Allostery between distant structural regions dictates selectivity in GPCR:G protein coupling.

Selectivity in GPCR-G protein coupling is widely regarded as a solved problem that derives from a combination of structural hotspot pairings and dynamics at the protein-protein interface. Here, using interpretable machine learning Bayesian Network model with Molecular Dynamics simulations and experiments, we reveal the influence of distant residue communities within the G protein core on coupling selectivity. We observed distinct cooperative hotspot residues across different G protein subtypes, including key regions such as the N-terminus, h4s6 loop, and H5 helix. These results demonstrate the intricate allosteric dependencies between the core and the H5 helix in stabilizing selective interactions. The functional significance of these cooperative regions is validated through subtype swapping mutations. By introducing targeted Gq like mutations in the Gs core, we successfully altered receptor coupling profile to signal through Gq. Our findings emphasize that cooperative interactions in the G core are not only crucial for selectivity but can also be leveraged to engineer G proteins with tailored coupling preferences. TeaserAllosteric interactions in the G protein core shape GPCR selectivity and can be engineered to rewire receptor signaling preferences.