Distinct Roles of 1 Metal Ion-dependent Adhesion Site (MIDAS), Adjacent to MIDAS (ADMIDAS), and Ligand-associated Metal-binding Site (LIMBS) Cation-binding Sites in Ligand Recognition by Integrin 2 1*

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 2 1, ligand recognition takes place exclusively at the subunit I domain. However, activation of the I domain depends on its interactionwith 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 2 1; in contrast mutation of the ADMIDAS reduced ligand recognition but this effect could be overcomeby the activatingmonoclonal antibodyTS2/16.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 Ca2 , Mg2 , and Mn2 on ligand binding to these mutants showed that theMIDAS is a ligand-competent site throughwhichMn2 stimulates ligand binding, whereas the LIMBS is a stimulatory Ca2 -binding site, occupancy of which increases the affinity of Mg2 for the MIDAS.