Structural Dynamics of Multi-drug Transporters

描述(由申请人提供)：将继续研究定义构象运动的长期目标，该构象运动将能量消耗与活性多药转运体中的底物转运相结合。临床上治疗细菌和真菌感染以及肿瘤化疗的多药耐药性可能与这些膜包埋的外排泵的过度表达有关，这些外排泵选择性地从细胞中排出细胞毒分子。下一个资助期的实验重点是两个超家族，三磷酸腺苷结合盒(ABC)和主要促进器(MFS)，它们占细菌多药耐药转运蛋白的大多数，代表两个能量转换基序，涵盖广泛的挤压药物。ABC转运蛋白利用ATP水解的能量进行能量运输，而MFS转运蛋白将底物转运与质子的内向运动结合起来。一种创新的实验设计结合了先进的自旋标记电子顺磁共振(EPR)方法的定量系综分析和二硫化物化学对远程运动的洞察，以推导描述每个转运体在运输周期不同阶段的构象状态的约束条件。我们将测试在前一个资助期开发的ATP诱导的构象变化模型是否描述了ABC药物外排转运体在类似本地环境中的运输周期。对于主要的促进剂超家族的多药物转运体，我们将研究质子/底物耦合的结构基础，并描绘转运体异构化的共同结构基序，从内向构象到外向构象。这些实验将利用两种技术--纳米圆盘磷脂双层和Q波段脉冲EPR的融合，通过双电子电子共振(DER)提高自旋标记之间的远程测量(高达70E)的吞吐量。所提出的方法的成功实施将为应用于绝对量和浓度更有限的真核膜蛋白奠定基础。结果将提供必要的动态维度，以弥合这些转运体的功能模型和静态晶体快照之间的分歧，静态晶体快照通常是机械上定义不清的。

DESCRIPTION (provided by applicant): Research will continue on the long term goal of defining the conformational motion that couples energy expenditure to substrate translocation in active multidrug transporters. Clinical multidrug resistance in the treatment of bacterial and fungal infections and chemotherapy of neoplasms can be associated with overexpression of these membrane-embedded efflux pumps that selectively extrude cytotoxic molecules from the cell. The experimental focus for the next funding period is on two superfamilies, the ATP binding cassettes (ABC) and the major facilitator (MFS), that account for the majority of bacterial multidrug resistance transporters, represent two energy conversion motifs and encompass a broad spectrum of extruded drugs. ABC transporters harness the energy of ATP hydrolysis to power transport while MFS transporters couple substrate translocation to inward movement of protons. An innovative experimental design combines quantitative ensemble analysis by advanced spin labeling electron paramagnetic resonance (EPR) methods with insight into long range motions by disulfide chemistry, to derive constraints that describe the conformational state of each transporter at different stages of the transport cycle. We will test whether a model of ATP-induced conformational changes, developed in the previous funding period, describes the transport cycle of ABC drug efflux transporters in a native-like environment. For multidrug transporters of the major facilitator superfamily, we will investigate the structural basis of proton/substrate coupling and delineate the common structural motifs underlying transporter isomerization from inward-facing to outward-facing conformations. The experiments will capitalize on the convergence of two technologies, Nanodiscs phospholipid bilayers and Q-band pulse EPR, to increase the throughput of long range distance measurements (up to 70E) between spin labels by double electron electron resonance (DEER). The successful implementation of the proposed methodology will set the stage for application to eukaryotic membrane proteins where absolute amounts and concentrations are more limited. The results will provide the dynamic dimension necessary to bridge the divide between functional models of these transporters and static crystallographic snapshots that are often mechanistically ill-defined.