Structural modulation of factor VIIa by full-length tissue factor (TF_1-263): implication of novel interactions between EGF2 domain and TF

Tissue factor (TF)-mediated factor VII (FVII) activation and a subsequent proteolytic TF-FVIIa binary complex formation is the key step initiating the coagulation cascade, with implications in various homeostatic and pathologic scenarios. TF binding allosterically modifies zymogen-like free FVIIa to its highly catalytically active form. As a result of unresolved crystal structure of the full-length TF_1-263-FVIIa binary complex and free FVIIa, allosteric alterations in FVIIa following its binding to full-length TF and the consequences of these on function are not entirely clear. The present study aims to map and identify structural alterations in FVIIa and TF resulting from full-length TF binding to FVIIa and the key events responsible for enhanced FVIIa activity in coagulation. We constructed the full-length TF_1-263-FVIIa membrane bound complex using computational modeling and subjected it to molecular dynamics (MD) simulations. MD simulations showed that TF alters the structure of each domain of FVIIa and these combined alterations contribute to enhanced TF-FVIIa activity. Detailed, domain-wise investigation revealed several new non-covalent interactions between TF and FVIIa that were not found in the truncated soluble TF-FVIIa crystal structure. The structural modulation of each FVIIa domain imparted by TF indicated that both inter and intra-domain communication is crucial for allosteric modulation of FVIIa. Our results suggest that these newly formed interactions can provide additional stability to the protease domain and regulate its activity profile by governing catalytic triad (CT) orientation and localization. The unexplored newly formed interactions between EGF2 and TF provides a possible explanation for TF-induced allosteric activation of FVIIa.组织因子（Tissue factor, TF）介导的凝血因子VII（factor VII, FVII）激活，以及后续形成蛋白水解型TF-FVIIa二元复合物，是启动凝血级联反应的关键步骤，在多种生理稳态与病理场景中均具有重要意义。TF通过变构作用将酶原样游离FVIIa转变为高催化活性形式。由于全长TF₁₋₂₆₃-FVIIa二元复合物与游离FVIIa的晶体结构尚未解析，FVIIa结合全长TF后发生的变构改变，以及这些改变对其功能的影响尚不明确。本研究旨在定位并阐明FVIIa与全长TF结合后引发的FVIIa及TF的结构改变，以及凝血过程中增强FVIIa活性的关键事件。我们通过计算建模构建了膜结合型全长TF₁₋₂₆₃-FVIIa复合物，并对其开展分子动力学（molecular dynamics, MD）模拟。MD模拟结果显示，TF可改变FVIIa各结构域的构象，这些协同构象改变共同促成了TF-FVIIa活性的增强。细致的结构域水平分析发现，TF与FVIIa之间存在数种此前在截短可溶性TF-FVIIa晶体结构中未被观测到的新型非共价相互作用。TF对FVIIa各结构域的构象调控表明，结构域间与结构域内的信号通讯对于FVIIa的变构调控均至关重要。我们的研究结果提示，这些新生相互作用可增强蛋白酶结构域的稳定性，并通过调控催化三联体（catalytic triad, CT）的取向与定位，调节其活性谱。此前未被报道的表皮生长因子样结构域2（EGF2）与TF之间的新生相互作用，为TF诱导的FVIIa变构激活提供了一种合理的解释。