Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex

Gerwin H. Westfield; Søren G. F. Rasmussen; Min Su; Somnath Dutta; Brian T. DeVree; Ka Young Chung; Diane Calinski; Gisselle Velez-Ruiz; Austin N. Oleskie; Els Pardon; Pil Seok Chae; Tong Liu; Sheng Li; Virgil L. Woods; Jan Steyaert; Brian K. Kobilka; Roger K. Sunahara; Georgios Skiniotis

doi:10.1073/pnas.1113645108

New Research In

Contributed by Brian K. Kobilka, August 19, 2011 (sent for review August 9, 2011)

Abstract

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β₂-adrenoceptor (β₂AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotide-dependent transition from a flexible to a conformationally stabilized state.

The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), which instigate intracellular signaling by activating their cognate heterotrimeric G proteins on the cytoplasmic side. GPCRs constitute the largest family of membrane proteins and play essential roles in regulating every aspect of normal physiology, thereby representing major pharmacological targets. 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. The β₂-adrenergic receptor (β₂AR) and its complex with heterotrimeric stimulatory G-protein Gs (Gαsβγ) represent an ideal model system for the large family of GPCRs activated by diffusible ligands. Agonist binding to the β₂AR promotes interactions with GDP-bound Gsαβγ heterotrimer, leading to the exchange of GDP for GTP, and the functional dissociation of Gs into Gα-GTP and Gβγ subunits. To examine the architecture of agonist occupied β₂AR in complex with Gαsβγ under different conditions, we used electron microscopy (EM) and single-particle analysis. Because of the limited size of the protein complex (∼148 kDa), we visualized specimens embedded in negative stain, which provides sufficient contrast from relatively small protein assemblies (1). This approach allowed us to obtain 2D projection averages and 3D reconstructions that provided new insights into dynamic features of the β₂AR-Gs complex, and helped guide a successful approach to crystallize the complex enabling a high-resolution structure (2).

Results and Discussion

In a first step, we sought to examine the architecture of complexes in the nucleotide-free state of Gαs. Before coupling with an agonist-bound receptor, the nucleotide binding pocket of the α-subunit of the Gαsβγ heterotrimer is occupied by GDP. Upon forming a complex with the β₂AR, GDP dissociates, and the resulting nucleotide-free β₂AR-Gs complex is highly stable (2). EM visualization of the nucleotide-free complex showed a monodisperse particle population (Fig. 1A, and SI Appendix, Fig. S1). Reference-free alignment and classification of ∼17,000 particle projections revealed characteristic class averages with an overall density that is in very good agreement with the crystal structure of the complex (2). Because of its shape, the complex adsorbs on the carbon support with small variations (± 20°) of mainly two diametrically opposite preferred orientations that generate practically identical, mirror-related 2D projections (SI Appendix, Figs. S2 and S3).

Fig. 1.

Two-dimensional projection analysis of the T4L-β₂AR-Gs complex in the nucleotide-free state. (A) Raw EM image of detergent-solubilized T4L-β₂AR-Gs complex embedded in negative stain. (Scale bar, 50 nm.) (B) Representative EM class averages of the nucleotide-free complex with the projection profile of the AH domain not visible (Left), or visible on the Ras domain (Right, AH indicated by arrow). The cartoon models represent the conformations reflected by the EM averages, with the one on the left depicting the variable positioning of the AH domain, suggesting flexibility or multiple conformations (the position of the detergent micelle is indicated by gray shaded arcs and labeled with “m”). (Scale bar, 10 nm.) (C) Reprojections (Upper) of the crystal structure (2) (Lower) in the same overall orientation as B reveal the identity of each EM density component. The crystal structure on the Right shows the AH domain in the same position (relative to the Ras-like domain) as the one determined in the crystal structure of Gαs-GTPγS alone (4). (D) Representative class averages of nucleotide-free complex with nanobody Nb37 bound on the AH domain (arrows) reveal its flexibility. (Scale bar, 10 nm.)

