β₂ adrenergic receptor-induced conformational changes in the heterotrimeric G protein Gs

The formation of a complex between a G protein-coupled receptor (GPCR) and a heterotrimeric G protein is responsible for the majority of transmembrane signaling in response to hormones and neurotransmitters. Heterotrimeric G proteins are composed of a nucleotide-binding α-subunit (Gα) and an obligate dimer of the β and γ-subunits (Gβγ). In their inactive form Gα-subunits are bound to GDP and tightly associated with Gβγ. Gα proteins are evolutionarily related to the ras family of G proteins but contain a small globular domain, referred to as the α-helical domain (GαsAH) (Fig 1a). The ras homology domain (GαsRas) contains most of the catalytic residues necessary for GTP hydrolysis, as well as the Gβγ and effector binding regions³. The structures within these regions differ between GTP- and GDP-bound states, and are appropriately termed switch regions^4,5.

The process of G protein activation involves formation of a complex (often referred to as a ternary complex) consisting of an agonist-occupied GPCR and a nucleotide-free heterotrimer. In a companion manuscript we present the β₂AR-Gs crystal structure² (Fig 1b), which provides the first high-resolution snapshot of such ternary complex. However, a static view of this structure provides only limited insight into the mechanism of GDP release, the first step in G protein activation. In an effort to provide dynamic insights into the mechanism of receptor-mediated activation of Gs, the stimulatory G protein for adenyly cyclase, we studied the effect of agonist-bound β₂AR on the structure of Gs using peptide amide hydrogen-deuterium exchange mass spectrometry (DXMS, see Supplementary Methods). The rates of exchange of the amide hydrogens in a protein are a function of the protein’s thermodynamic stability, most particularly the stability of the hydrogen bonds that each amide forms in the protein’s native structure. In general, an amide hydrogen involved in an intra-molecular hydrogen bond, such as those that occur in a β-strand or α-helix in a relatively stable region of the protein, exchanges more slowly than amide hydrogens forming less stable bonds ^6-11. Changes in exchange rates reflect the propensity to formation or disruption of these bonds, thereby providing information about protein structural changes and dynamics.

We examine the amide hydrogen exchange behavior of 1) the Gs heterotrimer alone, 2) Gs in complex with β₂AR, 3) following uncoupling (dissociation) of the β₂AR-Gs complex with transition state analogue, GDP/aluminum fluoride (AlF₃ or [AlF₄]⁻), and 4) when the β₂AR-Gs complex is exposed to GDP (Fig 2, supplemental data Fig S2-S9A). When comparing the Gs heterotrimer with the β₂AR-Gs complex we observe marked changes in exchange rates for Gαs, but little change in the exchange rates of Gβ or Gγ (Supplemental Fig.S2-S5). In addition, we do not report exchange behavior on the β₂AR itself since comparatively limited peptide probe sequence coverage was obtained for the receptor when we employed DXMS analysis conditions optimal for the G proteins.¹²

Transitioning from the GDP-bound heterotrimer to the nucleotide-free β₂AR-Gs complex is associated with changes in exchange rates throughout the Gα subunit (Fig 2a). There is a general increase in exchange in the segments that form the nucleotide binding pocket, consistent with a loss of polar interactions with GDP or as a result of alterations in protein structure following GDP dissociation. We observe reduced exchange at the C-terminal portion of the α5-helix when bound to β₂AR (Fig 2a and 3a,b). This is not surprising considering the crystal structures of opsin and metarhodopsin bound to a C-terminal peptide of transducin^13-15, and with Gs bound to β₂AR (ref 2), showing that the C-terminus of Gα subunits penetrates into the cytoplasmic core of the transmembrane bundle of GPCRs. This finding is also consistent with a plethora of biochemical and biophysical evidence (reviewed by Oldham et al 2006)¹⁶, as well as mutagenesis studies and peptide analysis that suggest that the C-terminus determines receptor coupling specificity¹⁷.

In contrast to the C-terminal region of α5, we observe a dramatic increase in exchange rates of peptide amides in the amino terminal segment of the α5-helix, suggesting an increase in the dynamics or disordering of this segment that is not evident in the structure of β₂AR-Gs complex (Fig 2a, 3a,b and Supplemental data Fig. S11a). The β6-α5 loop constitutes part of the nucleotide-binding pocket. Thus, increases in exchange at the amino terminal portion of the α5-helix likely reflect the sensitivity to nucleotide release (Fig 2a, 3b). The reintroduction of GDP reverses the effects observed on formation of the complex in this region (Fig 2c). These observations are consistent with EPR spectroscopy studies suggesting movement of the α5-helix of the G protein transducin upon interacting with photo-activated rhodopsin¹⁸.

As expected, uncoupling Gs from the β₂AR with GDP/AlF₃ reverses all exchange changes induced by receptor binding (Fig. 2b). GDP/AlF₃ is a transition state analogue that structurally and functionally mimics GTP when bound to G proteins. Interestingly, the overall effect of adding GDP on exchange rates was similar to the effect of adding GDP/AlF₃; however, we did not observe a comparable increase in exchange at the carboxyl terminal end of the α5-helix (Fig 2c). These results suggest that the carboxyl terminal peptide of GDP-bound Gαs may remain weakly coupled to the receptor and are consistent with crosslinking experiments performed under similar conditions showing persistent interactions between the β₂AR and Gs in the presence of GDP (Fig. S10). It is tempting to speculate that this GDP-bound β₂AR-Gs complex may represent a ‘pre-coupled’ receptor complex. Nevertheless, it is likely that the GDP-bound β₂AR-Gαs complex is not very stable since increased exchange at the α5-helix carboxyl terminus could be observed following longer exchange durations or incubations at higher (but still physiologic) temperature (see Supplemental Fig S2 and S10).

