Conformational activation of 2 integrins, their I domains, and regulation by small molecule antagonists

The inserted or I domain of the integrin LFA-1 ( L 2) mediates binding to ligands such as the IgSF member ICAM-1. Mutation has been used to introduce disulfide bonds that lock the L I domain in two distinct conformations termed open and closed. Wild type, resting L 2 requires activation for binding to ICAM-1. Locked closed L 2 is inactive and resistant to activation whereas locked open L 2 is constitutively active. I domains can be expressed in the absence of other integrin domains with artificial membrane anchors. In static adhesion assays, the locked open I domain is adhesive and the locked closed and wild type I domains are not. However, in flow chamber assays, the wild type I domain mediates rolling, whereas the locked open I domain mediates firm adhesion, reflecting how adhesive function in the vasculature is poised to mediate rolling under basal conditions and firm adhesion after activation. Real time ligand binding assays using surface plasmon resonance show that locking open the I domain increases its affinity for ICAM-1 by 9,000 fold, and that its kinetics and KD are comparable to measurements on active, intact L 2. Two classes of small molecule antagonists can be discriminated.