Molecular Modeling of the Human P-glycoprotein Transporter Protein

像许多跨膜蛋白一样，通过X射线晶体学确定P-gp的结构已被证明非常困难。这源于形成保持蛋白质的不同部分的天然生理化学环境以及因此保持天然构象的天然质量晶体所遇到的问题。经过多年的奋进，一个结构的密切相关的小鼠P-gp蛋白已成为去年。然而，许多问题仍然是如何密切的晶体结构与蛋白质在体内，以及如何构象变化的一部分，运输功能。为了解决这些问题，我们正在努力将所有可用的晶体学和间接实验数据与基于物理化学的数学方法相结合，以产生先进的结构模型。幸运的是，三十多年来的研究提供了丰富的P-gp信息，从中我们可以隐约看到结构信息。除了小鼠P-gp，晶体结构可从同源蛋白获得：特别是细菌Sav 1866和MsbA脂质翻转酶。有用的间接实验数据的实例包括定点诱变、天然存在的多态性和残基交联的影响。理论上的，基于生理化学的方法的例子包括检查模式的残基保守性和极性/疏水性的家庭密切相关的MDR蛋白和ABC转运蛋白的超家族内。这些信息有助于预测哪些残基暴露于膜的核心和头基层，哪些残基排列在孔中，以及哪些残基位于两个跨膜结构域的界面处。为此，我们正在开发同源家族和超家族的大序列比对。其结果还将能够确定相关突变的模式，这有助于鉴定蛋白质三维结构中邻近的残基组。最近，我们已经使用我们的3-D结构建模的人类P-gp，以确定在哪里把电子顺磁性探针实验确定不同的构象状态的蛋白质的功能周期。今年，我们还使用计算方法与我们的P-gp模型，以选择核苷酸类似物和标记剂相互作用，并进一步阐明结构和功能机制。

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 this last year. 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 crystallographic 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. Most recently 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. This year we have also used 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.