Mapping the conformational cycle of transmembrane transporters

绘制跨膜转运蛋白的构象循环图

• 批准号: 8933627
• 负责人: Robert M Stroud
• 金额: $ 210.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8933627
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; ATP-Binding Cassette Transporters; Adenylyl Imidodiphosphate; Antibodies; Antigen Presentation; Antigens; Bacteriophages; Binding; Biological; Biological Assay; Biology; Blood Platelets; Cell membrane; Cells; Chemicals; Clostridium perfringens; Complex; Coupling; Cryoelectron Microscopy; Crystallization; Crystallography; Cysteine; Data; Detergents; Disease; Disulfides; Drug resistance; Electrons; Energy Transfer; Energy-Generating Resources; Eukaryota; Fab Immunoglobulins; Fluorescence; Future; Goals; Health; Homologous Gene; Human; Image; Immune System Diseases; Immunity; In Vitro; Label; Lead; Libraries; Ligands; Light; Lipids; Location; Maps; Measures; Membrane; Membrane Biology; Membrane Proteins; Membrane Transport Proteins; Methods; Mission; Modeling; Molecular; Molecular Conformation; Multi-Drug Resistance; Mutation; Neuromyelitis Optica; Nucleotides; Nutrient; Pathway interactions; Peptide Transport; Peptides; Pharmaceutical Preparations; Physiologic pulse; Physiology; Play; Process; Production; Proteins; Pump; Resolution; Roentgen Rays; Role; Site; Solutions; Spectrum Analysis; Staging; Structure; TAP1 gene; Temperature; Therapeutic; Thermodynamics; Validation; Weight; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; crosslink; models and simulation; monomer; nanometer; programs; screening; simulation; single molecule; thermophilic bacteria; thermophilic organism; tool

DESCRIPTION (provided by applicant): The objective is to understand the basis for energy driven cycles that lead to pumping substrates against the gradient of their concentration, across membranes of the cell. The Program Project approach is to determine the structures of the distinct functional states in the multi-step transport cycles and the pathways between them without relying exclusively on crystallography, since crystallizing intermediate states of very different structure is often as difficult as crystallizing the initial structure. The Program stratgy combines crystallography, that provides an atomic resolution structures, with specific Fab fragments to aid in sub nanometer electron cryo- microscopy (cryo-EM), `temperature dependent' cryo-EM, super-resolution Fluorescence Energy Transfer (FRET) spectroscopy, double electron-electron Resonance (DEER), serial femtosecond x-ray diffraction (SFX), small angle X-ray scattering (SAXS), chemical and disulfide cross-linking, and integrative structure modeling methods. The project focuses on ABC transporters that use ATP binding at two sites and ATP hydrolysis as the energy source for transport of substrates. The Program aims are to define the mechanism of coupling ATP binding to transport in single transporters. To accomplish this the structures of a heteromeric exporter, a homodimeric peptide exporter, and a heteromeric multi-drug exporter are expressed and will be subject to structure determination. Each transporter will be stalled at certain states throughout the transport cycle with some 5-6 expected states verified by pumping assays, or trapped by femtosecond X-ray pulses synchronized to light flash activation. Antibody Fab fragments will be generated by screening libraries displayed in bacteriophage against stabilized states of the cycle. The Fab fragments provide additional orientation for high- resolution cryo-EM imaging that provides domain interactions, Fab locations, detergent and lipid locations. X- ray crystallography provides the atomic basis for interpreting the domains, which are placed accurately within cryo-EM images. Mutations are introduced to provide for distance-sensitive labels and spectroscopies that define distances between selected points through critical stages in the mechanism. These data are subject to integrative structure modeling that seeks to then produce the pathway between the states, revealing the currently undefined mechanism of ABC transporters at atomic level. In humans 48 ABC transporters coordinate normal physiology. Through understanding the structural basis for moving through many states new target conformations for human therapeutics will be uncovered. This integrative approach and simulations of the pumping cycle consistent with thermodynamics of the cycle will be applicable to many other large complexes of membrane proteins.

描述（由申请人提供）：目的是了解能量驱动循环的基础，该循环导致将底物逆着其浓度梯度泵送穿过细胞膜。计划项目的方法是确定多步传输循环中不同功能状态的结构以及它们之间的路径，而不完全依赖于晶体学，因为结晶结构非常不同的中间状态通常与结晶初始结构一样困难。该计划策略将提供原子分辨率结构的晶体学与特定Fab片段相结合，以辅助亚纳米电子冷冻显微镜（cryo-EM）、“温度依赖性”cryo-EM、超分辨率荧光能量转移（FRET）光谱学、双电子-电子共振（DEER）、系列飞秒X射线衍射（SFX）、小角度X射线散射（SAXS），化学和二硫键交联，以及综合结构建模方法。该项目的重点是ABC转运蛋白，使用ATP结合在两个网站和ATP水解作为能源运输的底物。该计划的目的是确定耦合ATP结合到单个转运蛋白中的转运机制。为了实现这一点，表达异聚体输出蛋白、同二聚体肽输出蛋白和异聚体多药物输出蛋白的结构，并进行结构测定。在整个运输周期中，每个转运蛋白都将停滞在某些状态，其中约5-6个预期状态通过泵送试验验证，或通过与闪光激活同步的飞秒X射线脉冲捕获。抗体Fab片段将通过针对循环的稳定状态筛选噬菌体中展示的文库来产生。Fab片段为高分辨率cryo-EM成像提供额外的定向，其提供结构域相互作用、Fab位置、去污剂和脂质位置。X射线晶体学提供了解释域的原子基础，这些域被准确地放置在冷冻EM图像内。引入突变以提供距离敏感标签和光谱，其通过机制中的关键阶段定义所选择的点之间的距离。这些数据受到综合结构建模的影响，该模型试图产生状态之间的路径，揭示原子水平上ABC转运蛋白目前未定义的机制。在人类中，48个ABC转运蛋白协调正常生理学。通过理解通过许多状态移动的结构基础，将发现人类治疗的新靶构象。这种综合的方法和模拟的泵送循环与热力学的循环将适用于许多其他大型复合物的膜蛋白。