Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 1:1 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

凝血因子V和VIII联合缺乏症（F5F8D）是一种常染色体隐性遗传病，其特征是因子V（FV）和因子VIII（FVIII）的血浆水平协同降低，在基因上与跨膜凝集素ERGIC - 53和可溶性钙结合蛋白MCFD2的突变有关。越来越多的证据表明，这两种蛋白在内质网（ER）和内质网 - 高尔基体中间腔室之间形成一种循环复合物，从而在FV和FVIII的早期分泌途径中作为一种货物受体发挥作用。为了更好地理解ERGIC - 53和MCFD2功能协同的潜在机制，我们在此通过X射线晶体学分析，并结合核磁共振和超速离心分析来表征它们之间的相互作用。对综合数据的研究表明，ERGIC - 53 - 碳水化合物识别结构域（CRD）通过其远离糖结合位点的分子表面与MCFD2结合，在溶液中形成1∶1的复合物。这种相互作用不依赖于ERGIC - 53的糖结合，并且涉及到目前在F5F8D中报道的MCFD2的大部分错义突变位点。与先前报道的每种蛋白质的未复合结构相比，表明在复合物形成时，MCFD2而非ERGIC - 53 - CRD发生显著的构象改变。我们的研究结果为ERGIC - 53和MCFD2在捕获FV和FVIII时的协同相互作用提供了结构基础。