Structure of the Regulator of G Protein Signaling 8 (RGS8)-Gα_q Complex

Abstract

Regulator of G protein signaling (RGS) proteins interact with activated Gα subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Gα_i/o and Gα_q/11 subunits as substrates, the R7 and R12 subfamilies select against Gα_q/11. In contrast, only one RGS protein, RGS2, is known to be selective for Gα_q/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Gα_q revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Gα subunit, or both. To resolve this ambiguity, the 2.6 Å crystal structure of RGS8, an R4 subfamily member, was determined in complex with Gα_q. RGS8 adopts the same pose on Gα_q as it does when bound to Gα_i3, indicating that the non-canonical interaction of RGS2 with Gα_q is due to unique features of RGS2. Based on the RGS8-Gα_q structure, residues in RGS8 that contact a unique α-helical domain loop of Gα_q were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Gα_q seems more likely determined by whether strong contacts can be maintained between α6 of the RGS domain and Switch III of Gα_q, regions of high sequence and conformational diversity in both protein families.

Introduction

Many transmembrane signaling events are transduced inside the cell by heterotrimeric G proteins, which are activated by cell surface G protein-coupled receptors. G protein-coupled receptors stimulate the exchange of bound GDP for GTP on the Gα subunit, which then separates from the Gβγ subunits and interacts with downstream effectors (1). After hydrolyzing GTP to GDP, the Gα subunit is deactivated and is rapidly sequestered by Gβγ. In biological processes, such as the visual response, deactivation of Gα has been observed at much faster rates than those measured for isolated Gα subunits in vitro (2, 3). This discrepancy helped lead to the discovery of a family of GTPase-activating proteins (GAPs),² now known as regulator of G protein signaling (RGS) proteins (4–6). RGS proteins contain a conserved helical domain called the RGS domain that directly binds to the three switch regions (SwI–III) of the Gα subunit and stabilizes them in a transition state conformation (7).

RGS domains are divided into four subfamilies based on sequence homology and substrate preference: RZ, R4, R7, and R12 (8). All utilize Gα_i/o subunits as substrates, although some RZ members seem selective for Gα_z subunits (9). A recent study using surface plasmon resonance indicated that the RGS domains that belong to the R7 and R12 subfamilies bind weakly or not at all to Gα_q, whereas the RZ and R4 subfamilies tend to interact with both Gα_i/o and Gα_q/11 (10). The exception is RGS2, an R4 subfamily member that is uniquely selective for Gα_q/11 (11). The underlying molecular mechanism dictating RGS selectivity has not been fully answered, and past research has focused mainly on RGS2 in part because of its strong link to hypertension and cardiac hypertrophy via its regulation of Gα_q signaling in vivo (12–16). RGS2 can be altered to enhance its selectivity for Gα_i/o by making mutations at three positions unique to RGS2 (Cys¹⁰⁶, Asn¹⁸⁴, and Glu¹⁹¹) that interact with the G protein, primarily near the SwI region (11). These residues are conserved as Ser, Asp, and Lys, respectively, in other RGS proteins. The crystal structure of an RGS2 mutant with conversion of these three residues to their equivalents in other RGS domains (RGS2^SDK) in complex with Gα_i confirmed that the mutant binds in the same canonical orientation as observed in other RGS-Gα_i/o complexes (17).

Subsequently, the structure of wild-type RGS2 in complex with Gα_q revealed a distinct binding mode (18). Because this was also the first RGS domain-Gα_q complex to be structurally characterized, it was unclear whether the significant tilt in the orientation of the RGS domain with respect to Gα was due to sequence differences in either RGS2 or Gα_q. Therefore, before one can address the molecular basis for why some RGS proteins select against Gα_q/11 subfamily members, structures of conventional R4 subfamily members in complex with Gα_q need to be determined.

Herein, we show that RGS8, an R4 subfamily member, binds to Gα_q in a manner similar to how other RGS proteins bind Gα_i/o, indicating that the distinct pose of RGS2 is driven by its unique switch binding residues. We investigate unique contacts formed between RGS8 and the Gα α-helical domain and demonstrate that although residues in the α-helical domain modulate GAP activity, the chief determinant of selectivity is found elsewhere, most likely SwIII. These results clarify the molecular determinants of RGS domain selectivity and, by extension, how RGS proteins impact signaling pathways in vivo.

