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

The leukocyte integrin, lymphocyte function-associated antigen 1 (LFA-1) (C D11a/CD18), mediates cell adhesion and signaling in inflammatory and immune responses. To support these functions, LFA-I must convert from a resting t o an activated state that avidly binds its ligands such as intercellular ad hesion molecule 1 (ICAM-1). Biochemical and x-ray studies of the Mac-1 (CD1 1b/CD18) I domain suggest that integrin activation could involve a conforma tional change of the C-terminal a-helix. We report the use of NMR spectrosc opy to identify CD11a I domain residues whose resonances are affected by bi nding 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 d omain site resulted in constitutively active LFA-1 binding to ICAM-1. Bindi ng data indicates that none of the 20 substitution mutants we tested at thi s second site form an essential ICAM-1 binding interface. We also demonstra te that residues in the I domain linker sequences can regulate LFA-1 bindin g. 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.