Cartoon models of integrin receptor conformational states and headpiece construct. (A) Three major conformational states of integrin receptor. (B-D) Integrin α_IIbβ₃ headpiece construct, protein purification, and protease digestion. The α_IIbβ₃ headpiece was first purified by Ni-NTA (B), then treated with chymotrypsin and further purified by Ni-NTA and gel filtration (C). It was then treated with carboxypeptidase in the presence of monoclonal antibody 10E5 Fab and finally purified by gel filtration (D). Protein molecular weight markers are in kDa.

The binding pocket of RUC-1 in the closed α_IIbβ₃ headpiece crystal structure. (A) Overview of RUC-1 binding site. α_IIb (light blue), β₃ (wheat), and Fab (gray) are shown as solvent-accessible surfaces. RUC-1 is shown as spheres with green carbons, red oxygens, blue nitrogens, and yellow sulfurs. (B) Close-up of the RUC-1 binding site. Selected side chain and backbone atoms are shown in stick with other regions in cartoon. Color code is as in panel A. A simulated annealing omit map for RUC-1 is shown at 1σ. Water molecules are small red spheres. (C) The tirofiban binding site (Protein Data Bank code 2VDM). Color code is same as in panel A. Ca²⁺ ions of the SyMBS or the ADMIDAS (yellow), and the Mg²⁺ ion of MIDAS (silver) are shown as spheres. (D, F, and G) Comparison of RUC-1, KQAGDV, and RGDV binding sites. Color codes are as above, except KQAGDV and RGDV are shown as sticks with cyan carbons, after superposition on the RUC-1 complex using super command in Pymol with the α_IIb β-propeller and β₃ βI domains. (E) Comparison of small molecule binding locations. Crystal structures (Protein Data Bank code in parentheses) containing the indicated small molecules were superimposed as above. The ligands from the structures are shown in exactly the same alignment, except for vertical separation on the page.

The α_IIb Y190F and D232H mutations increase the RUC-1 IC₅₀ for fibrinogen. Fibrinogen binding to cells expressing normal human α_IIbβ₃, the α_IIb Y190F mutant, or the α_IIb D232H mutant receptor in the presence of the activating monoclonal antibody PT25-2 was determined. The data were normalized and the mean ± standard error of the mean (SEM) for each concentration of RUC-1 tested is depicted.

The crystal structure of α_IIbβ₃ headpiece in closed conformation. (A) Cartoon diagram of the α_IIbβ₃ headpiece complex with 10E5 Fab. Ca²⁺ and Mg²⁺ ions are yellow and silver spheres, respectively. (B) Superposition of the closed headpiece structure found in this study (2 molecules in one asymmetric unit, red) with the indicated previously reported structures (number of molecules per asymmetric unit). The structures were aligned on the α_IIbβ-propeller and the β₃ βI domains by the super command of Pymol. (C) Superposition of β₃ βI domains of the closed α_IIbβ₃ headpiece structure in this study (red) and the closed headpiece structure of α_IIbβ₃ entire ectodomain (blue). Metal ions are shown as spheres. (D) 2Fo-Fc maps of metals and coordinating waters in β₃ βI domain in the presence of 5mM Mg²⁺ and 1mM Ca²⁺. The maps are contoured at 1.5 σ. Ca²⁺ (gold) and Mg²⁺ (green) ions are large spheres. Waters (red) are small spheres. Nitrogen atoms are blue and oxygen atoms are red. Metal coordination and hydrogen bonds are dashed. (E) The β₃ βI/Hybrid interface in the complete α_IIbβ₃ ectodomain structure. (F) The β₃ βI/Hybrid interface in the closed headpiece structure. The 2 structures were aligned on the β₃ βI domain.

The binding pocket of RUC-1 studied by molecular dynamics (MD) simulations. (A) Comparison of the binding site of RUC-1 in crystal structure (magenta) and in a representative conformation (at 8 nanoseconds) of the last 5 nanoseconds of a 10-nanosecond MD simulation (cyan). The structures were aligned on the α_IIb β-propeller domain by Pymol. α_IIb and β₃ subunits are shown in light blue and wheat, respectively. (B-D) Specific distance changes during 10-nanosecond MD simulations. Values for crystal structure are indicated by triangles. (B) Minimum distance between the basic nitrogen of RUC-1 and either of the 2 oxygens of the Asp-224 side chain (blue line). Minimum distance between either of the 2 oxygens of the Asp-224 side chain and the backbone nitrogen of Trp-162 (red line). (C) Distance between the oxygen of the Tyr-190 side chain and the backbone carbonyl oxygen of β₃ Arg-216 (blue line). Distance between the centroid of the aromatic ring of Tyr-190 side chain and the centroid of the RUC-1 fused ring (orange line). (D) Number of water molecules either forming direct interactions with the RUC-1 carbonyl oxygen or interacting with the carboxyl oxygens of the Asp-232 side chain.

Conformational change of integrin α_IIbβ₃ headpiece studied by gel filtration and dynamic light scattering. (A) Gel filtration profile of α_IIbβ₃ headpiece alone or bound with antagonists. The untagged α_IIbβ₃ headpiece was mixed with saturating amounts of RUC-1, tirofiban, or eptifibatide and incubated at room temperature for 1 hour before Superdex 200 chromatography in Tris-buffered saline with 1mM Ca²⁺/Mg²⁺. The elution volumes are shown in parentheses. (B) Stokes radius measured by gel filtration or dynamic light scattering. (C-D) The structures with surface representation indicate the putative conformational rearrangement after drug binding. The structures were generated by adding the α_IIb thigh domain from the complete ectodomain structure to the headpiece crystal structures. The Ca²⁺ and Mg²⁺ ions are shown as yellow and silver spheres, respectively. RUC-1 (C) and tirofiban (D) are shown as spheres.