Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118
Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118.
J Lipid Res. 2018 Feb;59(2):348-356. doi: 10.1194/jlr.M080986. Epub 2017 Dec 5.
ApoA-I activates LCAT that converts lipoprotein cholesterol to cholesteryl ester (CE). Molecular dynamic simulations suggested earlier that helices 5 of two antiparallel apoA-I molecules on discoidal HDL form an amphipathic tunnel for migration of acyl chains and unesterified cholesterol to the active sites of LCAT. Our recent crystal structure of Δ(185-243)apoA-I showed the tunnel formed by helices 5/5, with two positively charged residues arginine 123 positioned at the edge of the hydrophobic tunnel. We hypothesized that these uniquely positioned residues Arg123 are poised for interaction with fatty acids produced by LCAT hydrolysis of the sn-2 chains of phosphatidylcholine, thus positioning the fatty acids for esterification to cholesterol. To test the importance of Arg123 for LCAT phospholipid hydrolysis and CE formation, we generated apoA-I[R123A] and apoA-I[R123E] mutants and made discoidal HDL with the mutants and WT apoA-I. Neither mutation of Arg123 changed the particle composition or size, or the protein conformation or stability. However, both mutations of Arg123 significantly reduced LCAT catalytic efficiency and the apparent for CE formation without affecting LCAT phospholipid hydrolysis. A control mutation, apoA-I[R131A], did not affect LCAT phospholipid hydrolysis or CE formation. These data suggest that Arg123 of apoA-I on discoidal HDL participates in LCAT-mediated cholesterol esterification.
载脂蛋白 A-I 激活 LCAT，将脂蛋白胆固醇转化为胆固醇酯（CE）。早期的分子动力学模拟表明，两个反平行载脂蛋白 A-I 分子在圆盘状 HDL 上的螺旋 5 形成一个两亲性隧道，用于酰基链和未酯化胆固醇向 LCAT 的活性部位迁移。我们最近的 Δ(185-243)载脂蛋白 A-I 晶体结构显示，隧道由螺旋 5/5 形成，两个带正电荷的残基精氨酸 123 位于疏水性隧道的边缘。我们假设这些位置独特的残基 Arg123 准备与 LCAT 水解磷脂酰胆碱的 sn-2 链产生的脂肪酸相互作用，从而将脂肪酸定位进行胆固醇酯化。为了测试 Arg123 对 LCAT 磷脂水解和 CE 形成的重要性，我们生成了载脂蛋白 A-I[R123A]和载脂蛋白 A-I[R123E]突变体，并使用突变体和 WT 载脂蛋白 A-I 制备了圆盘状 HDL。Arg123 的突变均未改变颗粒组成或大小、蛋白构象或稳定性。然而，Arg123 的两个突变均显著降低了 LCAT 的催化效率和 CE 形成的表观 Km，而不影响 LCAT 磷脂水解。对照突变，载脂蛋白 A-I[R131A]，不影响 LCAT 磷脂水解或 CE 形成。这些数据表明，圆盘状 HDL 上的载脂蛋白 A-I 的 Arg123 参与了 LCAT 介导的胆固醇酯化。
