Modeling the structure and functional mechanisms of P-glycoprotein

像许多跨膜蛋白一样，尽管付出了很多努力，但通过X射线结晶学确定P-gp的结构已被证明是难以捉摸的。这是因为很难形成足够高质量的晶体，同时保持蛋白质不同部分的自然物理化学环境。因此，代替直接的实验确定，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。在广泛的范围内，我们知道该蛋白由两个同源结构域组成，每个结构域都有一个六段跨膜成分和一个核苷酸结合成分。到目前为止，关于P-gp结构信息最好的两个来源是同源细菌蛋白Sav1866的X射线晶体结构和人P-gp的低分辨率冷冻电子显微镜。此外，细菌脂翻转酶MSBA的修正X射线结构有望很快公布，它与P-gp的关系甚至比Sav1866更密切。因此，我们工作的一个主要重点是利用细菌蛋白的晶体结构作为模板来建立人P-gp的同源模型。虽然Peter Tielemans小组最近发表了这样的模型，但它只是基于P-gp和Sav1866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的定义。相反，我们的努力更深入地研究了密切相关的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。我们目前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。最后，我们将检查所得到的模型与所有实验数据的一致性，例如定点突变、自然发生的多态和交叉连接数据的影响。如果基于Sav1866模板的模型无法解释实验结果，我们将搜索替代构象以使其符合要求。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。这样做的好处是，结构数据直接来自目标蛋白人类P-gp，而不是来自可能在结构上有所不同的细菌同系物。这包括这样一个事实，即人P-gp的两个跨膜结构域在序列上不同，因此围绕大约两倍孔的轴是不对称的，而细菌同源蛋白只包含一个结构域，从而在膜上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，并获得了标准蛋白质螺旋和与电子密度相适应的核苷酸结合结构域的坐标。然而，由于数据的低分辨率，这些模型只指定了肽骨架，而不是每个位置的残基类型。为了解决这个问题，我们正在进行一个线程项目，该项目预测了骨架结构上的氨基酸序列的正确比对。为此，我们目前正在修改我们之前开发的线程软件，以适应P-gp蛋白。将根据多个标准对每个列举的序列与结构的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遵从性，不同类型残基的正确物理化学环境，以及保守残基和预测的簇是否在空间中邻近。此外，这种分析还将包括从膜蛋白的残基接触电势标度中计算出的能量。

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