The distinct features of the class averages in these preferred orientations allowed us to assign the negative stain projection profiles from specific components of the complex (Fig. 1 B and C). A central oval density represents the β₂AR in a detergent micelle, with a small protruding density corresponding to T4 lysozyme (T4L) that replaces the unstructured extracellular N terminus of the receptor and serves as an orienting landmark. This interpretation was confirmed by EM analysis of complexes lacking T4L (SI Appendix, Fig. S4). Some class averages of the T4L-β₂AR-Gs complex do not reveal a density corresponding to the T4L. Besides the presence of a relatively flexible linker connecting T4L and the β₂AR, this effect is mostly because T4L lies at an angle to the longitudinal axis of the complex, as shown in the X-ray structure (2). Because of this geometry, even a 10° variation in the way the particle adsorbs on the carbon support drastically reduces the visibility of the T4L projection profile, as demonstrated by projection simulation experiments (SI Appendix, Fig. S5). Thus, the visibility of the T4L projection profile is very sensitive to even limited out-of-plane particle tilting (e.g., because of particle “rock” and “roll” or because of variations in the flatness of the carbon support). Because we observe a single density corresponding to T4L, the detergent micelle contains only a single copy of the β₂AR, in agreement with the crystal structure. Therefore, the significant additional density around the receptor stems from the large micelle formed by the detergent (3). Diametrically opposite to the T4L domain, two main interacting densities representing the Gs trimer appear in close proximity to the receptor on its intracellular surface. One of the two domains appears to extensively interact with the receptor density, suggesting it corresponds to the Ras-like domain of Gαs, while its neighboring domain has a profile consistent with the side view of Gβγ. Interestingly however, several class averages revealed an additional small globular density bound on the Ras-like domain of Gαs (Fig. 1B, Right, and SI Appendix, Fig. S2). In this location, the additional density could only be attributed to the α-helical (AH) domain of Gαs, occupying a position expected from the crystal structure of Gαs-GTPγS alone (4) and the structure of the Gi heterotrimer (5) (Fig. 1C, and SI Appendix, Fig. S6), but in entirely different location from that observed in the crystal structure of the β₂AR-Gs complex (2). To assess the fraction of particles displaying the AH domain in this location, we selected and classified only projections clearly displaying the profiles of Ras-like, Gβγ, β₂AR, and T4L domain densities in the same position, thereby restricting the range of particle projection orientations. The classification revealed that the AH domain was ordered on the Ras-like domain in ∼35% of the particles, but in most other particle projections this density was absent (Fig. 1B, and SI Appendix, Fig. S3). It should also be noted that in contrast to the T4L domain, the projection profile of the AH domain in this position (SI Appendix, Fig. S3) is not sensitive to the relatively limited out-of-plane tilts (± 20°) of the preferred particle orientation on the carbon support (SI Appendix, Fig. S5). This EM analysis provided the initial evidence for a high degree of mobility of the AH domain relative to the Ras domain in the nucleotide-free β₂AR-Gs complex. Furthermore, the structural heterogeneity observed provided insights to the challenges in obtaining 3D crystals of the complex.

To promote complex stabilization for high-resolution structural studies, we generated and screened llama antibodies (nanobodies) to the purified complex (2). Nanobodies are small (∼15 kDa), clonable variable domains of a heavy chain-only antibody, obtained by immunizing a llama with purified detergent-solubilized β₂AR-Gs complex stabilized with a short homobifunctional crosslinker (2). By screening samples with negative-stain EM, we identified two nanobodies (Nb35 and Nb37) that bound to the complex, but not the receptor alone. Class averages of particles incubated with Nb35 indicated a homogeneous protein complex displaying increased density between Gαs and Gβγ. Even though the nanobody projection profile was not clearly distinguished in the preferred particle orientations, its presence appeared to enhance the uniformity in the disposition of Gαs-ras and Gβγ domains. The use of Nb35 indeed allowed us to obtain the crystal structure of the T4L-β₂AR-Gs complex, which showed that the nanobody binds at the interface of the Gαs-Ras and Gβγ. In this location, Nb35 would not be predicted to interact with or stabilize the AH domain (2). Accordingly, the classification of Nb35-bound complexes revealed a similar distribution of particles with an ordered AH domain on the Ras-like domain as in the absence of Nb35 (SI Appendix, Figs. S7–S9).

In contrast to Nb35, Nb37 appears bound directly to the AH domain [as also determined by deuterium-exchange MS (DXMS)]) (SI Appendix, Fig. S10) and could be distinguished in single-particle EM class averages of the β₂AR-Gs complex as an extension of the AH domain. Using Nb37 as a domain marker allowed us to track the variable positioning of the small AH region of Gαs (SI Appendix, Figs. S11 and 12). The 2D class averages from this preparation reveal an enhanced and elongated density adopting different orientations around the Ras-like domain, ranging from close proximity to the Gβγ module to extending much further out of the complex in the opposite direction (Fig. 1D and SI Appendix, Fig. S12). Collectively, these findings suggest that in the absence of nucleotide, the AH domain is flexible, thereby sampling different positions around the Ras-like domain. Deuterium-exchange studies are consistent with a dynamic interface between the Gαs Ras and AH domains (6). Therefore, the unexpected position of the “open” AH domain in the crystal structure (2) represents just one of the possible conformations.

To obtain a more detailed view of the complex architecture, we used the random conical tilt approach (7) to calculate initial 3D reconstructions of complexes with and without ordered AH domain on the Ras-like domain (SI Appendix, Fig. S13). These initial 3D models were subsequently used for multireference supervised alignment (8, 9) to separate particle projections from our entire dataset according to the AH positioning (see SI Appendix). This approach allowed us to obtain quality 3D reconstructions from particle projections with and without density corresponding to AH domain on the Ras-like domain. The reconstructions are in excellent agreement with the corresponding 2D averages (Fig. 2A and SI Appendix, Fig. S14). In 3D reconstructions of particles where density for AH domain is observed, its orientation relative to the Ras-like domain appears to be similar to that found in the crystal structure of Gαs-GTPγS (4) (Fig. 2A and SI Appendix, Fig. S6). In addition, we obtained 3D reconstructions of nucleotide-free complexes with bound Nb37 marking the positioning of the AH domain. The 3D maps clearly reveal that the Nb37-enhanced density of the AH domain can adopt different conformations around the Ras-like domain, indicative of a relative flexibility in the interaction between the two domains (Fig. 2B and SI Appendix, Figs. S15 and S16). This variability in the 3D conformation of the AH domain or the AH/Nb37 module around the Ras-like domain was further confirmed by cross-validating 3D reconstructions (SI Appendix, Fig. S17).

