Heterotrimeric G proteins serve as key membrane-associated signaling hubs, in concert with their cognate G protein-coupled receptors (GPCRs). Using site-directed labels located at four key allosteric sites within the Ras-homology domain of the stimulatory G protein α-subunit, Gsα, fluorine nuclear magnetic resonance spectroscopy (19F NMR) was employed to monitor the conformational equilibria of Gsα by itself, in the intact Gsαβγ heterotrimer, or in complex with either membrane alone or membrane-embedded adenosine A2A receptor (A2AR). The results reveal a concerted equilibrium which is strongly affected by nucleotide and interactions with the βγ-subunit, the lipid bilayer, and A2AR. 19F NMR spectra of the α1 helix of Gsα exhibit significant intermediate timescale dynamics, while those associated with the α4β6 loop and the α5 helix reflect respective membrane/receptor interactions and order/disorder transitions associated with G protein activation. The αN helix adopts a key functional state that serves as an allosteric conduit between the βγ-subunit and the receptor, while spectra in the presence of GTPγS reveal that a significant fraction of the ensemble remains tethered to the membrane and the receptor upon activation.
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Mapping the conformational landscape of the stimulatory heterotrimeric G protein
https://doi.org/10.21203/rs.3.rs-1409064/v1
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Heterotrimeric G proteins are membrane-anchored molecular switches that regulate intracellular signal cascades in response to activation by G protein-coupled receptors (GPCRs). The heterotrimer is composed of a nucleotide-binding α-subunit (Gα) and a dimer comprised of a β- and a γ-subunit (Gβγ). In its inactive state, Gα and Gβγ associate to form a heterotrimer (Gαβγ) where Gα is bound to guanosine diphosphate (GDP). Switching to the active state of Gα requires the exchange of GDP for GTP, which is mediated by guanine nucleotide exchange factors (GEFs) such as activated GPCRs. Here, GPCRs promote GDP release from Gα, facilitating rapid binding of guanosine triphosphate (GTP), which is present in high concentrations in the cell (Traut, 1994). GTP binding leads to functional dissociation of Gα•GTP from Gβγ and subsequent activation of downstream signaling pathways. Signaling is terminated upon hydrolysis of GTP to GDP by Gα, through its intrinsic GTPase activity or is accelerated by GTPase-activating proteins (GAPs), such as regulators of G protein signaling (RGSs). Thus, the functional ensemble of the Gα subunit is defined by conformations that enable complexation with the receptor in a GDP-bound form, the release of GDP, the binding of GTP and dissociation from Gbg, and finally the hydrolysis of GTP. Here, we use fluorine nuclear magnetic resonance spectroscopy (19F NMR) to delineate these functional conformers and better understand the activation pathway.
There are four classes of Gα subunits: Gs, Gi/o, Gq/11, and G12/13, delineated by their differing modulatory behavior on effectors. The four families share a common protein fold featuring the nucleotide binding pocket sandwiched between a Ras homology domain (RHD) and an α-helical domain (AHD). Nucleotide release requires separation of the two domains in addition to conformational changes of various switch regions that interact with the nucleotide’s guanine base and phosphate groups (Chung et al., 2011; Dror et al., 2015; Van Eps et al., 2011; Westfield et al., 2011). The stimulatory family of Gα (Gsα) activates adenylate cyclase, which produces the second messenger 3',5'-cyclic adenosine monophosphate (cAMP). High resolution structures of Gsα have been determined in various states along the GTPase cycle. These include GDP-bound Gsα in complex with Gβγ (Liu et al., 2019), nucleotide-free Gsαβγ heterotrimer complexed to a GPCR (Rasmussen et al., 2011), Gsα bound to GTPγS, a non-hydrolysable GTP analog (Sunahara et al., 1997), and Gsα•GTPγS in complex with adenylate cyclase (Qi et al., 2019). While these structures highlight the conformational changes between different Gα states and the interaction between Gα and its binding partners, the mechanisms of GPCR-G protein complex assembly, nucleotide exchange, and dissociation of the heterotrimer have yet to be fully elucidated.
To interrogate and dissect the Gsα activation steps, we focused on 19F-labeled Gsα, bound to GDP, GTPγS, or a transition state analog, GDP-[AlF4]− and we have used NMR spectroscopy to monitor the conformational equilibria and dynamics of the functional states. Each of these nucleotide conditions was applied to separate NMR studies of Gsα and Gsαβγ, in addition to Gsα and Gsαβγ in the presence of a phospholipid bilayer, Gsα and Gsαβγ in the presence of a ligand-free GPCR, the adenosine A2A receptor (A2AR) and finally, in the presence of the agonist-saturated A2AR. In this way, 19F NMR spectroscopy allows us to identify key features of the conformational ensemble of Gsα throughout the GTPase cycle. In this study, four different labeling sites were employed to address both local and global conformational responses. The results reveal a complex landscape that is generally characterized by two global conformational signatures, one of which is selectively stabilized by membrane, Gβγ, and receptor. Each labeling site also features distinct local dynamics in response to allosteric events. Upon GTP binding, Gsαβγ undergoes only partial physical dissociation where some locations within Gsα remain in a coupled-state while others appear dissociated from Gβγ. Finally, a distinct conformational signature is observed for the αN helix of Gsα in the combined presence of Gβγ and A2AR, highlighting αN’s involvement in GPCR coupling and in facilitating allosteric communication between receptor and Gβγ.
