Thermodynamic stabilization of von Willebrand factor A1 domain induces protein loss of function

Abstract

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 to sites of vascular lesions. vWF performs this essential homeostatic task via specific protein–protein interactions between the vWF A1 domain and the platelet receptor, the glycoprotein Ib alpha (GPIBα). The two naturally occurring vWF A1 domain mutations G1324A and G1324S, near the GPIBα binding site, induce a dramatic decrease in platelet adhesion, resulting in a bleeding disorder classified as type 2M vWD. However, the reason for the drastic phenotypic response induced by these two supposedly minor modifications remains unclear. We addressed this question using a combination of equilibrium‐molecular dynamics (MD) and nonequilibrium MD‐based free energy simulations. Our data confirms that both mutations maintain the highly stable Rossmann fold of the vWF A1 domain. G1324A and G1324S mutations hardly changed the per‐residue flexibility of the A1 domain but induced a global conformational change affecting the region near the binding site to GPIBα. 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 mutations increasing the melting temperature. Overall, our results support the idea of thermodynamic conformational restriction of A1—before the binding to GPIBα—as a crucial factor determining the loss‐of‐function of the G1324A(S) vWD mutants.

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“…The domain prediction software InterProscan was used to predict the domains of differentially expressed proteins. The number of proteins for each domains, including WD domain [35], G-beta repeat, Ras Family [36], collagen triple helix repeat (20 copies) [37], Protein kinase domain [38], Fibronectin type III domain [39], von Willebrand factor type A domain [40] and others, are presented as a bar chart (top 20, figure 3(D)). The Fisher's exact test was used for domain enrichment analysis of differentially expressed proteins (red color represents the lowest P-value, and the highest significance under the corresponding domain classification) (figure 3(E)).…”