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

 描述(申请人提供)：人类细菌病原体对多种药物产生抗药性的惊人速度是一个严重的全球卫生问题。在利用抗生素特定的药代动力学和药效学知识来给药正确剂量的同时，人们也在努力寻找新的抗菌化合物和细胞靶点，并加深对耐药机制的理解。其中一种机制涉及增加多药耐药外排泵的表达，以对抗药物通过细菌被膜的流入，从而对多种抗生素产生耐药性。这类药物外排泵属于耐药结节分化科(RND)家族，在所有革兰氏阴性细菌中都是一致保守的。组成表达的大肠杆菌RND泵复合体由AcrA、AcrB和TolC蛋白组成，当组装时，它们连接内膜和外膜。来自不同细菌物种的所有三种蛋白质的三维结构都得到了解决。虽然这使我们更接近于了解外排机制，但关于药物是如何被捕获和排出的，仍有许多需要了解。当结合质子从周质到细胞质的运动时，内膜的实际泵蛋白AcrB具有显著的能力与结构不同的抗生素和抑制剂结合，并将它们运输到外膜的通道蛋白TolC。AcrA是一种锚定在内膜上的周质脂蛋白，完成了三方外排泵的组装。这项应用的两个目标是将结构和建模数据与遗传、分子动力学模拟和生化数据相结合，以创建AcrB中药物结合和易位的详细地图，并揭示其结构-功能可塑性。鉴于AcrB类多药外排泵在人类病原体中的保守和积极作用，这一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.