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_i subfamily member Gα_o. 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_o. Reciprocally, replacing the putative disruptor residues in RGS18 (a member of the R4 subfamily that exhibited low activity toward Gα_o) with the corresponding residues from a high-activity R4 subfamily RGS protein increased its activity toward Gα_o. Furthermore, the high activity of the R4 subfamily toward Gα_o 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.

了解RGS（G蛋白信号调节剂）蛋白与异三聚体（αβγ）G蛋白之间相互作用特异性的分子基础，将能够操纵RGS-G蛋白相互作用，探索其功能，并有效地靶向治疗。RGS蛋白被分为四个亚家族（R4，R7，RZ和R12），并通过失活Gα亚基而充当G蛋白信号传导的负调节剂。我们发现R12亚家族RGS10和RGS14对G _i亚家族Gαo的活性低于大多数R4亚家族_。。使用具有多个Gα-RGS配合物的基于结构的能量计算，我们在预测可确定该亚家族不同活性的位置上鉴定了R12特异性残基。该分析预测，这些残基（我们称为“干扰残基”）与Gα螺旋结构域相互作用。我们设计了R12破碎残基引入高活性R4亚科的RGS结构域，发现这些改变的蛋白表现出减少的朝向Gα活性_Ò。往复，替换RGS18（R4的亚家族表现出朝向Gα活性低的构件推定的干扰物的残基_ö从高活性的相应残基）R4亚科RGS蛋白增加其活性朝向Gα _ö。此外，R4亚科朝向Gα的高活性_Ó是独立于同源位置到R12亚科和RGS18干扰物的残基的残基。因此，我们的结果表明，已鉴定的RGS干扰残基起负设计元素的作用，从而削弱了特定Gα蛋白的RGS活性。