Figure 5.
Stability of the D3 interface is modulated by pH and FVIII binding. (A) Midpoint force F1/2 for different buffer conditions (see supplemental Table 1 for details of the buffer compositions). The D3 interface is destabilized by decreasing pH, not affected by addition of EDTA (to remove the Ca2+ from the binding loop in VWD3) and stabilized by addition of FVIII. The difference between F1/2 at pH 7.4 and pH 6.2 is highly significant (P < .0041; 2-tailed t test for independent means). Similarly, the difference between F1/2 at pH 7.4 and pH 7.4 + FVIII is highly significant (P < .0078). (B) Distances Δz between states for the same conditions as in panel A. There are no significant differences in Δz between the conditions. Each point in panels A and B corresponds to an individual molecule. Boxes indicate the median and 25th and 75th percentile, and whiskers are the furthest datapoint outside the box, but within 1.5 times the box width. (C) Free energy differences between the open and closed state of the D3 interface. The free energy differences were obtained from the equilibrium data as F1/2⋅Δx and from the kinetics as ksT⋅log(τ0,closed/τ0,open). The data fall along the 45° line (dashed), indicating that the 2 estimates give consistent values. Comparison of the different conditions reveals a lower free energy difference for decreased pH of 6.2 and 5.5, indicating a destabilization of the domain at low pH. Small points correspond to data from individual molecules; large points with error bars indicate the mean ± standard deviation for each condition. (D) Average number of histidine contacts for several subdomains with the VWD3 domain, determined from equilibrium simulations. Two conditions were considered: histidines unprotonated (orange) or protonated (dark red). Contacts are defined as atoms with a distance < 0.35 nm. Statistically significant differences between the unprotonated and protonated histidine simulations are indicated by 2 stars corresponding to p < .01. (E) Crystallographic structure of the D′D3 domain with histidine residues that showed the strongest changes in average contacts indicated as spheres.

Stability of the D3 interface is modulated by pH and FVIII binding. (A) Midpoint force F1/2 for different buffer conditions (see supplemental Table 1 for details of the buffer compositions). The D3 interface is destabilized by decreasing pH, not affected by addition of EDTA (to remove the Ca2+ from the binding loop in VWD3) and stabilized by addition of FVIII. The difference between F1/2 at pH 7.4 and pH 6.2 is highly significant (P < .0041; 2-tailed t test for independent means). Similarly, the difference between F1/2 at pH 7.4 and pH 7.4 + FVIII is highly significant (P < .0078). (B) Distances Δz between states for the same conditions as in panel A. There are no significant differences in Δz between the conditions. Each point in panels A and B corresponds to an individual molecule. Boxes indicate the median and 25th and 75th percentile, and whiskers are the furthest datapoint outside the box, but within 1.5 times the box width. (C) Free energy differences between the open and closed state of the D3 interface. The free energy differences were obtained from the equilibrium data as F1/2⋅Δx and from the kinetics as ksT⋅log(τ0,closed/τ0,open). The data fall along the 45° line (dashed), indicating that the 2 estimates give consistent values. Comparison of the different conditions reveals a lower free energy difference for decreased pH of 6.2 and 5.5, indicating a destabilization of the domain at low pH. Small points correspond to data from individual molecules; large points with error bars indicate the mean ± standard deviation for each condition. (D) Average number of histidine contacts for several subdomains with the VWD3 domain, determined from equilibrium simulations. Two conditions were considered: histidines unprotonated (orange) or protonated (dark red). Contacts are defined as atoms with a distance < 0.35 nm. Statistically significant differences between the unprotonated and protonated histidine simulations are indicated by 2 stars corresponding to p < .01. (E) Crystallographic structure of the D′D3 domain with histidine residues that showed the strongest changes in average contacts indicated as spheres.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal