Mapping the conformational cycle of transmembrane transporters

描述(由申请人提供)：目标是了解能量驱动循环的基础，该循环导致泵送底物，使其与其浓度梯度相反，穿过细胞膜。方案项目方法是确定多步运输循环中不同功能状态的结构以及它们之间的路径，而不完全依赖于结晶学，因为结晶非常不同的结构的中间状态通常与结晶初始结构一样困难。该计划战略结合了结晶学，它提供了原子分辨率结构，与特定的FAB片段，以帮助亚纳米电子冷冻显微镜(冷冻-EM)，“温度依赖”冷冻-EM，超分辨率荧光能量转移(FRET)光谱，双电子-电子共振(DER)，系列飞秒X射线衍射(SFX)，小角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，它使用两个位点的ATP结合和ATP水解作为底物运输的能源。该计划的目的是确定在单一转运体中将ATP结合与运输相耦合的机制。为了实现这一点，表达了异构体导出体、同二聚体多肽导出体和异构体多药导出体的结构，并将对其进行结构测定。每个传送器在整个运输周期中将在某些状态下停滞，通过泵送测试验证大约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展示的文库来对抗循环的稳定状态而产生。Fab片段为高分辨率冷冻-EM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射线结晶学为解释这些区域提供了原子基础，这些区域被精确地放置在低温电子显微镜图像中。引入突变是为了提供距离敏感的标记和光谱分析，这些标记和光谱分析通过机制中的关键阶段定义选定点之间的距离。这些数据受到综合结构建模的影响，该模型寻求产生状态之间的路径，揭示目前未确定的ABC转运蛋白在原子水平上的机制。在人类中，48个ABC转运体协调正常的生理。通过了解通过许多状态移动的结构基础，人类治疗学的新靶点构象将被发现。这种综合的方法和与循环热力学一致的抽运循环模拟将适用于许多其他大型膜蛋白复合体。

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.