Transport across two membranes by AcrAB-TolC complex

描述(申请人提供)：革兰氏阴性菌产生抗药性的主要原因是多药外排转运体(MDR)转运体将药物主动外排出细胞。来自抗性结节细胞分裂(RND)超家族的多药耐药转运体具有惊人的底物专一性。RND泵的关键机械优势是它们捕获周质中的抗生素，并将它们排出革兰氏阴性细菌的外膜。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。对于革兰氏阴性菌的多重耐药来说，MFP是必不可少的。然而，MFP如何使药物外流仍不清楚。长期目标是了解革兰氏阴性菌药物外排的机制。本应用的目的是表征MFP的生化机制。我们的中心假设是，革兰氏阴性细菌中的MFP具有双重作用。一方面，MFP是转运蛋白的功能亚单位，是启动运输周期所必需的。另一方面，需要这些蛋白质来建立物理联系，并协调位于两个不同膜上的MDR复合体组件之间的作用。用来检验这一假说的方法是研究AcrA的机制特性，并将它们与MFP进行比较，这些MFP具有属于不同蛋白质家族的多药物外排转运体。我们将追求三个具体目标：(I)研究MFP依赖的转运反应的机制；(Ii)研究MFP与其同源转运体之间相互作用的稳定性和特异性；(Iii)研究结构不同的MFP与外膜的功能相互作用。在第一个目标下，我们将使用已证实的完整细胞运输和体外重建方法来表征天然和突变外排泵的动力学和能量学。在第二和第三个目标下，将使用表面等离子体共振和体内半胱氨酸可及性方法来表征MFP与药物外排复合体的另外两个组分：内膜转运体和外膜通道之间的功能相互作用。拟议研究的预期结果是从机制上理解MFP如何在底物跨革兰氏阴性细菌的两个膜被膜运输中发挥作用。这一贡献是重大的，因为MFP是抗生素耐药性的绝对必需的，它们的功能可能被靶向于开发有效的多药外排转运蛋白的抑制剂。

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.