Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets

Abstract

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located DD3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ib (GpIb). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIb on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1–GpIb binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIb. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIb, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1–GpIb complex and to indirectly destabilize the complex through the N-terminal linker.