Abstract
Glycoprotein Ib (GPIb) is a platelet receptor with a critical role in mediating the arrest of platelets at sites of vascular damage. GPIb binds to the A1 domain of von Willebrand factor (vWF-A1) at high blood shear, initiating platelet adhesion and contributing to the formation of a thrombus. To investigate the molecular basis of GPIb regulation and ligand binding, we have determined the structure of the N-terminal domain of the GPIb(alpha) chain (residues 1-279). This structure is the first determined from the cell adhesion/signaling class of leucine-rich repeat (LRR) proteins and reveals the topology of the characteristic disulfide-bonded flanking regions. The fold consists of an N-terminal beta-hairpin, eight leucine-rich repeats, a disulfide-bonded loop, and a C-terminal anionic region. The structure also demonstrates a novel LRR motif in the form of an M-shaped arrangement of three tandem beta-turns. Negatively charged binding surfaces on the LRR concave face and anionic region indicate two-step binding kinetics to vWF-A1, which can be regulated by an unmasking mechanism involving conformational change of a key loop. Using molecular docking of the GPIb and vWF-A1 crystal structures, we were also able to model the GPIb.vWF-A1 complex.
Highlights
The glycoprotein (GP)1 Ib1⁄7V1⁄7IX complex is a platelet membrane receptor complex with a critical role in adhesion to the damaged vessel wall under conditions of high shear stress [1]
The role of the leucine-rich repeat (LRR) concave face binding site is fundamental for binding the A1 domain as the mutations here or binding of antibodies with epitopes in this region block A1 binding in response to all reagents [20]
The A1 domain interaction with the Glycoprotein Ib (GPIb)␣ anionic peptide probably precedes binding to the concave face in multistepbinding kinetics that are reminiscent of the thrombin-hirudin interaction (Fig. 7) [39]
Summary
Crystallization and Data Collection—The GPIb␣ N-terminal domain was prepared from platelets and in recombinant form. Glycocalicin, the GPIb␣ extracellular domain, was isolated and purified from platelets as described previously [24]. This was cleaved to generate the N-terminal 45-kDa region (amino acids 1–288) and macroglycopeptide using 1:250 w/w Lys-C endoprotease treatment for 24 h at 4 °C. The N-terminal domain was separated from the macroglycopeptide by linear salt gradient (0.2–1 M) on a Mono Q column equilibrated with 25 mM Tris 7.5, 0.2 M NaCl. DNA corresponding to the GPIb␣ signal peptide and resi-
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