Structural, Functional, and Dynamic Characterization of the Binding Site of RUC-1, a Novel αIIb-Specific Inhibitor of Integrin αIIbβ3.
Authors: Zhu Jieqing , Zhu Jianghai , Negri Ana , Provasi Davide , Filizola Marta , Coller Barry S. , Springer Timothy A 
Published: October 2019

Abstract
Abstract 151
The platelet integrin αIIbβ3 mediates platelet aggregation by binding the adhesive plasma proteins fibrinogen and von Willebrand factor. We previously reported that co-crystallizing the αIIbβ3 headpiece with either of the two ligand mimetic drugs eptifibatide or tirofiban, or with a fibrinogen γ-chain peptide, results in the αIIbβ3 headpiece adopting an open conformation characterized by a swing-out motion of the β3 hybrid domain. In addition, all of the compounds bound both to the αIIb and β3 subunits, with the β3 contact mediated in part by coordinating the MIDAS metal ion. We recently reported on RUC-1, a novel αIIbβ3-specific small molecular antagonist that we predicted from molecular docking and biochemical data bound exclusively to αIIb. We have now determined the crystal structure of the αIIbβ3 headpiece in the absence of ligand and after soaking in RUC-1, and correlated the findings with advanced molecular dynamics simulations and functional studies of mutant receptors to define the mechanism of RUC-1 binding. The crystal structure shows that the αaIIbβ3 headpiece in the absence of RUC-1 or other ligand adopts a closed conformation with minimal β3 swing-out, similar to what we found in the full-length ectodomain of the bent receptor in the absence of ligand. Strikingly, RUC-1 only binds to the pocket formed by the αIIb β-propeller domain and has no contact with MIDAS metal ion or other sites in the β3 I domain. Standard molecular dynamics studies revealed a very similar binding pocket for RUC-1, which reflects the lowest energy binding state as assessed by metadynamics simulations. Both the crystal structure and the metadynamics studies identified αIIb Y190 and αIIb D232 as important for RUC-1 binding. RUC-1 is a much less potent inhibitor of mouse and rat αIIbβ3 than human αIIbβ; both mouse and rat have αIIb Y190F substitutions, and in addition, rat αIIb has a D232H substitution. A recombinant cell line expressing Y190F αIIbβ3 demonstrated an ∼80% reduction in RUC-1 affinity as judged by a 6-fold increase in IC50 for inhibiting fibrinogen binding and one expressing D232H αIIb β3 had more than a 95% decrease in affinity for RUC-1. To test whether RUC-1's exclusive binding to the αIIb subunit was associated with a reduced ability to induce a conformational change in αIIbβ3, we performed gel filtration and dynamic light scattering studies. In sharp contrast to the effects of eptifibatide and tirofiban, RUC-1 did not induce αIIbβ3 to adopt a less compact conformation in either system. These data suggest that ligand engagement of the β3 I domain, in particular coordinating the MIDAS metal ion and stabilizing the β1-α1 loop movement toward the MIDAS, is a likely trigger for β3 undergoing its swing-out motion. These data have potential therapeutic implications because they raise the possibility of inhibiting αIIbβ3 with an αIIb-specific antagonist that does not initiate the conformational changes in β3 that have been hypothesized to underlie the development of two major toxicities of αIIbβ3 antagonists, thrombocytopenia and paradoxical activation of the receptor.

Disclosures:
Filizola: Mount Sinai School of Medicine: Employment, Patents & Royalties. Coller:Rockefeller University: Employment, Patents & Royalties; Research Foundation of the State University of New York: Patents & Royalties; Centocor: Patents & Royalties; Accumetrics: Consultancy, Patents & Royalties.