Determination of the synergy site acceptor residue on α5 subunit. (A) Solid-phase equilibrium binding using immobilized full-length α5β1 ectodomain and solution-phase biotinylated Fn7–10. Binding experiments with WT (closed squares) or R1374A/P1376A/R1379A mutant (open circles) Fn7–10 fragments to four different α5β1 preparations were performed, and data from one representative of three independent experiments are shown. (B) Effect of synergy residue mutations on the binding kinetics studied by surface plasmon resonance analysis. WT (solid lines) and R1379A mutant (dotted lines) biotinylated Fn7–10 were immobilized separately onto a streptavidin-coated chip (∼1,000 resonance units [RU]). The surface was infused for 60 s with either WT (left) or D154A-purified (right) full-length recombinant α5β1 integrin at 20 nM in a running buffer containing 1 mM Mn²⁺, and the dissociation phase was followed for 100 s. The data are from a single representative experiment out of three repeats.