THE CATION-BINDING DOMAIN FROM THE ALPHA-SUBUNIT OF INTEGRIN ALPHA(5)BETA(1) IS A MINIMAL DOMAIN FOR FIBRONECTIN RECOGNITION

The cation-binding domain from the alpha subunit of human integrin alp ha(5)beta(1) was produced as a recombinant protein, alpha(5)-(229-448) . This protein displays a well defined fold with a content of 30-35% a lpha-helix and 20-25% beta-strand, based on circular dichroism. The bi nding of Ca2+ or Mg2+ to alpha(5)-(229-448) results in a biphasic conf ormational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities. The two classe s of sites are located in two conformationally independent lobes, as e stablished by a parallel study of two recombinant half-domains (Nand C -terminal) that also adopt stable folds. Upon saturation with divalent cations, alpha(5)-(229-448) binds an Arg-Gly-Asp (RGD)-containing fib ronectin ligand to form a 1:1 complex. Complex formation is associated with a specific conformational adaptation of the Ligand, suggesting a n induced fit mechanism. In contrast, neither of the half-domains is c ompetent for ligand binding. The alpha(5)-(229-448)-fibronectin comple x is dissociated in the presence of an RGD peptide, as well as of a si mple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the alpha(5) cation- binding domain.