Fig. 2.

Three-dimensional reconstructions of the T4L-β₂AR-Gs complex in the nucleotide-free state. (A) Representative class averages and corresponding 3D reconstructions of particles in each category show the variability in the positioning of the AH domain in the nucleotide-free complex. In the reconstruction to the left, the AH domain (orange ribbon) is shown in the same position as found in the docked crystal structure (2). Absence of sufficient density to accommodate this domain indicates that its position is highly variable in this particle population. In the reconstruction to the right, the AH domain is modeled within the available EM density right below the Ras-like domain of Gαs, as also suggested by the 2D averages. (B) Three-dimensional reconstructions of distinct conformations of nucleotide-free T4L-β₂AR-Gs complex with bound nanobody Nb37. The Nb37-enhanced density of the AH domain (marked with an oval) shows variable positioning around the Ras-like domain of Gαs. (Scale bars, 5 nm.)

As noted above, previous crystal structures of G proteins show that bound nucleotides contribute to the stability of interactions between the Ras and AH domains. We therefore investigated the positioning of the AH domain in the presence of guanine nucleotides and nucleotide fragments. Pyrophosphate (PPi), representing two phosphates in GTP or GDP, has been shown to bind Ras in a Mg²⁺-dependent manner, presumably at the β- and γ-phosphate positions (10). PPi and its chemically more stable analog, foscarnet, more known for its antiviral properties (11), also bind to heterotrimeric Gαs with an apparent affinity of ∼0.5 and 1.6 mM, respectively, as determined by competition binding with a fluorescent GTPγS probe (Bodipy-GTPγS) (SI Appendix, Fig. S18). Binding of PPi and foscarnet most likely substitutes for the α- and β-phosphates of GDP rather than the β- and γ-phosphates of GTP. Binding of β- and γ-phosphates would result in modification of the switch II domain with subsequent Gβγ dissociation and dissolution of the complex (12). However, PPi (with or without Mg²⁺) does not disrupt the receptor-G protein complex. This finding is in contrast to dissociation of the complex observed with the GTP mimetic GTPγS (2).

Although the presence of PPi does not appear to affect the AH domain positioning, in the presence of Mg²⁺⋅foscarnet we observe a significantly higher proportion (∼70%) of complexes with an ordered AH region on the Ras-like domain (Fig. 3A and SI Appendix, Figs. S19–S28). The observations in 2D class averages were also reproduced by individual 3D reconstructions for the different conformers in each state (SI Appendix, Figs. S24 and S28). These results further confirm that the variability in the visibility of the AH domain is indeed because of its variable positioning and not because of negative stain artifacts, such as incomplete embedding. The ability of foscarnet to stabilize the AH domain on the Ras domain suggests it is acting as a ligand fragment that binds to the nucleotide binding pocket. Given its low affinity, it is not surprising that the stabilization is incomplete.

Fig. 3.

Nucleotide-dependent positioning of the Gαs AH domain. (A) Distribution of particles with a distinct projection profile of the AH domain stabilized on the Ras-like domain across different conditions (Inset Right, marked with a white dot). A class average of a particle with a nonvisible AH domain is shown for comparison (Inset Left). The presence of foscarnet and GDP significantly increases the number of particles with stabilized AH domain. (B) Representative class averages of the T4L-β₂AR-Gs complex after rapid mixing with GTPγS (1 μM) and immediate stain embedding reveal both intact as well as partially or fully dissociated complexes. The fraction of corresponding subpopulations is indicated. (Scale bars, 10 nm.)

In contrast to PPi and foscarnet, addition of GDP or the nonhydrolyzable GTP analog GTPγS leads to dissociation of the β₂AR-Gs complex (2). To examine the effect of GDP and GTPγS, we rapidly mixed the complex with nucleotide and immediately fixed the sample by negative stain embedding. Addition of either of these nucleotides at concentrations above 10 μM resulted in significant amounts of partially dissociated complexes (SI Appendix, Fig. S29). This result is expected because a large excess of either of these nucleotides would uncouple the G protein from the receptor. However, short incubation with lower GDP concentrations (1 μM) and immediate sample fixation for EM allowed to us to examine intact complexes, revealing that the AH region was ordered in ∼60% of the intact particles (SI Appendix, Figs. S30–S32). In contrast, even low concentrations of GTPγS (1 μM) showed a significant amount of destabilized complexes, and we were able to capture an array of intermediate dissociation states (Fig. 3B and SI Appendix, Figs. S30 and S33). Collectively, these data strongly suggest that the presence of nucleotide, or nucleotide fragments such as foscarnet, results in AH domain stabilization against the Ras-like domain of Gαs. In the absence of nucleotide, the position of the AH domain is highly variable (Figs. 3 and 4).

