Molecular Modeling of the Human P-glycoprotein Transporter Protein

像许多跨膜蛋白一样，用X射线结晶学确定P-gp的结构被证明是非常困难的。这源于形成足够高质量的晶体所遇到的问题，这些晶体维护蛋白质不同部分的自然物理化学环境，从而保持自然构象。经过多年的努力，与之密切相关的小鼠P-gp蛋白的结构在去年终于出现了。然而，许多问题仍然存在，如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。为了解决这些问题，我们正在努力将所有可用的结晶学和间接实验数据与基于物理化学的数学方法相结合，以产生结构的高级模型。幸运的是，三十多年的研究已经提供了关于P-gp的丰富信息，从中我们可以闪耀出结构信息。除了小鼠的P-gp外，同源蛋白还具有结晶学结构：特别是细菌Sav1866和MSBA脂翻转酶。有用的间接实验数据的例子包括定点突变、自然发生的多态和残基交联的影响。理论、物理化学方法的例子包括研究密切相关的MDR蛋白家族和ABC转运蛋白超家族中的残基保守和极性/疏水性的模式。这些信息有助于预测哪些残基暴露在膜的核心层和头基层，哪些残基排列在孔内，哪些位于两个跨膜域的界面上。为此，我们正在开发同源家族和超家族的宏大序列比对。这一结果还将使相关突变模式的确定成为可能，这有助于识别蛋白质三维结构中邻近的残基群。我们使用我们的人P-gp的三维结构模型来确定在哪里放置电子顺势探针，以实验地确定蛋白质功能周期中的不同构象状态。此外，我们将使用我们的P-gp模型的计算方法来选择核苷酸类似物和标记剂来与之相互作用，并进一步阐明其结构和作用机制。最近，我们用模型解释了实验确定的ATP类似物5‘-氟磺酰基苯甲酰基-5’-腺苷(FSBA)对P-gp作用机制的结合效应。我们还使用了计算方法来更好地解释蛋白质上的膜环境。去年，这些模型被用于设计和解释实验，揭示了P-gp结合口袋的功能灵活性。也就是说，已知底物结合残基的突变确定了允许功能运输的替代亚口袋。

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. 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. This last year, 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.