Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

Abstract

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215-217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215-217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity.

Keywords: allosteric regulation; coagulation factor; molecular dynamics; serine protease; x-ray crystallography.

FIGURE 1.

Overview of the FVIIa-WT·sTF complex and variant nomenclature. A, full view of the FVIIa-WT·sTF (Protein Data Bank code 1dan) complex with the FVIIa protease domain in gray, light chain in blue, sTF in orange, Ca²⁺ ions in yellow, and the active site triad in magenta. The phospholipid-interactive γ-carboxyglutamic acid (Gla) domain and EGF-like domain are shown. B, FVIIa-WT protease domain with the 170 loop (residues 170–178), AL1 (residues 142–152), AL2 (residues 184–193), and AL3 (residues 220–225) in sand. Residues suggested to be involved in TF-induced enhancement in FVIIa activity through allosteric pathways I (blue) and II (green) are shown with connecting dotted lines. Common residues for both pathways from Tyr⁹⁴ in TF are shown in red. FFR is shown in black. New sections of the pathways suggested here are marked with red fully drawn lines. C, FFR inhibitor (black) shown in the active site of FVIIa with substrate subsites 1 and 2 marked. D, sequence alignment of FVIIa-WT, trypsin, and the FVIIa variants with mutations highlighted (orange).

FIGURE 2.

Cofactor binding and amidolytic activity of FVIIa variants. A, initial velocity (V_int) of 1 mm S-2288 hydrolysis by 15 nm FVIIa-WT (○) in black, FVIIa-Y_T (♢) in orange, FVIIa-S_T (□) in cyan, and FVIIa-F_T (▵) in green as a function of sTF concentration (0–3 μm). Data are presented as the mean with error bars showing the highest and lowest data points (n = 2) and were fitted with a quadratic equilibrium equation. B, sTF fractional saturation of FVIIa variants using S-2288 activity. Data were normalized and fitted with a quadratic equilibrium equation. Data are presented as the mean with error bars showing the highest/lowest data points (n = 2). C and D, initial velocity values for hydrolysis of S-2288 without or with 3 μm sTF. Data were fitted with a Michaelis-Menten equation using non-linear regression. Data are presented as the mean with error bars showing the highest and lowest data points (n = 2) at 25 °C.

FIGURE 3.

Functional characterization of FVIIa variants. A, titration of 10–100 nm FVIIa-WT (○) in black, FVIIa-Y_T (♢) in orange, FVIIa-S_T (□) in cyan, and FVIIa-F_T (▵) in green with 0–30 mm pABA inhibitor normalized to residual activity in the presence of 1 mm S-2288. Data were fitted with a competitive inhibition equation. Data are presented as the mean with error bars showing the highest and lowest data points (n = 2). Top panel, without sTF; bottom panel, with 3 μm sTF. B, carbamylation assay with 0.2 m KNCO monitoring residual S-2288 activity as a function of time fitted with a single exponential decay function. Data are presented as the mean with error bars showing the highest and lowest data points (n = 2) at 25 °C.

FIGURE 4.

Structural analysis reveals important role of Tyr¹⁷². A, comparison of FVIIa-WT (Protein Data Bank code 1dan; dark blue) and FVIIa-Y_T (orange) showing the insertion of Tyr¹⁷², which displaces HOH₁. Water molecules found in the region of AL2–3 in FVIIa-WT are shown as blue spheres with all density contoured at the 1.0 σ level. Shown are the crystal structures of FVIIa-Y_T (orange) (B), FVIIa-S_T (cyan) (C), and FVIIa-F_T (green) (D) in complex with sTF and an active site inhibitor (FFR). Water molecules in the variants around the AL2–3 region are shown as spheres in their respective colors and for FVIIa-WT in blue. Hydrogen bonding from the OH group of Tyr¹⁷² to Gln²¹⁷/Phe²²⁵ is shown with red dashed lines together with possible stacking interaction with Trp²¹⁵. The N terminus was found to be in place in all variants, illustrated by the red dotted line from Asp¹⁹⁴ to Ile¹⁶. The cacodylate ion present in the FVIIa-WT structure is found in FVIIa-S_T but not in FVIIa-Y_T and -F_T as the pocket is occupied by Tyr/Phe.

FIGURE 5.

