Fig. 4: Characterization of ORF45-RSK2 binding and its effect on ppERK2-RSK2 binding.
a Binding between RSK2 and ORF45 or ppERK2 analyzed by SPR. ORF45 or ppERK2 were injected over the immobilized RSK2 surface in two different concentrations for 5 min (association phase) and then the dissociation of the complex was monitored in time. RU response units. b Comparison of ternary complex formation with ERK2 or ppERK2. Results of kinetic binding experiment using nonphosphorylated ERK2 (upper panel) or ppERK2 (lower panel) where proteins were injected over the immobilized RSK2 surface alone (10 μM ORF45 in black or 1 μM ERK2 in blue) or together (in red). c Schematics of the ppERK2-RSK2-ORF45(16-76) ternary complex. The “closed” ternary complex, which could form from three different “open” complexes (but only one of them is shown on this panel), is formed by three linear motif (VF, FxFP, and D-motif) mediated protein-protein contacts. The binding affinity of the binary interactions between VF(ORF45):NTK(RSK2), FxFP(ORF45):ppERK2, and D-motif(RSK2):ppERK2 are indicated. Note that VF:NTK binding is ~1000-fold stronger and has low koff compared to the other two interactions. Classical vs “complex” allostery within the ternary complex are highlighted with green or gray arrows, respectively. In the simulation of SPR data, the difference between binding rates in the binary versus the ternary complex could be introduced by the global “a” and “d” values affecting the kon or the koff of complex formation, respectively. The lower panel shows the simulation results (dashed lines) overlaid with the corresponding experimental binding curve (solid black line) where the ternary kon and koff are the same as in the binary complexes (a = 1 and d = 1; red) or adjusted by the fitted a and d values (blue). The apparent kinetic binding parameters in the closed complex (kon_c or koff_c) are obtained by the multiplication of the binary binding constants by a or d. Source data are provided as Source data file.
