Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling

Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq. The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions. Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq. The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions. In recent years, there has been significant progress in structural and spectroscopic studies of Class A G protein-coupled receptors (GPCR), 3The abbreviations used are:GPCRG protein-coupled receptorV1ARV1A receptorβ2-ARβ2 adrenergic receptorMDmolecular dynamicsPDBProtein Data BankANOVAanalysis of varianceAVP[Arg8]vasopressinr.m.s.d.root mean square deviationRMSFroot mean square fluctuationD1Rdopamine receptor D1CB1cannabinoid receptor type 1TMtransmembrane2-AG2-arachidonylglycerolICLintracellular loop. which are energizing structure-based drug discovery efforts (1Bhattacharya S. Hall S.E. Li H. Vaidehi N. Ligand-stabilized conformational states of human β(2) adrenergic receptor: insight into G-protein-coupled receptor activation.Biophys. J. 2008; 94: 2027-2042Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 2Granier S. Kobilka B. A new era of GPCR structural and chemical biology.Nat. Chem. 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The Gα C terminus alone constitutes 76% of the atoms making contact in the GPCR/G protein interface in the β2-AR·Gs crystal structure (10Rasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. Mathiesen J.M. Shah S.T. Lyons J.A. Caffrey M. Gellman S.H. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2267) Google Scholar). Previous studies have shown that the Gα C terminus is essential for G protein activation by the GPCR (15Hamm H.E. Deretic D. Arendt A. Hargrave P.A. Koenig B. Hofmann K.P. Site of G protein binding to rhodopsin mapped with synthetic peptides from the α subunit.Science. 1988; 241: 832-835Crossref PubMed Scopus (394) Google Scholar, 18Lambright D.G. Noel J.P. Hamm H.E. Sigler P.B. Structural determinants for activation of the α-subunit of a heterotrimeric G protein.Nature. 1994; 369: 621-628Crossref PubMed Scopus (530) Google Scholar, 22Rasenick M.M. Watanabe M. Lazarevic M.B. Hatta S. Hamm H.E. Synthetic peptides as probes for G protein function. Carboxyl-terminal G α s peptides mimic Gs and evoke high affinity agonist binding to β-adrenergic receptors.J. Biol. Chem. 1994; 269: 21519-21525Abstract Full Text PDF PubMed Google Scholar, 28Yang C.S. Skiba N.P. Mazzoni M.R. Hamm H.E. Conformational changes at the carboxyl terminus of Gα occur during G protein activation.J. Biol. Chem. 1999; 274: 2379-2385Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 29Oldham W.M. Hamm H.E. Heterotrimeric G protein activation by G-protein-coupled receptors.Nat. Rev. Mol. Cell Biol. 2008; 9: 60-71Crossref PubMed Scopus (806) Google Scholar), and the last three residues of the Gα C terminus are important for selective G protein activation (13Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα.Nature. 1993; 363: 274-276Crossref PubMed Scopus (606) Google Scholar). We recently developed a FRET-based sensor to probe the interaction between the GPCR and the Gα C terminus in live cells (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The sensor is based on a technique termed SPASM (systematic protein affinity strength modulation) that involves tethering two proteins/protein domains by an ER/K linker flanked by a FRET pair (mCerulean, FRET donor, and mCitrine, FRET acceptor) (31Sivaramakrishnan S. Spudich J.A. Systematic control of protein interaction using a modular ER/K α-helix linker.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 20467-20472Crossref PubMed Scopus (56) Google Scholar). Sensor FRET correlates linearly with the fraction of the sensors in the bound state (31Sivaramakrishnan S. Spudich J.A. Systematic control of protein interaction using a modular ER/K α-helix linker.