Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.

Dickeson SK, Bhattacharyya-Pakrasi M, Mathis NL, Schlesinger PH, Santoro SA
Biochemistry. 1998 37 (32): 11280-8

PMID: 9698375 · DOI:10.1021/bi9727848

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.

MeSH Terms (16)

Binding Sites Blood Platelets Cations, Divalent Edetic Acid Energy Transfer Fluorescence Polarization Humans Integrins Ligands Peptide Fragments Procollagen Protein Binding Protein Structure, Tertiary Receptors, Collagen Spectrometry, Fluorescence Terbium

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