Conserved structural elements in Gα subunit switch regions are necessary for efficient inactivation by RGS proteins

Heterotrimeric G proteins (αβγ) function as molecular switches that regulate intracellular signaling downstream of G protein-coupled receptors (GPCRs). Gα subunits cycle between an inactive GDP-bound state and an active GTP-bound state that engages downstream effectors. The duration of Gα-mediated signaling is primarily governed by Regulators of G Protein Signaling (RGS) proteins, which accelerate GTP hydrolysis as GTPase-activating proteins (GAPs). To characterize these multi-specific Gα interactions at the individual residue level, we employed computational energy calculations across Gα-partner complexes, focusing on residues critical for RGS interactions. This analysis showed that specific residues within the Gα “switch regions”, conserved across the Gi and Gq subfamilies, play a central role in RGS recognition and GAP activity. Substantial energy contributions arise mainly from conserved residues within the switch I and II in the Gα GTPase domain. However, switch I interactions are dominated by electrostatic and non-polar contributions from the protein backbone, while switch II interactions are dominated by strong electrostatic side-chain contributions. Mutagenesis experiments confirmed that switch I side chains are not required for RGS GAP activity, but disrupting the tight switch I interface with RGSs abolished GAP activity. In contrast, alanine substitutions in individual switch II residues severely reduced RGS GAP activity, while switch III played only a minor role. Our findings offer residue-level insights into the conserved interactions underlying RGS/effector regulation of G proteins and provide a basis for engineering Gα subunits with tailored interaction profiles, developing inhibitors of Gα-RGS binding, or enable rewiring of G protein-coupled signaling pathways in vivo.