Reversibly locking a protein fold in an active conformation with a disulfide bond:: Integrin αL I domains with high affinity and antagonist activity in vivo

The integrin alphaL beta2 has three different domains in its headpiece that have been suggested to either bind ligand or to regulate ligand binding. One of these, the inserted or I domain, has a fold similar to that of small G proteins. The I domain of the alphaM and alpha subunits has been crystallized in both open and closed conformations; however, the cut I domain has been crystallized in only the closed conformation. We hypothesized that the cut domain also would have an open conformation, and that this would be the ligand binding conformation. Therefore, we introduced pairs of cysteine residues to form disulfides that would lock the at I domain in either the open or closed conformation. Locking the I domain open resulted in a 9,000-fold increase in affinity to intercellular adhesion molecule-1 (ICAM-1), which was reversed by disulfide reduction. By contrast, the affinity of the locked closed conformer was similar to wild type. Binding completely depended on Mg2+. Orders of affinity were ICAM-1 > ICAM-2 > ICAM-3. The k(on), k(off), and K-D Values for the locked open I domain were within l,5-fold of values previously determined for the alphaL beta2 complex, showing that the I domain is sufficient for full affinity binding to ICAM-1. The locked open I domain antagonized alphaL beta2-dependent adhesion in vitro, lymphocyte homing in vivo, and firm adhesion but not rolling on high endothelial venules. The ability to reversibly lock a protein fold in an active conformation with dramatically increased affinity opens vistas in therapeutics and proteomics.