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 20467-20472Crossref PubMed Scopus (56) Google Scholar). The ER/K linker also controls the effective concentration of the protein interaction. We combine the SPASM sensor-based measurements with molecular dynamics (MD) simulations in an iterative fashion to delineate the structural basis of selective interactions between the GPCR and the Gα C terminus. Using a combination of FRET sensors and MD simulations, we first demonstrate that the GPCR/Gα C-terminal interaction sufficiently captures the selectivity for the dominant signaling pathway for six different Class A GPCRs in live cells. The interaction energy calculated from MD simulation for the agonist-GPCR pair with the cognate Gα C-terminal peptide is favorable compared with the non-cognate peptide. Additionally, the intracellular region of the agonist-GPCR conformation ensemble shows reduced flexibility when bound to the cognate Gα C-terminal peptide. A combination of MD and sequence analysis reveals three “hot spot” residues in the Gα C terminus that contribute significantly to the interaction energy necessary for cognate Gα selection. Importantly, the distinct residues in Gαs and Gαq are conserved across species, suggesting these residues constitute a conserved structural mechanism for GPCRs to differentiate between G protein subtypes. Point mutations of hot spot residues within the Gα C terminus result in increased interaction of non-cognate GPCR/Gα C-terminal peptides and affect downstream signaling profiles, validating our structural observations in the cellular milieu. We previously reported a FRET sensor that probes the interaction between the GPCR and the C terminus of the Gα subunit and characterized it in live HEK-293T cells (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The sensor contains, as a single polypeptide, the following: a full-length GPCR, mCitrine, a 10-nm ER/K linker, mCerulean, and the C-terminal peptides of Gαs, Gαi, or Gαq, all of which are separated by (GSG)4 linkers (Fig. 1a). Our previous report showed an agonist-dependent selective interaction for the Gαs C terminus with β2-AR (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Here, we investigate whether other Gs-, Gi-, and Gq-coupled GPCRs show similar agonist-dependent selectivity for their cognate Gα C-terminal “peptides.” Fig. 1b shows that changes in FRET intensity exemplify the selectivity of canonical Gs-coupled receptors, β3-adrenergic receptor (β3-AR), and dopamine receptor D1 (D1R) to the s peptide. β3-AR was chosen as an additional s-coupled receptor as we have previously tested β2-AR (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Receptors tethered to the s, i, q, or no-peptide control are activated using full agonists (100 μm isoproterenol and 100 μm dopamine, respectively). For β3-AR, the s peptide shows the most significant change in FRET (ΔFRET = 0.0267) compared with no-peptide control (ΔFRET = 0.0020, p ≤ 0.0001). The s peptide also has a significant FRET change compared with the i peptide (ΔFRET = 0.0120, p ≤ 0.001). Analysis of variance (ANOVA) values are provided in supplemental Table 1 for comparison of all four receptor-peptide pairings. Tukey's multiple comparison test values are in supplemental Table 2 analyzing pairwise significance for all receptor-peptide pairings. This selection for the s peptide is also observed for D1R, where the s peptide change in FRET is most significant at ΔFRET = 0.0192 (p ≤ 0.0001). Unmarked bars are n.s. compared with no peptide. For these Gs-coupled receptors, the sensor detects a significant s peptide selection bias in the presence of full agonists. We further tested whether the Gα C terminus is sufficient for Gi- and Gq-coupled receptors to select for their cognate pathways using this tethered FRET system. Sensors were designed for Gi-coupled receptors, α2-adrenergic receptor (α2-AR), and cannabinoid receptor type 1 (CB1), and the same peptide constructs were tested with receptor-specific full agonists (100 μm epinephrine and 100 μm 2-AG) (Fig. 1c). Both α2-AR and CB1 show a significant change in FRET for i peptide compared with no-peptide control, s, or q peptide (p ≤ 0.0001). CB1 also has significant changes in FRET for s peptides (p ≤ 0.05) and q peptides (p ≤ 0.001) compared with no peptide. These changes may be a result of receptor promiscuity as well as a result of interactions with the receptor and endogenous G protein in the cells. The trend also holds for Gq-coupled receptors, α1-adrenergic receptor (α1-AR), and vasopressin 1A receptor (V1AR) (Fig. 1d). α1-AR significantly selects q peptide compared with no peptide (p ≤ 0.0001) upon stimulation with 100 μm phenylephrine as does V1AR (p ≤ 0.0001) with 100 μm AVP. V1AR also selects q peptide significantly compared with s or i peptides (p ≤ 0.0001). Although the Gα C terminus has repeatedly been shown as an important component for G protein selection (13Conklin B.R. Farfel Z. Lustig K.D. Julius D. Bourne H.R. Substitution of three amino acids switches receptor specificity of Gqα to that of Giα.Nature. 