Molecular Modeling of the Human P-glycoprotein Transporter Protein

人类 P-糖蛋白转运蛋白的分子模型

• 批准号: 8763757
• 负责人: Stewart Durell
• 金额: $ 7.02万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8763757
• 关键词: 3-Dimensional; ATP-Binding Cassette Transporters; Accounting; Address; Adenosine; Binding; Carrier Proteins; Cells; Chemotherapy-Oncologic Procedure; Computing Methodologies; Data; Effectiveness; Electrons; Environment; Family; Genetic Polymorphism; Goals; Homologous Protein; Human; Hydrolysis; Hydrophobicity; Integral Membrane Protein; Knowledge; Label; Lipids; Membrane; Methods; Modeling; Molecular Conformation; Molecular Models; Multi-Drug Resistance; Mus; Mutation; P-Glycoprotein; Pattern; Proteins; Pump; Sequence Alignment; Site-Directed Mutagenesis; Structural Models; Structure; Substrate Specificity; Transmembrane Domain; X-Ray Crystallography; analog; base; cancer therapy; chemotherapy; crosslink; design; in vivo; inhibitor/antagonist; member; molecular modeling; nucleotide analog; research study; small molecule; stem; three dimensional structure; tripolyphosphate

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.

与许多跨膜蛋白一样，通过 X 射线晶体学测定 P-gp 的结构已被证明非常困难。这是由于形成足够质量的晶体时遇到的问题，以维持蛋白质不同部分的天然理化环境，从而维持天然构象。经过多年的努力，去年，与小鼠密切相关的 P-gp 蛋白的结构已经获得。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，三十多年的研究提供了有关 P-gp 的丰富信息，我们可以从中了解结构信息。除了小鼠 P-gp 之外，还可以从同源蛋白质获得晶体结构：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定点诱变、天然存在的多态性和残基交联的影响。基于理论的、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 转运蛋白超家族内的残基保守性和极性/疏水性模式。该信息有助于预测哪些残基暴露于膜的核心层和头基层，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。该结果还将能够确定相关突变的模式，这有助于识别蛋白质 3 维结构中邻近的残基组。我们使用人类 P-gp 的 3D 结构模型来确定在何处放置电子顺动力探针，从而通过实验确定蛋白质功能循环中的不同构象状态。此外，我们将使用 P-gp 模型的计算方法来选择核苷酸类似物和标记剂来相互作用并进一步阐明其结构和功能机制。最近，我们使用这些模型来解释实验确定的 5'-氟磺酰基苯甲酰基-5'-腺苷 (FSBA)（一种 ATP 类似物）对 P-gp 功能机制的结合效应。我们还使用计算方法来更好地解释蛋白质的膜环境。