Guevara J, Spurlino J, Jan A Y, Yang C Y, Tulinsky A, Prasad B V, Gaubatz J W, Morrisett J D
Department of Medicine, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030.
Biophys J. 1993 Mar;64(3):686-700. doi: 10.1016/S0006-3495(93)81428-0.
The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.
人脂蛋白[a]的蛋白质成分主要由两种载脂蛋白组成，即载脂蛋白[a]和载脂蛋白B-100，它们通过一个或多个胱氨酸二硫键相连。在载脂蛋白B-100的氨基酸序列中，位于纤溶酶原kringle 4样重复序列（3991-4068）内的Cys4057被认为与载脂蛋白B-100中的一个特定半胱氨酸残基形成二硫键。我们的荧光标记实验和分子建模研究为该载脂蛋白[a] kringle类型与载脂蛋白B-100之间可能的相互作用提供了证据。荧光探针5-马来酰亚胺荧光素用于平行实验，以标记脂蛋白[a]和低密度脂蛋白（LDL）中的游离巯基部分。在LDL的载脂蛋白B-100中，Cys3734被该探针标记，但在自身的脂蛋白[a]中该位点未被标记。该结果强烈表明载脂蛋白B-100的Cys3734是与载脂蛋白[a]的Cys4057形成二硫键的残基。为了探索载脂蛋白B-100与载脂蛋白[a]之间可能的非共价相互作用，利用纤溶酶原kringle 4的晶体学坐标生成了载脂蛋白[a] kringle重复序列（3991-4068，LPaK9）的分子模型，这是载脂蛋白[a]中唯一的纤溶酶原kringle 4型重复序列，其具有一个不参与分子内二硫键的额外半胱氨酸残基。Cys4057残基（在LPaK9序列中此后指定为Cys67）被认为与载脂蛋白B-100的一个半胱氨酸形成分子间二硫键。在LPaK9的计算机图形分子模型中，Cys67位于kringle表面靠近赖氨酸配体结合位点处。对含有游离巯基半胱氨酸的LDL载脂蛋白B-100序列的选定片段进行能量最小化，并与LPaK9模型的配体结合位点及相邻区域进行对接。在对接实验中，载脂蛋白B-100片段3732-3745（PSCKLDFREIQIYK）显示出与LPaK9模型的最佳拟合以及最多的范德华接触。发现其他含有巯基半胱氨酸的载脂蛋白B-100肽在用该kringle进行最小化时兼容性较差。这些结果支持并扩展了先前提出的载脂蛋白[a]与载脂蛋白B-100之间复杂相互作用的机制，该机制涉及的不仅仅是一个简单的共价二硫键。