The most striking effect of β₂AR-Gs complex formation on amide hydrogen exchange was observed in the β1-strand, which links the second intracellular loop (ICL2) of the agonist-bound receptor to the P-loop that coordinates the β-phosphate of GDP in Gαs (Fig 2a, 3a,c). The large increase in exchange rate in the β1-strand is unexpected based on relatively small structural changes in β1-α1 loop observed when comparing the structures of Gαi bound to GDP with the β₂AR-Gs complex (Fig. 3c). The relatively minor differences observed in the crystal structures may not reflect the change required for GDP release, since the crystallization process often favors the lowest energy and most stable conformation. In contrast, DXMS experimentation interrogates the entire ensemble of native-state conformations under physiologic conditions (pH, salt, protein concentration and temperature)¹¹ . Such analysis reveals that the β1-P-loop-α1 region undergoes structural changes that markedly alter the stability of amide backbone hydrogen bonds.

The crystal structure of Gαs-GTPγS suggests that five amide hydrogens stabilize the backbone of the β1-strand of this peptidic fragment (Fig 3d and Supplemental data Fig S11b). Four of the five amides are contributed by the highly conserved R₄₂LLL₄₅ motif, found in all G protein α-subunits (See alignment in supplemental Fig S12). DXMS analysis indicates that 4-5 hydrogens rapidly exchange in this fragment. We were unable to detect fragments of the neighboring β3 and β4 strands by MS, but speculate that alterations in exchange might also be observed in these regions. The dramatic increase in exchange in this highly conserved motif suggests that formation of the β₂AR-Gs complex is associated with large changes in the structure and/or stability of the β-sheet formed by the β1-β5 strands.

The increased exchange in the β1-strand, the P-loop and the proximal α1-helix is compatible with the well-established mechanism of nucleotide exchange in the ras family of G proteins. Here the β-phosphate of GDP is wedged between the highly conserved ‘P-loop’ and α1-helix, as well as a single Mg²⁺ ion that is coordinated by residues within the P-loop. GDP release in ras-like G proteins is manifested simply by altering Mg²⁺ coordination to the β-phosphate⁴. Although heterotrimeric Gα proteins lack the dependence on Mg²⁺ for GDP binding, like all G proteins they display a 10⁵-10⁶-fold preference for GDP over GMP⁵. The structural similarities between the ras and heterotrimeric family of G proteins suggest that the mechanism for nucleotide exchange should be similar. Thus, a critical action of the agonist-bound receptor appears to be the engagement of the N-terminus of Gα, thereby altering the position and/or stability of the β1-strand with associated changes in the structure of the P-loop and α1-helix.

DXMS analysis also reveals regions with higher exchange that extend beyond the nucleotide-binding site and encompass the entire interface between the GαsAH and GαsRas (Fig 2, 4). These data are consistent with crystallographic and electron microscopy evidence on the β₂AR-Gs complex (ref 2 and Westfield et al, manuscript submitted). These studies and recent double electron-electron resonance analyses¹⁹ suggest that the GαsRas and GαsAH undergo a “clam shell-like” opening in the absence of nucleotide (see Fig 4). Nucleotide release appears to result in re-positioning of the GαsAH, but the observed slow rate of exchange within the core of the domain suggests that the domain itself is intact and folded. In the GDP- and GTPγS-bound forms of G proteins, a highly conserved glutamate residue in the P-loop (E50 in Gαs) tightly binds the highly conservative catalytic arginine residue (R201 in Gαs), which is located in the GαsAH (Fig 3c and see alignment in Supplement Fig S12). However, the side chain of E50 in Gαs in the receptor complex is poorly ordered in the crystal structure, displaying high B- or temperature factors. We propose that alterations in the P-loop structure from the loss of β-phosphate coordination disrupts the E50-R201 interaction and facilitates the dissociation of GαsAH from GαsRas.

In summary, DXMS analysis of the β₂AR-Gs complex complements the crystal structure and provides essential additional mechanistic insights into the structural events linking GPCR-G protein complex formation and GDP release. The amino and carboxyl termini of Gαs represent the principal structural conduits between the receptor and the nucleotide binding pocket (Fig. 5). Although it is not presently possible to determine the exact sequence of events leading to nucleotide dissociation and the formation of a stable complex, we speculate that the initial interaction may involve the carboxyl terminus of GDP-bound Gαs and agonist-bound β₂AR. Subsequent interactions between ICL2 of the β₂AR and the αN-helix of Gαs are associated with GDP release through dynamic changes in the β1-strand that involve disruption of a conserved hydrogen bond network and are coupled to structural changes in the highly conserved ‘P-loop’ that wraps around the β-phosphate of GDP. Thus, in a coordinated event, the agonist-bound receptor engages both the N-terminus and C-terminus to promote nucleotide release.