Salt-bridge modulates differential calcium-mediated ligand binding to integrin α1-and α2-I domains

Integrins are transmembrane cell-extracellular matrix adhesion receptors that impact many cellular functions. A subgroup of integrins contain an inserted (I) domain within the alpha-subunits (alpha I) that mediate ligand recognition where function is contingent on binding a divalent cation at the metal ion dependent adhesion site (MIDAS). Ca2+ is reported to promote alpha 1I but inhibit alpha 2I ligand binding. We co-crystallized individual I-domains with MIDAS-bound Ca2+ and report structures at 1.4 and 2.15 angstrom resolution, respectively. Both structures are in the "closed" ligand binding conformation where Ca2+ induces minimal global structural changes. Comparisons with Mg2+-bound structures reveal Mg2+ and Ca2+ bind alpha 1I in a manner sufficient to promote ligand binding. In contrast, Ca2+ is displaced in the alpha 2I domain MIDAS by 1.4 angstrom relative to Mg2+ and unable to directly coordinate all MIDAS residues. We identified an E152-R192 salt bridge hypothesized to limit the flexibility of the alpha 2I MIDAS, thus, reducing Ca2+ binding. A alpha 2I E152A construct resulted in a 10,000-fold increase in Mg2+ and Ca2+ binding affinity while increasing binding to collagen ligands 20%. These data indicate the E152-R192 salt bridge is a key distinction in the molecular mechanism of differential ion binding of these two I domains.