title: NMR and mutagenesis evidence for an I domain allosteric site that regulates lymphocyte function-associated antigen 1 ligand binding.
abstract: 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.
authors.affiliation: Abbott Laboratories, D47G, AP10, Abbott Park, IL 60064-6098, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Huth
authors.forename: E T
authors.initials: ET
authors.lastname: Olejniczak
authors.forename: R
authors.initials: R
authors.lastname: Mendoza
authors.forename: H
authors.initials: H
authors.lastname: Liang
authors.forename: E A
authors.initials: EA
authors.lastname: Harris
authors.forename: M L
authors.initials: ML
authors.lastname: Lupher
authors.forename: A E
authors.initials: AE
authors.lastname: Wilson
authors.forename: S W
authors.initials: SW
authors.lastname: Fesik
authors.forename: D E
authors.initials: DE
authors.lastname: Staunton
mesh_terms: D000495:Allosteric Site; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D018821:CD18 Antigens / Q000737:chemistry*; D019556:COS Cells; D002448:Cell Adhesion; D018360:Crystallography, X-Ray; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry*; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016177:Macrophage-1 Antigen / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D014162:Transfection
chemical_list: D018821:CD18 Antigens; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D016177:Macrophage-1 Antigen; D011994:Recombinant Proteins; D018799:Intercellular Adhesion Molecule-1