Transport across two membranes by AcrAB-ToIC complex

描述(申请人提供)：跨越两个膜的三组分蛋白质复合体广泛分布于革兰氏阴性细菌中，涉及多种运输功能，如向宿主运送毒力因子、分泌信号分子和保护细菌细胞免受结构上不同的抗菌剂的影响。这些转运蛋白的一个显着特征是底物通过两层膜直接转移到外部介质中，绕过周质空间。特殊的周质蛋白属于膜融合蛋白(MFP)家族，负责两种膜的偶联。本项目的主要目标是以来自大肠杆菌的多药外排转运体AcrAB-TolC为模型络合物，研究两种膜的偶联机理。 AcrAB-TolC从细胞中排出广泛的抗菌化合物，包括抗生素、洗涤剂、染料和有机溶剂。AcrB转运蛋白位于内膜，在内膜磷脂双层中捕获底物，并通过外膜通道ToIC将底物输送到外部介质中。AcrB和TolC之间的协同作用是由MFP蛋白AcrA介导的。生化研究表明，AcrA可能通过使内膜和外膜接近来协调复合体的功能。申请者打算批判性地检验这一假说，并研究AcrA的机制。其具体目的是：(1)研究AcrA对内外膜是否具有不同的亲和力；(2)利用有限的蛋白分解和化学交联，表征AcrA在体内和体外的构象；(3)表征AcrA的体外构象转变；(4)研究AcrA、AcrB和TolC之间的物理和功能相互作用。这些研究有望为AcrA介导的两种膜的偶联提供全面的图景。这些数据将有助于理解革兰氏阴性菌多药外排的机制，并将阐明MFP蛋白在协调两种膜运输中的作用。

DESCRIPTION (provided by applicant): Three-component protein complexes spanning two membranes are universally spread among Gram-negative bacteria and have been implicated in such diverse range of transport functions as delivery of virulence factors into the hosts, secretion of signaling molecules and protection of bacterial cells against structurally diverse antimicrobial agents. A remarkable feature of these transporters is that the substrate transfer occurs across two membranes directly into external medium bypassing the periplasmic space. Special periplasmic proteins, which belong to the Membrane Fusion Protein (MFP) family, are responsible for the coupling of two membranes. The major objective of this project will be to investigate the mechanism of coupling of two membranes using the multidrug efflux transporter AcrAB-TolC from E. coli as a model complex. The AcrAB-TolC extrudes out of the cell a broad range of antimicrobial compounds including antibiotics, detergents, dyes and organic solvents. Located in the inner membrane AcrB transporter captures its substrates within phospholipid bilayer of inner membrane and transports them into external medium via the outer membrane channel, ToIC. The cooperation between AcrB and TolC is mediated by the MFP protein, AcrA. Biochemical studies suggested that AcrA might coordinate the function of the complex by bringing the inner and outer membranes into proximity. The applicants intend to test this hypothesis critically and to investigate the mechanism of AcrA. The specific aims are: (1) investigate if AcrA has differential affinity for the inner and outer membranes; (2) using limited proteolysis and chemical cross-linking, characterize the conformation of AcrA in vivo and in vitro; (3) characterize conformational transitions of AcrA in vitro; and (4) investigate physical and functional interactions between AcrA, AcrB and TolC in vitro. These studies are expected to yield a comprehensive picture of AcrA-mediated coupling of two membranes. These data will contribute to an understanding of the mechanism of multidrug efflux in Gram-negative bacteria and will illuminate the role of MFP proteins in the coordination of transport across two membranes.