Dynamics of the AcrA-AcrB-TolC, a multi-drug efflux system of escherichia coli

大肠杆菌的AcrA-AcrB-TolC外排系统是大肠杆菌内在耐药的主要机制。大肠杆菌对大多数亲脂性抗生素、洗涤剂和染料的耐受性。AcrB是位于内膜（IM）的外排转运蛋白，AcrA是膜融合蛋白（MFP）家族的周质蛋白，而TolC是外膜（OM）通道。 这种三重复合物将IM处的能量（质子动力[PMF]）与底物的流出耦合，并允许系统将药物直接泵入介质中，使其在产生耐药性方面更加有效。最近，晶体结构AcrB和TolC，连同AcrA同系物，MexA，提供了显着的见解组装的外排复合物。然而，关于这三种蛋白质如何组装形成功能泵以及通道开口和底物运动如何发生的问题仍然存在。具体而言，AcrA的作用尚未完全理解，并且如何利用能量PMF来驱动底物的外排过程仍然未知。了解系统在外排过程中的动力学将有助于解决这些问题。在这个建议中，我们计划利用一个敏感的生物物理技术，定点自旋标记（SDSL）EPR（电子顺磁共振）光谱，研究AcrA-AcrB-TolC系统的动力学。使用这种技术，我们最近已经证明，AcrA经历pH诱导的构象变化，这表明AcrA可能作为一个动态的分子开关在流出过程中，而不是一个静态的适配器。在拟议的研究中，我们的短期目标是进一步研究AcrA的动力学及其在体内的功能相关性，以便更好地了解其在外排系统中的作用。 长期目标是利用SDSL技术研究外排过程中所有三种组分（AcrA，AcrB和TolC）的动力学，以充分了解革兰氏阴性菌中多药外排泵的作用机制。除了智力上的奖励，解开机制的细节将大大有助于对抗抗生素耐药性的斗争。

The AcrA-AcrB-TolC efflux system of Escherichia coli is largely responsible for the intrinsic resistance of E. coli to most lipophilic antibiotics, detergents, and dyes. AcrB is the efflux transporter located at the inner membrane (IM) and AcrA is a periplasmic protein of the membrane fusion protein (MFP) family, while TolC is an outer membrane (OM) channel.  This tripartite complex couples the energy (proton-motive-force [PMF]) at the IM with the efflux of substrates, and allows the system to pump out drugs directly into the medium, making it much more efficient in producing resistance. Recently, crystal structures AcrB and TolC, together with that of an AcrA homolog, MexA, have provided remarkable insights for the assembly of the efflux complex. However, questions remain as to how these three proteins assemble to form a functional pump and how channel openings and substrate movements occur. Specifically, the role of AcrA is not completely understood and how the energy PMF is utilized to drive the efflux process of substrates remains unknown. Knowledge on the dynamics of the system during the efflux process will help to address these questions. In this proposal, we plan to utilize a sensitive biophysical technique, the site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy, to study the dynamics of the AcrA-AcrB-TolC system. Using this technique, we have recently demonstrated that AcrA undergoes pH-induced conformational changes, suggesting that AcrA may act as a dynamic molecular switch during the efflux process, rather than a static adaptor. In the proposed research, our short-term goal is to further investigate the dynamics of AcrA and its functional relevance in vivo, so that its role in the efflux system can be better understood.  The long-term goal is to utilize the SDSL technique to investigate the dynamics of all three components (AcrA, AcrB and TolC) during the efflux process in order to fully understand the mechanism of action of multidrug efflux pumps in Gram-negative bacteria. Aside from being intellectually rewarding, unraveling the mechanistic details will greatly facilitate the fight to combat antibiotic resistance.