Experimental Procedures

Protein Expression and Purification

All constructs encoding RGS variants were confirmed by sequencing on both strands. Residues 42–173 spanning the RGS domain of RGS8 were expressed using the pQTEV vector (a kind gift from Dr. R. Neubig, Michigan State University). Residues 22–157 spanning the RGS domain of RGS10 were expressed using a pLIC-SGC1 vector obtained from the Structural Genomics Consortium (10). After cleaving the N-terminal His₆ tag with tobacco etch virus protease from RGS8 and RGS10, the exogenous sequence QSM is left on the N terminus.

For RGS8 variants, 1 liter of BL21 Rosetta cells grown in Terrific Broth was induced with 100 μm isopropyl-1-thio-β-d-galactopyranoside at 20 °C. The cells were pelleted by centrifugation at 3500 × g for 15 min and then resuspended in Buffer A (20 mm HEPES, pH 8.0, 500 mm NaCl, and 10 mm β-mercaptoethanol), supplemented with 7.6 μm leupeptin, 360 nm lima bean trypsin inhibitor, 1 mm PMSF, and 0.1 mm EDTA before being homogenized with a Dounce. Cells were then lysed using an EmulsiFlex-C3 homogenizer (Avestin). Cell debris was pelleted by centrifugation at 40,000 rpm (185,500 g) in a Type 45 Ti fixed angle rotor (Beckman-Coulter). The supernatant was passed over a 5-ml Ni-NTA affinity column pre-equilibrated with Buffer A. The column was then washed with 100 ml of Buffer A, followed by 100 ml of Buffer A with 10 mm imidazole, pH 8.0. RGS8 was eluted from the affinity column using 25 ml of Buffer A with 150 mm imidazole, pH 8.0, and then dialyzed into 20 mm sodium acetate, pH 5.5, 0.5 m NaCl, and 10 mm β-mercaptoethanol. The salt concentration was reduced by diluting 4-fold into Buffer B (20 mm sodium acetate, pH 5.5, and 2 mm DTT), and the protein was loaded onto an UnoS ion-exchange column (Bio-Rad) and eluted using Buffer B in an NaCl gradient increasing from 125 mm to 1 m. Fractions absorbing at 280 nm were verified using SDS-PAGE and pooled and concentrated to 4–9.5 mg/ml. For cleavage of the His tag, 2% (w/w) tobacco etch virus protease was added during a final dialysis into Buffer A.

RGS10 variants were expressed and purified similarly to RGS8 until after elution from the Ni-NTA affinity column. Cleavage of the His₆ tag was then performed as described above for RGS8, followed by passage over a second Ni-NTA affinity column to remove the cleaved tag and His₆-tagged protease. The flow-through was collected, concentrated to ∼7.5 mg/ml, and then buffer-exchanged on tandem Superdex 200 10/300 GL (GE Life Sciences) gel filtration columns into Buffer A with 5 mm DTT instead of β-mercaptoethanol. Fractions absorbing at 280 nm were verified using SDS-PAGE and then pooled and concentrated to 7.5–9 mg/ml.

The insect cell vector pFastBacHT expressing an N-terminally truncated variant of Mus musculus Gα_q spanning residues 35–359 (ΔN-Gα_q) was described previously (18). For purification, 6 liters of High Five^TM cells (BTI-TN-5B1-4) expressing ΔN-Gα_q were pelleted at 3000 × g for 20 min. The pellet was then resuspended in Buffer A (20 mm HEPES, pH 8.0, 100 mm NaCl, 10 mm β-mercaptoethanol, and 10 μm GDP, pH 8.0), 7.6 μm leupeptin, 360 nm lima bean trypsin inhibitor, 1 mm PMSF, 0.1 mm EDTA, and 3 mm MgCl₂. Cells were then homogenized, lysed, and pelleted as described for RGS8. The supernatant was then passed through a Ni-NTA agarose affinity column pre-equilibrated with Buffer A supplemented with 1 mm MgCl₂. The column was washed with 100 ml of Buffer A plus 1 mm MgCl₂, followed by 100 ml of Buffer A plus 1 mm MgCl₂ and 10 mm imidazole, pH 8.0, and then eluted with 25 ml of Elution Buffer (Buffer A with 1 mm MgCl₂ and 150 mm imidazole, pH 8.0). Gα_i/q-R183C was produced as described previously (19).

