Nguyen David, Dhanasekaran Padmaja, Phillips Michael C, Lund-Katz Sissel
Lipid Research Group, Division of Gastroenterology, Hepatology and Nutrition, The Children's Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-4318, USA.
Biochemistry. 2009 Apr 7;48(13):3025-32. doi: 10.1021/bi9000694.
The exchangeability of apolipoprotein (apo) E between lipoprotein particles such as very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) is critical for lipoprotein metabolism, but despite its importance, the kinetics and mechanism of apoE-lipoprotein interaction are not known. We have used surface plasmon resonance (SPR) to monitor in real time the reversible binding of apoE to human VLDL and HDL(3); biotinylated lipoproteins were immobilized on a streptavidin-coated SPR sensor chip, and solutions containing various human apoE molecules at different concentrations were passed across the surface. Analysis of the resultant sensorgrams indicated that the apoE3-lipoprotein interaction is a two-step process. After an initial interaction, the second slower step involves opening of the N-terminal helix bundle domain of the apoE molecule. Destabilization of this domain leads to more rapid interfacial rearrangement which is seen when the lipoprotein binding of apoE4 is compared to that of apoE3. The resultant differences in interfacial packing seem to underlie the differing abilities of apoE4 and apoE3 to bind to VLDL and HDL(3). The measured dissociation constants for apoE binding to these lipoprotein particles are in the micromolar range. C-Terminal truncations of apoE to remove the lipid binding region spanning residues 250-299 reduce the level of binding to both types of lipoprotein, but the effect is weaker with HDL(3); this suggests that protein-protein interactions are important for apoE binding to this lipoprotein while apoE-lipid interactions are more significant for VLDL binding. The two-step mechanism of lipoprotein binding exhibited by apoE is likely to apply to other members of the exchangeable apolipoprotein family.
载脂蛋白（apo）E在极低密度脂蛋白（VLDL）和高密度脂蛋白（HDL）等脂蛋白颗粒之间的交换能力对于脂蛋白代谢至关重要，但尽管其很重要，apoE与脂蛋白相互作用的动力学和机制仍不清楚。我们使用表面等离子体共振（SPR）实时监测apoE与人VLDL和HDL(3)的可逆结合；将生物素化的脂蛋白固定在链霉亲和素包被的SPR传感器芯片上，使含有不同浓度各种人apoE分子的溶液通过该表面。对所得传感图的分析表明，apoE3与脂蛋白的相互作用是一个两步过程。在初始相互作用之后，第二个较慢的步骤涉及apoE分子N端螺旋束结构域的打开。该结构域的不稳定导致更快速的界面重排，这在比较apoE4与apoE3的脂蛋白结合时可以看到。由此产生的界面堆积差异似乎是apoE4和apoE3与VLDL和HDL(3)结合能力不同的基础。测量得到的apoE与这些脂蛋白颗粒结合的解离常数在微摩尔范围内。对apoE进行C端截短以去除跨越250 - 299位残基的脂质结合区域，会降低与两种脂蛋白的结合水平，但对HDL(3)的影响较弱；这表明蛋白质 - 蛋白质相互作用对于apoE与这种脂蛋白的结合很重要，而apoE - 脂质相互作用对于VLDL结合更为显著。apoE表现出的脂蛋白结合两步机制可能适用于可交换载脂蛋白家族的其他成员。
