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：
7918582
负责人：
Hassane S Mchaourab
金额：
$ 31.31万
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 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7918582
关键词：
ATP Hydrolysis ATP-Binding Cassette Transporters Abbreviations Accounting Active Biological Transport Address Articular Range of Motion Bacteria Binding Biochemical Cells Chemistry Clinical 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 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. 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.

描述（由申请人提供）：研究将继续以长期目标定义构象运动，该构象运动将能量消耗耦合到主动多药品转运蛋白中的底物易位。临床多药在治疗细菌和真菌感染以及肿瘤化疗的抗药性可能与这些膜上包含的外排泵的过表达有关，这些外排泵从细胞中选择性地挤出了细胞毒性分子。下一个资金期间的实验重点是两个超家族，即ATP结合盒（ABC）和主要的辅助因子（MFS），这些超家族占大多数细菌多种耐药性转运蛋白，代表了两个能量转换基序，并包含了挤出药物的广泛范围。 ABC转运蛋白利用ATP水解为电力传输的能量，而MFS转运蛋白将底物置于质子向内移动。一种创新的实验设计结合了通过高级自旋标记电子顺磁共振（EPR）方法与对二硫化物化学对远距离运动的洞察力结合定量集合分析，从而得出了描述在运输周期不同阶段每个转运蛋白的构象状态的约束。我们将测试在上一个资金期间开发的ATP诱导构象变化模型是否描述了在类似天然的环境中ABC药物外排转运蛋白的运输周期。对于主要促进剂超家族的多重转运蛋白，我们将研究质子/底物耦合的结构基础，并描绘从内向构型到外向构型的基于转运蛋白异构化的共同结构基序。该实验将利用两种技术的收敛性，纳米盘磷脂双层和Q波段脉冲EPR，以通过双电子电子共振（DEER）在旋转标签之间增加远距离距离测量值（最高70E）的吞吐量。所提出的方法的成功实施将为对真核膜蛋白的应用奠定阶段，而绝对量和浓度更加有限。结果将提供所需的动态维度，以弥合这些转运蛋白的功能模型与静态晶体学快照之间的鸿沟，这些模型通常是机械上定义不明的。公共卫生相关性：世界卫生组织报告说，多药耐药细菌占全球所有医院获得感染的60％。这项研究的主要结果是学习多药转运蛋白如何利用矢量底物运动的能量输入的共同原则。这些是开发新的治疗策略的基本信息，以打击耐药性不断发展的流行病并克服对化学疗法的抵抗力。