Molecular Modeling of the Human P-glycoprotein Transporter Protein

像许多跨膜蛋白一样，用x射线晶体学测定P-gp的结构被证明是非常困难的。这源于形成足够质量的晶体所遇到的问题，以维持蛋白质不同部分的天然物理化学环境，从而维持天然构象。经过多年的努力，与之密切相关的小鼠P-gp蛋白的结构在去年得以实现。然而，关于晶体结构与体内蛋白质的关系有多密切，以及作为转运功能的一部分，构象如何变化，仍然存在许多问题。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，超过三十年的研究已经提供了关于P-gp的丰富信息，从中我们可以获得结构信息。除了小鼠P-gp外，同源蛋白的晶体学结构也可用：特别是细菌Sav1866和MsbA脂质翻转酶。有用的间接实验数据的例子包括位点定向诱变、自然发生的多态性和残基交联的影响。理论的、基于物理化学的方法的例子包括检查密切相关的MDR蛋白家族和ABC转运蛋白超家族中的残基保存和极性/疏水性模式。这些信息有助于预测哪些残基暴露在膜的核心和头群层，哪些残基排列在孔中，哪些在两个跨膜结构域的界面上。为此，我们正在开发同源家族和超家族的大序列比对。这一结果也将有助于确定相关突变的模式，这有助于识别蛋白质三维结构中近端的残基组。最近，我们使用人类P-gp的三维结构模型来确定在何处放置电子顺磁探针，以实验确定蛋白质在功能循环中的不同构象状态。

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.