The specificity and function of the metal-binding sites in the integrin beta3 A-domain.
The A-domains within integrin beta subunits contain three metal sites termed the metal ion-dependent adhesion site (MIDAS), site adjacent to the metal ion-dependent adhesion site (ADMIDAS), and ligand-induced metal-binding site (LIMBS), and these sites are involved in ligand engagement. The selectivity of these metal sites and their role in ligand binding have been investigated by expressing a fragment corresponding to the beta3 A-domain, beta3-(109-352), and single point mutants in which each of the cation-binding sites has been disabled. Equilibrium dialysis experiments identified three Mn2+- and two Ca2+-binding sites with the LIMBS being the site that did not bind Ca2+. Although the ADMIDAS could bind Ca2+, it did not bind Mg2+. These results indicate that the Ca2+-specific site that inhibits ligand binding is the ADMIDAS. Two different assay systems, surface plasmon resonance and a microtiter plate assay, demonstrated that the beta3 A-domain fragment bound fibrinogen in the presence of 0.1 mm Ca2+ but not in 3 mm Ca2+. This behavior recapitulated the effects of Ca2+ on fibrinogen binding to alphavbeta3 but not alphaIIbbeta3. Disabling any of the three cation-binding sites abrogated fibrinogen binding. These results indicate that the specificities of the three metal-binding sites for divalent cations are distinct and that each site can regulate the ligand binding potential of the beta3 A-domain.
Pesho
Michelle M
MM
Joseph J. Jacobs Center for Thrombosis and Vascular Biology and Department of Molecular Cardiology, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Bledzka
Kamila
K
Michalec
Lidia
L
Cierniewski
Czeslaw S
CS
Plow
Edward F
EF
D001665:Binding Sites; D018076:DNA, Complementary / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry; D006801:Humans; D039661:Integrin beta3 / Q000737:chemistry* / Q000502:physiology*; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D018076:DNA, Complementary; D039661:Integrin beta3; D008024:Ligands; D008670:Metals; D008345:Manganese; D005340:Fibrinogen; D008274:Magnesium