Fig. 4.

Model of conformational transitions in the β₂AR-Gs complex. The nucleotide-free β₂AR-Gs complex is characterized by a highly flexible AH domain (model 1). GTP binding promotes stabilization of the AH domain on the Ras-like domain of Gαs (model 2). In this model, the AH domain has the same position as the one observed in the crystal structure of Gαs-GTPγS (4). GTP binding results in subsequent uncoupling of Gs and β₂AR (model 3), with eventual dissociation of Gαs and Gβγ (model 4).

Our results are in agreement with a recent study of the complex formed by Gi and rhodopsin. Using double electron-electron resonance spectroscopy, Hamm, Hubbell, and colleagues documented large (up to 20 Å) changes in distance between nitroxide probes positioned on the Ras and AH domains of Gi upon formation of a complex with light-activated rhodopsin (13). The broad distance distributions observed for several labeling pairs are compatible with multiple conformations in dynamic equilibrium. Our findings are also consistent with results from DXMS that show increased deuterium exchange at both the nucleotide binding pocket and at sites of interaction between the Ras and AH domains upon formation of the β₂AR-Gs complex (6).

Support for the open conformation in vivo may come from studies on the action mechanism of the cholera toxin, the enterotoxin secreted by the pathogen Vibrio cholerae. Cholera toxin, together with ADP ribosylation factor (ARF), ADP ribosylates Gαs at R201, rendering the residue catalytically ineffective and the G protein GTPase-deficient (14). The ADP ribosylated and constitutively active Gαs will continue to stimulate adenylyl cyclase, cAMP production, and protein kinase A (PKA) activation. Activated PKA opens intestinal Cl⁻ channels and leads to increased water secretion that results in diarrhea (15). Crystallographic studies of G proteins in GDP and GTPγS-bound forms indicate that the catalytic arginine (R201 in Gs) is buried and likely inaccessible to cholera toxin and ARF (4). However, formation of the nucleotide-free form of Gαs and opening of the Ras and AH domains would facilitate accessibility to R201.

In conclusion, single-particle EM analysis of the β₂AR-Gs complex has provided novel structural insights into the dynamic nature of the assembly (Fig. 4). EM visualization of nanobody bound complexes allowed us to clearly reveal the variable positioning of the AH domain under nucleotide-free conditions and, additionally, to identify conditions that were key for the successful characterization of the complex by X-ray crystallography. Furthermore, single-particle EM examination of the β₂AR-Gs complexes in varying concentrations of GDP and GTPγS enabled us to capture transient intermediate dissociation states between β₂AR and Gs. This approach should prove useful for studying other signaling complexes involving GPCRs and other membrane proteins. The combination and integration of these technologies will be crucial for studying structural aspects of challenging macromolecular complexes at large.

Experimental Procedures

Specimen Preparation and EM Imaging of Negative-Stained Samples.

The T4L-β₂AR-Gs complex and nanobodies (Nb) were prepared as described in Rasmussen et al. (2). Specimens were visualized by EM in the following conditions: (i) T4L-β₂AR-Gs complex alone (nucleotide-free) (SI Appendix, Fig. S1); (ii) T4L-β₂AR-Gs in presence of Nb35 (SI Appendix, Fig. S7); (ii) T4L-β₂AR-Gs in presence of Nb37 (SI Appendix, Fig. S11); (iv) T4L-β₂AR-Gs in presence of 1 mM PPi (SI Appendix, Fig. S19 A and B); (v) T4L-β₂AR-Gs in the presence of 10 mM PPi (SI Appendix, Fig. S19 C and D); (vi) T4L-β₂AR-Gs in the presence of 10 mM foscarnet and 10 mM MgCl₂ (SI Appendix, Fig. S25); (vii) T4L-β₂AR-Gs in the presence of 1 μM GDP and 10 mM MgCl₂ (SI Appendix, Fig. S30 A and B); (viii) T4L-β₂AR-Gs in presence of 1 μM GTPγs and 10 mM MgCl₂ (SI Appendix, Fig. S30 C and D). All samples were prepared for EM using the conventional negative staining protocol (1). For nanobody labeling, the T4L-β₂AR-Gs complex was incubated for 15 min at room temperature with approximately equimolar concentrations of Nb35 or Nb37, and subsequently prepared by negative staining. For nucleotide (GDP or GTPγS) and nucleotide fragment (PPi or foscarnet) incubations, these components were rapidly mixed with the complex and the sample was immediately fixed by negative-stain embedding.

