Gupta Vinita, Narayanaswami Vasanthy, Budamagunta Madhu S, Yamamato Taichi, Voss John C, Ryan Robert O
Center for the Prevention of Obesity, Cardiovascular Disease and Diabetes, Children's Hospital Oakland Research Institute, Oakland, California 94609, USA.
J Biol Chem. 2006 Dec 22;281(51):39294-9. doi: 10.1074/jbc.M608085200. Epub 2006 Nov 1.
Apolipoprotein E (apoE) serves as a ligand for the low density lipoprotein receptor (LDLR) only when bound to lipid. The N-terminal domain of lipid-free apoE exists as globular 4-helix bundle that is conferred with LDLR recognition ability after undergoing a lipid binding-induced conformational change. To investigate the structural basis for this phenomenon, site-directed spin label electron paramagnetic resonance spectroscopy experiments were conducted, focusing on the region near the C-terminal end of helix 4 (Ala-164). Using C112S apoE-N-terminal as template, a series of single cysteine substitution variants (at sequence positions 161, 165, 169, 173, 176, and 181) were produced, isolated, and labeled with the nitroxide probe, methane thiosulfonate. Electron paramagnetic resonance analysis revealed that lipid association induced fixed secondary structure in a region of the molecule known to exist as random coil in the lipid-free state. In a complementary approach, site-directed fluorescence analysis using an environmentally sensitive probe indicated that the lipid-induced transition of this region of the protein to alpha helix was accompanied by relocation to a more hydrophobic environment. In studies with full-length apoE single Cys variants, a similar random coil to stable backbone transition was observed, consistent with the concept that lipid interaction induced an extension of helix 4 beyond the boundary defining its lipid-free conformation. This structural transition likely represents a key conformational change necessary for manifestation of the LDLR recognition properties of apoE.
载脂蛋白E（apoE）只有在与脂质结合时才作为低密度脂蛋白受体（LDLR）的配体。无脂质的apoE的N端结构域以球状4螺旋束形式存在，在经历脂质结合诱导的构象变化后获得LDLR识别能力。为了研究这一现象的结构基础，进行了定点自旋标记电子顺磁共振光谱实验，重点关注螺旋4（Ala-164）C端附近的区域。以C112S apoE-N端为模板，产生、分离了一系列单半胱氨酸替代变体（序列位置为161、165、169、173、176和181），并用氮氧化物探针甲硫基磺酸盐进行标记。电子顺磁共振分析表明，脂质结合在分子的一个区域诱导了固定的二级结构，该区域在无脂质状态下以无规卷曲形式存在。作为一种补充方法，使用环境敏感探针的定点荧光分析表明，脂质诱导的该蛋白质区域向α螺旋的转变伴随着向更疏水环境的重新定位。在对全长apoE单半胱氨酸变体的研究中，观察到了类似的从无规卷曲到稳定主链的转变，这与脂质相互作用诱导螺旋4延伸超出其无脂质构象定义边界的概念一致。这种结构转变可能代表了apoE表现出LDLR识别特性所必需的关键构象变化。
