Bridging the gap between structural bioinformatics and receptor research: the membrane-embedded, ligand-gated, P2X glycoprotein receptor.
Bridging the gap between structural bioinformatics and receptor research: the membrane-embedded, ligand-gated, P2X glycoprotein receptor.
复制标题
弥合结构生物信息学和受体研究之间的差距：膜嵌入、配体门控的 P2X 糖蛋白受体。
DOI：
10.2174/1568026043387197
复制
发表时间：
2004
影响因子：
3.4
通讯作者：
P. Illés
中科院分区：
文献类型：
--
作者：
P. Mager;A. Weber;P. Illés
动机
在原子分辨率水平上尚不清楚 P2X 受体结构的细节。使用基于比较同源性的分子建模和线程，尝试预测 P2X 受体的三维结构。然而，这一预测无法实现，因为 P2X 家族的重要特性与潜在模板蛋白的重要特性有很大不同。本文回顾了一种由三个研究领域组成的替代方法：生物信息学、结构建模和各种生物实验结果。
型号
起点是氨基酸序列。使用顺序数据，第一步是二级结构预测。由此产生的二级结构被转换成三维几何形状。然后，利用量子化学RHF/3-21G最小基本集和全原子分子力学AMBER96力场对二级和三级结构进行优化。通过合适的电介质模拟膜嵌入蛋白质的折叠。使用共轭梯度最小化器（Polak-Ribiere 方法的 Fletcher-Reeves 修改）对结构进行细化。通过拉曼钱德兰图、旋转异构体分析、全原子接触点和 C(β) 偏差检查几何优化的结果。作为模型构建的附加工具，使用多重比对分析和比较序列功能分析。该方法以膜嵌入的配体门控 P2X3 受体亚基为例，该亚基是一种单价-二价阳离子通道形成糖蛋白，由细胞外腺苷 5'-三磷酸激活。根据这些结果，提出了 P2X3 受体亚基的成孔基序的拓扑结构。据信，功能齐全的 P2X 通道需要 (i) 两个不同的肽模块、细胞外发生的 ATP 结合模块和包括长跨膜和短细胞内部分的孔模块、(ii) 与膜的相互作用表面、以及 (iii) 残基和水合阳离子的氢键力之间的精确耦合。此外，本文还论证了定量构效关系 (QSAR) 在 P2X 研究中的作用（野生型的钙离子渗透性和大鼠 P2X2 受体蛋白定点诱变后的钙离子渗透性，KN-62 类似物作为人 P2X7 受体的竞争性拮抗剂）。实验证明：这些预测可以通过实验进行检验，并且可以为文献中发表的实验观察结果提供额外的解释。特别是，几何优化的P2X3结构与通过神经生理学、生化、药理学和突变实验获得的实验提出的P2X受体模型具有良好的一致性。尽管大鼠 P2X3 受体亚基（397 个氨基酸）比 KcsA 蛋白（160 个氨基酸）更复杂，但肽主链原子的整体折叠相似。
局限性
为了避免语义混淆，应该注意“预测”是在概率意义上定义的。与通用规则的匹配并不意味着“这是真的”，而是“这可能是真的”。只有生物和化学知识才能确定这些预测是否有意义。因此，计算工具的结果是概率预测，需要进一步的实验验证。
可用性
学术研究人员可通过电子邮件请求（PDB 格式）免费获得几何优化的 P2X3 受体亚基。
MOTIVATION
No details on P2X receptor architecture had been known at the atomic resolution level. Using comparative homology-based molecular modelling and threading, it was attempted to predict the three-dimensional structure of P2X receptors. This prediction could not be carried out, however, because important properties of the P2X family differ considerably from that of the potential template proteins. This paper reviews an alternative approach consisting of three research fields: bioinformatics, structural modelling, and a variety of the results of biological experiments.
MODEL
Starting point is the amino acid sequence. Using the sequential data, the first step is a secondary structure prediction. The resulting secondary structure is converted into a three-dimensional geometry. Then, the secondary and tertiary structures are optimized by using the quantum chemistry RHF/3-21G minimal basic set and the all-atom molecular mechanics AMBER96 force field. The fold of the membrane-embedded protein is simulated by a suitable dielectricum. The structure is refined using a conjugate gradient minimizer (Fletcher-Reeves modification of the Polak-Ribiere method). The results of the geometry optimization were checked by a Ramanchandran plot, rotamer analysis, all-atom contact dots, and the C(beta) deviation. As additional tools for the model building, multiple alignment analysis and comparative sequence-function analysis were used. The approach is exemplified on the membrane-embedded, ligand-gated P2X3 receptor subunit, a monovalent-bivalent cation channel-forming glycoprotein that is activated by extracellular adenosine 5'-triphosphate. From these results, a topology of the pore-forming motif of the P2X3 receptor subunit was proposed. It is believed that a fully functional P2X channel requires a precise coupling between (i) two distinct peptide modules, an extracellularly occurring ATP-binding module and a pore module that includes a long transmembrane and short intracellular part, (ii) an interaction surface with membranes, and (iii) hydrogen bonding forces of the residues and hydrated cations. Furthermore, this paper demonstrates the role of quantitative structure-activity relationships (QSARs) in P2X research (calcium ion permeability of the wild-type and after site-directed mutagenesis of the rat P2X2 receptor protein, KN-62 analogs as competitive antagonists of the human P2X7 receptor). EXPERIMENTAL PROOFS: The predictions are experimentally testable and may provide an additional interpretation of experimental observations published in literature. In particular, there is the good agreement of the geometry optimized P2X3 structure with experimentally proposed P2X receptor models obtained by neurophysiological, biochemical, pharmacological, and mutation experiments. Although the rat P2X3 receptor subunit is more complex (397 amino acids) than the KcsA protein (160 amino acids), the overall folds of the peptide backbone atoms are similar.
LIMITATIONS
To avoid semantic confusion, it should be noted that "prediction" is defined in a probabilistic sense. Matches to generic rules do not mean "this is true" but rather "this might be true". Only biological and chemical knowledge can determine whether or not these predictions are meaningful. Thus, the results from the computational tools are probabilistic predictions and subject to further experimental verification.
AVAILABILITY
The geometry optimized P2X3 receptor subunit is freely available for academic researchers on e-mail request (PDB format).