Two-dimensional kinetics regulation of αLβ2-ICAM-1 interaction by conformational changes of the αL-inserted domain

The leukocyte integrin alpha(L)beta(2) mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alpha(L)beta(2) is regulated by or induces conformational changes in the inserted ( I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional ( 2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alpha(L)beta(2) or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by similar to 8000- and similar to 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alpha(L)beta(2), but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alpha(L)beta(2), is required for affinity and on-rate up-regulation.