Mapping the conformational cycle of transmembrane transporters

绘制跨膜转运蛋白的构象循环图

• 批准号: 9751878
• 负责人: Robert M Stroud
• 金额: $ 192.63万
• 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/9751878
• 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; Fluorescence Resonance Energy Transfer; Future; Goals; Homologous Gene; Human; Image; Immune System Diseases; Immunity; In Vitro; Label; Lead; Libraries; Ligands; Light; Lipids; Location; Maps; Measures; Membrane; Membrane Biology; Membrane 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; Spectrum Analysis; Structural Models; Structure; TAP1 gene; Temperature; Therapeutic; Thermodynamics; Validation; Weight; X ray diffraction analysis; X-Ray Crystallography; base; crosslink; models and simulation; monomer; nanometer; programs; public health relevance; screening; simulation; single-molecule FRET; 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，该循环导致底物沿浓度梯度泵送穿过细胞膜。Program Project的方法是在不完全依赖晶体学的情况下，确定多步输运循环中不同功能状态的结构及其之间的途径，因为结晶非常不同结构的中间状态通常与结晶初始结构一样困难。该计划策略结合了晶体学，提供原子分辨率结构，与特定的Fab片段，以帮助亚纳米电子冷冻显微镜（cryo- em），“温度依赖”的cryo- em，超分辨率荧光能量转移（FRET）光谱，双电子-电子共振（DEER），连续飞秒x射线衍射（SFX），小角度x射线散射（SAXS），化学和二硫交联，以及综合结构建模方法。该项目侧重于ABC转运体，它利用ATP在两个位点结合和ATP水解作为底物运输的能量来源。该计划的目的是确定ATP结合与单一转运体转运的耦合机制。为了实现这一目的，表达了异质输出体、同二聚体肽输出体和异质多药输出体的结构，并将进行结构确定。在整个传输周期中，每个转运体将停滞在特定的状态，通过泵送试验验证大约5-6个预期状态，或者被与闪光激活同步的飞秒x射线脉冲捕获。抗体Fab片段将通过筛选噬菌体中显示的抗稳定循环状态的文库来产生。Fab片段为高分辨率冷冻电镜成像提供了额外的方向，提供了结构域相互作用，Fab位置，洗涤剂和脂质位置。X射线晶体学为解释在低温电镜图像中精确放置的畴提供了原子基础。引入突变以提供距离敏感标签和光谱，通过机制中的关键阶段定义选定点之间的距离。这些数据需要进行综合结构建模，以寻求产生状态之间的途径，揭示目前未定义的ABC转运蛋白在原子水平上的机制。在人类中，48个ABC转运蛋白协调正常生理。通过了解通过许多状态移动的结构基础，人类治疗的新目标构象将被发现。这种综合方法和与循环热力学相一致的泵送循环模拟将适用于许多其他大型膜蛋白复合物。