The helical domain of Gα determines the family specificity

First, we conducted cell-based assays to quantitatively evaluate the Gαi/o-specific activation of GIRK, to identify the structural element of Gα that determines the specificity in the activation of GIRK. To date, the Gαi/o specificity in regulating GIRK has been mainly characterized by electrophysiological analyses observing GPCR agonist-induced GIRK currents, using cultured cells or oocytes expressing a GPCR, GIRK, and various Gα mutants10,13. In these experiments, the Gαi/o specificity in activating GIRK was mainly observed as the differences in the steady-state of GIRK current upon the addition of GPCR agonists, indicating that the preference of Gαi/o is mainly under thermodynamic control, rather than the kinetic control. This steady-state GIRK-current has been compared between Gα families to characterize the Gαi/o specificity, however, the observed GIRK currents are strongly affected by the extents of G protein activation; i.e., the amounts of Gβγ released upon the activation of GPCRs, which can significantly differ among the Gα mutants analyzed. Therefore, the Gα specificity in activating GIRK over other effector proteins has been difficult to compare in a quantitative manner. Accordingly, we conducted cell-based BRET experiments, in which we monitored the thermodynamic stability of the binding of Gβγ to several effector proteins, including GIRK23,24. We conducted two sets of BRET experiments for each Gα protein: one to observe the intermolecular BRET between Gβγ and GIRK that reflects the GPCR-mediated GIRK activation, and the other to observe the intermolecular BRET between Gβγ and the Gβγ-binding domain of G protein-coupled receptor kinase (hereafter referred to as GRK), which does not exhibit a Gα family preference and serves as a reporter of the G protein activation23,24,25. By normalizing the BRET signals observed between Gβγ–GIRK with those observed between Gβγ–GRK, we can quantitatively compare the Gα specificity in the activation of GIRK between different Gα families and mutants.

Fig. 1a–d shows the schema and representative results of the BRET experiments, using NLuc-tagged GRK and GIRK (GRK-Luc and GIRK-Luc). The expression of G proteins and GIRK on the plasma membrane was confirmed by fluorescence imaging (Supplementary Fig. 1). In HEK293T cells expressing Venus-tagged Gβγ and GRK-Luc, along with delta opioid receptor (DOR) and Gα, the addition of Met-enkephalin (DOR agonist) induced the activation of DOR and the subsequent association between Venus-Gβγ and GRK-Luc, resulting in energy transfer between them to increase the BRET signal. The observed increase in the BRET signal (ΔBRET) was reversibly decreased to the basal level by the addition of naloxone (DOR antagonist), showing that the observed BRET change reflects the binding between GRK and Gβγ, controlled by the DOR-mediated G protein signaling pathway. Similar changes in BRET signals were also observed in the cells expressing GIRK-Luc (Fig. 1c, d). These ligand-dependent changes in BRET were good indicators of the extents of GRK or GIRK activation. We confirmed that the effects of the basal activities on the measured BRET values were small, because further decreases in the BRET signal were not observed upon the application of inverse agonists of GPCR (Supplementary Table 1). The ΔBRET obtained using GIRK-Luc (ΔBRET GIRK ) was normalized by using GRK-Luc (ΔBRET GRK ), and we defined this ΔBRET GIRK /ΔBRET GRK ratio as a “specificity factor” to quantitatively compare the preference of Gα for activating GIRK over GRK.

Fig. 1 Measuring Gα specificity in Gβγ-GIRK binding. a Schematic representation of the BRET assay to measure G protein activation. Upon adding receptor agonists, the Venus-tagged Gβγ, the BRET acceptor, dissociates from Gα and then associates with the BRET donor GRK-Luc, leading to the increased BRET signal. b Representative traces of time-resolved BRET between Venus-Gβγ and GRK-Luc on cells expressing Gαi3 or Gαiqi along with DOR. The additions of the agonist Met-enkephalin (ENK) (10 μM) and the antagonist naloxone (NLX) (~83 μM) are indicated by bars. c Schematic representation of the BRET assay to measure GIRK-Gβγ binding. d Representative traces of time-resolved BRET between Venus-Gβγ and GIRK-Luc. e Top, the crystal structure of Gαi1 from Gαi1βγ (PDB ID: 1GP2)33. Bottom, topological representations of the Gα proteins used in this study. GD GTPase domain; HD helical domain. f The ΔBRET GIRK /ΔBRET GRK ratios, named specificity factors, for each combination of GPCR and Gα. Data are means ± SEM. The number of measurements taken from independently transfected cell batches is indicated in the bar. *p < 0.001 by one-way ANOVA with post hoc Tukey–Kramer’s test. Source data are provided as a Source Data File Full size image

