Figure 3

(A) Rapid kinetics of FPR binding to prothrombin (closed circles), prethrombin-1 (triangles) and prethrombin-2 (open circles) showing the slow relaxation α₂ from the reaction scheme in eqs 1 and 2. Continuous lines were drawn according to eq. 2 in the text with best-fit parameter values listed in Table 1. (B) Equilibrium titrations of FPR binding to prothrombin (closed circles), prethrombin-1 (triangles) and prethrombin-2 (open circles) consistent with a single-site binding interaction with best-fit values of K_d,app listed in Table 1. The kinetic profiles of prothrombin, prethrombin-1 and prethrombin-2 differ markedly, especially in the value of k₁₂. A progressive shift of the E*-E equilibrium toward the E conformational ensemble (Table 1) accounts for the increase in ligand binding affinity when auxiliary domains are removed in the zymogen from prothrombin to prethrombin-2. Experimental conditions are: 400 mM ChCl, 50 mM Tris, 0.1% PEG8000, pH 8.0, at 15 °C. Representative kinetic traces and residuals are shown in the Supplementary Information.