Mohammed Bassem M, Basore Katherine, Di Cera Enrico
Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO.
Washington University Center for Cellular Imaging, Washington University School of Medicine, St. Louis, MO.
Blood. 2025 Jun 26;145(26):3166-3177. doi: 10.1182/blood.2025028476.
Coagulation factor Va (FVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, FVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the FVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of FVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here, we report the cryo-EM structure of the FVa-APC complex at 3.15 Å resolution in which the protease domain of APC engages R506 in the A2 domain of FVa through electrostatic interactions between positively charged residues in the 30-loop and 70-loop of APC and an electronegative surface of FVa. The auxiliary γ-carboxyglutamic acid and epidermal growth factor domains of APC are highly dynamic and point to solvent, without making contacts with FVa. Binding of APC displaces a large portion of the A2 domain of FVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch," or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC, which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the FVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.
凝血因子Va（FVa）是凝血酶原酶复合物的辅因子成分，在凝血级联反应的倒数第二步中，它是从凝血酶原快速生成凝血酶所必需的。此外，FVa是活化蛋白C（APC）进行蛋白水解失活的靶点。与凝血级联反应中的其他蛋白质 - 蛋白质相互作用一样，FVa - APC相互作用长期以来一直是结构生物学的一个挑战，其分子基础仍然未知。最近FVa的低温电子显微镜（cryo - EM）结构揭示了其A1 - A2 - A3 - C1 - C2结构域的排列以及APC在R306和R506处切割位点的环境。在这里，我们报告了分辨率为3.15 Å的FVa - APC复合物的低温电子显微镜结构，其中APC的蛋白酶结构域通过APC的30环和70环中带正电的残基与FVa的A2结构域中的R506之间的静电相互作用与FVa结合。APC的辅助γ - 羧基谷氨酸和表皮生长因子结构域高度动态且指向溶剂，不与FVa接触。APC的结合取代了FVa的大部分A2结构域，并将654VKCIPDDDEDSYEIFEP670片段作为“闩锁”或外位点配体投射到酶的70环上。该闩锁诱导了APC自溶环的大构象变化，这反过来又促进了R506对接进入主要特异性口袋。FVa - APC复合物的低温电子显微镜结构验证了大量现有的生化数据，并为凝血级联反应的关键调节相互作用提供了分子背景。