The Escherichia coli vector pQE60 expressing a C-terminal, His₆-tagged Gα_i1 spanning residues 1–354 was provided courtesy of Dr. Barry Kreutz (University of Illinois at Chicago). Expression was carried out as described previously (20). Purification was similar to RGS8, with the following exceptions. The Lysis Buffer was 50 mm HEPES, pH 8.0, 1 mm EDTA, 2 mm DTT, 0.1 mm PMSF, 7.6 μm leupeptin, and 360 nm lima bean trypsin inhibitor. Buffer A was 50 mm HEPES, pH 8.0, and 2 mm DTT. After washing the Ni-NTA affinity column with Buffer A, the elution step was performed using Elution Buffer (50 mm HEPES, pH 8.0, 2 mm DTT, and 150 mm imidazole, pH 8.0). The eluate was then loaded onto a UnoQ anion exchange chromatography column (Bio-Rad) pre-equilibrated with Buffer A and eluted using a gradient of 0–250 mm NaCl in Buffer A. The integrity of Gα_i1 was confirmed by visualizing trypsin digests on SDS-PAGE as described previously (21).

Purification of the RGS8-Gα_q Complex

Purified ΔN-Gα_q was incubated with 30 μm AlCl₃, 10 mm NaF, and 1 mm MgCl₂ in a buffer also containing 10 μm GDP, pH 8.0, 20 mm HEPES, pH 8, 100 mm NaCl, and 2 mm DTT. It was then mixed with purified RGS8 in a 1:1 molar ratio based on the RGS8 concentration determined using a NanoDrop^TM ND-1000 spectrophotometer, and the ΔN-Gα_q concentration was determined using Bradford reagent. The proteins were incubated together for 30 min on ice before loading onto tandem Superdex 200 10/300 GL (GE Life Sciences) gel filtration columns equilibrated with 20 mm HEPES, pH 8.0, 100 mm NaCl, 2 mm DTT, 10 μm GDP, pH 8.0, and 1 mm MgCl₂. Fractions shown to contain 1:1 complex by SDS-PAGE were then concentrated to 5–7 mg/ml.

Crystallization and Cryoprotection

Crystals were grown in VDX plates (Hampton Research) using hanging drop vapor diffusion on glass cover slides. The RGS8-Gα_q complex (6.6 mg/ml) was mixed 1:1 with well solution to a final volume of 1 μl and suspended over 1 ml of well solution. Octahedral crystals grew in 2 weeks at 4 °C using a well solution containing 0.1 m ammonium acetate, 0.1 m Bis-Tris, pH 5.5, and 11% PEG 8000. Crystals were harvested by adding several μl of cryoprotectant (20 mm HEPES, pH 8.0, 100 mm Bis-Tris, pH 5.4, 200 mm ammonium acetate, 15% PEG 8000, 200 mm NaCl, 1 mm DTT, 50 μm GDP, pH 8.0, 20 μm AlCl₃, 10 mm NaF, and 5 mm MgCl₂) in 0.5-μl increments to the drop containing the crystal. The crystal was then transferred into 100% cryoprotectant and moved stepwise through mixtures of cryoprotectant plus glycerol until a final glycerol concentration of 24% (v/v). The crystal was then suspended in a nylon loop and frozen in liquid nitrogen.

Data Collection, Processing, and Model Building

X-ray diffraction data were collected at the Life Sciences Collaborative Access Team (LS-CAT) beamline 21-ID-D at the Advanced Photon Source (APS). Reflection intensities were integrated and scaled using HKL2000, and initial phases were determined by molecular replacement using PHASER and structures of ΔN-Gα_q from Protein Data Bank (PDB) entry 4EKD and RGS8 from PDB entry 2ODE as search models. Manual model building in Coot was alternated with TLS refinement with local non-crystallographic symmetry restraints in REFMAC5. Coordinates and structure factors were deposited with the Protein Data Bank as entry 5DO9. Figures were generated using The PyMOL Molecular Graphics System, Version 1.5.0.4 (Schrödinger, LLC).

