Integrating energy calculations with functional assays to decipher the specificity of G protein – RGS protein interactions

RGS proteins have a critical role in many G protein–dependent signaling pathways. RGS proteins ‘turn off ’ heterotrimeric (αβγ) G proteins and thereby determine the duration of G protein–mediated signaling events1–5. Like many signaling proteins, RGS proteins comprise a large and diverse family. In humans, about 20 ‘canonical’ RGS proteins downregulate activated G proteins of the Gi and Gq subfamilies6,7. In these RGS proteins, the ~120-residue RGS homology domain functions as a GTPase-activating protein (GAP) for GTP-bound Gα subunits3–5. RGS proteins have been implicated in a wide range of pathologies, including cancer, hypertension, arrhythmias, drug abuse and schizophrenia7–10, making them promising drug targets7,8. Therefore, identifying the determinants of G protein recognition by RGS proteins is essential for understanding these signaling pathways and for eventually manipulating them with drugs. Although multiple RGS proteins are often expressed in the same cell, only particular RGS proteins mediate a given biological function11–17. This has generated considerable interest in understanding the interaction specificity of RGS proteins. In many cases this specificity may originate from precise subcellular targeting, contributions from additional noncatalytic domains, adaptor proteins or participation in scaffolded protein complexes7,9,13,15,18,19. However, in some cases the ability to recognize a given G protein is defined by the RGS domain itself7,9,13,15. Nevertheless, the only two well-studied examples of RGS domain specificity are RGS9, whose specific recognition of Gαt requires the adaptor protein PDEγ18,20, and RGS2, which specifically downregulates G proteins of the Gq, but not Gi, subfamilies21,22 (compare ref. 23). The key determinants of RGS2 specificity have been identified22 by analysis of the multiple sequence alignment of RGS proteins in the context of the RGS4–Gαi1 crystal structure24. This alignment shows three crucial positions that are highly conserved in the RGS family, but are different in RGS2. Changing these three RGS2 residues to their counterparts in RGS4 yields a gain-of-function phenotype that enables RGS2 to efficiently downregulate Gαi. Additional studies showed that the GAP activity of individual RGS proteins toward a given Gα may vary (reviewed in refs. 6–8,13), but the molecular determinants for this selectivity have not been identified. Critical insights into the GAP activity of RGS proteins have been obtained using X-ray crystallography. So far, eight different structures of Gα subunits in complex with canonical RGS domains have been solved24–28. These studies, combined with biochemical examinations, have established that high-activity RGS domains bind Gα subunits and stabilize their catalytic residues allosterically in a conformation optimal for GTP hydrolysis6,24,29–31. RGS protein residues in the vicinity of the Gα–RGS domain interface show substantial diversity, suggesting that they may set interaction specificity. However, low sequence identity among RGS domains (as low as 30%; Supplementary Table 1) makes it difficult to pinpoint RGS domain residues that determine selective interaction with a specific Gα subunit27,32. In this study, we combined functional assays with structure-based computations to determine the structural features within a large array of human RGS proteins that control their ability to inactivate a representative G protein, Gαo (also known as GNAO1). We combined the experimental benchmark of the ability of ten RGS domains to activate Gαo GTPase with comparative structural analysis, electrostatic calculations of interaction energies using the finite-difference Poisson-Boltzmann (FDPB) method and in silico mutagenesis. Using a