The alternatively spliced type III connecting segment of fibronectin is a zinc-binding module.

Fibronectin (FN) is a prototypic adhesive glycoprotein that is widely expressed in extracellular matrices and body fluids. The fibronectin molecule is dimeric, and composed of a series of repeating polypeptide modules. A recombinant fragment of FN incorporating type III repeats 12-15, and including the alternatively-spliced type three connecting segment (IIICS), was found to bind Ni(2+), Cu(2+) and Zn(2+) divalent cations, whereas a similar fragment lacking the IIICS did not. Mutation of two pairs of histidine residues in separate spliced regions of the IIICS reduced cation binding to near the level of the variant lacking the IIICS, suggesting a zinc finger-like mode of cation coordination. Analysis of native FNs purified from plasma or amniotic fluid revealed significant levels of zinc associated with those isoforms that contain the complete IIICS. Taken together, these data demonstrate that the IIICS region of FN is a novel zinc-binding module.

Novel activating and inactivating mutations in the integrin beta1 subunit A domain.

The ligand-binding activity of integrins is regulated by shape changes that convert these receptors from a resting (or inactive) state to an active state. However, the precise conformational changes that take place in head region of integrins (the site of ligand binding) during activation are not well understood. The portion of the integrin beta subunit involved in ligand recognition contains a von Willebrand factor type A domain, which comprises a central beta-sheet surrounded by seven alpha helices (alpha1-alpha7). Using site-directed mutagenesis, we show here that point mutation of hydrophobic residues in the alpha1 and alpha7 helices (which would be predicted to increase the mobility of these helices) markedly increases the ligand-binding activity of both integrins alpha5beta1 and alpha4beta1. In contrast, mutation of a hydrophilic residue near the base of the alpha1 helix decreases activity and also suppresses exposure of activation epitopes on the underlying hybrid domain. Our results provide new evidence that shifts of the alpha1 and alpha7 helices are involved in activation of the A domain. Although these changes are grossly similar to those defined in the A domains found in some integrin alpha subunits, movement of the alpha1 helix appears to play a more prominent role in betaA domain activation.