Integrin-ligand interactions are regulated in a complex manner by divalent cations, and previous studies have identified ligand-competent, stimulatory, and inhibitory cation-binding sites. In collagen-binding integrins, such as ␣21, ligand recognition takes place exclusively at the ␣ subunit I domain. However, activation of the ␣I domain depends on its interaction with a structurally similar domain in the  subunit known as the I-like or I domain. The top face of the I domain contains three cation-binding sites: the metal-ion dependent adhesion site (MIDAS), the ADMIDAS (adjacent to MIDAS), and LIMBS (ligand-associated metal-binding site). The role of these sites in controlling ligand binding to the ␣I domain has yet to be elucidated. Mutation of the MIDAS or LIMBS completely blocked collagen binding to ␣21; in contrast mutation of the ADMI-DAS reduced ligand recognition but this effect could be overcome by the activating monoclonal antibody TS2/16. Hence, the MIDAS and LIMBS appear to be essential for the interaction between ␣I and I, whereas occupancy of the ADMIDAS has an allosteric effect on the conformation of I. An activating mutation in the ␣2 I domain partially restored ligand binding to the MIDAS and LIMBS mutants. Analysis of the effects of Ca 2؉ , Mg 2؉ , and Mn 2؉ on ligand binding to these mutants showed that the MIDAS is a ligand-competent site through which Mn 2؉ stimulates ligand binding, whereas the LIMBS is a stimulatory Ca 2؉ -binding site, occupancy of which increases the affinity of Mg 2؉ for the MIDAS.Integrins are a large family of receptors for extracellular and cell surface proteins (1, 2). Integrin-ligand binding is regulated by divalent ions and conformational changes (3, 4). The overall shape of an integrin is that of a large ligand-binding "head" on two long "legs" with flexible "knees." Integrins can exist in low affinity ("inactive") and high affinity (primed or "active") states; the balance between these states is regulated by both local and global conformational changes. The inactive form of the receptor has been shown to be bent with a closed headpiece, whereas the fully active form appears to be extended with an open headpiece (5, 6). Integrins are heterodimers containing non-covalently associated ␣ and  subunits. Eighteen ␣ subunits (␣1-␣11, ␣V, ␣IIb, ␣D, ␣E, ␣M, ␣L, and ␣X) and eight  subunits (1-8) have been identified in mammals. Two subfamilies can be identified depending on the presence or absence of an autonomous folding domain of ϳ200 amino acids, the inserted or "I" domain in the ␣ subunit ("␣I").2 The I domain comprises a central hydrophobic six-stranded -sheet surrounded by seven ␣-helices, with the characteristic divalent cation-binding sequence motif DXSXS forming part of the metal-ion dependent adhesion site (MIDAS) on the top face of the domain (7). In each of the nine ␣I-containing integrins (␣1, ␣2, ␣10, ␣11, ␣E, ␣D, ␣M, ␣L, and ␣X), the MIDAS is the key site for ligand binding (8 -10). I domains can exist in closed (inactive) or open (active) confor...