Specimens were imaged at room temperature with a Tecnai T12 electron microscope operated at 120 kV using low-dose procedures. Images were recorded at a magnification of 71,138× and a defocus value of ∼1.5 μm on a Gatan US4000 CCD camera (SI Appendix, Figs. S1, S7, S11, S19, S25, S29, and S30). All images were binned (2 × 2 pixels) to obtain a pixel size of 4.16 Å on the specimen level. Tilt-pair particles from 60° and 0° images were selected using WEB (16). Particles for only 2D classification of 0° projections were excised using Boxer (part of the EMAN 1.9 software suite) (17). The number of particles or tilt-pair particle projections per condition is provided in SI Appendix, Table S1.

Two-Dimensional Classifications and 3D Reconstructions of T4L-β₂AR-Gs.

The 2D reference-free alignment and classification of particle projections were performed using SPIDER (16). For all conditions, the 0° particle projections were iteratively classified into multiple classes for 10 cycles (SI Appendix, Figs. S2, S8, S12, S20, S22, S26, S31, and S33). SI Appendix, Table S1 provides the number of classes for each condition. For AH conformation assignments, we used the first classification to select only the particles from averages clearly displaying the profiles of Ras-like, Gβγ, β₂AR, and T4L domain densities in the same position, thereby restricting the range of particle projection orientations. These projections were pulled together and subjected to a second iterative classification (referred to as the “secondary” classification) (SI Appendix, Figs. S3, S9, S21, S23, S27, and S32). SI Appendix, Table S2 provides the number of classes and particle projections for each condition in the secondary classification. For counting the numbers of particles with and without stabilized ΑΗ domain on the Ras-like domain, three different operators examined each secondary classification and assigned each class average according to the projection profile of the specific region (SI Appendix, Figs. S3, S9, S21, S23, S27, S32, and S33). The assignment from the different operators was in good agreement, and the particle numbers belonging to individual classes were added to calculate percentages for each conformation. Assignments for each full individual dataset were done in addition to the secondary classification, and the results were in agreement. To test any bias, the particles from nucleotide-free, 1 mM PPi, 10 mM PPi, and foscarnet conditions were combined into a single dataset of 15,753 particles and were classified into 200 classes. The individual class averages were assigned as before according to the visibility of the AH domain, and the percentage of projections from each condition was determined according to the number of projections contributing to the assigned class averages (SI Appendix, Fig. S34). The results of this “blind” test showed very good agreement with our assignments from individual classifications.

For 3D reconstructions, in a first step we used the random conical tilt technique (7) to determine initial 3D maps by back-projection of tilted particle images belonging to individual classes. After a first round of angular refinement, corresponding particles from the images of the untilted specimen were added, and the images were subjected to another cycle of refinement. We thus generated reliable initial models for complexes with variability in the positioning of the ΑΗ domain of Gαs (SI Appendix, Fig. S13). After contrast transfer function (CTF) correction according to local defocus values obtained by CTFTILT (18), the full dataset from each condition was subjected to multiple reference-supervised alignment (8, 9) with the “multirefine” routine in EMAN (1.9) by using our initial models as reference maps. This approach allowed us to separate particles from the entire dataset (of each condition) according to the positioning of the ΑΗ domain of Gαs. The number of contributing particles in each condition and conformation is provided in SI Appendix, Table S3. For final maps, we used the separated datasets, as provided by the multiple reference-supervised alignment, and used FREALIGN (19) for further refinement of the orientation parameters and reconstruction (SI Appendix, Fig. S14–S16, S24, and S28). The resolution for each map was determined at FSC = 0.5 and is provided in SI Appendix, Table S3.

Molecular Modeling.

The crystal structure of T4L-β₂AR-Gs (2) was fit in the EM density as a rigid body. Because of the presence of the detergent micelle, which accounted for significant density surrounding β₂AR, all docking operations were performed manually with visual inspection of the best fit. The docking of the T4L-β₂AR-Gs complex revealed that the EM density corresponding to Gβγ was shifted further away from the receptor in all 3D maps compared with the crystal structure. The most likely cause for this variation is the presence of the planar lipid bilayer provided by the cubic lipid phase in the crystals, which most likely maintains a different interaction with Gβγ compared with the detergent micelle. However, it is also possible that this difference is attributed to the crystal packing, or a limited deformation of the complex because of the presence of the carbon support on the EM grid, or both. Accordingly, Gβγ was translated manually by 9 Å to best fit its density yet retain all interactions with Gαs. This final model is shown unmodified for all fittings in 3D reconstructions (Fig. 2, and SI Appendix, Figs. S14–S16, S24, and S28). For maps showing the ΑΗ domain of Gαs on the Ras-like domain, we manually modeled the ΑΗ domain in its corresponding position by taking into account steric constraints. In this conformation, the position of the ΑΗ domain is very similar to the one observed in the crystal structure of Gαs-GTPγS (4) (SI Appendix, Fig. S6).

Deuterium-Exchange MS.

