Identification and characterization of two cation binding sites in the integrin beta 3 subunit.
The midsegment of the beta(3) subunit has been implicated in the ligand and cation binding functions of the beta(3) 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 Ca(2+) supported ligand binding to both recombinant fragments, 1.0 mm Ca(2+) 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 Mg(2+) or Ca(2+) and one Ca(2+)-specific, whereas beta(3)-(95-301) lacked the Ca(2+)-specific site. Mutant forms of beta(3)-(95-373) 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 Ca(2+) could be docked onto a flexible loop in close proximity to the MIDAS. These results indicate that the ligand competent and Ca(2+)-specific inhibitory cation binding sites are distinct and reside in beta(3)-(95-373).
Cierniewska-Cieslak
Aleksandra
A
Center for Microbiology and Virology, Polish Academy of Sciences, Lodz, Poland.
Cierniewski
Czeslaw S
CS
Bledzka
Kamila
K
Blecka
Kamila
K
Papierak
Malgorzata
M
Michalec
Lidia
L
Zhang
Li
L
Haas
Thomas A
TA
Plow
Edward F
EF
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002412:Cations; D002942:Circular Dichroism; D017931:DNA Primers; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D039661:Integrin beta3; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D015703:Antigens, CD; D002412:Cations; D017931:DNA Primers; D039661:Integrin beta3; D008024:Ligands; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D005340:Fibrinogen