CATION-BINDING TO THE INTEGRIN CD11B I-DOMAIN AND ACTIVATION MODEL ASSESSMENT

Background: The integrin family of cell-surface receptors mediate cell adhesion through interactions with the extracellular matrix or other cell-surface receptors. The alpha chain of some integrin heterodimers includes an inserted 'I domain' of about 200 amino acids which binds d ivalent metal ions and is essential for integrin function. Lee et al. proposed that the I domain of the integrin CD11b adopts a unique 'acti ve' conformation when bound to its counter receptor. In addition, they proposed that the lack of adhesion in the presence of Ca2+ ion reflec ted the stabilization of an 'inactive' I-domain conformation. We set o ut to independently determine the structure of the CD11b I domain and to evaluate the structural effects of divalent ion binding to this pro tein. Results: We have determined the X-ray structure of a new crystal form of the CD11b I domain in the absence of added metal ions by mult iple isomorphous replacement (MIR). Metal ions were easily introduced into this crystal form allowing the straight-forward assessment of the structural effects of divalent cation binding at the metal ion depend ent adhesion site (MIDAS). The equilibrium binding constants for these ions were determined by titration calorimetry. The overall protein co nformation and metal-ion coordination of the I domain is the same as t hat observed for all previously reported CD11a I-domain structures and a CD11b I-domain complex with Mn2+. These structures define a majorit y conformation. Conclusions: Addition of the cations Mg2+, Mn2+ and Cd 2+ to the metal-free I domain does not induce conformational changes i n the crystalline environment. Moreover, we find that Ca2+ binds poorl y to the I domain which serves to explain its failure to support adhes ion. We show that the active conformation proposed by Lee et al. is li kely to be a construct artifact and we propose that the currently avai lable data do not support a dramatic structural transition for the I d omain during counter-receptor binding.