Rusinova and co-workers have previously reported that the helical domain of Gα confers the specificity for M2R-mediated GIRK activation13. Referring to this report, we compared the specificity factors when 3 different Gα proteins, Gαi3, Gαqi5, and Gαiqi, were used (Fig. 1e). Gαi3 belongs to the i/o family of Gα and is responsible for GIRK activation in biological processes; Gαqi5 refers to Gαq with the C-terminal 5 residues replaced by those of Gαi3 to couple with Gi/o-coupled GPCRs26; and Gαiqi is a chimeric protein consisting of the GTPase domain of Gαi3 (residues 1–62 and 176–354) and the helical domain of Gαq (residues 69–180)13. We confirmed that all of the Gα chimeric proteins used to calculate the specificity factor showed similar ΔBRET GRK values upon the addition of the GPCR agonists, indicating that they have comparable nucleotide binding properties and GPCR-coupling efficiencies (Supplementary Table 1–3). We also conducted competitive binding experiments, in which we monitored the decrease in the BRET signal caused by the displacement of Venus-Gβγ bound to GRK-Luc by increasing the amounts of Gα. In the cases of both Gαi3 and Gαiqi, the BRET signal decreased to a similar extent by increasing the amounts DNA encoding Gα, indicating that the replacement of the helical domain does not result in marked differences in the Gβγ-binding property in the inactive state (Supplementary Fig. 2A, B). Similar results were obtained when we expressed GIRK-Luc and monitored the ΔBRET GIRK values (Supplementary Fig. 2C, D).

When we used the Gi/o-coupled receptor DOR, the specificity factors were 0.286 ± 0.009 and 0.148 ± 0.008 in cells expressing Gαi3 and Gαiqi, respectively (n = 10), and the specificity factor of Gαiqi was significantly smaller than that of Gαi3 (p < 0.001) (Fig. 1f). We could not observe either ΔBRET GRK or ΔBRET GIRK in cells expressing Gαqi5 (ΔBRET < 0.003), indicating that Gαqi5 is not activated by DOR. We also compared the specificity factors using the Gi/o-coupled dopamine D 2 receptor (D2R), and obtained values of 0.303 ± 0.025, 0.091 ± 0.022, and 0.157 ± 0.015 for Gαi3, Gαqi5, and Gαiqi, respectively (n = 7–9), and the values obtained with Gαqi5 and Gαiqi were significantly smaller than that obtained with Gαi3 (p < 0.001) (Fig. 1f). To gain further insights into the role of the helical domain, we also prepared a chimeric Gα, Gαqiqi5, in which the helical domain of Gαqi5 (residues 69–180) is replaced with that from Gαi3 (residues 63–175), and found that the specificity factor of Gαqiqi5 (0.236 ± 0.016) was significantly larger than that of Gαqi5, and similar to that of Gαi3. These results show that the Gβγ dissociated from Gαi3 or Gαqiqi5 binds to GIRK with significantly higher specificity than the Gβγ dissociated from Gαiqi and Gαqi5, even though the released Gβγ is identical, and this preference of Gα is commonly observed in both the DOR-mediated and D2R-mediated pathways. Since the difference among these Gα proteins exists mainly in the helical domain, our results strongly support the hypothesis that the helical domain of Gα is the major determinant that confers the Gα specificity in the activation of GIRK.

