RGS9 N-terminal lobe. (a) Ribbon diagram of superposed RGS9 (orange) and Dishevelled (blue) Dishevelled/Egl-10/Pleckstrin homology (DEP) domains. RGS9-DEP domain secondary structure elements are labeled. (b) RGS9-DEP helical extension (RGS9-DHEX; maroon) domain with secondary structure elements labeled. (c) Specific contact residues between the RGS9-DEP domain (orange) and Gβ5 (blue) are represented as balls and sticks. (d) 2F_o – F_c electron density map of the RGS9 DHEX-Gγ–like (GGL) linker (dark gray) and N-terminal (light gray) elements with neighboring Gβ5 residues (blue) contoured at 1σ (dark-blue mesh).

Gγ-subunit–like (GGL) domains and Gγ subunits are structurally equivalent. (a) Structure-based sequence alignments of the RGS9-GGL domain with Gγ subunits and other mammalian R7-RGS-GGL domains. Residue numbers indicated above the R7-RGS–GGL domain sequences are for mouse RGS9. The helical elements of the RGS9-GGL domain are indicated above the R7-RGS-GGL domain alignments as cylinders. Residues contacting Gβ subunits17,18 are boxed in green. The residues that are identical or strongly conserved with RGS9-GGL residues are colored red, and residues that are weakly conserved are colored blue. RGS9-GGL domain residues supporting Gβ5-specific contacts are indicated with blue dots. These are defined as contacts that could not be reproduced in a complex with other Gβ subunits owing to chemical or steric conflicts. Residues proposed to contact an RGS-domain–bound Gα subunit are labeled with gold dots. (b) Backbone trace of the superposed RGS9-GGL (red) domain and Gγ1 (ref. 17; dark-blue) structures. GGL domain secondary structure elements are labeled. (c) RGS9-W270 binding pocket shown as a translucent surface with individual Gβ5 (blue) and GGL domain (red) residues shown as balls and sticks. The GGL domain backbone is represented as a transparent ribbon. A hydrogen-bonded water molecule (yellow sphere) is depicted. (d) Gγ1-F64 (red) and Gβ1 (blue) binding interface, depicted similarly to the figure in c.

Membrane orientation of the Gβ5–RGS9 complex. (a) A model of the membrane-relative orientation and interprotein interactions of the Gβ5–RGS9 complex. Gβ5 and RGS9, colored as in Figure 1, with Gαt/i (ref. 30; gold) docked and colored as in Figure 5d. A modeled N-terminal helix for Gαt/i is depicted as a cylinder, and a wheat-colored R9AP cartoon is positioned near the RGS9-Dishevelled/Egl-10/Pleckstrin homology (DEP) and RGS9-DEP helical extension (DHEX) domains. (b) A model of the Gβ5–RGS9 complex, colored and positioned as in a, with the DEP and DHEX domains arbitrarily repositioned to allow docking of a Gαt structure17 on Gβ5. The Gα subunit is colored as in Figure 1b. A cartoon depiction of a G protein–coupled receptor (GPCR)52 (cyan) is positioned in the membrane.

Structure of Gβ5–RGS9. (a) Stereo ribbon diagram of Gβ5 (blue) in dimeric complex with RGS9 including its N-terminal (light gray), Dishevelled/Egl-10/Pleckstrin homology (DEP; orange), DEP helical extension (DHEX; maroon), G protein γ (Gγ)-subunit–like (GGL; red), regulators of G-protein signaling (RGS; green) and DHEX-GGL linker (dark gray) regions. (b) Ribbon representation of the transducin Gαβγ heterotrimer17 (PDB 1GOT), with the Gβ subunit oriented similar to Gβ5 in a. Gα, gold; Gα-switch regions, magenta; Gβ, blue; Gγ, red; GDP, purple spheres. Structure figures were generated with PyMOL software (http://pymol.sourceforge.net).

Conserved Gα binding interface on Gβ5. (a) Surface representation of the N-terminal lobe (top) and regulators of G-protein signaling (RGS) domain (bottom) binding faces of Gβ5. Divergent residues as indicated in Supplementary Figure 1 are blue. A transparent ribbon representation of RGS9 is shown and colored according to Figure 1a. The DEP helical extension (DHEX) domain and RGS domain elements that have no Gβ5 contacts are deleted for clarity. (b) Surface representation of Gβ1 (ref. 17) oriented similarly to the top face of Gβ5 in a. The residues that align with divergent Gβ5 residues are colored blue for comparison with the figure in a, and the surfaces of Gβ1 that interface with Gα subunits are indicated with black outlines. (c) Superposed Gβ5 (blue) and Gβ1 (gold) structures with Gαt-binding residues of Gβ1 (refs. 17,23) and structurally equivalent Gβ5 residues depicted as balls and sticks.

The RGS9-RGS domain interfaces with Gβ5 and activated Gα subunits. (a) Ribbon diagram of the RGS9-RGS domain (green) interface with the Gγ-subunit–like (GGL) domain (red) and Gβ5 (blue). Gβ5 loop residues contacting the RGS domain are purple. Bundle and terminal RGS subdomains are indicated with green lines. (b) Transparent ribbon diagram of the RGS domain (green) and Gβ5 (blue) with interfacial contact residues depicted as balls and sticks. (c) Superimposed structures from the isolated RGS9-RGS domain (wheat) and RGS9-RGS domains in complexes with Gαt/i (coral) and Gβ5 (green) are depicted as transparent ribbon diagrams along with the Gβ5 (blue) structure. The Gαt/i contact residues from all three RGS domain structures and the Gβ5 residue (Glu207) that shares a common RGS domain contact (Lys397) with Gαt/i30 are depicted as balls and sticks. The isolated and Gαt/i-bound RGS9-RGS domain coordinates PDB accession numbers are 1FQI and 1FQJ, respectively. (d) A model of Gαt/i (ref. 30; gold) docked onto the RGS domain (green) of Gβ5–RGS9 is depicted and colored similarly to Figure 1. The GTPase and all-helical subdomains of the Gα subunit are indicated with gold lines. A region where the Gα subunit clashes with the GGL domain and Gβ5 is circled. The direction of a proposed reorientation of the RGS domain is indicated with a curved arrow. The orientation of this image is rotated clockwise ~90° around the y axis with respect to the image in a. (e) Magnified and ~90° rotated view of the encircled region of d, with sterically clashing residues shown as balls and sticks.