Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S
Conformational changes in activated protein C caused by binding of the first epidermal growth factor-like module of protein S
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DOI：
10.1042/bj3490757
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发表时间：
2000-08-01
影响因子：
4.1
通讯作者：
Griffin, JH
中科院分区：
文献类型：
--
作者：
Hackeng, TM;Yegneswaran, S;Griffin, JH
化学合成人血浆蛋白S的第一个表皮生长因子样模块（EGF 1，残基76-116），并测试其抑制蛋白S的抗凝剂辅因子活性（抗凝剂蛋白酶，活化蛋白C（APC））的能力。在血浆凝固试验中，EGF 1完全抑制蛋白S对APC活性的刺激，在约100 μ mol/L时抑制率为50%。1 μ M EGF 1，表明EGF 1与APC直接结合。为了研究EGFI和APC之间的直接相互作用，采用荧光共振能量转移（FRET）实验。以荧光素为供体在活性位点标记APC，含有十八烷基罗丹明作为受体的磷脂囊泡表明，EGF 1与APC的结合导致能量转移增加，这与APC活性位点从94埃（9.4 nm）的重新定位一致。到85埃以上的磷脂表面（假设kappa（2）= 2/3）。APC活性位点结合的荧光素和磷脂结合的罗丹明之间的能量转移的增加是相同的，获得后协会的蛋白S或蛋白S-C4 b结合蛋白复合物与APC。后者表明在磷脂表面上存在蛋白S-C4 b结合蛋白与APC的三元复合物。为了证实EGF 1与APC的直接相互作用，将罗丹明共价连接到EGF 1的α-N-末端，并使用FRET直接证明标记的EGF 1与APC的结合。数据表明APC的活性位点和EGF 1的α-N-末端之间的分离为76埃（kappa（2）= 2/3），将APC结合的蛋白S-EGF 1置于接近但高于磷脂表面和APC的两个EGF结构域附近。因此，我们提供了直接的证据，结合蛋白S-EGF 1 APC，并表明，它诱导APC的构象变化。
The first epidermal growth factor-like module of human plasma protein S (EGF1, residues 76-116) was chemically synthesized and tested for its ability to inhibit the anticoagulant cofactor activity of protein S for the anticoagulant protease, activated protein C (APC). EGF1 completely inhibited the stimulation of APC activity by protein S in plasma coagulation assays, with 50 % inhibition at approx. 1 mu M EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGFI and APC, fluorescence resonance energy transfer (FRET) experiments were employed. APC labelled in the active site with fluorescein as the donor, and phospholipid Vesicles containing octadecylrhodamine as the acceptor, showed that EGF1 association with APC caused an increase in energy transfer consistent with a relocation of the active site of APC from 94 Angstrom (9.4 nm) to 85 Angstrom above the phospholipid surface (assuming kappa(2) = 2/3). An identical increase in energy transfer between the APC active site-bound fluorescein and phospholipid-bound rhodamine was obtained upon association of protein S or protein S-C4b-binding protein complex with APC. The latter suggests the presence of a ternary complex of protein S-C4b-binding protein with APC on the phospholipid surface. To cofirm a direct interaction of EGF1 with APC, rhodamine was covalently attached to the alpha-N-terminus of EGF1, and binding of the labelled EGF1 to APC was directly demonstrated using FRET. The data suggested a separation between the active site of APC and the alpha-N-terminus of EGF1 of 76 Angstrom (kappa(2) = 2/3), placing the APC-bound protein S-EGF1 close to, but above, the phospholipid surface and near the two EGF domains of APC. Thus we provide direct evidence for binding of protein S-EGF1 to APC and show that it induces a conformational change in APC.