Structural Dynamics of Multi-drug Transporters

细菌的动态平衡和生存在很大程度上取决于防御机制， 停用或排出细胞毒性分子，如防腐剂和抗生素，被动地 沿着它们的浓度梯度穿过膜。一种无处不在且高度保守的 机制需要多特异膜转运蛋白的表达，称为多药 (MDR)转运体，它利用存储在离子电化学梯度中的吉布斯能量来 为广谱的细胞毒分子的上坡矢量清除提供动力。能量耦合 转运蛋白在多个中间体之间的异构化使得能够交替访问 底物结合部位从膜的一侧到另一侧。定义结构元素 在类脂质双层环境中，调节交替通路和解码能量转换机制是该领域令人兴奋的前沿，对于确定运输机制至关重要。 该提案将继续支持致力于解决这些问题的富有成效的研究计划 临床上涉及的两个离子偶联MDR转运蛋白家族的问题 抗药性。我们的方法充分利用了EPR波谱工具包 高分辨率结构，由功能研究提供信息，并通过 协作性分子建模工作。目标1旨在阐明离子衬底的原理 偶联，确定对离子和底物结合至关重要的残基，并揭示其特异性 转运蛋白-脂类相互作用在两种情况下塑造构象变化的能量格局 多药和毒素排出(Mate)家族的原型。目标2 寻求确定交替访问和离子-衬底耦合的保守元件 MDR转运体的主要促进者(MFS)家族。我们将测试一种详细的配体依赖的构象变化机制，该机制是在前一资金时期开发的，它整合了离子 结合特定的脂类相互作用，在建立良好的运输模型的背景下。 这两个目标将共同阐明涉及的运输者家庭的机械原理。 在耐药现象和基本的细菌防御策略。

Bacterial homeostasis and survival is critically dependent on defense mechanisms that modify, deactivate, or extrude cytotoxic molecules such as antiseptics and antibiotics, which passively cross the membrane down their concentration gradients. One ubiquitous and highly conserved mechanism entails the expression of polyspecific membrane transporters, referred to as multidrug (MDR) transporters, which harness the Gibbs energy stored in ion electrochemical gradients to power the uphill vectorial clearance of a broad spectrum of cytotoxic molecules. Energy-coupled isomerization of the transporter between multiple intermediates enables alternating access of the substrate binding site from one side of the membrane to the other. Defining the structural elements mediating alternating access and decoding the mechanism of energy conversion in a lipid bilayer-like environment are exciting frontiers in the field and critical for defining transport mechanisms. This proposal will continue support of a productive research program focused on addressing these questions for two families of ion-coupled MDR transporters that have been implicated in clinical drug resistance. Our approach capitalizes on the tool kit of EPR spectroscopy in the context of high resolution structures, is informed by functional studies, and is contextualized through collaborative molecular modeling efforts. Aim 1 seeks to elucidate principles of ion-substrate coupling, identify residues critical for ion and substrate binding, and reveal how specific transporter-lipids interactions shape the energy landscape of conformational changes in two archetypes of the Multidrug and Toxin Extrusion (MATE) family of multidrug transporters. Aim 2 seeks to identify conserved elements of alternating access and ion-substrate coupling for the major facilitator (MFS) family of MDR transporters. We will test a detailed mechanism of ligand-dependent conformational changes, developed in the previous funding period, that integrate ion coupling with specific lipid interactions in the context of a well-established transport model. Together, the two aims will illuminate mechanistic principles for families of transporters implicated in the phenomenon of drug resistance and basic bacterial defense strategies.