Molecular Modeling of the Human P-glycoprotein Transporter Protein

与许多跨膜蛋白一样，通过X射线测定P-gp的结构 晶体学已经证明是非常困难的。这源于成型时遇到的问题 保持天然的物理化学环境， 蛋白质的不同部分，以及天然构象。经过多年的 通过努力，已经获得了密切相关的小鼠P-gp蛋白的结构。然而，在这方面， 许多问题仍然是晶体结构与体内蛋白质的关系有多密切， 以及作为运输功能的一部分，构象如何变化。解决这些 问题，我们正在努力整合所有可用的X射线晶体和电子显微镜 确定和间接的实验数据与物理化学为基础的数学方法， 生产先进的结构模型。幸运的是，三十多年的研究 提供了丰富的关于P-gp的信息，我们可以从中获得结构信息。在 除小鼠P-gp外，还可从同源蛋白获得晶体结构： 特别是细菌Sav1866和MsbA脂质翻转酶。有用的间接例子 实验数据包括定点诱变、天然发生的 多态性和残基交联。理论的、基于物理化学的 方法包括检查残基保守性和极性/疏水性的模式 在密切相关的MDR蛋白家族和ABC转运蛋白超家族中。 该信息有助于预测哪些残基暴露于蛋白质的核心和头基层。 膜，残留物衬在孔中，并且位于两者的界面处 跨膜结构域。为此，我们正在开发一个大的同源序列比对， 家庭和超家庭。这样做的结果也将有助于确定 相关突变的模式，这有助于确定在基因组中邻近的残基组。 蛋白质的三维结构我们用我们的三维人体结构模型 P-gp决定在哪里放置电子顺磁探针， 在蛋白质的功能循环中的不同构象状态。另外我们 将使用计算方法与我们的P-gp模型来选择核苷酸类似物和标记 试剂相互作用，并进一步阐明结构和功能机制。最 最近，我们用这些模型来解释实验确定的结合效应， ATP类似物5'-氟磺酰基苯甲酰基-5'-腺苷（FSBA）的作用机制 的P-gp。我们还使用了计算方法来更好地解释膜 环境对蛋白质最近，这些模型被用来设计和解释实验 这揭示了P-gp结合口袋的功能灵活性。也就是说， 已知的底物结合残基鉴定了允许功能性结合的替代的子口袋， 运输我们还使用计算方法来预测抗体与 人Pgp的细胞外环。

Like many transmembrane proteins, determination of the structure of P-gp by X-ray crystallography has proven very difficult. This stems from the problems encountered forming sufficient-quality crystals that maintain the native physiochemical environments for the different parts of the protein, and thus the native conformations. After many years of endeavor, a structure of the closely-related mouse P-gp protein has become available. However, many questions remain as to how close the crystal structure relates to the protein in vivo, and how the conformation changes as part of the transport function. To address these questions, we are striving to integrate all available X-ray crystal and electron microscopy determined and indirect experimental data with physiochemically-based mathematical methods to produce advanced models of the structures. Fortunately, over three decades of study has provided a wealth of information about P-gp from which we can gleam structural information. In addition to mouse P-gp, crystallographic structures are available from homologous proteins: especially bacterial Sav1866 and the MsbA lipid flippase. Examples of useful indirect experimental data include the effects of site-directed mutagenesis, naturally occurring polymorphisms, and residue cross-linking. Examples of theoretical, physiochemically-based methods include examining the patterns of residue conservation and polarity/hydrophobicity within the family of closely related MDR proteins and the superfamily of ABC transporters. This information helps predict which residues are exposed to the core and headgroup layers of the membrane, which residues line the pore, and which are at the interfaces of the two transmembrane domains. To this end, we are developing a grand sequence alignment of homologous families and the superfamily. The results of this will also enable the determination of patterns of correlated mutations, which help identify groups of residues that are proximal in the 3-dimensional structure of the protein. We have used our 3-D structural modelling of human P-gp to determine where to put electron paramagentic probes to experimentally determine different conformational states over the functional cycle of the protein. Additionally, we will use computational methods with our P-gp models to select nucleotide analogs and labeling agents to interact with and further elucidate the structure and functional mechanisms. Most recently, we have use the models to explain the experimentally-determined binding-effects of 5'-fluorosulfonylbenzonyl-5'-adenosine (FSBA), an ATP analogue, on the functional mechanisms of P-gp. We have also used computational methods to better account for the membrane environment on the protein. Recently, the models were used to design and interpret experiments that revealed the functional flexibility of the P-gp binding pocket. That is, mutation of known substrate-binding residues identified alternative, sub-pockets that allow for functional transport. We have also used computational methods to predict the binding of antibodies to the extracellular loops of human Pgp.