Functional and computational studies of the ligand-associated metal binding site of β3 integrins

Authors

  • Marta Murcia,

    1. Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029
    2. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021
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  • Marketa Jirouskova,

    1. Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, New York 10021
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  • Jihong Li,

    1. Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, New York 10021
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  • Barry S. Coller,

    1. Laboratory of Blood and Vascular Biology, The Rockefeller University, New York, New York 10021
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  • Marta Filizola

    Corresponding author
    1. Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York 10029
    2. Department of Physiology and Biophysics, Weill Medical College of Cornell University, New York, New York 10021
    • Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Icahn Medical Institute Building, Room 16–20, 1425 Madison Avenue, Box 1677, New York, NY 10029-6574
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Abstract

A combination of experimental and computational approaches was used to provide a structural context for the role of the β3 integrin subunit ligand-associated metal binding site (LIMBS) in the binding of physiological ligands to β3 integrins. Specifically, we have carried out (1) adhesion assays on cells expressing normal αIIβ3, normal αVβ3, or the corresponding β3 D217A LIMBS mutants; and (2) equilibrium and nonequilibrium (steered) molecular dynamics (MD) simulations of eptifibatide in complex with either a fully hydrated normal αIIβ3 integrin fragment (αIIb β-propeller and the β3 βA (I-like), hybrid, and PSI domains) or the equivalent β3 D217A mutant. Normal αIIβ3 expressing cells adhered to immobilized fibrinogen and echistatin, whereas cells expressing the αIIβ3 D217A LIMBS mutant failed to adhere to either ligand. Similarly, the equivalent αVβ3 mutant was unable to support adhesion to vitronectin or fibrinogen. The αIIβ3 D217A mutation increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain, indicating that this mutation induced allosteric changes in the protein. Steered MD simulating the unbinding of eptifibatide from either normal αIIβ3 or the equivalent β3 D217A mutant suggested that the reduction in ligand binding caused by the LIMBS mutant required the loss of both the LIMBS and the metal ion-dependent adhesion site (MIDAS) metal ions. Our computational results indicate that the LIMBS plays a crucial role in ligand binding to αIIβ3 by virtue of its effects on the coordination of the MIDAS. Proteins 2008. © 2008 Wiley-Liss, Inc.

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