Divalent cations stabilize the alpha 1 beta 1 integrin I domain

Recent structural and functional analyses of a integrin subunit I domains i mplicate a region in cation and ligand binding referred to as the metal ion -dependent adhesion site (MIDAS). Although the molecular interactions betwe en Mn2+ and Mg2+ and the MIDAS region have been defined by crystallographic analyses, the role of cation in I domain function is not well understood. Recombinant alpha 1 beta 1 integrin I domain (alpha 1-I domain) binds colla gen in a cation-dependent manner, We have generated and characterized a pan el of antibodies directed against the alpha 1-I domain, and selected one (A JH10) that blocks alpha 1 beta 1 integrin function for further study. The e pitope of AJH10 was localized within the loop between the alpha 3 and alpha 4 helices which contributes one of the metal coordination sites of the MID AS structure. Kinetic analyses of antibody binding to the I domain demonstr ate that divalent cation is required to stabilize the epitope. Denaturation experiments demonstrate that cation has a dramatic effect on the stabiliza tion of the I domain structure. Mn2+ shifts the point at which the I domain denatures from 3.4 to 6.3 M urea in the presence of the denaturant, and fr om 49.5 to 58.6 degrees C following thermal denaturation, The structural st ability provided to the alpha 1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.