Stabilization of Interdomain Interactions in G protein α Subunits Determines Gα Subtype Signaling Specificity.

Highly homologous members of the Gα family, Gα, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gα effector, however, it is poorly activated by Gα. Here, in a proteomic proximity labeling screen we observed a strong preference for Gα relative to Gα with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gα into Gα or substitution of a single amino acid, A230 in Gα to the corresponding D in Gα, largely rescues PRG activation and interactions with other Gα targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gα relative to Gα and that mutation of A230 to D in Gα stabilizes HD-RLD interactions through formation of an ionic interaction with R145 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229 in Gα interacts with R144 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gα is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gα or Gα by GPCRs.