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

Biochemistry. 1998 Aug 11;37(32):11280-8. doi: 10.1021/bi9727848.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites / drug effects
  • Blood Platelets / metabolism
  • Cations, Divalent
  • Edetic Acid / pharmacology
  • Energy Transfer
  • Fluorescence Polarization
  • Humans
  • Integrins / chemistry
  • Integrins / metabolism*
  • Ligands
  • Peptide Fragments / metabolism*
  • Procollagen / metabolism*
  • Protein Binding / drug effects
  • Protein Structure, Tertiary
  • Receptors, Collagen
  • Spectrometry, Fluorescence
  • Terbium / metabolism*

Substances

  • Cations, Divalent
  • Integrins
  • Ligands
  • Peptide Fragments
  • Procollagen
  • Receptors, Collagen
  • Terbium
  • Edetic Acid