Structural analysis and rational design of orthogonal stacking system in an E. coli DegP PDZ1-peptide complex
Structural analysis and rational design of orthogonal stacking system in an E. coli DegP PDZ1-peptide complex
复制标题
大肠杆菌 DegP PDZ1-肽复合物正交堆叠系统的结构分析和合理设计
DOI：
10.1007/s11696-019-00797-8
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发表时间：
2019
期刊：
影响因子：
2.2
通讯作者：
Liu Lijun
中科院分区：
文献类型：
--
作者：
Zhang Dingwa;He Deyong;Pan Xiaoliang;Xu Yaping;Liu Lijun
DegP是清除大肠杆菌（Escherichia coli，E. coli）内膜和周质中变性或聚集成分的必需蛋白。coli）。该酶含有两个调节PDZ结构域，其已被证明通过与底物蛋白的C-末端疏水延伸结合而充当底物特异性决定簇。这里，E.利用肽移植、虚拟诱变和QM/MM计算对具有噬菌体展示的C3 H1五肽的coliDegP PDZ 1结构域进行建模和检查。在结构域-肽复合物界面处确定了正交堆积系统，其由结构域阳离子残基R325分别与肽芳香残基Trp− 1和Phe−4形成的T形阳离子-π堆积（t堆积）和非阳离子-π堆积（p堆积）组成。在-堆积和p-堆积之间观察到协同效应;π电子共轭是主要负责协同效应。随后，这两个肽的芳香族残基被系统地替换为其他芳香族氨基酸以及非芳香族丙氨酸以优化协同效应，由此野生型C3 H1肽和七种变体对E的结合亲和力。coliDegP PDZ 1结构域使用基于荧光的测定法在微摩尔水平下测定。实验结合亲和力和计算的结合能之间的良好的线性相关性推导出，与皮尔逊的相关系数为0.928。芳香取代的Phe− 4 Tyr可以显著提高肽结合效力8.7倍，而非芳香取代的每一个邻-堆叠和p-堆叠或两者都可以大大降低肽亲和力20.7倍（Phe−4Ala），82.1倍（Trp −1Ala）和110.7倍（Trp−1Ala/Phe−4Ala）。
The DegP is essential for clearance of denatured or aggregated components from the inner-membrane and periplasmic space inEscherichia coli(E. coli). The enzyme contains two regulatory PDZ domains that have been shown to act as substrate specificity determinant by binding to the C-terminal hydrophobic stretch of substrate proteins. Here, the complex structure ofE. coliDegP PDZ1 domain with a phage-displayed C3H1 pentapeptide is modeled and examined using peptide grafting, virtual mutagenesis, and QM/MM calculation. An orthogonal stacking system is identified at the domain–peptide complex interface, which consists of a T-shaped cation-π stacking (t-stacking) and a paralleled cation-π stacking (p-stacking) formed from domain cationic residue R325 to peptide aromatic residues Trp−1and Phe−4, respectively. A synergistic effect betweent-stacking andp-stacking is observed;π-electron conjugation is primarily responsible for the synergistic effect. Subsequently, the two peptide aromatic residues are systematically replaced by other aromatic amino acids as well as a non-aromatic alanine to optimize the synergistic effect, from which the binding affinities of wild-type C3H1 peptide and seven variants toE. coliDegP PDZ1 domain are determined at micromolar level using fluorescence-based assay. A good linear correlation between experimental binding affinities and calculated binding energies is derived, with a Pearson’s correlation coefficientrp= 0.928. The aromatic Phe−4Tyr substitution can considerably improve peptide binding potency by 8.7-fold, whereas the non-aromatic substitutions at each oft-stacking andp-stacking or both can largely impair the peptide affinity by 20.7-fold (Phe−4Ala), 82.1-fold (Trp−1Ala) and 110.7-fold (Trp−1Ala/Phe−4Ala).