Transport across two membranes by AcrAB-TolC complex

描述（由申请方提供）：革兰氏阴性菌抗生素耐药性的主要原因是多药外排（MDR）转运蛋白从细胞主动外排药物。耐药-结瘤-细胞分裂（RND）超家族的MDR转运蛋白具有惊人的底物特异性。RND泵的关键机械优势是它们捕获周质中的抗生素并将它们挤出革兰氏阴性菌的外膜。这种活性可能是由于RND泵和属于膜融合蛋白（MFP）家族的蛋白质的协同作用。MFP是革兰氏阴性病原体多重耐药的绝对必要条件。然而，MFP如何使药物外排仍不清楚。长期目标是了解革兰氏阴性菌中药物外排的机制。本申请的目的是表征MFP的生化机制。我们的中心假设是，在革兰氏阴性菌的MFP发挥双重作用。一方面，MFP是转运蛋白的功能亚基，并且需要启动转运循环。另一方面，这些蛋白质需要在位于两个不同膜中的MDR复合物的组分之间建立物理连接和协调作用。用于测试这一假设的方法是研究AcrA的机械特性，并将其与属于不同蛋白质家族的多药外排转运蛋白的MFP功能进行比较。我们将追求三个具体的目标：（i）调查MFP-dependent运输反应的机制;（ii）调查MFPs和它们的同源转运蛋白之间的相互作用的稳定性和特异性;（iii）调查结构多样的MFPs与外膜的功能相互作用。在第一个目标下，我们将使用已经证明的完整细胞和体外重建方法中的运输来表征天然和突变体外排泵的动力学和能量学。根据第二和第三个目标，表面等离子体共振和体内半胱氨酸可及性方法将被用来表征MFP和药物外排复合物的其他两个组件之间的功能相互作用：内膜转运蛋白和外膜通道。所提出的研究的预期结果是理解MFP如何在革兰氏阴性菌的两个膜包膜中转运底物的机制。这一贡献是重要的，因为MFP是抗生素耐药性绝对需要的，并且它们的功能可以在开发多药外排转运蛋白的有效抑制剂中被靶向。 公共卫生相关性：该应用程序的重点是细菌中最令人不安的抗生素耐药性形式-多药耐药性，这是由外排转运蛋白的活动引起的。多药外排转运蛋白是药物发现和开发的重要靶点。了解这些转运蛋白的生化机制将极大地促进对抗多药耐药细菌的新策略的发展。

DESCRIPTION (provided by applicant): The main cause of antibiotic resistance of Gram-negative bacteria is active efflux of drugs from cells by multidrug efflux (MDR) transporters. MDR transporters from Resistance-Nodulation-cell Division (RND) superfamily possess an astonishing breadth of substrate specificity. The key mechanistic advantage of RND pumps is that they capture antibiotics in the periplasm and extrude them across the outer membrane of Gram-negative bacteria. This activity is possible due to the concerted action of the RND pumps and proteins belonging to the Membrane Fusion Protein (MFP) family. MFPs are absolutely required for multidrug resistance of Gram-negative pathogens. However, how MFPs enable drug efflux remains unclear. The long term goal is to understand the mechanism of drug efflux in Gram-negative bacteria. The objective of this application is to characterize the biochemical mechanism of MFPs. Our central hypothesis is that MFPs in Gram-negative bacteria play a dual role. On one hand, MFPs are functional subunits of transporters and are required to initiate transport cycles. On the other hand, these proteins are needed to create a physical link and coordinate actions between components of MDR complexes located in two different membranes. The approach used to test this hypothesis is to investigate the mechanistic properties of AcrA and compare them to MFPs functioning with multidrug efflux transporters belonging to different families of proteins. We will pursue three specific aims: (i) Investigate the mechanism of MFP-dependent transport reaction; (ii) Investigate the stability and specificity of interactions between MFPs and their cognate transporters; (iii) Investigate functional interactions of structurally diverse MFPs with the outer membrane. Under the first aim, we will characterize the kinetics and energetics of native and mutant efflux pumps using already proven transport in intact cells and in vitro reconstitution approaches. Under the second and third aims, surface plasmon resonance and in vivo cysteine accessibility approaches will be used to characterize functional interactions between MFPs and two other components of drug efflux complexes: the inner membrane transporters and the outer membrane channels. The expected outcome of the proposed studies is the mechanistic understanding how MFPs function in transport of substrates across two membrane envelope of Gram-negative bacteria. This contribution is significant because MFPs are absolutely required for antibiotic resistance and their function could be targeted in development of effective inhibitors of multidrug efflux transporters. PUBLIC HEALTH RELEVANCE: This application is focused on the most troubling form of antibiotic resistance in bacteria - multidrug resistance, which is caused by activities of efflux transporters. Multidrug efflux transporters are important targets in drug discovery and development programs. Understanding the biochemical mechanism of these transporters will greatly facilitate the development of new strategies to combat multidrug resistant bacteria.