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

Highly homologous members of the G alpha i family, G alpha 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 G alpha i1 effector that is poorly activated by G alpha i2. In a proteomic proximity labeling screen we observed a strong preference for G alpha i1 relative to G alpha 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 G alpha i1 into G alpha i2 or substitution of a single amino acid, A230 in G alpha i2 with the corresponding D in G alpha i1, largely rescues PRG activation and interactions with other potential G alpha i targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HDRas-like domain (RLD) interface is more pronounced in G alpha i2 than G alpha i1. Mutation of A230 to D in G alpha 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 G alpha i1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in G alpha 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 G alpha i1 or G alpha i2 by G protein-coupled receptors.