Transport across two membranes by AcrAB-ToIC complex

描述（由申请人提供）：跨越两层膜的三组分蛋白复合物在革兰氏阴性菌中普遍传播，并参与多种转运功能，如将毒力因子递送至宿主、分泌信号分子和保护细菌细胞免受结构多样的抗菌剂的侵害。 这些转运蛋白的一个显著特征是底物转移发生在两个膜上，绕过周质空间直接进入外部介质。 属于膜融合蛋白（MFP）家族的特殊周质蛋白负责两个膜的偶联。 本课题的主要目的是研究利用大肠杆菌多药外排转运蛋白AcrAB-TolC偶联两种膜的机制。大肠杆菌作为模型复合物。 AcrAB-TolC从细胞中挤出广泛的抗微生物化合物，包括抗生素，洗涤剂，染料和有机溶剂。 位于内膜的AcrB转运蛋白将其底物捕获在内膜的磷脂双分子层内，并通过外膜通道ToIC转运到外部介质中。 AcrB和TolC之间的合作由MFP蛋白AcrA介导。 生化研究表明，AcrA可能通过使内膜和外膜接近来协调复合物的功能。 申请人打算批判性地检验这一假设并研究AcrA的机制。 具体目标是：（1）研究AcrA是否对内膜和外膜具有不同的亲和力;（2）使用有限的蛋白水解和化学交联，表征AcrA在体内和体外的构象;（3）表征AcrA在体外的构象转变;和（4）研究AcrA、AcrB和TolC之间在体外的物理和功能相互作用。 这些研究预计将产生一个全面的图片AcrA介导的耦合两个膜。 这些数据将有助于了解革兰氏阴性菌中的多药外排机制，并将阐明MFP蛋白在两种膜间运输协调中的作用。

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.