Hackeng T M, Yegneswaran S, Johnson A E, Griffin J H
Departments of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037, USA.
Biochem J. 2000 Aug 1;349 Pt 3(Pt 3):757-64. doi: 10.1042/bj3490757.
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 microM+ EGF1, suggesting direct binding of EGF1 to APC. To investigate a direct interaction between EGF1 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 A (9.4 nm) to 85 A 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 confirm 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 N-terminus of EGF1 of 76 A (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.
对人血浆蛋白S的首个表皮生长因子样结构域（EGF1，第76至116位氨基酸残基）进行了化学合成，并测试了其抑制蛋白S作为抗凝蛋白酶即活化蛋白C（APC）的抗凝辅因子活性的能力。在血浆凝固试验中，EGF1完全抑制了蛋白S对APC活性的刺激作用，在约1 microM + EGF1时抑制率达50%，这表明EGF1与APC直接结合。为了研究EGF1与APC之间的直接相互作用，采用了荧光共振能量转移（FRET）实验。用荧光素作为供体标记活性位点的APC，以及含有十八烷基罗丹明作为受体的磷脂囊泡，结果表明EGF1与APC的结合导致能量转移增加，这与APC活性位点从磷脂表面上方94 Å（9.4 nm）重新定位到85 Å一致（假设κ(2)=2/3）。当蛋白S或蛋白S - C4b结合蛋白复合物与APC结合时，APC活性位点结合的荧光素与磷脂结合的罗丹明之间的能量转移也出现相同程度的增加。后者表明在磷脂表面存在蛋白S - C4b结合蛋白与APC的三元复合物。为了证实EGF1与APC的直接相互作用，将罗丹明共价连接到EGF1的α - N末端，并使用FRET直接证明了标记的EGF1与APC的结合。数据表明APC活性位点与EGF1的N末端之间的距离为76 Å（κ(2)=2/3），使得结合了APC的蛋白S - EGF1靠近但高于磷脂表面，且靠近APC的两个EGF结构域。因此，我们提供了蛋白S - EGF1与APC结合的直接证据，并表明它会诱导APC发生构象变化。