Together with the fact that the helical domain of Gαi/o(GTP) is involved in the binding to GIRK21, we hypothesized that the Gαi/o specificity in the GIRK activation is attributable to the formation of a complex comprised of GIRK and Gαi/oβγ, in which the activation of GIRK is enhanced by the increased availability of Gβγ provided by the Gαi/oβγ that is colocalized with GIRK. This notion is further supported by the observation that the differences in the specificity factors between Gαi3 and Gαiqi markedly decreased in the cells expressing larger amounts of Gαβγ, where non-specific protein-protein encounters are facilitated and the formation of non-specific Gαiqiβγ–GIRK complexes tends to occur more frequently (Supplementary Fig. 3).

NMR spectral changes of Gαi3βγ upon interaction with GIRK

To determine whether the binding between Gαi/oβγ–GIRK actually occurs and contributes to the Gαi/o specificity, we set out to characterize the direct interaction between Gαi/oβγ and GIRK in an in vitro reconstituted system. In these analyses, we used a chimeric channel of GIRK1 (GIRK chimera), in which three-fourths of the transmembrane region were replaced with the pore of prokaryotic KirBac1.327. The structure of the GIRK chimera is quite similar to that of the mammalian GIRK228, and the cytoplasmic region of the GIRK chimera is identical to that of the mammalian GIRK1. Since Gαi/oβγ is anchored to the cytoplasmic side of the membrane, the interaction with GIRK, if any, would occur on the cytoplasmic region of GIRK. Hence, the interaction between GIRK1 and Gαi/oβγ could be characterized by using the GIRK chimera. The GIRK chimera was reconstituted into phospholipid bilayer nanodiscs to mimic the physiologically relevant Gαi/oβγ–GIRK interaction that takes place on cell membrane29. We analyzed the interaction by using solution NMR techniques, which can characterize weak protein–protein interactions in physiological solution environments. In the analyses, we used a recombinant Gαi3βγ that lacks the lipid modification, which is partially localized to the lipid bilayer surface of the nanodiscs via an N-terminal polybasic region30,31. The experiments were conducted under physiologically-relevant ionic conditions (KCl = 150 mM), to suppress the non-specific binding mediated by this polybasic, positively charged regions.