Structural effects of 170 loop substitution. A, alignment of the 170 loop from FVIIa-WT and FVIIa-Y_T showing removal of the possible Gln¹⁷¹ clash in FVIIa-Y_T by replacement to a serine at position 171. B, alignment of the 170 loop main chain of FVIIa-WT, -Y_T, -S_T, and-F_T showing changes to the main chain hydrogen bonding network of the TF-binding α-helices in the variants. Red dashed lines depict the backbone hydrogen bonding network of the FVIIa-WT α-helix. C, calculated φ/ψ angles for amino acids Thr¹⁶⁵–Cys¹⁶⁸ in the beginning of the TF-binding helix with gray shaded areas being ideal φ/ψ angles as published by the European Molecular Biology Laboratory and * marking angles outside the ideal region.

FIGURE 6.

Trp²¹⁵ location in relation to the active site. Representative conformations of segment 215–217 measured as Trp²¹⁵ relative distances to the catalytic triad are shown. A, open; B, intermediate; C, collapsed. Each state is depicted on the scatter plots with their respective letter. D, scatter plots of Trp²¹⁵ distance to the active residues His⁵⁷ and Ser¹⁹⁵. Data from 0 to 33 ns are in light red, from 33 to 67 ns are in light blue, and from 67 to 100 ns are in black. Single data points from 1 ns are shown as a blue filled dot, from 50 ns are shown in green, and from 100 ns are shown in red. The calculated trajectories are available as supplemental data (supplemental Movies S1–S5).

FIGURE 7.

Evaluation of tryptophan surface accessibility. A, conformations of Trp²¹⁵ in FVIIa-WT during MD simulations with van der Waals surface area in red in an open (0 ns), intermediate (73 ns), and collapsed conformations with collapsed S1 pocket (99 ns). B, plot of the calculated SASA values for all tryptophans of the protease domain during the simulation of FVIIa-WT. Trp²⁹ (black), Trp⁵¹ (orange), Trp⁶¹ (green), Trp¹⁴¹ (blue), Trp²⁰⁷ (purple), Trp²¹⁵ (red), and Trp²³⁷ (gray) are shown. C, calculated SASA values of Trp²¹⁵ for all FVIIa variants during 100-ns MD simulations smoothened using a Savistsky and Golay algorithm. FVIIa-WT (black), FVIIa-WT·sTF (blue), FVIIa-Y_T (orange), FVIIa-S_T (cyan), and FVIIa-F_T (green) are shown. D, Stern-Volmer plot for the FVIIa variants as a function of acrylamide concentration. Shown are FVIIa-WT (○; black), FVIIa-Y_T (♢; orange), FVIIa-S_T (□; cyan) and FVIIa-F_T (▵; green) in the presence (dotted line) or absence (full line) of FFR. Data are shown as mean ± S.D. (error bars) (n = 2–4) at 15 °C. E–G, scatter plots and correlation between the estimated K_sv constants and trajectory-averaged SASA values for Trp⁶¹, Trp²⁰⁷, and Trp²¹⁵ for each individual FVIIa variant. SASA values with FFR present were calculated from the obtained crystal structures. The error bars indicating the standard deviation are shown for both abscissa and ordinate. A linear regression has been overlaid for Trp²¹⁵ to illustrate correlation, which was statistically evaluated using a Pearson test.

FIGURE 8.

Overview of 170 loop-mediated active site stabilization in trypsin-like serine proteases. A, alignment of the selected proteases with residues corresponding to Tyr¹⁷², Trp²¹⁵, Asp²¹⁷, and Pro²²⁵ in trypsin shown in red. Structural overviews of plausible 170 loop stabilization of the 215–217 segment in factor VIIa·sTF (B; Protein Data Bank code 1dan) with cacodylate (CAC), factor IXa (C; Protein Data Bank code 2wph in dark orange and Protein Data Bank code 2wpk in light orange with ethylene glycol (EG) in gray), factor Xa (D; Protein Data Bank code 2jkh) with Na⁺ in gray, thrombin (E; Protein Data Bank code 1sgi in light red and Protein Data Bank code 1sg8 in dark red) with Na⁺ in gray and coordinated waters in red, trypsin (F; Protein Data Bank code 1trn), and mouse chymotrypsin (G; Protein Data Bank code 2gch) are shown. Water molecules for all structures are shown as blue spheres with electron density contoured at σ = 0.7 with water hydrogen bonds (2.5–3.5 Å) as red dotted lines and ethylene glycol in gray.