Epitope-guided engineering of monobody binders for in vivo inhibition of Erk-2 signaling

Abstract

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 (~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.

Figure 1. Engineering monobodies to target the Erk-2 CD domain

a. The Erk-2 residues within 5 Å of bound D-peptide (2GPH) are colored orange. The activation loop residues are colored in blue to show the separation between the docking domain and the active site. An Fn3 structure is shown to the scale on the left and is depicted as binding the docking surface. The randomized loops are shown in color: BC (green), DE (pink), FG (brown). b. The yeast displayed Fn3 library was sorted using streptavidin-coated magnetic beads and FACS. During FACS, the cells were labeled with a cMyc antibody and Erk-2 to normalize binding with protein expression. i) Unsorted cells labeled with 1 μM of Erk-2. ii) Cells that have been sorted twice by FACS and then labeled with 250 nM of Erk-2. iii) Cells labeled with Erk-2(NHN) during negative sorting (Round 6). iv) Cells after the final round of FACS labeled with 10 nM of Erk-2. The percentages indicate the fraction of the cells within the indicated gates. c. A representative selected clone, F7.9, binds 10 nM wt Erk-2 but not Erk-2(NHN). The lack of binding to the mutant indicates that the monobody epitope includes one or more of the mutated residues.

Figure 2. Binding characteristics of selected monobodies

a. The yeast displaying various Fn3 clones were labeled with purified Erk-2, and the MFI of the displaying population was fitted to a binding curve to extract the apparent binding constant K_d. All measurements were done in triplicates or more to compute the mean and standard deviation. The loop sequences correspond to the Fn3 residues that were randomized in the library. The stability of F6.2 and F7.9 was measured by circular dichroism spectroscopy. The specificity of Erk-2 binding was tested by labeling yeast cells with 1 μM biotinylated Erk-2, p38, or JNK; and SA-PE. The MFI of the displaying population was measured.

Figure 3. Functional assays of engineered Fn3

a. The components of mammalian ERK pathway. The binding of the MEK2 D-peptide mediates Erk-2 phosphorylation, and the binding of the Elk1 D-peptide mediates Elk1 phosphorylation. The engineered monobodies should disrupt both interactions. b. GST-pepHePTP was used to pull down Erk-2 in the presence of Fn3 competitors. The precipitated Erk-2 was visualized by Western blot using M2. F0.1 is a control monobody that binds MBP (22). Competitor concentrations in Lanes 1 – 7: 0, 0.016, 0.08, 0.4, 2, 10, and 50 μM. c. In vitro phosphorylation of Elk1(307-428) by Erk-2 in the presence of Fn3 competitors was analyzed by Western blot. Competitor concentrations: (For F0.1 and F6.2) 0, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM. (For F7.1, F7.4, and F7.9) 0, 0.0013, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM. The phospho-Elk1 band was quantified using ImageJ and normalized to Lane 1. Only the intensities corresponding to three highest Fn3 concentrations and Lane 1 are plotted for clarity. d. In vitro phosphorylation of Erk-2 by MEK2 in the presence of Fn3 competitors. Competitor concentrations: 0, 0.0064, 0.032, 0.16, 0.8, 4 and 20 μM. The intensity of the phospho-Erk-2 bands was quantified and normalized to Lane 1. Only select intensities are plotted for clarity.

Figure 3. Functional assays of engineered Fn3

a. The components of mammalian ERK pathway. The binding of the MEK2 D-peptide mediates Erk-2 phosphorylation, and the binding of the Elk1 D-peptide mediates Elk1 phosphorylation. The engineered monobodies should disrupt both interactions. b. GST-pepHePTP was used to pull down Erk-2 in the presence of Fn3 competitors. The precipitated Erk-2 was visualized by Western blot using M2. F0.1 is a control monobody that binds MBP (22). Competitor concentrations in Lanes 1 – 7: 0, 0.016, 0.08, 0.4, 2, 10, and 50 μM. c. In vitro phosphorylation of Elk1(307-428) by Erk-2 in the presence of Fn3 competitors was analyzed by Western blot. Competitor concentrations: (For F0.1 and F6.2) 0, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM. (For F7.1, F7.4, and F7.9) 0, 0.0013, 0.0064, 0.032, 0.16, 0.8, 4, and 20 μM. The phospho-Elk1 band was quantified using ImageJ and normalized to Lane 1. Only the intensities corresponding to three highest Fn3 concentrations and Lane 1 are plotted for clarity. d. In vitro phosphorylation of Erk-2 by MEK2 in the presence of Fn3 competitors. Competitor concentrations: 0, 0.0064, 0.032, 0.16, 0.8, 4 and 20 μM. The intensity of the phospho-Erk-2 bands was quantified and normalized to Lane 1. Only select intensities are plotted for clarity.

Figure 4. Activity of engineered Fn3 in HEK293 cells

a. HEK293 cells were transiently transfected with pCMVFn3-IEGFP-Elk1(307-428) and stimulated with EGF to activate the ERK-2 pathway. in the resulting Elk1 phosphorylation was inhibited by F6.2, F7.1, and F7.9. The stable expression of EGFP-Elk1 was checked by fluorescence microscopy and by Western blot with anti-GFP antibody (Fig. S7). Fn3 expression was checked using 9E10 against the cMyc tag. The number of cells used in the analysis was normalized using GAPDH. b. HEK293 cells expressing various Fn3 clones were stimulated with 100 mM NaCl to activate the p38 pathway. The monobodies had little or no effect on the p38-dependent phosphorylation of Elk1.

Figure 5. Specificity of monobody activity in yeast

Yeast were transformed with reporter vectors to measure the activity of three yeast MAPK pathways that control a. mating, b. filamentous growth, and c. hyperosmolarity responses. The yeast cells were further transformed with an Fn3 expression vector (F0.1, F6.2, F7.1, F7.4, F7.8, and F7.9) or not (“No Fn3”). The cells were stimulated to activate each of the three pathways, and the resulting reporter activity (in arbitrary unit) was measured.

Figure 6. Monobodies inhibit MAP kinase function in C. elegans

a. Nomarski images of wild-type (N2) and Ce-Ras let-60(gof) C. elegans. Whereas wt animals consistently display a single, properly positioned and functional vulva (black arrow), let-60(gof) animals are multivulval and display a range of additional ectopic pseudovulvae (white arrows). b. Distribution of Muv phenotype among different strains and transgenic worms. The percentage of each population displaying a given number of vulvae is shown in pie charts. let-60(gof);mpk-1(lof) are significantly non-Muv compared to let-60(gof) worms (p = 2.5 × 10⁻⁷² based on χ² statistics), indicating the let-60(gof) Muv phenotype is mediated primarily through downstream Ce-MPK-1 activity. Similarly, expression of F6.2 and F7.9 in let-60(gof) animals using the VPC-specific lin-31 promoter significantly reduced the prevalence of ectopic pseudovulvae compared to either let-60(gof) animals or the transgenic worms expressing the control monobody, F0.1 (p = 1.6 × 10⁻⁴⁷ − 8.5 × 10⁻²²). The combined data of two independent lines is shown. The total number of animals analyzed is n > 150 for control strains and n > 750 for each set of transgenic let-60(gof) worms.