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

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• 作者：Kathleen 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 ; Renee Zindell
• 刊名：Journal of the American Chemical Society
• 出版年：2001
• 出版时间：June 20, 2001
• 年：2001
• 卷：123
• 期：24
• 页码：5643 - 5650
• 全文大小：444K
• 年卷期：v.123,no.24(June 20, 2001)
• ISSN：1520-5126

文摘

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 withthe intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizingcell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integratedimmunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domainwere blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding siteresides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysisof the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to thephotoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed forthe 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 crystalstructure of the complex.