Locking in alternate conformations of the integrin αLβ2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin alphaL beta2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins, mAbs to the alphaL I domain and beta2 I-like domain inhibit adhesion of wild-type alphaL beta2 to intercellular adhesion molecule-1. However, with alphaL beta2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and tin that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, alphaL beta2 containing a locked open I domain was completely resistant to inhibition by mAbs to the beta2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type alphaL beta2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the beta2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the beta2 stalk region. Furthermore, locked open I domains, in alphaL beta2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates alphaL beta2 by binding to a site other than the I domain, most likely the I-like domain of beta2.