Transport Mechanism of the Multidrug Resistance Efflux Protein, EmrE

项目总结/摘要 由于多药耐药（MDR）外排泵的活性导致的抗生素耐药性是一个重要的 细菌耐药机制。EmrE是已知最小的MDR转运蛋白之一， 成为质子耦合反输运的原型。EmrE利用质子导入驱动聚芳族阳离子 E.大肠杆菌，从而赋予耐药性的一个广泛的药物。我们之前已经进行了第一次 转运体中打开和打开状态之间构象交换的定量测量 通过核磁共振。我们的最新结果，EmrE的不对称质子化，直接反驳了长期接受的单位点 药物和质子耦合反向转运的交替存取模型。它表明了还有多少是未知的 关于这个看似简单的转运蛋白，并表明众所周知的MDR外排泵的混杂性 甚至可能更大。该项目使用NMR光谱和广泛的脂质体转运测定， 研究EmrE活性的详细分子机制。核磁共振提供了一个独特的工具，因为蛋白质 构象交换和质子化事件可以分别和同时监测， 对质子和药物运输如何耦合的新见解。我们的目标是将联合收割机定量生物物理学 定义EmrE活性的pH依赖性和底物性质的数据和功能评估， 开发一种新的质子耦合反输运模型。我们还将测试不同的假设如何质子- 对于具有不同亲和性和传输速率的这些不同底物，实现了偶联的反向转运。的 我们获得的见解将有助于未来的努力，以打击细菌抗生素耐药性，由于多药耐药外流。

Project Summary/Abstract Antibiotic resistance due to the activity of multidrug resistance (MDR) efflux pumps is one important mechanism of bacterial drug resistance. EmrE is one of the smallest known MDR transporters, and it has become a prototype for proton-coupled antiport. EmrE harnesses proton import to drive polyaromatic cation efflux in E. coli, thus conferring resistance to a broad range of drugs. We have previously performed the first quantitative measurement of conformational exchange between open-in and open-out states in a transporter by NMR. Our newest result, asymmetric protonation of EmrE, directly contradicts the long-accepted single-site alternating access model for coupled antiport of drugs and protons. It demonstrates how much is still unknown about this deceptively simply transporter and suggests that the well-known promiscuity of MDR efflux pumps may even be even greater. This project uses NMR spectroscopy and extensive liposomal transport assays to investigate the detailed molecular mechanism of EmrE activity. NMR offers a unique tool because protein conformational exchange and protonation events can be monitored separately and simultaneously, allowing novel insight into how proton and drug transport are coupled. Our goal is to combine quantitative biophysical data and functional assessment of the pH dependence and substrates properties that define EmrE activity to develop a novel model for proton-coupled antiport. We will also test different hypotheses for how proton- coupled antiport is achieved for such diverse substrates with different affinities and transport rates. The insights we gain will aid future efforts to combat bacterial antibiotic resistance due to MDR efflux.