Molecular architecture of Galphao and the structural basis for RGS16-mediated deactivation

Abstract

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an alpha subunit with intrinsic GTPase activity and a betagamma heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the alpha subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Galpha subunit's intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Galpha subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 A crystal structure of the enigmatic, neuronal G protein Galpha(o) in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 A structure of apo-RGS16, also presented here, reveals plasticity upon Galpha(o) binding, the determinants for GAP activity, and the structurally unique features of Galpha(o) that likely distinguish it physiologically from other members of the larger Galpha(i) family, affording insight to receptor, GAP and effector specificity.

Fig. 1.

Structure of the Gα_o·GDP·AlF₄⁻·RGS16 complex. The Gα_o·GDP·AlF₄⁻·RGS16 complex is represented in ribbons format. RGS16 is indicated in gray; Gα_o GTPase domain is in green with nucleotide-dependent switch regions highlighted in orange; Gα_o helical domain is in purple. Structure at Right is rotated 90° about the y axis relative to the structure at Left.

Fig. 2.

Gα_o–RGS16 contacts and the RGS domain GAP mechanism. (A) Stick-and-ribbons diagram of the Gα_o GTP binding pocket occupied by the transition state analog of GTP hydrolysis; GDP·AlF₄⁻ is shown with Mg²⁺ and the attacking water. Gα_o is shown in green and orange (switch regions). RGS16 is shown in purple. RGS16 residues do not contact the GTP or attacking water directly; instead they buttress the endogenous catalytic residues of Gα_o, stabilizing their conformation in the transition state. (B) Comparative <4 Å electrostatic interaction matrix between RGS16 and Gα subunits. Electrostatic interactions between mouse Gα_o and mouse RGS16 are indicated in green. Electrostatic interactions between human Gα_i1 and human RGS16 are indicated in yellow (PDB ID code 2IK8; see ref. 18). Gα switch residues are boxed in orange; helical domain residues are boxed in purple. Interactions that occur in one or both crystallographic protomers are included for both Gα_o–RGS16 and Gα_i1–RGS16.

Fig. 3.

Determinants of RGS16 binding and conformational plasticity. Structural alignment of mouse RGS16 in the free (gray) and Gα_o-bound (slate) states. The Cα trace is presented for both structures, with key residues used in the Gα_o interaction represented in stick format. The structural alignment was performed with PyMol.

Fig. 4.

Unique structural determinants in the Gα_o helical domain. (A) Structural alignment of Gα_o and Gα_i1 bound to GDP·AlF₄⁻, coordinates taken from their respective complex structures with RGS16 and RGS4 [PDB ID code 1AGR (5)] and displayed as a Cα trace. Gα_o is colored as in Fig. 1A except that the divergent αB–αC loop is shown in red. Gα_i1 is shown in gray except for the comparative αB–αC loop, which is shown in blue. The structural alignment was performed with PyMol. (B) Structural alignment as in A, zoomed in on the αB–αC loop region. Gα_i/12 [PDB ID code 1ZCA (36)] is included, with its respective αBd–αC loop region shown in yellow. (C) Representation of Gα_o in ribbon format (Left, colored as in Fig. 1A) for orientation and in CPK (Right) with the GTPase domain, helical domain, and switch regions colored white, light gray, and dark gray respectively. Residues identical across Gα_i1-3 and Gα_t but not identical in Gα_o are mapped in red. Residues identical across Gα_i1-3 but not identical in Gα_o are mapped in blue. Two orientations, related by 180° rotations, are displayed vertically.