Designer proteins that competitively inhibit Gα(q) by targeting its effector site

During signal transduction, the G protein, Gα(q), binds and activates phospholipase C-β isozymes. Several diseases have been shown to manifest upon constitutively activating mutation of Gα(q), such as uveal melanoma. Therefore, methods are needed to directly inhibit Gα(q). Previously, we demonstrated that a peptide derived from a helix-turn-helix (HTH) region of PLC-β3 (residues 852–878) binds Gα(q) with low micromolar affinity and inhibits Gα(q) by competing with full-length PLC-β isozymes for binding. Since the HTH peptide is unstructured in the absence of Gα(q), we hypothesized that embedding the HTH in a folded protein might stabilize the binding-competent conformation and further improve the potency of inhibition. Using the molecular modeling software Rosetta, we searched the Protein Data Bank for proteins with similar HTH structures near their surface. The candidate proteins were computationally docked against Gα(q), and their surfaces were redesigned to stabilize this interaction. We then used yeast surface display to affinity mature the designs. The most potent design bound Gα(q/i) with high affinity in vitro (K(D) = 18 nM) and inhibited activation of PLC-β isozymes in HEK293 cells. We anticipate that our genetically encoded inhibitor will help interrogate the role of Gα(q) in healthy and disease model systems. Our work demonstrates that grafting interaction motifs into folded proteins is a powerful approach for generating inhibitors of protein–protein interactions.