Stabilization of Interdomain Interactions in G protein αiSubunits Determines GαiSubtype Signaling Specificity

Abstract

AbstractHighly homologous members of the Gαifamily, Gαi1-3, 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 Gαi1effector, however, it is poorly activated by Gαi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gαi1relative to Gαi2with 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 Gαi1into Gαi2or substitution of a single amino acid, A230 in Gαi2to the corresponding D in Gαi1, largely rescues PRG activation and interactions with other Gαitargets. 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 Gαi2relative to Gαi1and that mutation of A230s4h3.3to D in Gαi2stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3in Gαi1interacts with R144HD.11stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2is 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 Gαi1or Gαi2by GPCRs.