Collagen-binding analysis of the mutant Y285F/E318W-I domain. (A) The coordinates for the isolated α2-I domain (1DZI) and α2-I domain in complex with the collagen peptide (1AOX) were obtained from the Protein Data Bank (PDB). The closed or “low-affinity” conformation adopted by the isolated I domain and the open or “high-affinity” conformation found in the I domain–collagen complex (the collagen triple helix is shown in dark gray) were constructed using the program Rasmol (University of Massachusetts, Amherst, MA). Black spheres indicate the position of the 2 residues mutated in the recombinant Y285F/E318W-I domain. (B) Analysis of the binding of recombinant α2-I variants to collagen type I from human placenta by surface plasmon resonance (SPR). Binding curves of the wild-type (WT) or Y285F/E318W mutant were obtained perfusing different concentrations of each purified I domain protein over the collagen surface as described in “Materials and methods.” The figures depicted overlay plots of sensograms observed for the interaction at different concentrations. RU indicates resonance units. Dissociation constant obtained for each α2-I variant was 1.0 ± 0.2 μM for WT; 2.0 ± 0.15 μM for Y285F, and 0.02 ± 0.09 μM for Y285F/E318W.