Identification and characterization of two cation binding sites in the integrin β3 subunit

The midsegment of the beta(3) subunit has been implicated in the ligand and cation binding functions of the 93 integrins. This region may contain a metal ion-dependent adhesion site (MIDAS) and fold into an I domain-like structure. Two recombinant fragments, beta(3)-(95-373) and beta(3)-(95-301), were expressed and found to bind fibrinogen. Whereas 0.1 mM Ca2+ supported ligand binding to both recombinant fragments, 1.0 m-m Ca2+ suppressed binding to the longer but not the shorter fragment. These properties suggest that beta(3)-(95-373) contains both the ligand-competent (LC) and inhibitory (I) cation binding sites, and beta(3)-(95-301) lacks the I site. In equilibrium dialysis experiments, beta(3)-(95-373) contained two divalent cation binding sites, one reactive with either Mg2+ or Ca2+ and one Ca2+-specific, whereas beta(3)-(95-301) lacked the Ca2+-specific site. Mutant forms of 133495373) suggested that the LC site is a MIDAS motif involving Asp(119), Ser(121), Ser(123), Asp(217), and/or Glu(220) as coordination sites, and the I site was dependent upon residues within beta(3)-(301-323). In a molecular model of beta(3)-(95-373), a second Ca2+ could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca2+ specific inhibitory cation binding sites are distinct and reside in beta(3)-(95-373).