GAP Assays

3 and 4 mg/ml stocks of Gα_i/q-R183C and Gα_i1, respectively, were incubated for 10 min with 10 mm EDTA, and then diluted to 0.3 μm final concentration in 300 μl of Incubation Buffer (50 mm HEPES, pH 8.0, 1 mm DTT, 1 mm EDTA, 100 μg/ml albumin, 5.5 mm CHAPS, 5% glycerol, and 37.5 μm ammonium sulfate) plus 33.3 μCi/ml [γ-³²P]GTP (PerkinElmer, EasyTide) and enough cold GTP, pH 8.0, to make the total GTP concentration 6.25 μm. The reaction was then incubated at room temperature for 3 h (Gα_i/q-R183C) or 30 min (Gα_i1). Samples were buffer-exchanged into fresh Incubation Buffer using a pre-equilibrated Micro Bio-Spin^TM chromatography column (Bio-Rad) and stored on ice for the duration of the assay. Each assay was initiated by adding 20 μl of the buffer-exchanged Gα subunit to a tube containing 100 nm RGS protein in 180 μl of Assay Buffer (20 mm HEPES, pH 8.0, 80 mm NaCl, 1 mm DTT, 1 mm EDTA, 0.9 mm MgCl₂, 1 mm cold GTP, pH 8.0, 10 μg/ml albumin, and 0.20% w/v cholate) on ice. 40-μl aliquots of the reaction were quenched at various time points by vortexing with 750 μl of ice-cold Quenching Buffer (10 mm sodium phosphate, pH 2.0, and 5% (w/v) activated charcoal). The quenched reaction was spun for 25 min at 6500 × g at 4 °C. Afterward, 200 μl of the supernatant was added to 3 ml of MicroScint^TM 40 scintillation mixture (Perkin-Elmer) and read on a liquid scintillation counter instrument measuring ³²P cpm. Each RGS variant was tested in triplicate in three separate experiments. Data were processed in Prism 6 using a non-linear exponential fit with a time lag for Gα_i1 or a straight line fit for Gα_i/q-R183C.

Sequence Alignment and Structure Comparisons

Human RGS sequences from UniProt were aligned using Clustal Omega at the European Molecular Biology Laboratory-European Bioinformatics Institute (22). RMSD calculations were performed using Superpose from the CCP4 software suite (23, 24). Calculation of buried surface area for complexes was performed using PISA (25).

Results

Crystal Structure of the RGS8-Gα_q Complex

To determine whether the altered pose of RGS2 on Gα_q was due to the unique switch interface residues Cys¹⁰⁶, Asn¹⁸⁴, and Glu¹⁹¹ of RGS2 or to unique residues in the switch regions of Gα_q relative to Gα_i, the crystal structure of the RGS domain of RGS8, an R4 subfamily member selective for both Gα_q and Gα_i, was determined in complex with N-terminally truncated Gα_q (ΔN-G_q). RGS8 was used because it readily crystallizes, and the structure of its complex with Gα_i3 was previously reported (10). The final structure was refined to 2.6 Å spacings (Table 1, Fig. 1A). Residues 42–173 of RGS8 with two N-terminal exogenous residues are visible in all complexes, as well as residues 38–350 of ΔN-Gα_q. Three complexes of RGS8-Gα_q exist in the asymmetric unit, with their overall RMSD in Cα positions varying by less than 0.6 Å. Comparisons with the RGS8-Gα_i3 and RGS2^SDK-Gα_i3 complexes give RMSD values of 0.9 Å for 432 and 426 Cα atoms, respectively, whereas comparison with the RGS2-Gα_q complex gives an RMSD of 1.2 Å for 439 Cα atoms. This indicates that RGS8 binds Gα_q in a manner most similar to how RGS proteins have previously been shown to bind Gα_i/o subfamily members (Fig. 1B). Thus, the unique substitutions (Fig. 2A) in the Gα-binding interface of RGS2 are primarily responsible for its altered pose when bound to Gα_q.

