Muller Melanie P, Mortenson Alex, Sedzro Josepha C, Wen Po-Chao, Morrissey James H, Tajkhorshid Emad
Theoretical and Computational Biophysics Group, National Institutes of Health Center for Macromolecular Modeling and Visualization, Beckman Institute for Advanced Science and Technology, Department of Biochemistry, Center for Biophysics and Quantitative Biology, University of Illinois Urbana-Champaign, Urbana, IL.
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI.
Blood Adv. 2025 Feb 25;9(4):729-740. doi: 10.1182/bloodadvances.2024014845.
Formation of the extrinsic complex (EC) on cell surfaces is the event that triggers the coagulation cascade. Tissue factor (TF) and factor VIIa (FVIIa) form the EC together with FX on phosphatidylserine-containing membranes, leading to FX activation by TF:FVIIa. This lipid dependence has made experimental characterization of the EC structure challenging. Using a novel computational methodology combining rigid-body protein-protein docking and extensive nonequilibrium molecular dynamics simulations in the explicit presence of a membrane, we developed, to our knowledge, the first atomic-level model of the EC, taking full account of the role of the membrane. Rigid-body docking generated 1 000 000 protein-only structures that predict the binding of key EC domains. Residue-residue contact information was then used in nonequilibrium simulations to drive the formation of the EC on a phosphatidylserine/phosphatidylcholine membrane surface, providing, to our knowledge, the first membrane-bound model for the EC. Strikingly, in our model, FX makes contact with TF:FVIIa chiefly via its γ-carboxyglutamate-rich (GLA) domain and protease domain, with the majority of the FX light chain (ie, its 2 epidermal growth factor-like domains) out in the solvent, making no direct contact with TF:FVIIa. The TF exosite makes substantial contacts with both the FX- and FVIIa-GLA domains, in which TF residue K165 engages directly with the FVIIa-GLA domain, whereas K166 plays a central role in binding to the FX-GLA domain. These findings underscore the substrate-binding exosite of TF as being pivotal in the formation of the EC, serving as a critical interface linking the GLA domains of both FVIIa and FX.
细胞表面外在复合物（EC）的形成是触发凝血级联反应的事件。组织因子（TF）和因子VIIa（FVIIa）与FX在含磷脂酰丝氨酸的膜上共同形成EC，导致TF:FVIIa激活FX。这种对脂质的依赖性使得对EC结构进行实验表征具有挑战性。我们运用一种新颖的计算方法，将刚体蛋白-蛋白对接与在膜明确存在的情况下进行的广泛非平衡分子动力学模拟相结合，据我们所知，开发出了首个充分考虑膜作用的EC原子水平模型。刚体对接生成了100万个仅含蛋白质的结构，这些结构预测了关键EC结构域的结合。然后，残基-残基接触信息被用于非平衡模拟，以驱动EC在磷脂酰丝氨酸/磷脂酰胆碱膜表面的形成，据我们所知，这提供了首个EC的膜结合模型。引人注目的是，在我们的模型中，FX主要通过其富含γ-羧基谷氨酸（GLA）的结构域和蛋白酶结构域与TF:FVIIa接触，FX轻链的大部分（即其2个表皮生长因子样结构域）位于溶剂中，不与TF:FVIIa直接接触。TF外部位点与FX和FVIIa的GLA结构域都有大量接触，其中TF残基K165直接与FVIIa的GLA结构域结合，而K166在与FX的GLA结构域结合中起核心作用。这些发现强调了TF的底物结合外部位点在EC形成中起关键作用，是连接FVIIa和FX的GLA结构域的关键界面。
