“Disruptor” residues in the regulator of G protein signaling (RGS) R12 subfamily attenuate the inactivation of Gα subunits

Abstract

Understanding the molecular basis of interaction specificity between RGS (regulator of G protein signaling) proteins and heterotrimeric (αβγ) G proteins would enable the manipulation of RGS-G protein interactions, explore their functions, and effectively target them therapeutically. RGS proteins are classified into four subfamilies (R4, R7, RZ, and R12) and function as negative regulators of G protein signaling by inactivating Gα subunits. We found that the R12 subfamily members RGS10 and RGS14 had lower activity than most R4 subfamily members toward the G subfamily member Gα Using structure-based energy calculations with multiple Gα-RGS complexes, we identified R12-specific residues in positions that are predicted to determine the divergent activity of this subfamily. This analysis predicted that these residues, which we call "disruptor residues," interact with the Gα helical domain. We engineered the R12 disruptor residues into the RGS domains of the high-activity R4 subfamily and found that these altered proteins exhibited reduced activity toward Gα Reciprocally, replacing the putative disruptor residues in RGS18 (a member of the R4 subfamily that exhibited low activity toward Gα) with the corresponding residues from a high-activity R4 subfamily RGS protein increased its activity toward Gα Furthermore, the high activity of the R4 subfamily toward Gα was independent of the residues in the homologous positions to the R12 subfamily and RGS18 disruptor residues. Thus, our results suggest that the identified RGS disruptor residues function as negative design elements that attenuate RGS activity for specific Gα proteins.

Structural design principles that underlie the multi-specific interactions of Gαq with dissimilar partners

“…4b). The electrostatic dominance in interactions of RGS domains with Gα subunits was also observed in interactions of Gα o and Gα i with various RGS domains 46 . The number of Gα q residues that contribute to interactions with RGS2 and RGS8 are 27 and 25, respectively.…”

“…This region was shown to participate in the binding of Gα subunits to effectors, such as the binding of Gα s to adenylyl cyclase 43 . RGS proteins were shown to substantially interact with the Gα GTPase domain, yet were also shown to interact with the Gα helical domain 37,38,44–46 . More recently, it was suggested that the helical domain is a major determinant of the specific interactions between Gα q and RGS2 47 .…”

Residue‐level determinants of angiopoietin‐2 interactions with its receptor Tie2

“…We followed the methodology described previously to analyze the per‐residue contributions of Ang2 and Tie2 residues to intermolecular interactions in the complex (Supporting Information Figure S1). The finite difference Poisson‐Boltzmann (FDPB) method was used to calculate the net electrostatic and polar contributions (ΔΔG elec ) of each residue that is within 15 Å of the dimer interface.…”

“…We followed the methodology described previously 32,[36][37][38][39][40] to analyze the per-residue contributions of Ang2 and Tie2 residues to intermolecular interactions in the complex (Supporting Information Figure S1).…”

Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins

“…To map the individual residues that contribute to colicin/pyocin-immunity protein interactions, we analyzed the representative X-ray structures of colicins bound to their immunity proteins, using an energybased computational methodology developed previously by our lab [55][56][57][58][59][60][61] . As described in the "Materials and methods" sectio, we calculate the net electrostatic/polar contributions (ΔΔG elec ) of each residue ≤ 15 Å of the colicin-immunity protein interfaces.…”