Labeling sites to probe the conformational dynamics of Gsα
Previous NMR studies of G proteins have focused on Giα, using mainly two-dimensional 1H-15N or 13C-methyl-based experiments (Abdulaev et al., 2006, 2005; Goricanec et al., 2016; Goricanec and Hagn, 2019; Knight et al., 2021; Ridge et al., 2006; Smrcka et al., 2010; Toyama et al., 2017). Truncation of the αN helix is common to enhance protein stability and yield. However, several studies identify strong roles of the Gα N-terminus in efficient G protein coupling and activation (Chung et al., 2011; Herrmann et al., 2006; Medkova et al., 2002). As our goals in the current investigation were to study Gsα in complex with the receptor and Gβγ, we regarded αN as critical to establishing a fully functional complex. Given that this compromises expression yield and protein stability, 19F NMR offers an advantage since a trifluoromethyl probe provides relatively good sensitivity and protein expression can be accomplished in rich media, avoiding complicated biosynthetic labeling steps. All 19F NMR studies were performed on Gsα using the thiol-specific probe, 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA). The fluorine chemical shift is extremely sensitive to the local electronic environment and allows the delineation of conformers in the ensemble, arising from subtle changes in protein conformation and dynamics (Danielson and Falke, 1996).
To achieve site-specific labeling, we first replaced all solvent-exposed cysteine residues in Gsα (C3S, C200T, C237S, C359I, C365A, C379V). Similar constructs have been made previously for Giα and Gqα, and have been shown to retain native-like activity and fold (Hu et al., 2010; Kaya et al., 2014; Medkova et al., 2002). This ΔCys construct was subsequently used to generate four separate single-cysteine mutants – K34C, R61C, Y358C, and E370C (Fig. 1A). These labeling sites provide a comprehensive view of both site-specific and global conformational space of the RHD of Gsα without interfering with conserved sequences or switch regions that are known to play a role in receptor coupling or nucleotide exchange. We note that residue C365, which had been replaced by an alanine in the ΔCys construct, belongs to the conserved guanine-interacting TCAT/V motif. Mutation at this motif reduces the affinity of Gα for GDP, which leads to increased rates of GDP release and exchange of nucleotide (Iiri et al., 1994; Johnston et al., 2008; Posner et al., 1998; Thomas et al., 1993). We performed GTPase activity assays to assess the functionality of each Gsα mutant (Fig. 1F). This assay measures cumulative GTP hydrolysis in a 90-minute period, which depends on both the rate of nucleotide exchange and GTPase activity. GTP turnover is similar across all the mutants in the absence of Gβγ. In the presence of the βγ subunit, those harboring the C365A mutation exhibit enhanced GTP turnover, likely due to their reduced affinities for GDP and higher nucleotide exchange rates, consistent with previous reports. All mutants retain the ability to couple to A2AR, as shown by increased GTP hydrolysis in the presence of agonist-bound A2AR.
The four labeling sites are each situated at an allosterically sensitive site within the Gsα RHD (Fig. 1A). K34C is located on αN near the αNβ1 hinge. αN is reversibly palmitoylated in eukaryotes and helps target Gsα to the plasma membrane (Kleuss and Krause, 2003; Linder et al., 1993; Tsutsumi et al., 2009). It also forms part of the interaction surface with both Gβ and the receptor and becomes disordered in the absence of Gβγ (Fig. 1D). Note that the Gsα used in this study was recombinantly produced in E. coli and was therefore not lipidated. E370C is situated at the base of α5 above the conserved β6α5 loop containing the nucleotide-binding TCAT/V motif. The site is part of an ordered helix when Gsα is loaded with nucleotide and becomes disordered upon complexation with the receptor and nucleotide release (Fig. 1A-C). Y358C is located on the α4β6 loop at the membrane interface. This loop interacts with intracellular loop 3 (ICL3) (Rasmussen et al., 2011) and helix-8 (Hu et al., 2010) of GPCRs, making Y358C a suitable probe for assessing Gsα binding to the membrane or the receptor. For instance, the corresponding site in Giα experiences reduced motion upon coupling to rhodopsin (Oldham et al., 2006). R61C is positioned on the α1-helix near the α1αA hinge connecting the RHD and the AHD. The site becomes disordered during receptor-catalyzed nucleotide release (Fig. 1C), making R61C a potentially sensitive probe for AHD motion and large allosteric network rearrangements within Gsα.
