NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.

Huth JR, Olejniczak ET, Mendoza R, Liang H, Harris EA, Lupher ML, Wilson AE, Fesik SW, Staunton DE
Proc Natl Acad Sci U S A. 2000 97 (10): 5231-6

PMID: 10805782 · PMCID: PMC25811 · DOI:10.1073/pnas.97.10.5231

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-1 must convert from a resting to an activated state that avidly binds its ligands such as intercellular adhesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD11b/CD18) I domain suggest that integrin activation could involve a conformational change of the C-terminal alpha-helix. We report the use of NMR spectroscopy to identify CD11a I domain residues whose resonances are affected by binding to ICAM-1. We observed two distinct sites in the CD11a I domain that were affected. As expected from previous mutagenesis studies, a cluster of residues localized around the metal ion-dependent adhesion site (MIDAS) was severely perturbed on ICAM-1 binding. A second cluster of residues distal to the MIDAS that included the C-terminal alpha-helix of the CD11a I domain was also affected. Substitution of residues in the core of this second I domain site resulted in constitutively active LFA-1 binding to ICAM-1. Binding data indicates that none of the 20 substitution mutants we tested at this second site form an essential ICAM-1 binding interface. We also demonstrate that residues in the I domain linker sequences can regulate LFA-1 binding. These results indicate that LFA-1 binding to ICAM-1 is regulated by an I domain allosteric site (IDAS) and that this site is structurally linked to the MIDAS.

MeSH Terms (19)

Allosteric Site Amino Acid Substitution Animals Binding Sites CD18 Antigens Cell Adhesion COS Cells Crystallography, X-Ray Humans Intercellular Adhesion Molecule-1 Ligands Lymphocyte Function-Associated Antigen-1 Macrophage-1 Antigen Models, Molecular Mutagenesis, Site-Directed Nuclear Magnetic Resonance, Biomolecular Protein Structure, Secondary Recombinant Proteins Transfection

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