Ligand binding to integrin α(IIb)β₃ is dependent on a MIDAS-like domain in the β₃ subunit

Substitution of β₃ residue Asp¹¹⁹, Ser¹²¹, or Ser¹²³ results in a loss of the ligand binding function of integrin α(IIb)β₃. Homologous residues in other integrin β subunits are similarly critical for ligand binding function. This DXSXS motif is also present in the I domain of certain integrin α subunits, where it constitutes a portion of the unique metal ion- dependent adhesion site (MIDAS). In this report, we have utilized the crystal structure of the recombinant α(M) I domain to produce a three-dimensional model of the homologous region in the integrin β₃ subunit. We performed mutagenesis of candidate amino acid residues predicted from this model to be involved in cation coordination and ligand binding. We report the identification of Asp¹²⁷ and Glu²²⁰ as residues essential for the ligand binding function of α(IIb)β₃. Alanine substitution of these residues did not affect receptor expression but abolished the binding of activation-dependent (PAC1) and -independent (OPG2) ligand mimetic antibodies. In our proposed model, β₃ Asp²¹⁷ is analogous to a metal- coordinating residue in the α(M) MIDAS domain, while Glu²²⁰ does not correspond to a functional MIDAS domain residue. Substitution of the highly conserved β₃ residue Thr¹⁹⁷ corresponding to a critical MIDAS metal- coordinating Thr residue did not affect ligand binding function, suggesting that this region of β₃ adopts a structure that is very similar to but not identical to that of the MIDAS domain. These data support a functional linkage between these two sequences and further define a common feature of ligand binding to integrins.