We employed rigidity-transmission allostery (RTA) analysis to assess the potential for these chosen labeling sites to respond to perturbation at the Gsα nucleotide binding pocket. RTA is a computational method based on the mathematical rigidity theory and is used to identify allosteric networks within proteins (Jacobs et al., 2001; Sljoka, 2021; Whiteley, 2005). The algorithm was carried out on a model derived from the crystal structure of GDP-bound Gsαβγ (Liu et al., 2019). This involved computationally rigidifying the bound GDP, then quantifying the changes in conformational degrees of freedom across the system. The results are plotted in Fig. 2 for each residue of Gsα, where a larger change in degrees of freedom signifies stronger allosteric transmission from the nucleotide binding site. All labeling sites except for K34 were found to be allosterically engaged to the bound GDP. The greatest allosteric transmission is observed at the phosphate-interacting P-loop (β1α1 loop) and the N-terminal portion of the α1 helix. This is consistent with recent mutagenesis studies identifying the P-loop to be most critical for stabilizing GDP binding (Ham et al., 2021), as well as bioinformatics analysis that highlighted the role of α1 as a structural hub (Flock et al., 2015).
Gsα undergoes global and local conformational changes as a function of membrane, Gβγ, nucleotide, and receptor
To monitor the conformational changes of Gsα at different stages of the GTPase cycle, we acquired 19F NMR spectra of BTFMA-labeled Gsα as a function of Gβγ, nucleotide, membrane without receptor, and A2AR reconstituted in discoidal high-density lipoprotein particles (rHDLs, commonly known as nanodiscs). To create a membranous environment, empty nanodiscs were employed. The corresponding 19F NMR spectra, shown in Fig. 3, generally reveal two sets of NMR resonances, denoted A and B. Interestingly, the response of the A/B equilibria as a function of condition are similar among all 4 labeling sites. In the absence of membrane and Gβγ, Gsα predominantly populates state A. Shifts in equilibria toward B are observed for GDP-bound or nucleotide-free Gsα in the presence of either membrane, Gβγ, apo A2AR, or A2AR bound to the full agonist NECA. The addition of GTPγS saw a return of population A in some but not all labeling sites, suggesting a partial uncoupling of Gsα from the membrane or Gβγ. Taken together, the 19F NMR spectra point to the presence of two broad energy basins in the conformational landscape of Gsα (A and B), whose equilibrium is jointly modulated by the phospholipid bilayer, the Gβγ subunit, the receptor, and nucleotides. While nucleotide binding stabilizes A, the presence of phospholipid, Gβγ, and receptor all bias the ensemble toward B.
In addition to coordinated global changes, the 19F NMR spectra from the four labeling sites reveal differences in local dynamics and their response to environmental perturbation. For instance, a series of broad NMR signatures (~ 600 Hz line width) in the R61C spectra point to conformational exchange events on an intermediate (µs-ms) timescale. On the other hand, the narrower resonances (40–50 Hz) observed in the spectra of K34C suggest much faster motional averaging. Other site-specific observations are addressed below.
K34C (αN helix)
The K34C spectral series is characterized by two narrow upfield resonances at -61.72 ppm and − 61.89 ppm, and a broader downfield resonance at -60.87 ppm that only appears in the combined presence of Gβγ and A2AR (Fig. 3). Exchange between the two upfield peaks coincide with transitions between the two global conformational states discussed above. The K34C labeling site is located on the N-terminal helix of Gsα, approximately one turn away from the αNβ1 hinge (Fig. 4A). This helix is mostly disordered in the absence of Gβγ, as shown by electron paramagnetic resonance spectroscopy and through missing electron densities in the crystal structures of Gsα•GTPγS and Giα•GDP (Coleman and Sprang, 1998; Medkova et al., 2002; Sunahara et al., 1997). Therefore, the two upfield resonances (A and B) are likely reporting on conformational disorder/order at the labeling site, where helix formation (B) is induced upon contact with the membrane. Phospholipid binding may be facilitated through sidechains of multiple lysine residues on αN, which can form electrostatic interactions with negatively-charged lipid headgroups.
