Structural Dynamics of Multi-drug Transporters

多药物转运蛋白的结构动力学

基本信息

批准号：
8445258
负责人：
Hassane S Mchaourab
金额：
$ 30.11万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-03-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8445258
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Abbreviations Accounting Active Biological Transport Address Articular Range of Motion Bacteria Bacterial Infections Binding Biochemical Cells Chemistry Clinical Coupled Couples Coupling Cysteine Data Data Set Development Dimensions Disulfides Drug Efflux Drug Transport Drug resistance Electron Spin Resonance Spectroscopy Electrons Energy Metabolism Environment Epidemic Escherichia coli Experimental Designs Fingerprint Funding Goals Homology Modeling Learning Ligands Lipid A Lipid Bilayers Liposomes Malignant Neoplasms Maps Measurement Measures Membrane Membrane Proteins Methodology Methods Modeling Molecular Conformation Monitor Motion Movement Multi-Drug Resistance Mycoses Nature Nosocomial Infections Nucleotides Outcomes Research Pharmaceutical Preparations Phospholipids Physiologic pulse Proton-Motive Force Protons Reporting Request for Proposals Research Resistance Side Site Spin Labels Staging Structure System Technology Testing Vanadates Vesicle Water Work World Health Organization base chemotherapy combat crosslink cytotoxic efflux pump extracellular frontier global environment innovation insight multi drug transporter multidrug transport nanodisk neoplasm chemotherapy novel therapeutics overexpression programs public health relevance research study response tumor

项目摘要

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.

描述（由申请人提供）：研究将继续以定义构象运动为长期目标，该构象运动将能量消耗与活性多药物转运蛋白中的底物易位结合起来。细菌和真菌感染治疗以及肿瘤化疗中的临床多药耐药性可能与这些膜嵌入的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，即 ATP 结合盒（ABC）和主要促进子（MFS），它们占细菌多药耐药性转运蛋白的大多数，代表两种能量转换基序，涵盖广泛的挤出的药物。 ABC 转运蛋白利用 ATP 水解的能量来为运输提供动力，而 MFS 转运蛋白则将底物易位与质子向内运动结合起来。创新的实验设计将先进的自旋标记电子顺磁共振（EPR）方法的定量系综分析与二硫化物化学对长程运动的洞察相结合，得出描述每个转运蛋白在转运周期不同阶段的构象状态的约束。我们将测试上一个资助期开发的 ATP 诱导构象变化模型是否描述了 ABC 药物外排转运蛋白在类似天然环境中的转运周期。对于主要促进子超家族的多药物转运蛋白，我们将研究质子/底物偶联的结构基础，并描绘转运蛋白从内向构象到外向构象异构化的共同结构基序。该实验将利用 Nanodiscs 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) 提高自旋标记之间的长距离距离测量（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加有限的真核膜蛋白奠定基础。结果将提供必要的动态维度，以弥合这些转运蛋白的功能模型和静态晶体快照之间的鸿沟，而静态晶体快照通常在机械上是不明确的。