Structural design principles that underlie the multi-specific interactions of Gα with dissimilar partners.

Gα is a ubiquitous molecular switch that activates the effectors phospholipase-C-β3 (PLC-β3) and Rho guanine-nucleotide exchange factors. Gα is inactivated by regulators of G protein signaling proteins, as well as by PLC-β3. Gα further interacts with G protein-coupled receptor kinase 2 (GRK2), although the functional role of this interaction is debated. While X-ray structures of Gα bound to representatives of these partners have revealed details of their interactions, the mechanistic basis for differential Gα interactions with multiple partners (i.e., Gα multi-specificity) has not been elucidated at the individual residue resolution. Here, we map the structural determinants of Gα multi-specificity using structure-based energy calculations. We delineate regions that specifically interact with GTPase Activating Proteins (GAPs) and residues that exclusively contribute to effector interactions, showing that only the Gα "Switch II" region interacts with all partners. Our analysis further suggests that Gα-GRK2 interactions are consistent with GRK2 functioning as an effector, rather than a GAP. Our multi-specificity analysis pinpoints Gα residues that uniquely contribute to interactions with particular partners, enabling precise manipulation of these cascades. As such, we dissect the molecular basis of Gα function as a central signaling hub, which can be used to target Gα-mediated signaling in therapeutic interventions.