Epitope-Guided Engineering of Monobody Binders for in Vivo Inhibition of Erk-2 Signaling

Although the affinity optimization of protein binders is straightforward, engineering epitope specificity is more challenging. Targeting a specific surface patch is important because the biological relevance of protein binders depends on how they interact with the target. They are particularly useful to test hypotheses motivated by biochemical and structural studies. We used yeast display to engineer monobodies that bind a defined surface patch on the mitogen activated protein kinase (MAPK) Erk-2. The targeted area (CD domain) is known to control the specificity and catalytic efficiency of phosphorylation by the kinase by binding a linear peptide (D peptide) on substrates and regulators. An inhibitor of the interaction should thus be useful for regulating Erk-2 signaling in vivo. Although the CD domain constitutes only a small percentage of the surface area of the enzyme (similar to 5%), sorting a yeast displayed monobody library with wild type (wt) Erk-2 and a rationally designed mutant led to isolation of high affinity clones with desired epitope specificity. The engineered binders inhibited the activity of Erk-2 in vitro and in mammalian cells. Furthermore, they specifically inhibited the activity of Erk-2 orthologs in yeast and suppressed a mutant phenotype in round worms caused by overactive MAPK signaling. The study therefore shows that positive and negative screening can be used to bias the evolution of epitope specificity and predictably design inhibitors of biologically relevant protein-protein interaction.