Dong L M, Wilson C, Wardell M R, Simmons T, Mahley R W, Weisgraber K H, Agard D A
Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 94141-9100.
J Biol Chem. 1994 Sep 2;269(35):22358-65.
Human apolipoprotein (apo) E4 (arginine at residue 112) preferentially associates with very low density lipoproteins (VLDL), and apoE3 (cysteine at 112) associates with high density lipoproteins. It has been postulated that the amino-terminal domain, which contains residue 112, influences the lipoprotein preference by interacting with the carboxyl-terminal domain, which contains the lipid-binding region. To delineate the region in the carboxyl-terminal domain mediating lipoprotein binding and involved in isoform preference, we produced truncated apoE3 and apoE4 variants (terminating at residues 251, 260, 266, or 272) in Escherichia coli and assessed them for lipoprotein association. This analysis suggested that residues 260-272 contain important determinants for complete lipoprotein association and isoform preferences. To determine whether positive charge at residue 112 was an absolute requirement for the apoE4 VLDL preference, we compared the distributions of rabbit apoE (equivalent to apoE3, with cysteine at a position corresponding to 112), canine apoE (arginine at the corresponding site), and cysteamine-treated rabbit apoE (cysteine converted to a positively charged residue). Surprisingly, all distributed like human apoE3, suggesting that positive charge at a position corresponding to 112 was not directly responsible for the isoform preference and that other residues in the amino-terminal domain were involved. To determine which residues were involved, the structure of the apoE4 22-kDa fragment (the amino-terminal two-thirds of the molecule) was determined to 2.5 A by x-ray crystallography. Compared with the known four-helix bundle structure of apoE3, the only significant differences in the apoE4 structure were that glutamic acid 109 formed a salt bridge with arginine 112 and that the arginine 61 side chain was displaced to a new position. Site-directed mutagenesis of glutamic acid 109 in apoE3 and arginine 61 in apoE4 demonstrated that the position of the arginine 61 side chain in apoE4 was critical in determining apoE4 lipoprotein distribution, suggesting that arginine 61 interacted with the carboxyl-terminal domain to direct binding to VLDL.
人类载脂蛋白（apo）E4（第112位残基为精氨酸）优先与极低密度脂蛋白（VLDL）结合，而apoE3（第112位残基为半胱氨酸）则与高密度脂蛋白结合。据推测，包含第112位残基的氨基末端结构域通过与包含脂质结合区域的羧基末端结构域相互作用，影响脂蛋白偏好。为了确定羧基末端结构域中介导脂蛋白结合并参与异构体偏好的区域，我们在大肠杆菌中产生了截短的apoE3和apoE4变体（在第251、260、266或272位残基处终止），并评估它们与脂蛋白的结合情况。该分析表明，第260 - 272位残基包含完全脂蛋白结合和异构体偏好的重要决定因素。为了确定第112位残基处的正电荷是否是apoE4对VLDL偏好的绝对必要条件，我们比较了兔apoE（等同于apoE3，在对应于112的位置为半胱氨酸）、犬apoE（在相应位点为精氨酸）和半胱胺处理的兔apoE（半胱氨酸转化为带正电荷的残基）的分布情况。令人惊讶的是，它们的分布都与人类apoE3相似，这表明对应于112位置的正电荷并非异构体偏好的直接原因，且氨基末端结构域中的其他残基也参与其中。为了确定哪些残基参与其中，通过X射线晶体学将apoE4 22 kDa片段（分子的氨基末端三分之二）的结构解析到了2.5 Å。与已知的apoE3四螺旋束结构相比，apoE4结构中唯一显著的差异是谷氨酸109与精氨酸112形成了盐桥，且精氨酸61的侧链移位到了一个新位置。对apoE3中的谷氨酸109和apoE4中的精氨酸61进行定点诱变表明，apoE4中精氨酸61侧链的位置对于确定apoE4脂蛋白分布至关重要，这表明精氨酸61与羧基末端结构域相互作用以指导与VLDL的结合。
