Lund-Katz S, Zaiou M, Wehrli S, Dhanasekaran P, Baldwin F, Weisgraber K H, Phillips M C
Joseph Stokes Jr. Research Institute, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104-4318, USA.
J Biol Chem. 2000 Nov 3;275(44):34459-64. doi: 10.1074/jbc.M005265200.
Lysines in apolipoprotein (apo) E are key factors in the binding of apoE to the low density lipoprotein receptor, and high affinity binding requires that apoE be associated with lipid. To gain insight into this effect, we examined the microenvironments of the eight lysines in the 22-kDa fragment of apoE3 (residues 1-191) in the lipid-free and lipid-associated states. As shown by (1)H,(13)C heteronuclear single quantum coherence nuclear magnetic resonance, lysine resonances in the lipid-free fragment were poorly resolved over a wide pH range, whereas in apoE3.dimyristoyl phosphatidylcholine (DMPC) discs, the lysine microenvironments and protein conformation were significantly altered. Sequence-specific assignments of the lysine resonances in the spectrum of the lipidated 22-kDa fragment were made. In the lipid-free protein, six lysines could be resolved, and all had pK(a) values above 10. In apoE3.DMPC complexes, however, all eight lysines were resolved, and the pK(a) values were 9.2-11.1. Lys-143 and Lys-146, both in the receptor binding region in helix 4, had unusually low pK(a) values of 9.5 and 9.2, respectively, likely as a result of local increases in positive electrostatic potential with lipid association. Shift reagent experiments with potassium ferricyanide showed that Lys-143 and Lys-146 were much more accessible to the ferricyanide anion in the apoE3.DMPC complex than in the lipid-free state. The angle of the nonpolar face of helix 4 is smaller than the angles of helices 1, 2, and 3, suggesting that helix 4 cannot penetrate as deeply into the DMPC acyl chains at the edge of the complex and that its polar face protrudes from the edge of the disc. This increased exposure and the greater positive electrostatic potential created by interaction with DMPC may explain why lipid association is required for high affinity binding of apoE to the low density lipoprotein receptor.
载脂蛋白(apo)E中的赖氨酸是apoE与低密度脂蛋白受体结合的关键因素,而高亲和力结合要求apoE与脂质相关。为深入了解这种效应,我们研究了apoE3(残基1 - 191)22 kDa片段中八个赖氨酸在无脂质和脂质结合状态下的微环境。如通过(1)H、(13)C异核单量子相干核磁共振所示,在宽pH范围内,无脂质片段中的赖氨酸共振峰分辨率较差,而在apoE3.二肉豆蔻酰磷脂酰胆碱(DMPC)圆盘状结构中,赖氨酸微环境和蛋白质构象发生了显著改变。对脂质化22 kDa片段光谱中的赖氨酸共振峰进行了序列特异性归属。在无脂质蛋白中,六个赖氨酸可以分辨,且所有赖氨酸的pK(a)值均高于10。然而,在apoE3.DMPC复合物中,所有八个赖氨酸都能分辨,pK(a)值为9.2 - 11.1。位于螺旋4受体结合区域的Lys - 143和Lys - 146的pK(a)值异常低,分别为9.5和9.2,这可能是由于与脂质结合后局部正静电势增加所致。用铁氰化钾进行的位移试剂实验表明,在apoE3.DMPC复合物中,Lys - 143和Lys - 146比在无脂质状态下更容易被铁氰根阴离子接近。螺旋4非极性面的角度小于螺旋1、2和3的角度,这表明螺旋4在复合物边缘不能像其他螺旋那样深入DMPC酰基链,且其极性面从圆盘边缘突出。这种增加的暴露以及与DMPC相互作用产生的更大正静电势可能解释了为什么apoE与低密度脂蛋白受体的高亲和力结合需要脂质结合。