We observed the NMR spectra of Gαi3βγ in the absence and presence of the GIRK chimera-nanodiscs to investigate whether Gαi3βγ interacts with the GIRK chimera and identify the regions that are affected upon the interaction. Due to the large molecular weights of Gαi3βγ (87 K) and the GIRK chimera-nanodisc (~380 K), we adopted selective methyl-labeling strategies and applied methyl-TROSY techniques22. We focused on observing the Gα subunit, since it confers the specificity, and prepared a selectively labeled {ul-[2H, 15N]; Alaβ, Ileδ1, Leuδ, Valγ-[13CH 3 ]} Gαi3 complexed with [non-labeled]βγ (Gαi3[ILVA]βγ). We observed the 1H–13C HMQC spectrum of Gαi3[ILVA]βγ, and assigned the methyl signals based on the nuclear Overhauser effect spectroscopy and mutagenesis experiments (Supplementary Fig. 4). By comparing the HMQC spectrum with that of Gαi3 alone, which we previously reported32, we confirmed the formation of the Gαi3βγ complex that is consistent with the reported crystal structure33 (Supplementary Fig. 5). Upon the addition of 2 equivalents of the GIRK chimera-nanodiscs to Gαi3[ILVA]βγ, most of the signals exhibited intensity reductions with relative intensities lower than 0.9, and the signals from L5δ1, L5δ2, A12β, V13γ1, V13γ2, A30β, A31β (N-terminal helix), L36δ1, L36δ2, L37δ1, L37δ2 (β1 strand), A41β (β1-α1 loop), I127δ1(αC helix), L148δ1(αD-αE loop), L159δ2 (αE helix), V218γ2 (α2-β4 loop), I221δ1(β4 strand), L232δ2, L234δ2 (β4-α3 loop), L249δ1, L249δ2, I253δ1 (α3 helix), I264δ1, I265δ1 (β5 strand), I278δ1, L283δ1 (αG-α4 loop), and L348δ1 (C-terminus) exhibited further reduced intensities lower than 0.8 (Fig. 2a–c), while the observed chemical shift changes were very small (<0.01 ppm). When we added the empty nanodiscs, we did not observe significant intensity reductions, showing that the observed intensity reductions upon the addition of the GIRK chimera-nanodiscs are mainly triggered by the specific binding of Gαi3[ILVA]βγ to the GIRK chimera (Fig. 2c). The overall intensity reductions are caused by slower tumbling, due to an increased average molecular weight, indicating that a fraction of Gαi3[ILVA]βγ forms a complex with the GIRK chimera-nanodiscs. The further intensity reductions are caused by differential line broadening, which results from the chemical shift changes in an intermediate-to-fast exchange regime between the free and the bound states, and/or the effect of the anisotropic tumbling induced by the binding, although the effect of the anisotropic tumbling was estimated to be relatively small for membrane proteins in nanodiscs34. Since the total molecular weight of the complex is quite large for NMR observation (>400 K) and the interaction is relatively weak, the binding effects are mainly observed as reductions in the signal intensities, caused by the differential line broadening35, in a similar manner to the interaction between the cytoplasmic region of GIRK and Gαi3(GTP)21. Assuming that the overall intensity reduction (~0.1) reflects the apparent increase in the molecular weight as a function of the bound population, we estimated the apparent K d to be larger than 200 μM. The residues with significant intensity reductions were located on the N-terminal and C-terminal regions, the Gβγ-binding site within the GTPase domain, and the helical domain of Gαi3 (Fig. 2d), suggesting that these regions exhibit chemical shift differences caused by the direct contact with the GIRK chimera-nanodiscs, and/or by the conformational changes that occur upon the interaction. This estimation of the K d value is also consistent with the site-specific intensity reductions that were as large as 0.3, if we assume that the on-rate is diffusion limited (k on ~107 M−1 s−1) and the 1H chemical shift difference between the free-state and bound-state is around 0.05–0.1 ppm. Since Gαi3 is anchored to the membrane at its N-terminus under physiological conditions, the spectral changes observed in the N-terminal region and the neighboring C-terminal region may reflect the binding of Gαi3βγ to the membrane lipids of the nanodiscs. As the N-terminal region of Gαi3 simultaneously interacts with Gβγ, the Gβγ-binding site might be slightly affected upon membrane-anchoring via the N-terminal region, resulting in the intensity reduction observed on the Gβγ-binding site. The helical domain of Gαi3 is distant from the membrane-binding site, so the chemical shift differences in this domain might be caused by interactions with the GIRK chimera. Together, these spectral changes strongly suggest the direct interaction of Gαi3βγ with the GIRK chimera-nanodiscs.

Fig. 2 NMR spectral changes of Gαi3[ILVA]βγ induced by the GIRK chimera-nanodiscs. a Overlay of the 1H–13C HMQC spectra of Gαi3[ILVA]βγ in the presence (red) and absence (black) of 2 eq. of the GIRK chimera-nanodiscs. b Close-up views and cross-sections of V13γ2, L148δ1, and L310δ2 as representative signals. For comparison, the corresponding signals of Gαiqi[ILVA]βγ in the presence (blue) and absence (black) of the GIRK chimera-nanodiscs are shown on the right. c Plots of relative intensities of Gαi3[ILVA]βγ (top) and Gαiqi[ILVA]βγ (middle) in the presence of 2 eq. of the GIRK chimera-nanodiscs. A plot of the relative intensities of Gαi3[ILVA]βγ in the presence of empty nanodiscs is also shown (bottom). The error bars are calculated based on the signal-to-noise ratios. Methyl groups with relative intensities lower than 0.80 are colored according to their values. d Mapping of the methyl groups of Gαi3βγ with significant intensity reductions on the structure of Gαi1βγ (PDB ID: 1GP2)33. Methyl groups are shown as spheres and colored according to their relative intensity values. Source data are provided as a Source Data File Full size image

We performed the same experiment using Gαiqi[ILVA]βγ (Supplementary Fig. 6). In contrast to Gαi3[ILVA]βγ, we did not observe an overall intensity reduction upon the addition of the GIRK chimera-nanodiscs, and no signals exhibited reduced intensities lower than 0.8 (Fig. 2b, c). These results demonstrated that Gαiqiβγ has significantly lower affinity for the GIRK chimera-nanodiscs than Gαi3βγ. Together with the results of our cell-based BRET experiments, in which Gαiqi did not efficiently provoke the activation of GIRK, we concluded that Gαi3βγ directly interacts with GIRK through its helical domain, and the interaction is responsible for the Gαi/o-specific GIRK activation.

