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Competition between intercellular adhesion molecule‐1 and a small‐molecule antagonist for a common binding site on the αl subunit of lymphocyte function‐associated antigen‐1
Author(s) -
Keating Susan M.,
Clark Kevin R.,
Stefanich Lisa D.,
Arellano Fred,
Edwards Caroline P.,
Bodary Sarah C.,
Spencer Steven A.,
Gadek Thomas R.,
Marsters James C.,
Beresini Maureen H.
Publication year - 2006
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.051583406
Subject(s) - lymphocyte function associated antigen 1 , intercellular adhesion molecule 1 , binding site , allosteric regulation , cell adhesion molecule , chemistry , intracellular , intercellular adhesion molecule , small molecule , ligand (biochemistry) , biophysics , stereochemistry , biology , biochemistry , receptor , cell adhesion , microbiology and biotechnology , cell
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 α 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 α subunit of LFA‐1, which has previously been localized to the I domain.