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

大肠杆菌的AcrA-AcrB-TolC外排系统在很大程度上是导致其对大多数亲脂性抗生素、洗涤剂和染料产生内在抗药性的原因。AcrB是位于内膜(IM)的外流转运体，AcrA是膜融合蛋白(MFP)家族的周质蛋白，而TolC是外膜(OM)通道。这个三方复合体将IM处的能量(质子动力[PMF])与底物的外流耦合，允许系统将药物直接泵入介质，使其在产生耐药性方面更加高效。最近，AcrB和TolC的晶体结构，以及Acra同系物MexA的晶体结构，为外排复合体的组装提供了非凡的见解。然而，这三种蛋白质如何组装形成一个功能泵，以及通道开口和底物运动如何发生，这些问题仍然存在。具体地说，AcrA的作用还不完全清楚，如何利用能量PMF来驱动底物的外流过程仍是未知的。有关外排过程中系统动态的知识将有助于解决这些问题。在这个提议中，我们计划利用一种灵敏的生物物理技术--定点自旋标记(SDSL)EPR(电子顺磁共振)谱来研究AcrA-AcrB-TolC系统的动力学。利用这一技术，我们最近证明了AcrA经历了pH诱导的构象变化，这表明AcrA可能在外排过程中作为一个动态的分子开关，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成分(AcrA、AcrB和TolC)的动态，以全面了解多药外排泵在革兰氏阴性菌中的作用机制。除了在智力上有回报外，解开机制细节将极大地促进抗击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.