Structural diversity in the RGS domain and its interaction with heterotrimeric G protein alpha-subunits

Abstract

Regulator of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits and thus facilitate termination of signaling initiated by G protein-coupled receptors (GPCRs). RGS proteins hold great promise as disease intervention points, given their signature role as negative regulators of GPCRs-receptors to which the largest fraction of approved medications are currently directed. RGS proteins share a hallmark RGS domain that interacts most avidly with Galpha when in its transition state for GTP hydrolysis; by binding and stabilizing switch regions I and II of Galpha, RGS domain binding consequently accelerates Galpha-mediated GTP hydrolysis. The human genome encodes more than three dozen RGS domain-containing proteins with varied Galpha substrate specificities. To facilitate their exploitation as drug-discovery targets, we have taken a systematic structural biology approach toward cataloging the structural diversity present among RGS domains and identifying molecular determinants of their differential Galpha selectivities. Here, we determined 14 structures derived from NMR and x-ray crystallography of members of the R4, R7, R12, and RZ subfamilies of RGS proteins, including 10 uncomplexed RGS domains and 4 RGS domain/Galpha complexes. Heterogeneity observed in the structural architecture of the RGS domain, as well as in engagement of switch III and the all-helical domain of the Galpha substrate, suggests that unique structural determinants specific to particular RGS protein/Galpha pairings exist and could be used to achieve selective inhibition by small molecules.

Fig. 1.

Heterogeneity in the αV–αVII regions of R12 subfamily RGS domains versus the canonical RGS domain fold of R4, R7, and RZ subfamily members. (A) Apo-RGS domains of R4 subfamily member RGS8 (green; PDB ID 2IHD), R7 subfamily member RGS9 (orange; PDB ID 1FQI), and RZ subfamily member RGS19 (gray; PDB ID 1CMZ) were aligned along helices αIV and αV and superimposed by using PyMOL. (B–D) Apo-RGS domains of RGS14 (B) (blue; PDB ID 2JNU), RGS10 from this study (C) (salmon; PDB ID 2I59), and RGS10 from Yokoyama et al. (D) (light purple; PDB ID 2DLR) are presented to highlight differences in the αV–αVI–αVII region. The heterogeneous αVI regions are specifically highlighted in cyan (B), red (C), and magenta (D), respectively.

Fig. 2.

Predicted structural determinants of Gα selectivity by RGS2. (A) RGS1 (gray-blue) bound to Gα_i1 (α1 helix in light red; switch I in orange) is presented to highlight the Gα switch-I interaction interface (PDB ID 2GTP). Asp-172 of RGS1 is within hydrogen-bonding distance of the backbone amine of Thr-182 in Gα_i1 and additionally stabilized by the terminal amines of the highly conserved Arg-176 in the RGS1 αVII helix. Ser-95 is placed within close proximity (≤4.0 Å) of three Gα_i1 residues (Thr-182, Gly-183, and Lys-210). (B) Residues 170–190 of RGS2 (PDB ID 2AF0) were superimposed on residues 159–179 of RGS1 from the RGS1/Gα_i1 complex (PDB ID 2GTP) with an r.m.s.d. of 0.5 Å. RGS1 is not shown, RGS2 is presented in green, and Gα_i1 is rendered in light red (α1 helix) and orange (switch I). Asparagine at position 184 in RGS2 (normally an aspartate in R4 subfamily members) does not allow for the hydrogen bond to the peptide bond amine of Thr-182 in Gα_i1; however, Asn-184 can potentially form a hydrogen bond with the backbone carbonyl of Lys-180. The increased atomic radius of Cys-106 in RGS2 (versus serine in RGS1) may cause steric hindrance with the switch-I backbone and the side-chain of Lys-210.

Fig. 3.

Heterogeneity in RGS-domain interactions with the Gα all-helical domain. All RGS domain/Gα complexes were aligned and superimposed on the RGS1/Gα_i1 structure (PDB ID 2GTP) by using PyMOL. The Gα Ras-like domain is colored in shades of red, the all-helical domain is colored in shades of blue, and switch regions are highlighted in orange. GDP, Mg²⁺, and AlF₄⁻ are shown in magenta, yellow, and cyan, respectively. All RGS domain residues within 4.0 Å of residues from the Gα all-helical domain are in yellow sticks. (A) RGS4/Gα_i1 complex (PDB ID 1AGR). Glu-161, Lys-162, and Arg-166 in the RGS4 αVII helix are within 4.0 Å of the Gα_i1 all-helical domain residues Ser-75 or Glu-116. (B) RGS9/Gα_t/i1 complex (PDB ID 1FQK). Three lysine residues in the RGS9 αVII helix at positions 397, 398, and 406 are all within 4.0 Å of Glu-64, Ala-67, and Glu-112 of the Gα_t/i1 all-helical domain. (C) RGS8/Gα_i3 complex (PDB ID 2ODE). The αVII helix residues Lys-156 and Arg-164 interact with Glu-65 and Ser-75 within the αA helix of the Gα_i3 all-helical domain. (D) RGS10/Gα_i3 complex (PDB ID 2IHB). Residues Lys-131 and Tyr-132 within the RGS10 αVII helix are within 4.0 Å of Ser-75 and Glu-115 of the Gα_i3 all-helical domain.