Modeling the structure and functional mechanisms of P-glycoprotein

P-糖蛋白的结构和功能机制建模

• 批准号: 7592960
• 负责人: HOMER ROBERT GUY
• 金额: $ 19.9万
• 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/7592960
• 关键词: 3-Dimensional; ATP Hydrolysis; ATP-Binding Cassette Transporters; Amino Acid Sequence; Antineoplastic Agents; Bacterial Proteins; Binding; Cells; Cellular biology; Chemotherapy-Oncologic Procedure; Computer software; Crystallization; Data; Depth; Effectiveness; Electron Microscopy; Electrons; Environment; Family; Genetic Polymorphism; Goals; Head; Helix (Snails); Homologous Gene; Homology Modeling; Human; Integral Membrane Protein; Knowledge; Laboratories; Lipids; Maps; Measures; Membrane; Membrane Proteins; Methods; Microscopy; Modeling; Molecular Conformation; Multi-Drug Resistance; Mutation; Nucleotides; Numbers; P-Glycoprotein; P-Glycoproteins; Pattern; Peptides; Positioning Attribute; Process; Proteins; Publishing; Resistance; Resolution; Roentgen Rays; Score; Sequence Alignment; Site-Directed Mutagenesis; Source; Specific qualifier value; Standards of Weights and Measures; Structure; Techniques; Testing; Transmembrane Domain; Vertebral column; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; crosslink; data modeling; density; design; ear helix; efflux pump; electron density; experience; inhibitor/antagonist; member; molecular modeling; research study; small molecule; stem; three dimensional structure

Like many transmembrane proteins, determination of the structure of P-gp by X-ray crystallography has proven elusive despite much effort. This stems from the difficulty of forming sufficient-quality crystals that also maintain the native physiochemical environments for the different parts of the protein. Thus, in lieu of the direct, experimental determination, we strive to integrate all available (often indirect) experimental data with physiochemically-based mathematical methods to produce one or more physically realistic models of the structure. Fortunately, over three decades of study has provided a wealth of information about P-gp from which we can gleam structural information. On a broad scale, it is known that the protein is composed of two homologous domains, each with a six-segment transmembrane component and a nucleotide-binding component. To date, the best two sources of structural information about P-gp are an X-ray crystal structure of the homologous bacterial protein Sav1866, and low-resolution cryo-electron micrographs of human P-gp. In addition, the corrected X-ray structure of the bacterial lipid flippase MsbA is expected soon, which is even closely related to P-gp than is Sav1866. Thus, one major focus of our work is to develop a homology model of human P-gp using the crystal structures of the bacterial proteins as templates. While such a model has recently been published by Peter Tielemans group, it was only based on a simple alignment of the P-gp and Sav1866 sequences, and unfortunately, this is overall not well defined for the transmembrane segments. Rather, our efforts go deeper into examining the patterns of residue conservation 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. We are currently in the process of 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. Finally, we will examine the resultant model for consistency with all the experimental data, such as the effects of site-directed mutagenesis, naturally occurring polymorphisms, and cross-linking data. Where the model based on the Sav1866 template fails to explain the experimental results, we will search for alternate conformations that bring it into compliance. The other major focus is to develop models from the density maps obtained from electron microscopy. This has the advantage that the structural data is directly from human P-gp, the target protein, and not from a bacterial homolog, which likely differs in structure to some degree. This includes the fact that the two transmembrane domains of human P-gp are different in sequence, and thus are asymmetrical around the approximate two-fold axis of the pore, while the bacterial homologs only contain one domain, and thus form perfectly symmetrical homodimers in the membrane. We have already contacted the group that published the microscopy data, and have obtained their coordinates for standard protein helices and nucleotide-binding domains fitted to the electron density. However, due to the low resolution of the data, these models only specify the peptide backbone, and not the type of the residue at each position. To solve this, we are embarking on a threading project, which predicts the correct alignment of the amino acid sequence on the structure of the backbone. To this end, we are currently adapting our previously developed threading software to the P-gp protein. Each enumerated alignment of sequence to structure will be scored and ranked according to a number of criteria. As described above, this will again measure compliance with experimental data, correct physiochemical environments for the different types of residues, and whether conserved residues and predicted clusters are proximal in space. In addition, this analysis will include the calculated energy from scales of residue contact potentials specifically derived for membrane proteins

像许多跨膜蛋白一样，尽管付出了很多努力，但通过X射线晶体学确定P-gp的结构已被证明是难以捉摸的。这是因为很难形成具有良好品质的晶体，同时也很难为蛋白质的不同部分保持天然的理化环境。因此，代替直接的实验测定，我们努力将所有可用的（通常是间接的）实验数据与基于物理化学的数学方法相结合，以产生一个或多个物理上真实的结构模型。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。在广泛的范围内，已知该蛋白由两个同源结构域组成，每个结构域具有六段跨膜组分和核苷酸结合组分。迄今为止，关于P-gp的结构信息的最佳两个来源是同源细菌蛋白Sav 1866的X射线晶体结构和人P-gp的低分辨率冷冻电子显微照片。此外，预计不久将获得细菌脂质翻转酶MsbA的校正X射线结构，其与P-gp的关系甚至比Sav 1866更密切。因此，我们的工作的一个主要重点是开发一个同源模型的人P-gp使用的晶体结构的细菌蛋白作为模板。虽然Peter Tielemans小组最近发表了这样的模型，但它仅基于P-gp和Sav 1866序列的简单比对，不幸的是，这对于跨膜片段总体上没有很好地定义。相反，我们的努力更深入地研究密切相关的MDR蛋白家族和ABC转运蛋白超家族内的残基保守模式。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。我们目前正在开发一个大的同源家族和超家族的序列比对的过程中。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。最后，我们将检查所得模型与所有实验数据的一致性，如定点诱变，自然发生的多态性和交联数据的影响。当基于Sav 1866模板的模型无法解释实验结果时，我们将寻找使其符合的替代构象。另一个主要的焦点是从电子显微镜获得的密度图开发模型。其优点是结构数据直接来自人P-gp（靶蛋白），而不是来自细菌同源物，后者可能在结构上存在一定程度的差异。这包括以下事实：人P-gp的两个跨膜结构域在序列上是不同的，因此围绕孔的近似两倍轴是不对称的，而细菌同源物仅包含一个结构域，因此在膜中形成完全对称的同源二聚体。我们已经联系了发表显微镜数据的小组，并获得了他们的标准蛋白质螺旋和符合电子密度的核苷酸结合结构域的坐标。然而，由于数据的低分辨率，这些模型仅指定肽骨架，而不是每个位置处的残基类型。为了解决这个问题，我们正在进行一个线程项目，该项目预测骨架结构上氨基酸序列的正确对齐。为此，我们目前正在调整我们以前开发的线程软件的P-gp蛋白。将根据多个标准对序列与结构的每个枚举比对进行评分和排序。如上所述，这将再次测量与实验数据的符合性，针对不同类型的残基校正生理化学环境，以及保守残基和预测簇在空间上是否接近。此外，该分析还将包括从膜蛋白特异性衍生的残基接触电位标度计算的能量