Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the alphal subunit of lymphocyte function-associated antigen-1

Protein Sci. 2006 Feb;15(2):290-303. doi: 10.1110/ps.051583406. Epub 2005 Dec 29.

Abstract

The lymphocyte function-associated antigen-1 (LFA-1) binding of a unique class of small-molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule-1 (sICAM-1) and A-286982, which respectively define direct and allosteric competitive binding sites within LFA-1's inserted (I) domain. All three molecules antagonized LFA-1 binding to ICAM-1-Immunoglobulin G fusion (ICAM-1-Ig) in a competition ELISA, but only compound 3 and sICAM-1 inhibited the binding of a fluorescein-labeled analog of compound 3 to LFA-1. Compound 3 and sICAM-1 displayed classical direct competitive binding behavior with ICAM-1. Their antagonism of LFA-1 was surmountable by both ICAM-1-Ig and a fluorescein-labeled compound 3 analog. The competition of both sICAM-1 and compound 3 with ICAM-1-Ig for LFA-1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross-linking studies with a photoactivated analog of compound 3 localized the high-affinity small-molecule binding site to the N-terminal 507 amino acid segment of the alpha chain of LFA-1, a region that includes the I domain. In addition, cells transfected with a variant of LFA-1 lacking this I domain showed no significant binding of a fluorescein-labeled analog of compound 3 or ICAM-1-Ig. These results demonstrate that compound 3 inhibits the LFA-1/ICAM-1 binding interaction in a directly competitive manner by binding to a high-affinity site on LFA-1. This binding site overlaps with the ICAM-1 binding site on the alpha subunit of LFA-1, which has previously been localized to the I domain.

MeSH terms

  • Allosteric Regulation
  • Binding Sites
  • Binding, Competitive
  • Cross-Linking Reagents
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Immunoglobulin G / metabolism
  • Intercellular Adhesion Molecule-1 / chemistry*
  • Intercellular Adhesion Molecule-1 / genetics
  • Intercellular Adhesion Molecule-1 / metabolism*
  • Kidney / metabolism
  • Ligands
  • Lymphocyte Function-Associated Antigen-1 / chemistry*
  • Lymphocyte Function-Associated Antigen-1 / genetics
  • Lymphocyte Function-Associated Antigen-1 / metabolism*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Protein Structure, Tertiary
  • Protein Subunits
  • Recombinant Fusion Proteins / antagonists & inhibitors
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • Cross-Linking Reagents
  • Immunoglobulin G
  • Ligands
  • Lymphocyte Function-Associated Antigen-1
  • Peptide Fragments
  • Protein Subunits
  • Recombinant Fusion Proteins
  • Intercellular Adhesion Molecule-1