Drug, Nucleotide, and Lipid Interactions with P-glycoprotein

项目摘要 P-糖蛋白通过部分特征化的 依赖于ATP的机制。由于P-gp的极端底物混杂，它有助于 几乎所有小分子药物的处置以及药物与药物的相互作用。P-gp可能是 在癌细胞耐药中尤其重要，因为它在几个 癌症。这项提议的目的是填补P-GP三个不同方面的知识空白 机制。每个目标都共享构象动力学的共同机制元素。 第一个目标是定义低ATP占有率下的P-gp构象，以便理解 它们如何控制底物外流所必需的下游构象变化，以及 它们在有无底物的情况下有何不同。这些构象 假设差异对于将ATP的浪费支出降至最低至关重要 没有可运输的底物，并控制下游的适当构象访问 台阶。这些差异将通过快速、预平衡、动力学方法进行识别，状态为- 最先进的动力学建模和氢/氢交换质谱学(H/DX) 脂类纳米盘中的P-gp。 第二个目的是了解P-gp对其脂质环境的极端敏感性。这个 脂质纳米盘平台提供了对P-gp所在的脂质双层的精细控制。P-GP将 被掺入具有不同头基或酰基的不同比例的脂类的纳米盘中 链提供了不同的表面电荷的双层流动性。基板和基板 不同脂质环境中依赖的ATPase活性将与 由H/DX确定构象。 第三个目标是确定P-gp能够与如此广泛的 底物和缓蚀剂的范围。底物乱交是一种越来越受欢迎的行为 对于生物学中的许多蛋白质，但分子基础还没有明确的定义。在这里，前 将使用平衡结合动力学和单分子荧光方法进行比较 结合参数在P-gp系综与单个P-gp纳米盘中的分布 确定P-GP是利用相互转换的构象还是持久的DISTINCT 构象以招募和保留不同结构的药物。

Project Summary P-glycoprotein pumps drugs, xenobiotics and nutrients out of cells via a partially characterized ATP-dependent mechanism. Due to the extreme substrate promiscuity of P-gp, it contributes to the disposition of nearly all small molecule drugs and to drug-drug interactions. P-gp may be particularly important in cancer cell drug resistance due to its over expression in several cancers. The aims of this proposal are to fill knowledge gaps in three distinct aspects of P-gp mechanism. Each aim shares the common mechanistic element of conformational dynamics. The first aim is to define the P-gp conformations at low ATP occupancy in order to understand how they control downstream conformational changes that are essential for substrate efflux, and how they are different in the presence vs. the absence of substrates. These conformational differences are hypothesized to be essential for minimizing wasteful expenditure of ATP in the absence of transportable substrate and to control proper conformational access in downstream steps. These differences will be identified by rapid, pre-equilibrium, kinetic methods with state- of-the-art kinetic modeling and Hydrogen/Deuterium Exchange Mass Spectrometry (H/DX) with P-gp in lipid nanodiscs. The second aim is to understand the extreme sensitivity of P-gp to its lipid environment. The lipid nanodisc platform provides fine control of the lipid bilayer in which P-gp resides. P-gp will be incorporated in nanodiscs with varying ratios of lipids with different head groups or acyl chains that provide different surface charge of bilayer fluidity. The basal- and substrate dependent ATPase activity in the different lipid environments will be correlated with changes in conformation determined by H/DX. The third aim is to determine the mechanism by which P-gp is able to interact with such a wide range of substrates and inhibitors. Substrate promiscuity is an increasingly appreciated behavior for many proteins throughout biology but the molecular basis is poorly defined. Here, pre equilibrium binding kinetics and single molecule fluorescence methods will be used to compare the distribution of binding parameters in P- gp ensembles vs. single P-gp nanodiscs in order to determine whether P-gp exploits interconverting conformations or persistent distinct conformations to recruit and retain drugs of varying structure.