Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody
Crystal structure of the calcium-stabilized human factor IX Gla domain bound to a conformation-specific anti-factor IX antibody
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DOI：
10.1074/jbc.m314011200
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发表时间：
2004-04-02
影响因子：
4.8
通讯作者：
Furie, B
中科院分区：
文献类型：
--
作者：
Huang, MD;Furie, BC;Furie, B
凝血过程中因子IX与膜的结合由N-末端γ-羧基谷氨酸富集（Gla）结构域介导，Gla结构域是维生素K依赖性凝血和调节蛋白上发现的膜锚定结构域。构象特异性抗因子IX抗体针对钙稳定的Gla结构域并干扰因子IX-膜相互作用。一种这样的抗体10 C12识别因子IX的Gla结构域的钙稳定形式。我们通过固相肽合成制备了因子IX的完全羧化的Gla结构域，并使与10 C12抗体的Fab片段复合的因子IX-（1- 47）结晶。在2.2埃下，因子IX-（1 - 47）-抗体复合物中Gla结构域的总体结构与在钙离子存在下因子IX Gla结构域的结构相似，如通过NMR光谱测定的（Freedman，S. J.，Furie，B. C.的方法，Furie，B.，Baleja，J. D.（1995）Biochemistry 34，12126 - 12137）和X射线晶体学（Shikamoto，Y.，Morita，T.，Fujimoto，Z.，和Mizuno，H.（2003）J.Biol.Chem.278，24090 - 24094）。复合物结构显示10 C12抗体的互补决定区环形成疏水口袋以容纳由Leu-6、Phe-9和瓦尔-10组成的Gla结构域的疏水补丁。极性相互作用在抗体-抗原识别中也起重要作用。此外，因子IX Gla结构域的钙配位网络不同于其他维生素K依赖性蛋白的Gla结构域结构。我们的结论是，这种抗体是针对在因子IX的Ω环的膜结合位点，并通过抑制其与膜的相互作用来阻断因子IX的功能。
The binding of Factor IX to membranes during blood coagulation is mediated by the N-terminal gamma-carboxyglutamic acid-rich (Gla) domain, a membrane-anchoring domain found on vitamin K-dependent blood coagulation and regulatory proteins. Conformation-specific anti-Factor IX antibodies are directed at the calcium-stabilized Gla domain and interfere with Factor IX-membrane interaction. One such antibody, 10C12, recognizes the calcium-stabilized form of the Gla domain of Factor IX. We prepared the fully carboxylated Gla domain of Factor IX by solid phase peptide synthesis and crystallized Factor IX-(1- 47) in complex with Fab fragments of the 10C12 antibody. The overall structure of the Gla domain in the Factor IX-(1 - 47)-antibody complex at 2.2 Angstrom is similar to the structure of the Factor IX Gla domain in the presence of calcium ions as determined by NMR spectroscopy (Freedman, S. J., Furie, B. C., Furie, B., and Baleja, J. D. (1995) Biochemistry 34, 12126 - 12137) and by x-ray crystallography (Shikamoto, Y., Morita, T., Fujimoto, Z., and Mizuno, H. (2003) J. Biol. Chem. 278, 24090 - 24094). The complex structure shows that the complementarity determining region loops of the 10C12 antibody form a hydrophobic pocket to accommodate the hydrophobic patch of the Gla domain consisting of Leu-6, Phe-9, and Val-10. Polar interactions also play an important role in the antibody-antigen recognition. Furthermore, the calcium coordination network of the Factor IX Gla domain is different than in Gla domain structures of other vitamin K-dependent proteins. We conclude that this antibody is directed at the membrane binding site in the omega loop of Factor IX and blocks Factor IX function by inhibiting its interaction with membranes.