Mechanisms of Allostery and Molecular Recognition in the Small Multidrug Resistance Family

项目摘要 细菌耐药性是一个世界性的问题，限制了抗生素在临床上的有效性。而当 有几种分子机制导致耐药表型，众所周知 外排泵在病原菌中扮演着重要的角色。事实上，多药转运体构成了一种 细菌在有毒化合物存在时通过结合和结合而生存的基本机制 运输一系列结构不同的化合物。这个项目的长期目标是发现 多药物转运体使用的新机制，并利用这一知识来预测和控制功能。 在这场竞争性的更新中，我们现在准备应对在了解如何 外排泵可实现广泛的药物特异性，从而产生多药耐药性。为了实现这一目标， 我们需要对顺从的外排泵系统的催化循环建立一个全面的理解。 到详细的生物、生化和生物物理研究。出于这个原因，我们的提案将使用 SMR家族作为模型药物转运体，因为它体现了最低水平的复杂性，同时保留了 所有二次主动外排泵共有的关键特性。目标1将测试一种遮挡状态理论，即我们 假说被外排泵广泛用于药物结合。目标2将寻求定义分子基础 底物诱导的动力学激活与抑制剂诱导的动力学抑制，以及 开发用于预测结合和运输的计算平台。最后，目标3将着手于 通过对两者的比较分析，确定结合专一性与混杂的分子基础 SMR家族中具有明显不同特异性特征的亚家族。这些目标中的每一个都有效 协同实现我们的长期目标，即阐明新的运输机制并应用我们的知识 开发预测函数的模型。该项目的一大优势是整体性 该方法利用重要的协作以及生物学、生物物理学和 旨在揭示由自然设计并共享的通用传输机制的计算方法 其他多种药物外排泵。这项研究的结果将对我们的理解产生重大影响 外排介导的多药耐药，所开发的途径和方法将可转化为 在其他外流系统中的知识发现。

Project Summary Bacterial drug resistance is a worldwide problem that limits the effectiveness of antibiotics in the clinic. While there are several molecular mechanisms that contribute to drug resistant phenotypes, it is well established that efflux pumps play a prominent role in pathogenic bacteria. Indeed, multidrug transporters constitute a fundamental mechanism used by bacteria to survive in the presence of toxic compounds by binding and transporting a broad array of structurally diverse compounds. The long-term goals of this project are to discover novel mechanisms used by multidrug transporters and to harness this knowledge to predict and control function. In this competitive renewal, we are now poised to tackle the major challenge in the field of understanding how efflux pumps achieve broad drug specificity required for conferring multidrug resistance. To accomplish this goal, we need to establish a comprehensive understanding of the catalytic cycle for an efflux pump system amenable to detailed biological, biochemical and biophysical studies. For this reason, our proposal will use EmrE from the SMR family as the model drug transporter since it embodies the minimal level of complexity while retaining the key features shared among all secondary active efflux pumps. Aim 1 will test an occluded-state theory that we hypothesize is widely used by efflux pumps for drug binding. Aim 2 will seek to define the molecular basis for substrate-induced activation of dynamics versus inhibitor-induced repression of dynamics, as well as development of a computational platform for predicting binding and transport. Finally, Aim 3 will set out to determine the molecular basis of binding specificity versus promiscuity through a comparative analysis of two subfamilies within the SMR family that have markedly different specificity profiles. Each of these Aims works synergistically toward our long-term goal of articulating novel transport mechanisms and applying our knowledge to develop models for making predictions about function. A major strength of this project is the integrated nature of the approach which utilizes significant collaboration and a combination of biological, biophysical, and computational methods aimed at unveiling general transport mechanisms designed by nature and shared among other multidrug efflux pumps. The outcomes of this research will make a significant impact in understanding efflux-mediated multidrug resistance, and the approaches and methods developed will be translatable to knowledge discovery in other efflux systems.