Kemball-Cook G, Johnson D J, Tuddenham E G, Harlos K
MRC Clinical Sciences Centre, Imperial College School of Medicine, Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom.
J Struct Biol. 1999 Oct;127(3):213-23. doi: 10.1006/jsbi.1999.4158.
Factor VIIa (FVIIa) is a crucial haemostatic protease consisting of four distinct domains termed the Gla, epidermal growth factor-1 (EGF-1), EGF-2, and protease domains (from N- to C-terminus). The crystal structure of human FVIIa inhibited at the active site with 1, 5-dansyl-Glu-Gly-Arg-chloromethyl ketone and lacking the Gla domain has been solved to a resolution of 2.28 A. The EGF-2 and protease domains were well resolved, whereas no electron density for the EGF-1 domain was observed, suggesting a flexible arrangement or disorder within the crystal. Superposition of the protease domain of the present structure with that previously resolved in the tissue factor (TF)/FVIIai complex revealed that although overall the domain structures are similar, the EGF-2 domain is rotated by 7.5 degrees relative to the protease domain on binding TF. A single cleavage in the protease domain was found, between Arg315 and Lys316 (chymotrypsin numbering 170C-170D) in a FVII-specific insertion loop: this cleavage appeared to be essential for crystallisation. Insertion of the heavy chain N-terminal Ile153 is essentially identical in the two structures, as is the geometry of the active site residues and the inhibitor C-terminal arginine residue. Some differences are seen in the cleaved loop, but changes in TF-contact residues are generally minor. This structure supports the hypothesis that TF binding enables spatial domain arrangements in the flexible FVIIa molecule necessary for procoagulant function and furthermore that active site occupancy induces FVIIa active conformation via N-terminal insertion.
凝血因子VIIa(FVIIa)是一种关键的止血蛋白酶,由四个不同的结构域组成,从N端到C端依次为γ-羧基谷氨酸(Gla)结构域、表皮生长因子-1(EGF-1)结构域、EGF-2结构域和蛋白酶结构域。人FVIIa在活性位点被1,5-丹磺酰基-L-谷氨酸-L-甘氨酸-L-精氨酸氯甲基酮抑制且缺乏Gla结构域的晶体结构已解析到2.28埃的分辨率。EGF-2和蛋白酶结构域解析良好,而未观察到EGF-1结构域的电子密度,这表明在晶体中该结构域排列灵活或无序。将本结构的蛋白酶结构域与先前在组织因子(TF)/FVIIa抑制物复合物中解析的结构叠加显示,虽然总体结构域结构相似,但结合TF时,EGF-2结构域相对于蛋白酶结构域旋转了7.5度。在FVII特异性插入环中,蛋白酶结构域在精氨酸315和赖氨酸316之间(胰凝乳蛋白酶编号170C - 170D)发现了一处切割:这种切割似乎对结晶至关重要。重链N端异亮氨酸153的插入在两种结构中基本相同,活性位点残基和抑制剂C端精氨酸残基的几何结构也是如此。在切割环中可见一些差异,但TF接触残基的变化通常较小。该结构支持以下假设:TF结合使灵活的FVIIa分子中形成促凝血功能所需的空间结构域排列,而且活性位点被占据通过N端插入诱导FVIIa形成活性构象。