The tissue factor region that interacts with substrates factor IX and factor X
The tissue factor region that interacts with substrates factor IX and factor X
复制标题
DOI：
10.1021/bi000182
复制
发表时间：
2000-06-27
期刊：
影响因子：
2.9
通讯作者：
Kelley, RF
中科院分区：
文献类型：
--
作者：
Kirchhofer, D;Lipari, MT;Kelley, RF
凝血因子 VIIa 的酶活性由其细胞辅助因子组织因子 (TF) 控制。 TF 以高亲和力结合 VIIa 因子，此外，还通过其 C 端纤连蛋白 III 型结构域参与底物相互作用。我们分析了 C 端 TF 结构域中表面暴露的残基，以更全面地确定 TF 上对底物激活重要的区域。可溶性 TF (sTF) 突变体在大肠杆菌中表达，并在磷脂囊泡或 SW-13 细胞膜存在的情况下测量它们支持因子 VIIa 依赖性底物激活的能力。结果表明，因子 IX 和因子 X 与位于假定磷脂表面附近的相同 TF 区域相互作用。根据sTF突变体的活性丧失程度，该TF区域可分为主区域（残基Tyr157、Lys159、Ser163、Gly164、Lys165、Lys166、Tyr185），形成488埃的溶剂暴露斑块（2）和包含额外7-8个残基（包括位于远端的Asn199）的扩展区域， Arg200 和 Asp204。一些已鉴定的 TF 残基，例如 Trp158 和环 Lys159-Lys165 内的残基，位于因子 VIIa γ-羧基谷氨酸 (Gla) 结构域附近，表明因子 VIIa Gla 结构域也可能参与底物相互作用。此外，被认为对底物相互作用重要的表面带有净正电荷，这表明电荷相互作用可能对 TF-底物结合有显着贡献。计算得出的该基底相互作用区域的表面暴露面积约为 1100 埃 (2)，大约是与因子 VIIa 接触的 TF 区域大小的一半。因此，TF 表面的很大一部分（3000 埃（2））在底物催化过程中参与蛋白质-蛋白质相互作用。
The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 Angstrom(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 Angstrom(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 Angstrom(2)) is engaged in protein-protein interactions during substrate catalysis.