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

• Published in: The Journal of biological chemistry 2017-11, Vol.292 (45), p.18608-18617
• Main Authors: Interlandi, Gianluca, Yakovenko, Olga, Tu, An-Yue, Harris, Jeff, Le, Jennie, Chen, Junmei, López, José A., Thomas, Wendy E.
• Format: Article
• Language: English
• Subjects: allosteric regulation; Amino Acid Substitution; Aspartic Acid - chemistry; Binding Sites; Blood Flow Velocity; Blood Platelets - metabolism; Cell Adhesion; dynamic flow assay; Gene Deletion; hemostasis; Humans; Microspheres; Models, Molecular; Molecular Bases of Disease; Molecular Docking Simulation; molecular dynamics; Molecular Dynamics Simulation; parasteric inhibition; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Platelet Glycoprotein GPIb-IX Complex - antagonists & inhibitors; Platelet Glycoprotein GPIb-IX Complex - chemistry; Platelet Glycoprotein GPIb-IX Complex - genetics; Platelet Glycoprotein GPIb-IX Complex - metabolism; Point Mutation; protein binding; Protein Conformation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Static Electricity; thrombosis; von Willebrand factor; von Willebrand Factor - antagonists & inhibitors; von Willebrand Factor - chemistry; von Willebrand Factor - genetics; von Willebrand Factor - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M117.797456

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 D′D3 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 Ibα (GpIbα). 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 GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1–GpIbα 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 GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, 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–GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.