Structural Dynamics of Multi-drug Transporters

描述（由申请人提供）：研究将继续确定活性多药转运体中能量消耗与底物转运耦合的构象运动的长期目标。在治疗细菌和真菌感染以及肿瘤化疗中的临床多药耐药可能与这些膜内外排泵的过度表达有关，这些外排泵选择性地从细胞中挤出细胞毒性分子。下一个资助期的实验重点是两个超家族，ATP结合盒（ABC）和主要促进剂（MFS），它们占细菌多药耐药转运体的大多数，代表两种能量转换基基，并涵盖广泛的挤压药物。ABC转运体利用ATP水解的能量来驱动转运，而MFS转运体将底物易位与质子向内运动结合起来。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对二硫化学的远程运动的洞察，得出了描述每个转运子在转运周期不同阶段的构象状态的约束。我们将测试在上一个资助期开发的atp诱导构象变化模型是否描述了ABC药物外溢转运体在原生环境中的转运周期。对于主要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，并描述转运体从内向到外向异构化的共同结构基序。实验将利用纳米圆盘磷脂双层和q波段脉冲EPR两种技术的融合，通过双电子电子共振（DEER）增加自旋标签之间远距离测量（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数量和浓度更有限的真核膜蛋白奠定基础。这些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鸿沟，这些模型通常在机械上定义不清。

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. PUBLIC HEALTH RELEVANCE: The World Health Organization has reported that multidrug-resistant bacteria account for up to 60% of all hospital-acquired infections globally. The major outcome of this research is to learn common principles of how multidrug transporters harness energy input for vectorial substrate movement. These are fundamental information for the development of new therapeutic strategies to combat the evolving epidemic of drug resistance and overcome tumor resistance to chemotherapy.