Interacting sites of Gαi3βγ–GIRK complex revealed by PRE

In order to gain insight into the structure of the Gαi/oβγ–GIRK complex, we conducted paramagnetic relaxation enhancement (PRE) experiments. PRE arises from the magnetic dipolar interaction between a nuclear spin and an unpaired electron of the paramagnetic center, resulting in line-broadening of the NMR signal of the nuclear spin, depending on the distance from the paramagnetic center. The distance information within the complex can be obtained from the PREs observed in the free state signals, since PREs are transferred from the transiently-formed bound state to the free state in the fast-exchanging system36,37.

To collect the distance information, we site-specifically labeled the GIRK chimera with a spin-labeling reagent, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), which can be covalently ligated to cysteine side chains, and measured the PREs observed on Gαi3[ILVA]βγ. We first constructed a mutant of the GIRK chimera (C53S/C310T), which has no reactive cysteine residue. Using this mutant as a template, Q344, V351, and L366 were separately replaced with cysteine for the site-specific spin labeling of the GIRK chimera. These three residues are distributed across the entire cytoplasmic domain of the GIRK chimera, and thus they would allow us to identify the relative position of Gαβγ to the GIRK chimera. Q344 and V351 are located on the βM-βN loop and the βN-C-terminal helix loop, respectively, which are both within the highly structured β-strand region, while L366 is located on the C-terminal helix of the GIRK chimera (Fig. 3a). The PRE contributions to the transverse relaxation rates, Γ 2 , were measured using the signal intensities of Gαi3[ILVA]βγ in the presence of the spin-labeled GIRK chimera-nanodiscs, before and after the paramagnetic center of 4-maleimido-TEMPO was reduced by ascorbate.

Fig. 3 Observed and calculated PREs between the GIRK chimera and Gαi3βγ. a The locations of Q344, V351, and L366 are shown on the structure of the GIRK chimera (modified from PDB ID: 2QKS; see methods)27. b The methyl groups with significant PREs (Γ 2 obs > 5 s–1) in the experiment using L366C-TEMPO are shown as red spheres on the crystal structure of Gαi1βγ (PDB ID: 1GP2)33. c Γ 2 rates observed in the PRE experiments, Γ 2 obs (bars), and those back-calculated from the ensemble structure, Γ 2 calc (orange lines) are shown. The signals with significant PREs (Γ 2 obs > 5 s–1) from L366C-TEMPO are labeled. The error bars are calculated based on the signal-to-noise ratios. d The distribution of Gαi3βγ relative to the GIRK chimera in the obtained ensemble structure. An atomic probability density map is displayed as a surface representation at the contour level of ρ = 0.05. One orientation in which Gαi3βγ is directed toward the membrane is shown in the ribbon diagram. Source data are provided as a Source Data File Full size image

The results are summarized in Fig. 3. Significant Γ 2 values over 5 s−1 caused by L366C-TEMPO were observed for the signals of I85δ1, L130δ1, L130δ2, and V136γ2 of Gαi3. These methyl groups are clustered around the αA and αB helices in the helical domain of Gαi3 (Fig. 3b). A few signals (V126γ2 and A299β) exhibited Γ 2 larger than 5 s−1 caused by Q344C-TEMPO and V351C-TEMPO, and these methyl groups are not clustered on the structure (Fig. 3c). Based on these PRE patterns, we concluded that the C-terminal helix of GIRK, where L366 is located, is proximate to the helical domain of Gαi3 in the Gαi3βγ–GIRK complex, while the β-strand regions of the cytoplasmic region of GIRK do not form stable interactions with Gαi3βγ.

