Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.
The alpha 2 beta 1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the alpha 2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 +/- 1.7 microM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a Kd of 27 +/- 4 microM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than Mg2+. The alpha 2 beta 1 integrin also binds the pro-alpha 1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha 2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.
From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
S K
SK
Dickeson
M
M
Bhattacharyya-Pakrasi
N L
NL
Mathis
P H
PH
Schlesinger
S A
SA
Santoro
D001665:Binding Sites / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism; D002413:Cations, Divalent; D004492:Edetic Acid / Q000494:pharmacology; D004735:Energy Transfer; D005454:Fluorescence Polarization; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D010446:Peptide Fragments / Q000378:metabolism*; D011347:Procollagen / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D038981:Receptors, Collagen; D013050:Spectrometry, Fluorescence; D013725:Terbium / Q000378:metabolism*
D002413:Cations, Divalent; D016023:Integrins; D008024:Ligands; D010446:Peptide Fragments; D011347:Procollagen; D038981:Receptors, Collagen; D013725:Terbium; D004492:Edetic Acid