A broad downfield signature is observed for GDP-bound and nucleotide-free K34C in the joint presence of Gβγ and A2AR, which is abolished upon the addition of GTPγS (Fig. 4B). This resonance likely represents a distinct conformation or local environment at the N-terminal helix that arises from complexation with both Gβγ and the receptor. In the structures of β2AR-bound Gsαβγ and A2AR-bound mini-Gs heterotrimer, the intracellular loop 2 (ICL2) of both receptors form a 2-turn helix that pack against the αNβ1 hinge (García-Nafría et al., 2018; Rasmussen et al., 2011). Residue K34 is located near this hinge and is “sandwiched” between the receptor and the Gβ subunit during receptor coupling (Fig. 1B & 1C). Interestingly, this “sandwiched” state constitutes a minor population of the ensemble, suggesting that αN of Gsαβγ remains somewhat dynamic while coupled to the receptor. The sandwiched state is abolished by GTPγS, whose addition resulted in the stabilization of two new resonances at -61.79 ppm and approximately − 62.09 ppm (Fig. 4B). The peak at -61.79 ppm could be the result of a new conformer, or a shift in the original A state through exchange averaging with the broader upfield resonance at -62.09 ppm. The resolution of these new peaks, combined with the fact that binding by GTPγS did not return the equilibrium toward the original A (Gsα only) state, suggest that Gsα is not fully dissociated from the receptor in response to GTPγS but adopts a different set of conformations compared to Gsα•GTPγS alone (Figs. 3 & 4B).
Another possible explanation for the distinct downfield state at K34 may be related to the AHD-αN interaction that is observed in the Gs-β2AR complex structure. In the crystal structure of the nucleotide-free state, the AHD interacts with both Gβ as well as with αN, thus stabilizing the open conformation of the Gsα. On the other hand, structures of Gα bound to GTPgS or GDP•[AlF4]− show an AHD-RHD closed conformation suggesting that the chemical shift at K34 may result from the proximity of AHD to αN in the apo and GDP-bound states of Gα. This effect is only observed in phospholipid, underlying the strong influence of the bilayer on the structure of αN.
R61C (α1 helix)
The spectrum of Gsα•GDP at R61C reveals a broad A state resonance that sharpens significantly upon the replacement of GDP for GTPγS (Fig. 3). The broader linewidth indicates intermediate (µs-ms) timescale conformational exchange at the labeling site, which is located on α1 near the α1αA hinge that connects the RHD and AHD (Fig. 5A). This may be a result of spontaneous domain separation, which has been shown by molecular dynamics simulations to occur on a µs-timescale in the GDP-bound G protein (Dror et al., 2015). Structural and bioinformatic analysis across the four Gα families found α1 to be an allosteric hot spot and a structural hub linking α5, GDP, and regions of the AHD (Flock et al., 2015). These contacts are lost upon receptor binding, which causes partial disordering of the α1-helix in the absence of nucleotide (Figs. 1 and 5A). The transition from state A to state B at R61C may be indicative of this loss in α1 integrity or other structural rearrangements within Gsα. The B state resonance is broad, suggesting that α1 remains dynamic and undergoes intermediate timescale motion when coupled to Gβγ or the receptor, possibly reflecting the dynamic motion of AHD.
E370C (β6α5 loop/α5 helix)
E370C is located at the end of the β6α5 loop, which connects to α5. This junction undergoes an order-to-disorder transition during receptor coupling as the C-terminal end of α5 inserts into the receptor’s intracellular cavity, leading to nucleotide release (Figs. 1C and 5B) (Chung et al., 2011). The labeling site is characterized by at least three underlying resonances in the 19F NMR spectra: the A state at -61.65 ppm, the B state at -61.99 ppm, and a downfield shoulder at -61.50 ppm whose population is slightly enhanced in the presence of the membrane. The A and B states may signify the labeling site being part of an ordered helix (A) or a disordered loop (B) (Fig. 5B). While the ordered helix (state A) is predominantly favored for Gsα•GDP and Gsα•GTPγS, state B is best stabilized for nucleotide-free Gsα that is coupled to the receptor. Interestingly, the presence of membrane or Gβγ also promoted the B state in addition to the downfield resonance at -61.50 ppm. This third resonance may be a consequence of the interaction between the α5 helix and the phospholipid bilayer and represents an intermediate state that is sampled prior to stable complexation with the receptor (Du et al., 2019; Kim et al., 2021; Liu et al., 2019).
Y358C (a4b6 loop)
In comparison to other labeling sites, Y358C shows a clearer delineation of nucleotide binding states. The chemical shift of state A is -61.19 ppm for Gsα•GDP and − 61.33 ppm for Gsα•GTPγS (Figs. 3 & 6A), pointing to conformational or environmental differences at the α4β6 loop where Y358C is located. The α4β6 loop is away from any nucleotide-binding elements or switch regions, with the Y358 sidechain overlaying almost completely between the Gsαβγ•GDP and Gsα•GTPγS crystal structures (Fig. 6B). However, this residue is also part of the crystal packing interface (in the Gsα•GTPgS structure), which experiences hampered conformational dynamics. The observed chemical shift sensitivity toward nucleotide in our 19F NMR spectra and allosteric propensity analysis (Fig. 2) suggests that the structure of the α4β6 loop may be quite different in solution from that of the crystals. Interestingly, the chemical shift of state B is not sensitive to the identity of the nucleotide. We wondered whether state B is simply a consequence of the probe being brought in close proximity to the phospholipid bilayer via heterotrimer assembly or receptor complexation. However, the addition of Gβγ without empty nanodiscs also shifted the equilibrium toward B, suggesting that this pose is allosterically induced and can be stabilized independently by either Gβγ or phospholipid (Figs. 3 & 6A).
