Nano‐G s Protein Peptidomimetics: Rational Design of Gα C‐Terminus‐Derived Peptides Mimicking Key Components of G s ‐β ₂ AR Interactions

G protein-coupled receptors (GPCRs) are involved in most human physiological processes and one of the largest families of approved drug-targeted proteins. Heterotrimeric Gαβɣ proteins bind to the intracellular cavity of the activated receptor mainly through the C-terminal α5 helix of the Gα subunit (GαCT). Modulation of GPCR activity through intracellular GPCR binding sites is emerging. Here we develop highly active GαsCT-derived peptidomimetics that stabilize the β₂ adrenergic receptor (β₂AR) in an active-like conformation when the helical conformation of GαsCT is preserved by a covalent tether ("staple"). By rational design, integrating the information of two crystal structures showing different binding modes of GαsCT with β₂AR, an appropriate staple position was identified. The key interactions observed in the two distinct β₂AR structures (β₂AR-Gs(empty)) and β₂AR-T4L-GsCT-CC), namely Y391 and E392, were substituted with non-canonical amino acids to improve activity. Optimization resulted in the identification of a potent peptidomimetic capable of stabilizing an active-like receptor conformation, whilst blocking receptor-mediated cAMP formation. Molecular dynamics simulations indicated a peptidomimetic binding mode that may represent another intermediate state preceding that of β₂AR-Gs(empty). We envision this approach to be useful for further structural and functional exploration of other GPCRs or as a tool in drug discovery.