1993; 363: 274-276Crossref PubMed Scopus (606) Google Scholar, 16Hamm H.E. Rarick H.M. Specific peptide probes for G-protein interactions with receptors.Methods Enzymol. 1994; 237: 423-436Crossref PubMed Scopus (16) Google Scholar), our measurements here show it is minimally sufficient for selection across many GPCRs (Fig. 1e). Computational techniques were used to better understand the molecular basis of GPCR selectivity for the Gα C-terminal peptide, observed in the FRET sensor measurements. Gs- and Gq-coupled receptors were chosen for modeling because their signaling profiles involve different secondary messengers. Although this approach can also be applied to Gi-coupled receptors, the dual influence of Gs and Gi on adenylyl cyclase, and consequently cAMP levels, complicates interpretation of second messenger profiles. Nonetheless, future studies will address the selection between Gs, Gi, and Gq pathways. We modeled an ensemble of conformations for the isoproterenol·β2-AR·s peptide complex and the AVP·V1AR·q peptide complex as detailed under “Experimental Procedures.” β2-AR was chosen due to the availability of the Gαs-bound fully active state crystal structure (10Rasmussen S.G. DeVree B.T. Zou Y. Kruse A.C. Chung K.Y. Kobilka T.S. Thian F.S. Chae P.S. Pardon E. Calinski D. Mathiesen J.M. Shah S.T. Lyons J.A. Caffrey M. Gellman S.H. et al.Crystal structure of the β2 adrenergic receptor-Gs protein complex.Nature. 2011; 477: 549-555Crossref PubMed Scopus (2267) Google Scholar). This structure provides an accurate starting model for computational studies. Additionally, previous FRET sensor measurements also show the selective interaction of β2-AR and Gαs peptide (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A Gq-coupled receptor was used to discern the differences in s and q peptide binding. For this receptor, we generated a homology model for the Gq-coupled receptor, V1AR, derived from the nanobody-bound active state structure of the μ-opioid receptor (32Huang W. Manglik A. Venkatakrishnan A.J. Laeremans T. Feinberg E.N. Sanborn A.L. Kato H.E. Livingston K.E. Thorsen T.S. Kling R.C. Granier S. Gmeiner P. Husbands S.M. Traynor J.R. Weis W.I. et al.Structural insights into micro-opioid receptor activation.Nature. 2015; 524: 315-321Crossref PubMed Scopus (575) Google Scholar). Fig. 2a shows the transmembrane (panels i and iv) and intracellular views (panels ii and v) of the ensemble of conformations and different orientations the Gα C-terminal peptides extracted from the MD simulations as follows: s peptide in β2-AR (left) and q peptide in V1AR (right). The s peptide inserts into β2-AR in a region encompassing transmembrane helix 3 (TM3)/ICL2/TM5 and extends toward TM7/H8; however, in V1AR, the q peptide inserts between TM3/TM5 and extends toward TM2. Fig. 2a (panels iii and vi) show the distinct conformations from the ensemble of β2-AR and V1AR bound to their cognate peptides. The overall root mean square fluctuation (RMSF) of β2-AR and V1AR does not show significant differences between cognate and non-cognate peptide-bound complexes throughout the simulations (supplemental Fig. 1). We further assessed conformational flexibility in the GPCR·peptide complexes by focusing on fluctuations in the intracellular regions of the receptor. For this, we measured the distance between the intracellular regions of TM3 and TM6 and plotted the distribution of this distance from each simulation (Fig. 2b). The outward movement of the intracellular portion of TM6 away from the intracellular portion of TM3 reflects a critical conformational change required for GPCR activation and binding to G protein (33Kobilka B.K. G protein coupled receptor structure and activation.Biochim. Biophys. Acta. 2007; 1768: 794-807Crossref PubMed Scopus (428) Google Scholar). As depicted, the range of TM3–TM6 distances is ∼3.9 Å for β2-AR with s peptide and 5.7 Å for both i and q peptides. Similarly, we observed a range of 3.7 Å for V1AR with q peptide but 5.0 and 4.2 Å for s and i peptides, respectively. Measures of central tendency for each distribution are included in supplemental Table 3. We assessed the similarity between the distributions of TM3–TM6 distances for each receptor-peptide