Gsαβγ undergoes partial dissociation with GDP-[AlF4]- and GTPγS
Classically, activation of the G protein by a GPCR involves GTP binding followed by subunit dissociation of the heterotrimer (Hamm, 1998). However, multiple lines of evidence suggest that the α- and the βγ-subunits do not completely dissociate upon activation (Adjobo-Hermans et al., 2011; Bünemann et al., 2003; Digby et al., 2008; Galés et al., 2006; Hillenbrand et al., 2015; Klein et al., 2000; Ridge et al., 2006; Tang et al., 2006; Wang et al., 2008). Instead, the heterotrimer undergoes structural rearrangements that would allow the α- and the βγ-subunits to remain loosely associated while interacting with their respective effector proteins (Bünemann et al., 2003; Galés et al., 2006; Kano et al., 2019; Rebois et al., 2006; Tang et al., 2006). GTPγS and GDP-aluminum fluoride (GDP-[AlF4]−) are widely used GTP analogs in the study of GTPases. GDP-[AlF4]− is also a transition state analog for the GTP hydrolysis reaction (Wittinghofer, 1997). In the structures of GDP-[AlF4]−-bound Giα and Gtα, the [AlF4]− moiety occupies the space that is normally taken up by the γ-phosphate of GTP and mimics the transition state of phosphoryl transfer (Coleman et al., 1994; Sondek et al., 1994; Sprang, 2016).
We examined the effects of both GTPγS and GDP-[AlF4]− on Gsα as a function of membrane and Gβγ. The 19F NMR spectra of Gsα•GDP, Gsα•GDP-[AlF4]−, and Gsα•GTPγS show predominantly the A state for all four labeling sites in the absence of the membrane (Fig. 7). There is a general bias toward sampling of the B state for Gsα•GDP when membrane is added. GDP-[AlF4]− and GTPγS were able to revert this effect for K34C and E370C but not for Y358C, suggesting a partial uncoupling of Gsα from the membrane by GTP analogs. In the case of R61C, a return of the sharp A state resonance is stabilized by GTPγS but not by GDP-[AlF4]−, indicating that GDP-[AlF4]− gives rise to an intermediate of Gsα between the GDP- and GTP-bound forms. The combined presence of Gβγ and membrane further reinforced state B for all four labeling sites. While the exchange of GDP for GTPγS partially reverted this equilibrium toward state A for K34C and E370C, no change is observed for Y358C and R61C. Consistent with a transition-state analog, GDP-[AlF4]− saw an intermediate A state population for K34C but otherwise similar spectra as Gsαβγ•GDP for E370C, Y358C, and R61C.
The above observations suggest that in the heterotrimeric form, the α- and βγ-subunits remain associated and membrane-attached throughout the GTPase cycle. The exchange of GDP for GTP triggers structural rearrangement within Gsα, leading to a partial dissociation of the heterotrimer where some portions of the α-subunit (such as the α4β6 loop and the α1 helix) remain in a coupled conformation while other locations (such as αN and α5) become decoupled. Finally, although near-identical structures were obtained for Giα•GDP-[AlF4]− and Giα•GTPγS in solution (Coleman et al., 1994), our NMR data indicate that in the presence of the membrane, GDP-[AlF4]− gives rise to a conformational ensemble of Gsα where the relative population of each state falls between that of the GDP- and GTP-bound conditions.
