Collagen and fibronectin (Fn) are two key extracellular matrix proteins, which are known to interact and jointly shape matrix structure and function. Most proteins that interact with collagen bind only to the native triple-helical form, whereas Fn is unusual in binding strongly to denatured collagen and more weakly to native collagen. The consequences of replacing a Gly by Ser at each position in the required (Gly-Xaa-Yaa)6 Fn-binding sequence are probed here, using model peptides and a recombinant bacterial collagen system. Fluorescence polarization and solid-state assays indicated that Gly replacements at four sites within the Fn-binding sequence led to decreased Fn binding to denatured collagen. Molecular dynamics simulations showed these Gly replacements interfered with the interaction of a collagen β-strand with the β-sheet structure of Fn modules seen in the high resolution crystal structure. Whereas previous studies showed that Gly to Ser mutations within an integrin-binding site caused no major structural perturbations, mutations within the Fn-binding site caused the triple helix to become highly sensitive to trypsin digestion. This trypsin susceptibility is consistent with the significant local unfolding and loss of hydrogen bonding seen in molecular dynamics simulations. Protease sensitivity resulting from mutations in the Fn-binding sequence could lead to degradation of type I collagen, early embryonic lethality, and the scarcity of reported osteogenesis imperfecta mutations in this region.
Keywords: collagen; extracellular matrix; extracellular matrix protein; fibronectin; molecular dynamics; mutation; osteogenesis imperfecta; peptides; recombinant protein expression.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.