Protein Frustration Reveals Active Sites in Co-Evolved GPCR:G Protein Complexes and in Engineered Targeted Degrader Complexes.

The folded structure of a protein is understood to be an optimal energy state. However, previous studies have shown that certain amino acid residue positions that play a critical role in protein function are often in a suboptimal energy state or "frustrated". Here, we leverage over 1200 three-dimensional structures of G protein-coupled receptors (GPCRs) to demonstrate that residues at the interface between GPCR and its ligand or G protein contain a higher density of frustrated residues compared to other structural regions in the receptor. Likewise, the Gα subunit of the trimeric G proteins shows multiple clusters of highly frustrated residues on its surface that overlap with their effector protein (Gβγ, RGS, Adenyl cyclase, Ric8) binding interfaces. Compared to the co-evolved GPCR:G protein complexes, engineered protein complexes, such as those facilitated by molecular degraders, show a much greater density of highly frustrated residues in the degrader interface. Our study highlights the use of protein frustration as an invaluable tool to evaluate both native protein-protein interfaces and design strategies to facilitate engineered protein complexes.