Constructing a model structure of the Gαi3βγ-GIRK complex

We sought to visualize the structure of the Gαi3βγ-GIRK complex by structural calculation using the observed PREs as distance restraints. However, our initial attempt to obtain a single complex structure that simultaneously satisfies the PRE patterns from Q344C-TEMPO, V351C-TEMPO, and L366C-TEMPO failed, as indicated by the relatively large Q-factor38 of 0.71, even in the best fit result. This result indicates that the relative orientation between Gαi3βγ and GIRK in the complex is inherently flexible, and we must use an ensemble of structures to explain the observed PREs. Therefore, we calculated an ensemble of multiple structures that explains the experimental PRE data. The calculations were performed in two steps: First, we docked the C-terminal helix of GIRK to Gαi3, based on the major PREs obtained from L366C-TEMPO. Second, to recapitulate the relatively minor PRE patterns from Q344C-TEMPO and V351C-TEMPO, we generated 30,000 possible structures considering the conformational flexibility of the GIRK region (residues 352 to 357) connecting the β-strand region with the C-terminal helix, and then optimized the weight of each structure. The calculated PREs from the weighted ensemble of the selected 1000 structures agreed with the experimental PREs from Q344C-TEMPO, V351C-TEMPO, and L366C-TEMPO, with an overall Q-factor of 0.421 (Fig. 3c orange lines and Supplementary Fig. 7), indicating that the ensemble illustrates the interaction mode between the GIRK chimera and Gαi3βγ under the experimental conditions.

To visualize the spatial distribution of Gαi3βγ relative to the GIRK chimera, we calculated the weighted atomic probability density39 (Fig. 3d). In the obtained ensemble consisting of 1000 orientations, the location of Gαi3βγ ranged from beside the membrane to below the β-strand region of GIRK, while retaining the interaction between the C-terminal helix of GIRK and the helical domain of Gαi3. Gαi3βγ distribution in the ensemble was different from the randomly generated distributions (Supplementary Fig. 8), so the calculated ensemble is likely to represent the orientations of Gαi3βγ while interacting with the GIRK chimera-nanodiscs. Notably, the ensemble included several orientations in which the N-terminus of Gαi3 and the C-terminus of Gγ are directed toward the membrane (Fig. 4a and Supplementary Fig. 9). These orientations are consistent with the membrane anchoring of lipidated Gαβγ in vivo, and thus we concluded that these orientations represent the physiological interaction mode of Gαi/oβγ–GIRK (Fig. 4a and Supplementary Fig. 9). In the ensemble structures, the C-terminal helix of GIRK and the αA helix in the helical domain of Gαi3 form a major binding surface (Fig. 4a). While the amino acid sequences of the helical domain, especially those of the αA, αB, and αC helices, are less conserved among G protein families (Fig. 4b), our structural model suggested that the αA helix is the key structural element that mediates the formation of the Gαi/oβγ–GIRK complex, and hence determines the Gαi/o specificity in the activation of GIRK. To verify this model, we conducted a structure-guided mutational analysis. We constructed a chimeric Gαi3 in which the αA helix (residues 71–90) was replaced by that of Gαq (Gαi3-q(αA)), and tested the effect on the specificity factor by BRET assays. For comparison, we also used chimeras in which other structural elements, the αB (residues 100–110) and αE (residues 151–163) helices, were replaced with those of Gαq (Gαi3-q(αB) and Gαi3-q(αE)). The specificity factors of these chimeras are shown in Fig. 4c. The specificity factor for Gαi3-q(αA) was 0.159 ± 0.008 (n = 4), which was significantly smaller than that of the wild-type Gαi3 (0.286 ± 0.009, p < 0.001). In contrast, the specificity factors of Gαi3-q(αB) and Gαi3-q(αE) were 0.248 ± 0.023 (n = 3, p = 0.041 vs. Gαi3) and 0.249 ± 0.011 (n = 5, p = 0.022 vs. Gαi3) respectively, which were statistically not significantly different from that of the wild-type Gαi3. We also conducted NMR experiments to observe Gαi3-q(αA)[ILVA]βγ, and significant intensity reductions were not found upon the addition of the GIRK chimera-nanodiscs, indicating that the specific binding to the GIRK chimera was diminished in Gαi3-q(αA)[ILVA]βγ (Supplementary Fig. 10). From these results, we concluded that the αA helix is the key structural element of Gαi/o that couples specifically with GIRK.