Interplay between negative and positive design elements in Gα helical domains of G proteins determines interaction specificity toward RGS2

Regulators of G protein signaling (RGS) proteins inactivate Gα subunits, thereby controlling G protein-coupled signaling networks. Among all RGS proteins, RGS2 is unique in interacting only with the Gα _q but not with the Gα _i subfamily. Previous studies suggested that this specificity is determined by the RGS domain and, in particular, by three RGS2-specific residues that lead to a unique mode of interaction with Gα _q . This interaction was further proposed to act through contacts with the Gα GTPase domain. Here, we combined energy calculations and GTPase activity measurements to determine which Gα residues dictate specificity toward RGS2. We identified putative specificity-determining residues in the Gα helical domain, which among G proteins is found only in Gα subunits. Replacing these helical domain residues in Gα _i with their Gα _q counterparts resulted in a dramatic specificity switch toward RGS2. We further show that Gα–RGS2 specificity is set by Gα _i residues that perturb interactions with RGS2, and by Gα _q residues that enhance these interactions. These results show, for the first time, that the Gα helical domain is central to dictating specificity toward RGS2, suggesting that this domain plays a general role in governing Gα-RGS specificity. Our insights provide new options for manipulating RGS–G protein interactions in vivo, for better understanding of their ‘wiring’ into signaling networks, and for devising novel drugs targeting such interactions.