In this study, 19F NMR spectroscopy was used to map the conformational landscape of Gsα throughout the GTPase cycle. Four distinct labeling sites were examined as a function of Gβγ, A2AR, and nucleotides which included GDP, GTPγS, and GDP-[AlF4]−, in the presence and absence of a membrane-like environment. Two global states, A and B, were identified where transitions between them are coordinated across all four labeling sites. While the binding of a nucleotide generally stabilizes state A, the presence of phospholipid, Gβγ, and receptor all independently bias the Gsα equilibrium toward state B. It is conceivable that these two states reflect the crystallographic end points of Gsα, with A representing an RHD-AHD “closed” conformation as depicted in the structure of Gsα•GTPγS, and B corresponding to an “open” conformation, as represented by the nucleotide-free structures of Gsαβγ bound to GPCRs where the RHD and the AHD are maximally separated. This does not necessarily indicate a nucleotide-free Gsα, since domain separation alone is not sufficient for GDP release (Dror et al., 2015). Regardless of the global structural interpretations of A and B, each labeling site is ultimately a probe of the local environment and is influenced differently by the nucleotides, Gβγ, and the lipid bilayer in comparison to other locations within the protein. Therefore, site-specific interpretation of the NMR spectra may provide more reliable, albeit regional, insights into the protein’s conformation and dynamics under each of the given conditions. For example, the exchange line broadening observed for R61C is indicative of µs-ms timescale dynamics, potentially due to spontaneous domain separations or rearrangements of internal contact networks. The A-B transitions for K34C, R61C, and E370C may reflect changes in the conformational order/disorder at αN, α1, and α5, respectively. Y358C gave rise to a large chemical shift difference between Gsα•GDP and Gsα•GTPγS, revealing a clear allosteric connection from the α4β6 loop to the nucleotide binding pocket. Finally, multiple resonances are observed for K34C and E370C in addition to states A and B, which points to a more complex mechanism underlying the regulation of Gsα.
The phospholipid bilayer appears to play a significant role in shaping the conformational landscape of Gsα. This is not surprising for K34C, given that the Gsα N-terminus is generally palmitoylated and anchored to the membrane (Kleuss and Krause, 2003; Linder et al., 1993; Tsutsumi et al., 2009). Our NMR data show that αN interacts with the phospholipid bilayer in the absence of a lipid anchor. The recent structure of the dopamine D2-Gi complex resolved in a phospholipid bilayer revealed significant electrostatic interactions between the phospholipid headgroups and the αN helix of Giα, in addition to their interactions with the putative acylated N-terminal lipid anchor (Yin et al., 2020). The organization of αN at the phospholipid interface may give rise to subsequent conformational changes at distal sites of the protein, thereby lowering the barrier for Gsα to adopt an overall conformation (e.g. state B) that is more complementary for receptor coupling. Similarly, the βγ-subunit, in addition to stabilizing the Gsα-membrane interaction through an additional lipid anchor on Gγ and enabling interaction with the receptor (Herrmann et al., 2006), reinforces the allosteric changes brought about by the membrane and further shift the Gsα conformational ensemble to a receptor coupling state. As seen in Fig. 3, though A2AR is more effective at stabilizing state B in comparison to the membrane alone, the combined effects of membrane and Gβγ are nearly equivalent to that of the receptor plus Gβγ. The exception is K34C, which revealed a distinct conformational signature in the joint presence of A2AR and Gβγ. This “sandwiched” state is reinforced by the A2AR full agonist NECA and may be indicative of the strength of receptor-G protein coupling within the ternary complex. It has recently been shown that the βγ-subunit, though not directly in contact with A2AR, plays a role in transmitting ligand efficacy (Huang et al., 2021). This may be relayed through αN, which is held at the membrane interface between the receptor and Gβ. These NMR data are further evidence for N-terminal helix involvement in receptor coupling and its role of bridging the allosteric communication between the receptor and Gβγ. We also cannot rule out the potential contributions of the interaction between αN and the AHD in the open conformation that is stabilized in the nucleotide-free state of Gα, as revealed in the Gs•β2AR complex structure.
We have previously shown that the activation of A2AR proceeds through a conformational selection mechanism (Huang et al., 2021; Ye et al., 2016). Furthermore, coupling between an apo, partial agonist-bound, or full agonist-bound A2AR with heterotrimeric G protein each stabilizes a distinct activation state of the receptor. We hypothesized that through reciprocal conformational selection, the G protein might also adopt distinct conformations specific to the state of the receptor and whose populations and lifetimes are determined by intrinsic ligand efficacy. This is not observed in our data with these probes, which show near identical spectra for the G protein coupled to an apo or a full agonist-bound A2AR (Fig. 3). Our labeling sites may not have provided the sensitivity required to resolve such conformers, being relatively distant from the receptor’s intracellular cavity. Better differentiation of the receptor states may be afforded by probes located closer to the receptor along α5, such as F376, R389, and E392 (residue 8, 21, and 24 on α5) (Kaya et al., 2014; Liu et al., 2019), provided that mutagenesis and labeling do not significantly change the nature of the interaction with receptor or the signal transmission pathway.
The current study provided strong evidence for a continued physical engagement of the Gα- and Gβγ-subunits upon binding by GTPγS or [AlF4]−. The results corroborate the hypothesis that activated heterotrimeric G proteins undergo internal structural rearrangements, rather than complete physical dissociation, during GTP turnover. This “functional dissociation” would expose potential effector-binding surfaces on each of the subunits to allow for downstream signaling, regulation, and rapid re-formation of the inactive heterotrimer upon GTP hydrolysis. The lipid bilayer is expected to play a key role in these signaling hubs both as an orienting scaffold and a modulator of each protein’s conformational landscapes.
