Thermodynamic stabilization of the von Willebrand Factor A1 domain due to loss-of-function disease-related mutations.

The von Willebrand disease (vWD) is the most common hereditary bleeding disorder, caused by defects of the von Willebrand Factor (vWF), a large extracellular protein in charge of adhering platelets at sites of vascular lesion. vWF carries out this essential homeostatic task, via the specific protein-protein interaction between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIB). Upon the vWF activation triggered by the shear of the flowing blood. The two naturally occurring mutations G1324A and G1324S at the A1 domain, near the GPIB binding site, result in a dramatic decrease of platelets adhesion, a bleeding disorder classified as type 2M vWD. However, it remained unclear how these two supposedly minor modifications lead to this drastic phenotypic response. We addressed this question using a combination of equilibrium-molecular dynamics (MD) and non-equilibrium MD-based free energy simulations. Our data confirm that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. These mutations locally diminished the flexibility of the binding site to GPIB and induced a conformational change that affected the nearby secondary structure elements. Furthermore, we observed two significant changes in the vWF A1 domain upon mutation, the global redistribution of the internal mechanical stress and the increased thermodynamic stability of the A1 domain. These observations are consistent with previously-reported mutation-augmented melting temperatures. Overall, our results support the idea of thermodynamic conformational restriction of A1-- before the binding to GPIB--as a crucial factor determining the loss-of-function of the G1324A(S) vWD mutants.