TABLE 1.

Crystallographic and refinement statistics for the RGS8-Gα_q complex

Values in parentheses correspond to the highest resolution shell of data.

	RGS8-Gαq
Data collection
X-ray source	APS 21 ID-D
Wavelength (Å)	1.0383
Dmin (Å)	30.0–2.60 (2.64–2.60)
Space group	C121
Cell dimensions
a, b, c (Å)	174.0, 95.9, 112.9
β (°)	94.3
Total reflections	211,145
Unique reflections	56,869
Rsym (%)	12.5 (58.5)
Completeness (%)	99.3 (92.3)
〈I/σI〉	7.6 (4.1)
Redundancy	3.7 (3.6)
CC½a	(71.5)
Refinement
Refinement resolution (Å)	30.0–2.60 (2.66–2.60)
Total reflections used	54,036 (3705)
RMSD bond lengths (Å)	0.013
RMSD bond angles (°)	1.584
Estimated coordinate error (Å)	0.219
Ramachandran plot
Favored (%)	98.35
Outliers (%)	0.00
Rwork/Rfree (%)	17.8/22.5 (27.0/31.0)
Protein atoms	11,092
Ligand atoms	102
Solvent molecules	231
Average B-factor (Å2):	47.8
Protein	48.2
Ligand	27.1
Solvent	37.7
Wilson B factor (Å2)	37.5
MolProbity score	1.39 (100th percentile)
PDB entry	5DO9

^a CC½, Pearson correlation coefficient.

FIGURE 1.

Structure of RGS8 in complex with Gα_q reveals a canonical tilt. A, the 2.6 Å crystal structure of the RGS8 GAP domain in complex with ΔN-Gα_q. The αB-αC loop exhibits structural differences between Gα_i and Gα_q that could dictate the selectivity of RGS proteins. RGS8 is cyan, Gα_q is yellow, the three switch regions are red, GDP is black, AlF₄⁻ is green, Mg²⁺ is orange, and the αB-αC loop of Gα_i is shown in pink (PDB code 2ODE). B, RGS2 adopts a unique tilt when bound to Gα_q. The Gα subunits of the RGS2-Gα_q complex (PDB code 4EKD), RGS8-Gα_i complex (PDB code 2ODE), and RGS8-Gα_q complex (PDB code 5DO9) were superimposed to compare the position of the RGS domain in each complex. Gα_q is shown in yellow, Gα_q-bound RGS8 is shown in pale cyan, Gα_i-bound RGS8 is shown in orange, and RGS2 is shown in dark blue.

FIGURE 2.

Sequence conservation of RGS8 residues in α6 and α7 suggests selectivity mechanisms. A, sequence alignment of the α6–9 regions of the R4, R7, and R12 subfamilies. Alignments were performed with Clustal Omega using human sequences. Residue positions important in Gα_i interactions are in dark gray, discussed SwIII interacting residues are in purple, and the dyad that interacts with the α-helical domain is in green. B, SwIII interface for the RGS8-Gα_q complex (PDB code 5DO9). RGS8 is in cyan, and Gα_q is in yellow. C, SwIII interface for the RGS10-Gα_i complex (PDB code 2IHB). RGS10 is in blue, and Gα_i is in pink. The disordered α6 region of RGS10 is depicted as a dashed line.

