Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri, USA.
J Thromb Haemost. 2024 Oct;22(10):2767-2772. doi: 10.1016/j.jtha.2024.07.003. Epub 2024 Jul 11.
All current X-ray structures of factor (F)Xa are devoid of the γ-carboxyglutamate (Gla) domain and fail to reveal the overall conformation of the free protein. The recent cryogenic electron microscopy (cryo-EM) structure of FXa in the prothrombinase complex is the only structure of full-length FXa and shows that the Gla domain is positioned at an angle relative to the epidermal growth factor 1 domain.
Establish if the curved conformation of FXa revealed by cryo-EM is also present in solution.
The conformation of FXa in solution was studied by single-molecule Förster resonance energy transfer.
The conformation of full-length FXa in solution is resolved for the first time. The conformation is curved and extremely sensitive to Ca. It does not differ significantly from its zymogen form or from that present in the prothrombinase complex free or bound to the physiologic substrates prothrombin and meizothrombin.
Measurements by single-molecule Förster resonance energy transfer reveal that FXa has a curved conformation in solution, free or bound to physiologic ligands, and validate the recent cryo-EM structures of prothrombinase. The drastic conformational changes observed in the absence of Ca suggest that the structural architecture of FXa changes upon administration of vitamin K antagonists that perturb the interaction of the Gla domain with divalent cations.
所有目前的因子（F）Xa X 射线结构均缺乏 γ-羧基谷氨酸（Gla）结构域，无法揭示游离蛋白的整体构象。最近凝血酶原酶复合物中 FXa 的低温电子显微镜（cryo-EM）结构是唯一全长 FXa 的结构，表明 Gla 结构域相对于表皮生长因子 1 结构域呈一定角度定位。
确定 cryo-EM 揭示的 FXa 弯曲构象是否也存在于溶液中。
通过单分子Förster 共振能量转移研究 FXa 在溶液中的构象。
首次解析了溶液中全长 FXa 的构象。该构象是弯曲的，对 Ca 极其敏感。它与酶原形式或游离或与生理底物凝血酶原和中间凝血酶结合的凝血酶原酶复合物中的形式没有显著差异。
单分子Förster 共振能量转移的测量结果表明，FXa 在溶液中具有弯曲的构象，无论游离或结合生理配体，均可验证最近的凝血酶原酶复合物的 cryo-EM 结构。在没有 Ca 的情况下观察到的剧烈构象变化表明，FXa 的结构架构在给予维生素 K 拮抗剂时会发生变化，这些拮抗剂会干扰 Gla 结构域与二价阳离子的相互作用。
