Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

Abstract

G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide hydrogen-deuterium exchange mass spectrometry to probe changes in the structure of the heterotrimeric bovine G protein, Gs (the stimulatory G protein for adenylyl cyclase) on formation of a complex with agonist-bound human β(2) adrenergic receptor (β(2)AR). Here we report structural links between the receptor-binding surface and the nucleotide-binding pocket of Gs that undergo higher levels of hydrogen-deuterium exchange than would be predicted from the crystal structure of the β(2)AR-Gs complex. Together with X-ray crystallographic and electron microscopic data of the β(2)AR-Gs complex (from refs 2, 3), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P-loop stabilization and β-phosphate coordination are key determinants of GDP (and GTP) binding affinity.

Figure 1. Receptor-mediated activation of G proteins through promoting GDP release

Agonist (BI-167107)-bound receptor-mediated interaction with heterotrimeric G proteins promotes nucleotide (GDP) release in the G protein α-subunit. A) Structures of the β₂AR (green) and G protein heterotrimer (Gαsβγ). The nucleotide-binding Gα-subunit (gray) is composed of a ras-homology domain (GαsRas) and an α-helical domain (GαsAH). B) The crystal structure of BI-167107 (agonist)-bound β₂AR with Gs reveals large domain movements in the α-subunit (yellow) of Gs that are associated with nucleotide release.

Figure 2. DXMS reveals conformational changes in the Gαs when in complex with agonist-bound β₂AR in solution

Pairwise comparisons of DXMS of Gαs under different conditions. (A) Changes in DXMS of Gαs following formation of β₂AR-Gs relative to Gαs in the Gs heterotrimer. (B) Changes in DXMS of Gαs in the β₂AR-Gs complex following dissociation of the complex with GDP/AlF₃. (C) Changes in DXMS of Gαs in the β₂AR-Gs complex following the addition of GDP alone. The changes in amide hydrogen-deuterium exchange, given as changes in the percentage of the theoretical maximum number of deuterons incorporated per peptide, were mapped on to the crystal structure of Gαs based on the GTPγS-bound form (PDB:1AZT). Residues displaying increases (magenta) and decreases (blue) in deuterium incorporation when comparing different conditions were plotted according to the indicated heat map. Regions that are not covered are indicated in. Among 3 time points analyzed (see Figure 2S), 100 sec time points are presented.

Figure 3. Agonist-bound β₂AR-mediated activation of GDP release revealed by DXMS

Illustrated are the conformational changes in the GαsRas of Gs when in complex with agonist-bound β₂AR (mauve). Highlighted are the changes in deuterium exchange (HX), according to the indicated heat map, of key regions of GαsRas mapped onto the crystal structure of the complex (ref 2). (B and C) Comparison of the structure of Gαs in complex with β₂AR with that of Gαi (the inhibitory G protein for adenylyl cyclase bound to GDP) highlighting regions of increases or decreases in HX in the β6-strand-α5 helix (B) or in the β1-strand, P-loop and α1-helix (C). As above, Gαs (in B and C) is colored according to the indicated heat map representing changes in HX when comparing Gs heterotrimer with the nucleotide-free β₂AR-Gs complex. Residues where no mass information was obtained are colored charcoal gray. The superimposed structure of Gαi bound to GDP (transparent gray) is based on the heterotrimeric GDP-bound form, but illustrating the conserved glutamate (E50 in Gαs) in the GαsRas and arginine (R102 in Gαs) located in the GαsAH. D) Five amide hydrogens, contributed by highly conserved ‘RLLL’ motif, are involved in stabilizing the β1-strand in a peptide fragment of Gαs that displays high HX.

Figure 4. Deuterium exchange at the interface between the α-helical and ras homology domains

A) Changes in HX at the interface between the GαsAH (ribbon diagram) and GαsRas (surface rendering) are mapped onto the “open” conformation of Gαs observed in the β₂AR-Gs complex (inset). In this “open” conformation, GαsAH and GαsRas are colored according to the indicated heat map. Also shown is a ribbon diagram of GαsAH in a “closed” position (gray) similar to that observed in the crystal structure of the GDP-bound Gαi heterotrimer. The location of the GDP binding site is shown as spheres. B) Surface rendering of panel A) rotated back 90° to show the cytoplasmic side of the “open” conformation of nucleotide-free Gαs.

Figure 5. Mechanism for receptor-catalyzed nucleotide exchange in heterotrimeric G protein α-subunits

The step-wise dissociation of GDP from Gαs (orange) by agonist-activated β₂AR that involves the engagement of both the N-and C-terminus of Gαs. The activation of Gs through an activated β₂AR (green) results in GDP release and subsequent GTP binding. The activated receptor engages the C-terminus of the α5-helix of Gαs which undergoes a rigid-body translation upward into the receptor core and reorganizes the β6-α5 loop, a region that participates in purine ring binding. In a simultaneous or sequential event, ICL2 of the β₂AR engages the N-terminus of the Gαs leading to reorganization of its β1-strand/P-loop, the loss of coordination of the β-phosphate of GDP (blue), and subsequently GDP release. The position of the N-terminal helix is aided by the Gβγ-subunits (not shown). The concomitant disruption of the interaction between the P-loop and GαsAH, primarily through the highly conserved R201 in the GαsAH and E50 in the P-loop, opens GαsAH allowing GDP to freely dissociate. Formation of the nucleotide-free form allows GTP (gray) to bind, resulting in reformation of the “closed” conformation, and activation of the G protein through functionally dissociating from Gβγ and uncoupling from β₂AR.