Fig. 5: Rationally designed MBP-16E6 mutations disrupt its binding to E6AP.
a Surface plasmon resonance (SPR) measurements of increasing concentrations of mutant MBP-16E6 binding to biotinylated E6AP. Mutations were rationally designed to disrupt the 16E6 and E6AP interaction, reducing the picomolar interaction observed in wild-type protein. The Figure shows measured binding responses (black) and curve fits to a 1:1 interaction model (red). Plots are representative from at least three independent experiments with similar results. RU, response units; KD, dissociation constant; ka, association rate; kd, dissociation rate. b p53 ubiquitination assays wherein recombinant MBP-16E6, E6AP, and p53 were incubated in the presence or absence of ATP over the indicated time, then resolved by SDS-PAGE and visualized for p53 (top panel) or E6AP (bottom panel) by Western blot. c Assay was set up as in (b) except wild-type MBP-16E6 was substituted with mutant protein and the unmodified p53 (left panels) or unmodified E6AP (right panels) were isolated. Reduced signal represents modification of p53 or E6AP by ubiquitination. Full blots with molecular weight markers can be found in Supplementary Fig. 20. d Quantification of unmodified p53 or (e) unmodified E6AP from experiments in (c). Values represent the mean ± SEM of three independent experiments for all mutant MBP-16E6 (4C4S) proteins, and six independent experiments for WT MBP-16E6 (4C4S) protein.