Molecular Basis for RGS Subfamily Selectivity

Next, the structures of the RGS8-Gα_i and RGS8-Gα_q complexes were compared to identify RGS domain contacts with Gα that are distinct between the Gα_i/o and Gα_q/11 subfamilies. The structural element that differs most is the αB-αC loop in the α-helical domain. In the RGS8-Gα_q complex, the αB-αC loop is less ordered when compared with the RGS8-Gα_i complex based on temperature factors, but extends closer to the RGS protein, which creates additional buried surface area (Fig. 1A). In fact, RGS2 seems to exploit this surface to maintain greater buried accessible surface area with Gα_q (2050 Ų) than does RGS8 (1900 Ų), or than does RGS8 in complex with Gα_i (1650 Ų). RGS residues that would contact this loop exhibit sequence heterogeneity among the various RGS subfamilies (Fig. 2A). The R4 family has a conserved Glu-Lys dyad in the α7 helix (RGS8 residues 155–156), whereas RGS10, an R12 member, has Lys-Tyr (residues 131–132) (Figs. 2A and 3, A and B). Superposition of the Gα subunits in the RGS8-Gα_q (Fig. 3A) and RGS10-Gα_i (Fig. 3B) complexes suggests that charge repulsion and/or steric hindrance by this dyad could discourage binding of R12 family members to Gα_q, as there is a charge reversal in the first position and introduction of a bulkier Tyr residue for Lys in the second (Fig. 3C). In comparison, modeling Glu-Lys for the Lys-Tyr dyad of RGS10 anticipates no overt issues with Gα_i binding (Fig. 3D). R7 subfamily members instead have a Lys-(Lys/Ser) dyad (Fig. 2A). The subfamily-specific sequences of these dyads could therefore contribute to Gα selectivity. In support of this hypothesis, a previous study found that mutating these positions contributes to differences in GAP activity of various R4 family members on Gα_o (26).

FIGURE 3.

Structural models of interactions between RGS α7 and the α-helical domain of Gα. A, contacts of the RGS8 α7 helix with the α-helical domain of Gα_q. B, contacts of the RGS10 α7 helix with Gα_i (PDB entry 2IHB) C, contacts of the RGS10 α7 helix when modeled in complex with Gα_q. For this model, the Gα subunits of the RGS10 (2IHB) and RGS8 (5DO9) complexes were superimposed to portray RGS10 in complex with Gα_q. D, RGS10 K131E/Y132K double mutant modeled in complex with Gα_i. RGS10 Lys¹³¹ and Tyr¹³² were mutated to their corresponding residues in RGS8 using PyMOL to show the hypothetical complex. RGS8 is shown in cyan, RGS10 is shown in blue, Gα_q is shown in yellow, and Gα_i is shown in pink. Oxygens are depicted in red, and nitrogens are depicted in cyan.

Functional Analysis of the α-Helical Domain Interface

The aforementioned α-helical domain interface was tested by site-directed mutagenesis followed by single turnover GTPase assays using [γ-³²P]GTP (Table 2, Fig. 4). RGS8-Glu¹⁵⁵ and/or Lys¹⁵⁶ were converted to their analogous residues in RGS10 (Lys and Tyr, respectively). Complementary mutations were introduced in RGS10, mutating Lys¹³¹ and/or Tyr¹³² to Glu and Lys, respectively. If RGS selectivity for Gα subunits was achieved via ionic repulsion with the α-helical domain of Gα_q, then mutation at the first position (E155K in RGS8, K131E in RGS10) would result in a selectivity switch. Selectivity achieved through steric pressure would be potentially altered by mutation at the second position (K156Y in RGS8, Y132K in RGS10). If both sterics and charge were necessary to affect a selectivity switch, then both point mutations (E155K/K156Y in RGS8 and K131E/Y132K in RGS10) would be required.

TABLE 2.

Single turnover GTPase assays using RGS8 and RGS10 variants

Values correspond to three experiments performed in triplicate for each mutant with each Gα subunit ± S.D. Prism 6 was used to calculate k values using one-phase association for Gα_i and to calculate steady state rates using a straight line fit for Gα_i/q-R183C.

	Gαi1	Gαi/q-R183C
	s−1	fmol/s
No GAP	0.013 ± 0.002	<0.01
RGS8 WT	0.042 ± 0.023	0.07 ± 0.018
RGS8 E155K	0.051 ± 0.030	0.19 ± 0.0080
RGS8 K156Y	0.063 ± 0.037	0.15 ± 0.012
RGS8 E155K/K156Y	0.051 ± 0.007	0.29 ± 0.043
No GAP	0.007 ± 0.003	<0.01
RGS10 WT	0.034 ± 0.005	<0.01
RGS10 K131E	0.021 ± 0.004	<0.01
RGS10 Y132K	0.007 ± 0.001	<0.01
RGS10 K131E/Y132K	0.029 ± 0.008	<0.01

FIGURE 4.

