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Binding Site Elucidation of Hydantoin-Based Antagonists of LFA-1 Using Multidisciplinary Technologies: Evidence for the Allosteric Inhibition of a Protein−Protein Interaction

2001; American Chemical Society; Volume: 123; Issue: 24 Linguagem: Inglês

10.1021/ja0104249

1943-2984

K Last-Barney, Walter Davidson, Mario Cardozo, Leah L. Frye, Christine A. Grygon, Jerry L. Hopkins, Deborah D. Jeanfavre, Susan Pav, Chungeng Qian, James M. Stevenson, Liang Tong, Renée Zindell, Terence A. Kelly,

Glycosylation and Glycoproteins Research

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.