Acknowledgements: This work was supported by the Canadian Institutes of Health Research (CIHR) Operating Grant MOP-43998 to R.S.P., CIHR Operating Grant PJT-159464 to O.P.E. and N.V.E., the National Institute of General Medical Sciences grants GM106990 and GM083118 to R.K.S. S.K.H. was supported by Alexander Graham Bell Canada Graduate Scholarship-Doctoral from NSERC. A.S. was supported by CREST, Japan Science and Technology Agency (JST), Japan, JPMJCR1402. We thank Dmitry Pichugin for NMR maintenance and Lu Chen for technical support.
Author contributions: S.K.H and R.S.P. designed the research. S.K.H. and R.S.M.R. carried out expression and purification of various Gsα constructs. S.K.H. carried out expression and purification of A2AR. S.K.H and A.P. carried out expression and purification of Gβγ. N.V.E., R.S.M.R., and S.K.H. carried out cloning for the various Gsα constructs. S.K.H. and R.S.M.R. performed the NMR experiments and the GTP hydrolysis experiments. A.S. performed the RTA analysis. R.K.S. provided assistance with data interpretation and the manuscript. S.K.H and R.S.P prepared the manuscript. R.S.P. and O.P.E. supervised the project.
Declaration of interests: The authors declare no competing interests.
Protein cloning, expression, and purification
Cloning of the wild type Gsα construct has been described previously (Huang et al., 2022). The human GNAS2 gene was subcloned into a pET15b vector immediately downstream of an open reading frame for hexahistidine-tagged maltose binding protein (MBP) and tobacco etch virus (TEV) cleavage site. The ΔCys construct (C3S, C200T, C237S, C359I, C365A, C379V) and additional single-cysteine mutants (ΔCys-K34C, ΔCys-R61C, ΔCys-Y358C, ΔCys-E370C) were generated via site-directed mutagenesis using the pET15b-Gsα wild type or ΔCys plasmid as a template. All mutations were confirmed by DNA sequencing at the Centre for Applied Genomics (TCAG), the Hospital for Sick Children (Toronto, Canada).
The expression and purification of Gsα (identical for the wild type and all mutant constructs) has been described previously (Huang et al., 2021). Briefly, Escherichia coli (E. coli) BL21 (DE3) cells containing the vector carrying the desired Gsα construct were grown in LB Miller medium at 25 °C to an optical density at 600 nm (OD600) of 0.3. Cells were induced overnight at 19 °C with isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 50 µM, then harvested by centrifugation at 6,000 g. Cell pellets were resuspended lysis buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole, 50 µM GDP, 2 mM MgCl2, 0.5 mM TCEP, 5 mM 6-aminocaproic acid, 5 mM benzamidine, 0.4 mg/mL lysozyme, 2 µg/mL DNase I, 10% glycerol) and lysed via sonication. The lysate was centrifuged at 20,000 g for 30 minutes, and the resulting supernatant was incubated with TALON resins (Takara Bio) for 3 h at 4 °C with gentle mixing. For 19F labeling, the TALON resins were washed with 3 bed-volumes of labeling buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.5 mM TCEP, 10% glycerol), then resuspended in 2 bed-volumes of labeling buffer containing 200 µM 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide (BTFMA, Apollo Scientific). The reaction was allowed to proceed overnight at 4 °C with gentle mixing. A second aliquot of BTFMA was added and incubated for 6 h prior to loading the resin onto a gravity column. The resins were washed with lysis buffer containing 20 mM imidazole, then eluted with elution buffer (50 mM sodium phosphate (pH 8.0), 100 mM NaCl, 50 µM GDP, 2 mM MgCl2, 0.5 mM TCEP, 10% glycerol, 250 mM imidazole). The eluted MBP-Gsα was buffer exchanged using a centrifugal filter to remove imidazole, then incubated with 10 µg/mL of TEV protease (produced in-house) at 4 °C overnight without agitation. The sample was loaded onto a Ni-NTA column and the flow-through was collected and purified on a HiLoad 16/600 Superdex 200 prep grade size exclusion column equilibrated with 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 5 µM GDP, and 10% glycerol. The eluted fractions containing Gsα were pooled, concentrated, and buffer-exchanged to final storage buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 100 µM GDP, 10% glycerol). The proteins are flash-frozen in liquid nitrogen and stored at -80 °C until needed.
