Dual Specificity PDZ- and 14-3-3-Binding Motifs: A Structural and Interactomics Study

Protein-protein interaction motifs are often alterable by post-translational modifications. For example, 19% of predicted human PDZ domain-binding motifs (PBMs) have been experimentally proven to be phosphorylated, and up to 82% are theoretically phosphorylatable. Phosphorylation of PBMs may drastically rewire their interactomes, by altering their affinities for PDZ domains and 14-3-3 proteins. The effect of phosphorylation is often analyzed by performing "phosphomimetic" mutations. Here, we focused on the PBMs of HPV16-E6 viral oncoprotein and human RSK1 kinase. We measured the binding affinities of native, phosphorylated, and phosphomimetic variants of both PBMs toward the 266 human PDZ domains. We co-crystallized all the motif variants with a selected PDZ domain to characterize the structural consequence of the different modifications. Finally, we elucidated the structural basis of PBM capture by 14-3-3 proteins. This study provides novel atomic and interactomic insights into phosphorylatable dual specificity motifs and the differential effects of phosphorylation and phosphomimetic approaches. [Display omitted] •A large proportion of PDZ-binding motifs are phosphorylatable•Phosphorylated and phosphomimetic PBMs bind differently to PDZs and 14-3-3 proteins•These differences are demonstrated by X-ray analysis and affinity profiling Gogl et al. studied the effects of phosphorylation of PDZ domain-binding motifs. They demonstrated that there are many phosphorylatable and phosphorylated motifs, some of which are also putative binding targets of 14-3-3 proteins. Using quantitative interactomic assays and crystallography they showed how phosphorylation and phosphomimetic substitution alters their binding properties.