Human tissue factor (TF, residues 1−263) is a cell-surface receptor consisting of three domains: an extracellular domain (residues 1−219 = soluble form TF = sTF) composed of two C2-type immunoglobulin-like modules (N-module with residues 1−106 and C-module with residues 107−219), a transmembrane (TM) domain (residues 220−242) and a cytoplasmic domain (residues 245−263) . After vessel injury, TF activates the blood coagulation cascade by binding a serine protease factor VIIa (FVIIa). Full-length TF acts as a cofactor to substantially increase the activity of FVIIa for a substrate factor X (FX) in the presence of an anionic phospholipid membrane and Ca2+. sTF shows 4% of the activity of full-length TF . The mechanism of enhancement of activity by TF, whether soluble or full length, is still elusive at the molecular level. Recent pioneering MD simulations by Ohkubo et al.  provided a new understanding of the dynamic features, structure, and atomic interaction of the proteins (sTF1-213 and FVIIa) and their binary complex (sTF1-213/FVIIa) on a lipid bilayer composed of 100% DOPS (1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]). In this letter, we have modeled a transmembrane (TM) domain of TF to a linker region which connects the truncated sTF210 in the X-ray crystal structure (PDB code: 1DAN)  with the TM domain (see Supporting Information, SI). The newly modeled TF1-242/FVIIa is incorporated onto a 4:1 POPC/POPS bilayer model with additional Ca2+ ions present beyond those bound to FVIIa. In building the initial structures, we adopted four criteria: (i) that the TM domain of TF penetrates the lipid bilayer, (ii) that three hydrophobic residues in the gamma-carboxyglutamate-rich (GLA) domain of FVIIa (Phe4, Leu5, and Leu8) are located in the hydrophobic region immediately beneath the phosphate groups in POPC/POPS, (iii) that the three charged residues in the linker of the TF (Lys214, Glu216 and Glu219) are placed on the charged surface of the lipid membrane and (iv) that the initial orientation of TF1-242/FVIIa on the lipid membrane is determined from a putative ternary complex of sTF/FVIIa/FXa . For the initial structure based on these four criteria, we estimated the distance between Cα of Ser195 in an active site of FVIIa and the nearest P atom to be ∼80 Å.
We performed two independent simulations employing the Amber11  and NAMD2.8  packages with independent force fields. The details of the molecular dynamics (MD) simulations are given in more detail in SI. Even though the four criteria for the initial structure set-up were applied to both simulations, the MD simulation environments differed somewhat (see Table S1). Interestingly, the separate RMSD profiles of TF1-242/FVIIa, FVIIa, sTF and the GLA domain obtained by the independent methods are similar (see Fig. S1). The last snapshot (115 ns) of the Amber simulation is shown in Fig. 1. We averaged the shortest distance between any phosphate atom and Cα of Ser195 for 100 ps snapshots during the last 10 ns of simulations by the Amber and NAMD packages and find these distances to be 77.96 Å (± 1.58) for Amber and 76.86 Å (± 2.22) for NAMD (Fig. S2). We thus obtained consistent values for the active site height irrespective of the method, force field and the locally different simulation environments. These consensus values of the height of Ser195 from the lipid membrane estimated by the two distinct simulations are comparable to the experimental estimates made using TM-containing TF (74 ± 2 Å , 75.0 ± 1.8 Å , 76 ± 3 Å ). Ohkubo et al.  defined the reference point of the lipid surface as the average z-coordinate of all carboxy oxygens in the head groups of DOPS of the proximal lipid leaflet, but found significantly larger distances of the active site of FVIIa above the surface for sTF1-213/FVIIa. These may reflect the differences in lipid composition, concentration of ions (Ca2+, Na+, Cl−) or the effect of the TM domain addition. We also investigated the dynamic local interaction between the GLA domain of FVIIa, the TF linker region and sTF1-213 (Table S2) with the bilayer. From the last 10 ns trajectories, we observed significant interactions involving residues Ser162 and Lys181 of TF in both the Amber and NAMD simulations. The involvement of these residues was also reported in Ohkubo et al.’s  pure DOPS bilayer simulation. Both Amber and NAMD simulations show new interactions between the TF transmembrane helix and the linker region with the bilayer that cannot be present for simulations with sTF (See Table S2). These new interactions may contribute to the smaller active site height that we observe. This concept is consistent with the desGLA-FVIIa/TF, FVIIa, FVIIa/TF comparative experiments , which suggest that it is TF, not FVIIa, that plays a dominant role in orienting FVIIa for the optimal position of the active site on the phospholipid; while interactions involving the FVIIa GLA domain, the FX GLA domain, and phospholipid are critical for efficient proteolysis of FX on a membrane surface . The FVIIa active site location estimated in the current simulations may provide an optimal height and orientation for the interaction with a substrate FX, and therefore be a significant factor in the enhancement of the FVIIa catalysis provided by TF. In a docking of the final snapshots from the Amber and NAMD simulations with a ternary model of sTF/FVIIa/FXa , both show reasonable ternary structures on the lipid bilayer by minimal adjustment of the linker region between the EGF-1 and EGF-2 domains of FXa (See Fig. S3). Finally, Ca(POPS)2 structures, similar to those proposed from site-resolved multidimensional solid-state NMR (SSNMR) experiments, are dynamically generated in both simulations . The final snapshot structures of the simulations are available on request from the authors.