Gβγ, and A2AR were expressed and purified as previously described (Huang et al., 2021). Empty nanodiscs were prepared as previously described (Huang et al., 2022). Both the A2AR and the empty nanodiscs were reconstituted using the MSPΔH5 membrane scaffold protein (Hagn et al., 2013) and a 3:2 ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The protein or nanodisc samples were flash-frozen in liquid nitrogen and stored at -80 °C in the following storage buffers until use. For Gβγ: 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 0.0125% n-Dodecyl β-D-maltoside (DDM), 100 µM TCEP, 100 µM GDP, 10% glycerol. For A2AR and empty nanodiscs: 50 mM HEPES, pH 7.4, 100 mM NaCl.
NMR experiments
The purified protein stocks, stored in their respective storage buffers as described above, were combined such that the final NMR sample contained 30-80 µM BTFMA-labeled Gsα. The samples were supplemented with a final concentration of 10% D2O and 20 µM sodium trifluoroacetate (TFA) as the 19F chemical shift reference. 1.2-fold excess of Gβγ, 3-fold excess of A2AR, or 3.5-fold excess of empty nanodiscs, relative to the Gsα concentration, were added where applicable. Where needed, the sample also contained a final concentration of 100 µM GDP (GDP or GDP-AlF4- conditions), 50 µM AlCl3 and 5 mM NaF (GDP-[AlF4]- conditions), 2 U/mL apyrase (nucleotide-free conditions), 2 U/mL apyrase and 1 mM GTPγS (GTPγS conditions), or 1 mM NECA (A2AR + NECA conditions). We note that by itself, nucleotide-free Gα is unstable and tend to aggregate within an hour at concentrations suitable for an NMR experiment. In the presence of membrane, Gβγ, or receptor, samples are stable for at least several hours to several days depending on the mutant and the temperature of storage. We also note that due to large quantities of proteins that would be required, NMR of the Gsαβγ+A2AR+NECA+GTPgS condition was only carried out for the K34C construct. All samples were prepared in Shigemi tubes with a final volume of 200-350 µL. NMR experiments were acquired at 20 °C on either a 600 MHz Varian Inova spectrometer equipped with a 19F-tunable triple-resonance cryoprobe or a 500 MHz Varian Inova spectrometer equipped with a 5 mm room temperature inverse HFX probe. A typical 1D fluorine experiment included a 100-300 ms recycle delay, a 5.5-7 μs (45°) excitation pulse, and a 500-600 ms acquisition time. Each experiment was acquired using 24,000-200,000 scans, yielding a S/N of approximately 30-100. Spectra were processed using MestReNova (Mestrelab Research S.L.) employing chemical shift referencing (-75.6 ppm for TFA), zero filling, baseline correction, and exponential apodization equivalent to a 20-30 Hz line broadening.
GTP hydrolysis assays
GTP hydrolysis assays were performed using the GTPase-GloTM assay kit (Promega, Madison, WI, USA) following the manufacturer’s protocol (Mondal et al., 2015). Reactions were carried out at room temperature in a buffer containing 50 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM MgCl2, 10% glycerol, 1 μM GDP, and 4 μM GTP. Each reaction contained either Gsα alone (250 nM), Gsαβγ (250 nM Gsα + 300 nM Gβγ), or Gsαβγ with A2AR (250 nM Gsα + 300 nM Gβγ + 250 nM A2AR + 100 µM NECA). All reaction mixtures were pre-incubated for 1 hour prior to the addition of GTP, which initiated the reaction. A control was also carried out with only buffer and no G protein. After 90 minutes, unhydrolyzed GTP was converted to ATP and detected using a reagent containing luciferase. The resulting luminescence is proportional to the amount of unreacted GTP and measured using a TECAN Spark multi-mode plate reader with an integration time of 1 minute. The amount of GTP hydrolysis for each condition (x) can be represented by its difference in luminescence with the control:
Computational rigidity-transmission allostery predictions
Starting with the structure of GDP-bound Gsαβγ heterotrimer (PDB: 6EG8), we measured the allosteric propensity from the GDP binding pocket across the complex by applying rigidity-transmission allostery (RTA) analysis (Sljoka, 2021). The RTA algorithm, whose details have been previously described (Sljoka, 2021; Ye et al., 2018), is a computational tool based on the mathematical rigidity theory which identifies allosteric networks within structures of proteins and protein complexes (Jacobs et al., 2001; Sljoka, 2021; Whiteley, 2005, Ye 2018, Mehrabi 2019). The algorithm was verified with NMR chemical shift data and predicts how changes in the conformational flexibility of one region in the protein are transmitted to distal sites by quantifying the resulting regiospecific change in conformational degrees of freedom. We applied the RTA algorithm on the GDP-bound Gsαβγ heterotrimer by rigidifying the bound GDP. The available conformational degrees of freedom were quantified for each residue before and after GDP rigidification. The resulting change in degrees of freedom was then extracted for each residue and represents the extent of allosteric transmission from the GDP binding pocket.
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