pair using a two-sample Kolmogorov-Smirnov test (supplemental Table 4), which indicates that all the distributions are significantly different. For both receptors, binding to cognate Gα peptides reduces the receptor's intracellular flexibility and results in a narrower range of distances sampled between TM3–TM6, leading to tighter binding by the GPCR to the cognate Gα peptide. The variance in TM3–TM6 distance is wider for the non-cognate Gα peptides leading to weaker binding. This is highlighted in Fig. 2a, (panels iii and iv) in which conformations with the maximum (‡) and minimum (*) deviations in the positions of TM6 have been marked. To test the accuracy of our dynamic ensembles in capturing cognate binding, we calculated the non-bonded Coulombic and van der Waals components of the GPCR/peptide interaction energies and compared this to previously published experimental FRET measurements (30Malik R.U. Ritt M. DeVree B.T. Neubig R.R. Sunahara R.K. Sivaramakrishnan S. Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.J. Biol. Chem. 2013; 288: 17167-17178Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and those reported here (Fig. 1d). Fig. 2c shows a strong correlation between the strength of GPCR·peptide binding energy and ΔFRET. Favorable non-bonded interactions that stabilize the cognate GPCR·peptide also reduce TM3–TM6 flexibility in the Gα peptide-bound conformation (Fig. 2b). To further explore which structural components of the GPCR/G protein interface contribute to peptide selection, we analyzed the nature of the residues located in the surface of the peptide binding grooves in the GPCR after optimization of peptide binding using MD. Fig. 3a shows the nature of different residues within the binding groove of β2-AR and V1AR when bound to their cognate peptides, s peptide in red and q peptide in blue. The nature of the residues in the binding surface of the receptor is represented by the colored surface as follows: yellow (non-charged polar residues); red (anionic residues); blue (cationic residues); and white (hydrophobic residues). Most notably, the characteristics and the location of the residues in the binding groove of β2-AR and V1AR with which the cognate peptide interacts vary significantly. In β2-AR the outer edge and center of the binding groove are populated with polar residues. V1AR shows a more hydrophobic binding groove, particularly near the outer edge of the interface (Fig. 3a), and most of its polar residues clustered in the center of the interface (Fig. 3a). The cognate peptides orient and bind differently between the two receptors, despite starting the MD simulations from the same initial orientations. The center of each peptide interacts in a similar groove of the receptor interface, always between TM5 and TM3/ICL2, but the extreme C terminus of the peptide orients toward different positions in the receptor interface. The s peptide points toward TM7 and helix 8 in β2-AR, whereas the q peptide points toward TM2 in V1AR. To identify residues in the C-terminal peptides that contribute significantly to the binding of the GPCR, we calculated the interaction energies of the residues in the s peptide to the residues in the binding groove in β2-AR, and the same for the q peptide to V1AR. Fig. 3b shows the increasing favorable interaction energies mapped as colored gradation in the s peptide (white to red) and q peptide (white to blue) with β2-AR and V1AR, respectively. Residues in the s and q peptides making tighter contacts with favorable interaction energies are represented with deeper shading. About eight amino acids in the N terminus “head” region (Fig. 3b) of both the s and q peptides are disordered and show no significant interactions with their respective receptors. It is evident that the s and q peptides interact with the receptor in different ways. The s peptide has its strongest interactions both at the C-terminal “tail” and “neck” of the peptide, denoted in Fig. 3b. The q peptide, in contrast, makes its strongest contacts at the C-terminal tail. To identify the residues that are “hot spots” in the interaction of the peptides with their respective GPCRs, we did two types of analyses as follows: (a) sequence alignment of the s and q peptides shown in Fig. 3c, with (from left to right) the head, neck, and tail demarcated by dashed lines, and (b) identify the residues that make tight contact