Membrane-bound model of the ternary complex between factor VIIa/tissue factor and factor X

• The structure of the ternary TF:FVIIa:FX complex, which forms on the surface of anionic membranes, is unknown.

• Through a combination of advanced computational techniques guided by experimental data, we construct the extrinsic complex on a membrane.

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 factor X (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 (MD) simulations in the explicit presence of a membrane, we developed 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 the first membrane-bound model for the EC. Strikingly, in our model FX makes contact with TF:FVIIa chiefly via its GLA (γ-carboxyglutamate-rich) domain and protease domain, with the majority of the FX light chain (i.e., its two 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, while 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.