Interplay between negative and positive design determines how Regulators of G Protein Signaling (RGS) proteins modulate the inactivation of Ga subunits

The molecular basis of interaction specificity between Regulators of G protein Signaling (RGS) proteins and the heterotrimeric (αβγ) G protein switches they inactivate is essential for controlling numerous signaling cascades, yet remains poorly understood. This knowledge gap hinders our ability to manipulate RGS-G protein interactions in vivo, to understand their “wiring” into signaling networks, and to influence them with drugs.

We investigate the structural determinants for the specific recognition of Gα subunits by the RGS family using a combination of structure-based energy calculations and experimental measurements. We show that high RGS activity can be set by different combinations of positive design elements that enhance interactions and vary across the RGS family. Extrapolating to the entire family level, we identify specific RGS residues that are predicted to restrict interaction specificity by functioning as negative design elements. Indeed, grafting these “putative RGS disruptor residues” into high-activity RGS proteins reduces their activity, while reciprocal substitutions resulted in a gain-of-function. We further show that the residues in these positions are not necessary for high activity in particular RGS proteins, indicating that they function as structural negative design elements that attenuate RGS activity in a specific fashion. This multi-tiered specificity system combines positive and negative design elements to modulate RGS-Gα interactions in a specific fashion.