Stabilization of interdomain interactions in G protein α subunits as a determinant of Gα_i subtype signaling specificity

Highly homologous members of the Gα_i family, Gα_i1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gα_i1 effector that is poorly activated by Gα_i2. In a proteomic proximity labeling screen we observed a strong preference for Gα_i1 relative to Gα_i2 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_i1 into Gα_i2 or substitution of a single amino acid, A230 in Gα_i2 with the corresponding D in Gα_i1, largely rescues PRG activation and interactions with other potential Gα_i targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gα_i2 than Gα_i1. Mutation of A230 to D in Gα_i2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gα_i1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gα_i2 is unable 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_i1 or Gα_i2 by G protein-coupled receptors.