to gain a deeper understanding of the mechanism of drug resistance. One such mechanism involves the elevated expression of multidrug resistance efflux pumps to combat the drug influx across the bacterial envelope, thereby conferring resistance to a multitude of antibiotics. A major class of such drug efflux pumps, uniformly conserved in all Gram-negative human bacterial pathogens, belongs to the Resistance-Nodulation-Division (RND) family. A constitutively expressed RND pump complex of Escherichia coli comprises of AcrA, AcrB, and TolC proteins, which when assembled, bridge the inner and outer membranes. Three- dimensional structures of all three proteins from various bacterial species have been solved. Although this has brought us closer to understanding the efflux mechanism, much remains to be learned as to precisely how drugs are captured and expelled. When coupled to the movement of protons from periplasm to the cytoplasm, AcrB, the actual pump protein of the inner membrane, has the remarkable ability to bind to structurally diverse group of antibiotics and inhibitors and transport them to the channel protein TolC in the outer membrane. AcrA, a periplasmic lipoprotein anchored to the inner membrane, completes the assembly of the tripartite efflux pump. The two aims of this application will integrate structural and modeling dat with genetic, molecular dynamics simulations, and biochemical data to create a detailed map of drug binding and translocation sites in AcrB and reveal its structure-function plasticity. Given th conservation and active role of AcrB-like multidrug efflux pumps in human pathogens, the outcome of this application will have a broad and important impact.