For DXMS, 1.5 mL of R:G complex or 1.5 mL of R:G:NB was mixed with 4.5 mL of D₂O buffer (20 mM Hepes, pH 7.5, 100 mM NaCl, 10 mM BI-167107, 100 mM TCEP, 0.0015% MNG-3 in D₂O) and incubated for 10, 100, 1,000, and 10,000 s on ice. At the indicated times, the sample was quenched by 15 mL of ice-cold Quench solution (0.1 M NaH₂PO₄, 20 mM TCEP, 16.6% glycerol, pH 2.4), immediately frozen on dry ice, and stored at –80 °C. Nondeuterated control was prepared in H₂O buffer (20 mM Hepes, pH 7.5, 100 mM NaCl, 10 μM BI-167107, 100 μM TCEP, 0.0015% MNG-3 in H₂O), mixed with Quench solution, and snap-frozen on dry ice. Samples were thawed and immediately passed through an immobilized porcine pepsin column (16 mL bed volume) at a flow rate of 20 mL/min of 0.05% trifluoroacetic acid. Peptide fragments were collected contemporaneously on a C18 trap column for desalting and separated by a Magic C18AQ column (Michrom BioResources Inc.) using a linear gradient of acetonitrile from 6.4% to 38.4% over 30 min. MS analysis was performed using LCQ Classic mass spectrometer from Thermo Finnigan, with capillary temperature of 20 °C. Deuterium quantification data were collected in MS1 profile mode, and peptide identification data were collected in data-dependent MS/MS mode. Recovered peptide identification and analysis were carried out using DXMS Explorer (Sierra Analytics Inc.), a software specialized in processing DXMS data (SI Appendix, Fig. S10).

Bodipy-GTPγS Binding.

The effect of foscarnet and PPi was measured using 100 nM bodipy-GTPγS-FL (Invitrogen). Fluorescence intensity of bodipy-GTPγS-FL (l_ex ∼470 nm) increases upon G-protein binding, as demonstrated by McEwan et al. (20). A wavelength scan of bodipy-GTPγS-FL (100 nM) in the absence (dotted) or presence (solid) of a molar excess of purified Gαs (1 mM) was determined to assess optimal spectroscopy conditions. The capacity of PPi and the chemically stable pyrophosphate analog of PPi, foscarnet, to inhibit bodipy-GTPγS-FL (l_ex∼470 nm, l_em∼515 nm) was measured as described in SI Appendix, Fig. S18 B and C in both Gαs (B and C) and heterotrimeric Gαsβγ (C). The fluorescence of 100 nM bodipy-GTPγS-FL was measured in the presence of 1 mM G protein. PPi or foscarnet were added together with bodipy-GTPγS-FL and initiated by the addition of G protein (1 mM) in 20 mM Tris-HCl, pH 8.0, 3 mM MgCl₂, 1 mM DTT in a final volume of 200 mL bodipy-GTPγS-FL binding to heterotrimeric G protein included 0.1% dodecylmaltoside (final). Fluorescence was measured on a short time scale (600 s) to minimize the accumulation of hydrolysis product bodipy-phosphate (21). Hydrolysis, which also appears as an increase in fluorescence, was determined simply by chelating Mg²⁺ with 10 mM EDTA following the 600-s incubation. Inhibition of bodipy-GTPγS-FL by PPi and foscarnet can be reversed with the subsequent addition of high Mg²⁺ (25 mM), which enhances bodipy-GTPγS-FL binding, indicating that PPi and foscarnet are not irreversibly binding or denaturing the G protein. Gαs was purified as described in Sunahara et al. (4). Gαsβγ was purified as described by Rasmussen et al. (2). Fluorescence was measured in a 96-well microtiter plate format on a M5 fluorescence plate reader (Molecular Precision).

Acknowledgments

We thank J. Tesmer for suggestions. This work was supported by the Lundbeck Foundation Junior Group Leader Fellowship (to S.G.F.R.); the Fund for Scientific Research of Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen) and the Institute for the encouragement of Scientific Research and Innovation of Brussels (to E.P. and J.S.); the National Institute of General Medical Sciences (NIGMS) Molecular Biophysics Training Grant GM008270 (to G.H.W. and B.T.D.); the National Institute of Neural Disorders and Stroke Grant R01-NS28471 (to B.K.K.); the Mather Charitable Foundation (to B.K.K.); NIGMS Grants R01-GM083118 (to B.K.K. and R.K.S.) and R01-GM068603 (to R.K.S.); National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grant R01-DK090165 (to G.S.); National Institutes of Health Grants AI076961, AI08192, AI2008031, CA118595, GM20501, GM066170, GM093325, and RR029388 (to V.L.W.); Michigan Diabetes Research and Training Center Grant, NIDDK, P60DK-20572 (to R.K.S.); and the University of Michigan Biological Sciences Scholars Program (R.K.S. and G.S). G.S. is a Pew Scholar of Biomedical Sciences.

Footnotes

↵¹G.H.W., S.G.F.R., M.S., and S.D. contributed equally to this work.
²To whom correspondence may be addressed. E-mail: kobilka{at}stanford.edu, sunahara{at}umich.edu, or skinioti{at}umich.edu.

