The cation-binding domain from the α subunit of integrin α5β1 is a minimal domain for fibronectin recognition

The cation-binding domain from the alpha subunit of human integrin alpha(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% alpha-helix and 20-25% beta-strand, based on circular dichroism. The binding of Ca2+ or Mg2+ to alpha(5)-(229-448) results in a biphasic conformational rearrangement consistent with the occurrence of two classes of cation-binding sites differing by their affinities. The two classes of sites are located in two conformationally independent lobes, as established 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 fibronectin ligand to form a 1:1 complex. Complex formation is associated with a specific conformational adaptation of the Ligand, suggesting an induced fit mechanism. In contrast, neither of the half-domains is competent for ligand binding. The alpha(5)-(229-448)-fibronectin complex is dissociated in the presence of an RGD peptide, as well as of a simple carboxylic acid, suggesting that the RGD aspartyl carboxylate is an essential element that directly interacts with the alpha(5) cation-binding domain.