The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles - A mass spectrometry study
The spatial organization of apolipoprotein A-I on the edge of discoidal high density lipoprotein particles - A mass spectrometry study
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DOI：
10.1074/jbc.m302764200
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发表时间：
2003-07-18
影响因子：
4.8
通讯作者：
Hilliard, GM
中科院分区：
文献类型：
--
作者：
Davidson, WS;Hilliard, GM
在过去的25年里，人们对新生的盘状HDL颗粒上的人apoA-I的三维结构进行了广泛的讨论。最近的证据表明apoA-I的α-螺旋结构域以带状取向排列，螺旋的长轴垂直于盘边缘上的磷脂酰基链。然而，缺乏关于光盘上apoA-I分子之间空间关系的实验信息。为了解决这个问题，我们利用了质谱技术的最新进展，结合可裂解的交联化学，以获得一组适合于测试apoA-I结构模型的距离限制。我们产生了高度均匀的，重组HDL颗粒含有两个分子的apoA-I。这些用硫醇可裂解的交联剂处理，所述交联剂共价连接椎间盘中的apoA-I分子内或之间的紧密接近的赖氨酸残基。然后将交联的盘彻底胰蛋白酶化以产生肽的离散群体。在交联裂解之前和之后通过液相色谱/质谱法分析所得肽，并基于apoA-I的理论胰蛋白酶裂解鉴定所得峰。我们确定了至少8个分子内和7个分子间的颗粒中的交联。利用距离约束分析了apoA-I结构的三种现有模型。结果强烈支持存在的盐桥相互作用，预测发生在apoA-I的“双带”模型，但螺旋发夹模型包含相同的盐桥对接接口也与数据一致。
The three-dimensional structure of human apoA-I on nascent, discoidal HDL particles has been debated extensively over the past 25 years. Recent evidence has demonstrated that the alpha-helical domains of apoA-I are arranged in a belt-like orientation with the long axis of the helices perpendicular to the phospholipid acyl chains on the disc edge. However, experimental information on the spatial relationships between apoA-I molecules on the disc is lacking. To address this issue, we have taken advantage of recent advances in mass spectrometry technology combined with cleavable crosslinking chemistry to derive a set of distance constraints suitable for testing apoA-I structural models. We generated highly homogeneous, reconstituted HDL particles containing two molecules of apoA-I. These were treated with a thiol-cleavable cross-linking agent, which covalently joined Lys residues in close proximity within or between molecules of apoA-I in the disc. The cross-linked discs were then exhaustively trypsinized to generate a discrete population of peptides. The resulting peptides were analyzed by liquid chromatography/mass spectrometry before and after cleavage of the cross-links, and resulting peaks were identified based on the theoretical tryptic cleavage of apoA-I. We identified at least 8 intramolecular and 7 intermolecular cross-links in the particle. The distance constraints are used to analyze three current models of apoA-I structure. The results strongly support the presence of the salt-bridge interactions that were predicted to occur in the "double belt" model of apoA-I, but a helical hairpin model containing the same salt-bridge docking interface is also consistent with the data.