Author contributions: V.L.W., J.S., B.K.K., R.K.S., and G.S. designed research; G.H.W., S.G.F.R., M.S., S.D., B.T.D., K.Y.C., D.C., G.V.-R., A.N.O., E.P., P.S.C., T.L., S.L., and G.S. performed research; and B.K.K., R.K.S., and G.S. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113645108/-/DCSupplemental.

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4. Walz T
(2004) Negative staining and image classification—Powerful tools in modern electron microscopy. Biol Proced Online 6(1):23–34.
OpenUrl CrossRef PubMed
↵
1. Rasmussen SG,
2. et al.
(2011) Crystal structure of the β(2) adrenergic receptor-Gs protein complex. Nature, 10.1038/nature10361.
↵
1. Rubinstein JL
(2007) Structural analysis of membrane protein complexes by single particle electron microscopy. Methods 41:409–416.
OpenUrl CrossRef PubMed
↵
1. Sunahara RK,
2. Tesmer JJ,
3. Gilman AG,
4. Sprang SR
(1997) Crystal structure of the adenylyl cyclase activator Gsalpha. Science 278:1943–1947.
OpenUrl Abstract/FREE Full Text
↵
1. Wall MA,
2. et al.
(1995) The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell 83:1047–1058.
OpenUrl CrossRef PubMed
↵
1. Chung KY,
2. et al.
(2011) beta(2) adrenergic receptor-induced conformational changes in the heterotrimeric G protein Gs. Nature, 10.1038/nature10488.
↵
1. Radermacher M,
2. Wagenknecht T,
3. Verschoor A,
4. Frank J
(1987) Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J Microsc 146(1):113–136.
OpenUrl PubMed
↵
1. Brink J,
2. et al.
(2004) Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis. Structure 12(2):185–191.
OpenUrl PubMed
↵
1. Ménétret JF,
2. et al.
(2005) Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 348:445–457.
OpenUrl CrossRef PubMed
↵
1. Zhang B,
2. Zhang Y,
3. Shacter E,
4. Zheng Y
(2005) Mechanism of the guanine nucleotide exchange reaction of Ras GTPase—Evidence for a GTP/GDP displacement model. Biochemistry 44:2566–2576.
OpenUrl CrossRef PubMed
↵
1. Sundquist B,
2. Oberg B
(1979) Phosphonoformate inhibits reverse transcriptase. J Gen Virol 45:273–281.
OpenUrl Abstract/FREE Full Text
↵
1. Sprang SR
(1997) G protein mechanisms: Insights from structural analysis. Annu Rev Biochem 66:639–678.
OpenUrl CrossRef PubMed
↵
1. Van Eps N,
2. et al.
(2011) Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proc Natl Acad Sci USA 108:9420–9424.
OpenUrl Abstract/FREE Full Text
↵
1. Freissmuth M,
2. Gilman AG
(1989) Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity. J Biol Chem 264:21907–21914.
OpenUrl Abstract/FREE Full Text
↵
1. Gabriel SE,
2. Brigman KN,
3. Koller BH,
4. Boucher RC,
5. Stutts MJ
(1994) Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 266(5182):107–109.
OpenUrl Abstract/FREE Full Text
↵
1. Frank J,
2. et al.
(1996) SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116:190–199.
OpenUrl CrossRef PubMed
↵
1. Ludtke SJ,
2. Baldwin PR,
3. Chiu W
(1999) EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128(1):82–97.
OpenUrl CrossRef PubMed
↵
1. Mindell JA,
2. Grigorieff N
(2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334–347.
OpenUrl CrossRef PubMed
↵
1. Grigorieff N
(2007) FREALIGN: High-resolution refinement of single particle structures. J Struct Biol 157(1):117–125.
OpenUrl CrossRef PubMed
↵
1. McEwen DP,
2. Gee KR,
3. Kang HC,
4. Neubig RR
(2001) Fluorescent BODIPY-GTP analogs: Real-time measurement of nucleotide binding to G proteins. Anal Biochem 291(1):109–117.
OpenUrl CrossRef PubMed
↵
1. Jameson EE,
2. et al.
(2005) Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues. J Biol Chem 280:7712–7719.
OpenUrl Abstract/FREE Full Text

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Proceedings of the National Academy of Sciences: 108 (38)

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Article Classifications

Biological Sciences
Pharmacology

[1] ↵
Ohi M,
Li Y,
Cheng Y,
Walz T
(2004) Negative staining and image classification—Powerful tools in modern electron microscopy. Biol Proced Online 6(1):23–34.
OpenUrl CrossRef PubMed

[2] Ohi M,

[3] Li Y,

[4] Cheng Y,

[5] Walz T

[6] ↵
Rasmussen SG,
et al.
(2011) Crystal structure of the β(2) adrenergic receptor-Gs protein complex. Nature, 10.1038/nature10361.

[7] Rasmussen SG,

[8] et al.

