Zhu Jian, Wei Sen, Huang Linchen, Zhao Qi, Zhu Haichao, Zhang Anwei
Department of Vascular Surgery, The Affiliated Hospital of Jiangsu University (Kunshan 1st People's Hospital), Kunshan, 215300, China.
Department of Vascular Surgery, The Affiliated Hospital of Jiangsu University (Kunshan 1st People's Hospital), Kunshan, 215300, China.
J Mol Graph Model. 2020 Jan;94:107455. doi: 10.1016/j.jmgm.2019.107455. Epub 2019 Sep 25.
The human plasma cholesteryl ester transfer protein (CETP) collects triglycerides from very-/low-density lipoproteins (V/LDL) and exchanges them for cholesteryl esters from high-density lipoproteins (HDL), which has recognized as an important therapeutic target for atherosclerosis. The protein has a C-terminal amphipathic α-helix that serves as self-binding peptide to fulfill biological function by dynamically binding to/unbinding from its cognate site (termed self-binding site) in the same protein. Previously, we successfully derived and halogenated the helical peptide to competitively disrupt the self-binding behavior of CETP C-terminal tail. However, the halogenated peptides have only a limited affinity increase as compared to native helical peptide (∼3-fold), thus exhibiting only a moderate competitive potency. Here, instead of optimizing the direct intermolecular interaction of peptide with CETP self-binding site we attempt to further improve the peptide competitive potency by reducing its conformational flexibility with hydrocarbon-stapling technique. Computational analysis reveals that the helical peptide has large intrinsic disorder in unbound free state, which would incur a considerable entropy penalty upon rebinding to the self-binding site. All-hydrocarbon bridge is designed and optimized on native and halogenated peptides in terms of the helical pattern and binding mode of self-binding peptide. Dynamics simulation and circular dichroism indicate that the stapling can considerably reduce peptide disorder in free state. Energetics calculation and fluorescence assay conform that the binding affinity of stapled/halogenated peptides is improved substantially (by > 5-fold), thus exhibiting an effective competition potency with native peptide for the self-binding site. Structural examination suggests that the binding modes and nonbonded interactions of native and halogenated peptides are not influenced essentially due to the stapling.
人血浆胆固醇酯转运蛋白（CETP）从极低密度脂蛋白（V/LDL）中收集甘油三酯，并将其与高密度脂蛋白（HDL）中的胆固醇酯进行交换，CETP已被认为是动脉粥样硬化的一个重要治疗靶点。该蛋白具有一个C端两亲性α螺旋，作为自身结合肽，通过在同一蛋白中与其同源位点（称为自身结合位点）动态结合/解离来实现生物学功能。此前，我们成功地衍生并卤化了该螺旋肽，以竞争性地破坏CETP C端尾部的自身结合行为。然而，与天然螺旋肽相比，卤化肽的亲和力仅有限增加（约3倍），因此仅表现出中等的竞争效力。在这里，我们不是优化肽与CETP自身结合位点的直接分子间相互作用，而是尝试通过烃钉合技术降低其构象灵活性来进一步提高肽的竞争效力。计算分析表明，螺旋肽在未结合的自由状态下具有很大的内在无序性，重新结合到自身结合位点时会产生相当大的熵罚。根据自身结合肽的螺旋模式和结合模式，在天然肽和卤化肽上设计并优化了全烃桥。动力学模拟和圆二色性表明，钉合可以显著降低自由状态下肽的无序性。能量计算和荧光测定证实，钉合/卤化肽的结合亲和力得到了显著提高（>5倍），因此在自身结合位点上与天然肽表现出有效的竞争效力。结构检查表明，由于钉合，天然肽和卤化肽的结合模式和非键相互作用基本不受影响。
