Transfer of a protein binding epitope to a minimal designed peptide

Quan, Clifford; Skelton, Nicholas J.; Jackson, David Y.; Renz, Mark; Chiu, Henry; Keating, Susan M.; Beresini, Maureen H.; Fong, Sherman; Artis, Dean R.

doi:10.1002/(sici)1097-0282(1998)47:4<265::aid-bip2>3.0.co;2-k

Cited by 9 publications

(1 citation statement)

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“…Small molecules and sICAM-1 were assayed for the ability to disrupt binding of ICAM-1-Ig or a fluorescein-labeled smallmolecule antagonist, compound 2B, to LFA-1 in a competitive format (Quan et al 1998;Burdick 1999;Gadek et al 2002). Compound 2B is similar to compound 1, but with a longer linker between the small molecule and fluorescein to maximize the binding of the anti-fluorescein detection antibody.…”

Section: Antagonist Competitionmentioning

confidence: 99%

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

Keating¹

2006

Protein Science

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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 fluoresceinlabeled 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 smallmolecule binding site to the N-terminal 507 amino acid segment of the a 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 a subunit of LFA-1, which has previously been localized to the I domain. Abbreviations: LFA-1, lymphocyte function-associated antigen-1; ICAM-1, intercellular adhesion molecule-1; I domain, inserted domain; MIDAS, metal ion-dependent adhesion site; IDAS, I domain allosteric site; ICAM-1-Ig, ICAM-1-Immunoglobulin G fusion; sICAM-1, soluble ICAM-1; HRP, horseradish peroxidase; FITC, fluorescein isothiocyante; FP, fluorescence polarization; BGG, bovine g globulins; PBS, phosphatebuffered saline; BSA, bovine serum albumin; TMB, tetramethylbenzidine; GuHCl, guanidine hydrochloride; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

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Section: Antagonist Competitionmentioning

confidence: 99%