[9] ↵
Rubinstein JL
(2007) Structural analysis of membrane protein complexes by single particle electron microscopy. Methods 41:409–416.
OpenUrl CrossRef PubMed

[10] Rubinstein JL

[11] ↵
Sunahara RK,
Tesmer JJ,
Gilman AG,
Sprang SR
(1997) Crystal structure of the adenylyl cyclase activator Gsalpha. Science 278:1943–1947.
OpenUrl Abstract/FREE Full Text

[12] Sunahara RK,

[13] Tesmer JJ,

[14] Gilman AG,

[15] Sprang SR

[16] ↵
Wall MA,
et al.
(1995) The structure of the G protein heterotrimer Gi alpha 1 beta 1 gamma 2. Cell 83:1047–1058.
OpenUrl CrossRef PubMed

[17] Wall MA,

[18] et al.

[19] ↵
Chung KY,
et al.
(2011) beta(2) adrenergic receptor-induced conformational changes in the heterotrimeric G protein Gs. Nature, 10.1038/nature10488.

[20] Chung KY,

[21] et al.

[22] ↵
Radermacher M,
Wagenknecht T,
Verschoor A,
Frank J
(1987) Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J Microsc 146(1):113–136.
OpenUrl PubMed

[23] Radermacher M,

[24] Wagenknecht T,

[25] Verschoor A,

[26] Frank J

[27] ↵
Brink J,
et al.
(2004) Experimental verification of conformational variation of human fatty acid synthase as predicted by normal mode analysis. Structure 12(2):185–191.
OpenUrl PubMed

[28] Brink J,

[29] et al.

[30] ↵
Ménétret JF,
et al.
(2005) Architecture of the ribosome-channel complex derived from native membranes. J Mol Biol 348:445–457.
OpenUrl CrossRef PubMed

[31] Ménétret JF,

[32] et al.

[33] ↵
Zhang B,
Zhang Y,
Shacter E,
Zheng Y
(2005) Mechanism of the guanine nucleotide exchange reaction of Ras GTPase—Evidence for a GTP/GDP displacement model. Biochemistry 44:2566–2576.
OpenUrl CrossRef PubMed

[34] Zhang B,

[35] Zhang Y,

[36] Shacter E,

[37] Zheng Y

[38] ↵
Sundquist B,
Oberg B
(1979) Phosphonoformate inhibits reverse transcriptase. J Gen Virol 45:273–281.
OpenUrl Abstract/FREE Full Text

[39] Sundquist B,

[40] Oberg B

[41] ↵
Sprang SR
(1997) G protein mechanisms: Insights from structural analysis. Annu Rev Biochem 66:639–678.
OpenUrl CrossRef PubMed

[42] Sprang SR

[43] ↵
Van Eps N,
et al.
(2011) Interaction of a G protein with an activated receptor opens the interdomain interface in the alpha subunit. Proc Natl Acad Sci USA 108:9420–9424.
OpenUrl Abstract/FREE Full Text

[44] Van Eps N,

[45] et al.

[46] ↵
Freissmuth M,
Gilman AG
(1989) Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity. J Biol Chem 264:21907–21914.
OpenUrl Abstract/FREE Full Text

[47] Freissmuth M,

[48] Gilman AG

[49] ↵
Gabriel SE,
Brigman KN,
Koller BH,
Boucher RC,
Stutts MJ
(1994) Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model. Science 266(5182):107–109.
OpenUrl Abstract/FREE Full Text

[50] Gabriel SE,

[51] Brigman KN,

[52] Koller BH,

[53] Boucher RC,

[54] Stutts MJ

[55] ↵
Frank J,
et al.
(1996) SPIDER and WEB: Processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116:190–199.
OpenUrl CrossRef PubMed

[56] Frank J,

[57] et al.

[58] ↵
Ludtke SJ,
Baldwin PR,
Chiu W
(1999) EMAN: Semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128(1):82–97.
OpenUrl CrossRef PubMed

[59] Ludtke SJ,

[60] Baldwin PR,

[61] Chiu W

[62] ↵
Mindell JA,
Grigorieff N
(2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334–347.
OpenUrl CrossRef PubMed

[63] Mindell JA,

[64] Grigorieff N

[65] ↵
Grigorieff N
(2007) FREALIGN: High-resolution refinement of single particle structures. J Struct Biol 157(1):117–125.
OpenUrl CrossRef PubMed

[66] Grigorieff N

[67] ↵
McEwen DP,
Gee KR,
Kang HC,
Neubig RR
(2001) Fluorescent BODIPY-GTP analogs: Real-time measurement of nucleotide binding to G proteins. Anal Biochem 291(1):109–117.
OpenUrl CrossRef PubMed

[68] McEwen DP,

[69] Gee KR,

[70] Kang HC,

[71] Neubig RR

[72] ↵
Jameson EE,
et al.
(2005) Real-time detection of basal and stimulated G protein GTPase activity using fluorescent GTP analogues. J Biol Chem 280:7712–7719.
OpenUrl Abstract/FREE Full Text

[73] Jameson EE,

[74] et al.

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Structural flexibility of the Gαs α-helical domain in the β₂-adrenoceptor Gs complex

Abstract

Results and Discussion

Experimental Procedures

Specimen Preparation and EM Imaging of Negative-Stained Samples.