Single turnover GAP assays of RGS variants reveal modulation by contacts with the α-helical domain. A, representative data from three experiments performed in triplicate of RGS8 variants using Gα_i as a substrate. Points were fit with a non-linear regression using one-phase association with a time lag. B, representative data from RGS8 variants using Gα_q as a substrate. Points were fit with a single steady state rate. C, representative data from RGS10 variants using Gα_i as a substrate. Error bars correspond to standard deviations. GTPase activity of the Gα subunit alone is indicated in pink, wild-type RGS is indicated in blue, RGS8 E155K or RGS10 K131E is indicated in green, RGS8 K156Y or RGS10 Y132K is indicated in orange, and RGS8 E155K/K156Y or RGS10 K131E/Y132K is indicated in red.

Wild-type Gα_i1 and the slow-hydrolyzing mutant Gα_i/q-R183C were used as substrates for each RGS variant. As expected, wild-type RGS8 showed robust GAP activity on both Gα_i1 (Fig. 4A) and Gα_i/q-R183C (Fig. 4B), whereas wild-type RGS10 only showed GAP activity on Gα_i1 (Fig. 4C, Table 2). All three mutants of RGS8 retained their activity on Gα_i1, but also retained wild-type, if not higher, activity on Gα_i/q-R183C. The RGS10 double mutant and K131E single mutant also retained activity on Gα_i3. Interestingly, the Y132K mutant did not. None of the RGS10 mutants showed GΑP activity on Gα_i/q-R183C. These results indicate that the RGS domain dyad that contacts the αB-αC loop is not responsible for RGS10 being inactive on Gα_q, as RGS10 and RGS8 mutants had no increase or loss, respectively, in selectivity for Gα_i/q-R183C.

Discussion

RGS proteins range from being relatively small proteins with little more than the RGS domain, to complex multi-domain entities with multiple signaling domains. However, even in simple RGS proteins such as RGS2, RGS4, and RGS8, the regions outside the RGS domain can play important roles such as targeting these enzymes to membranes, G protein-coupled receptors, or effector enzymes (27–30). Thus, when one considers the selectivity of an RGS protein for a particular Gα signaling pathway, there are many levels at which this can occur. However, the most fundamental aspect of selectivity is imposed by the direct interaction of the RGS domain with Gα to promote acceleration of GTP hydrolysis. Consequently, this study focused solely on the interaction of the RGS domain found in RGS proteins with Gα_i and Gα_q subunits. Moreover, previous studies have shown that isolated RGS domains exhibit selectivity for individual Gα subfamilies (10).

Previous structural analysis of the RGS2 complex with Gα_q suggested that RGS2 has a distinct tilt relative to the Gα subunit (Fig. 1B) that allows it to bury more surface area with Gα_q than it could with Gα_i. Moreover, the conformationally flexible α6 helix of RGS2 allows it to maintain optimal contacts with SwIII, despite the unique pose of the RGS domain (18). When the Cys¹⁰⁶, Asn¹⁸⁴, and Glu¹⁹¹ interface residues are mutated to their equivalents in other RGS proteins in RGS2^SDK, it can bind to Gα_i in a canonical fashion (17), but does not lose activity against Gα_q, suggesting that interactions with SwI are not directly responsible for Gα_q selectivity (11, 17). It was further demonstrated that altering interactions between the α7 helix of the RGS2 domain and the α-helical domain of Gα can dramatically promote or inhibit GAP activity (18), but the molecular basis for selectivity against Gα_q observed in other RGS subfamilies remained unclear.

In this work, it was shown that an R4 family member, RGS8, binds to Gα_q in a canonical fashion, permitting a more precise comparison of the interactions between RGS proteins and these two Gα subfamilies. The tilt of RGS2 in complex with Gα_q can thus be attributed to interfacial differences dictated by unique interfacial residues in RGS2. Two regions, in particular the αB-αC loop of the α-helical domain, stand out as a potential selectivity determinant. The G protein α-helical domain has previously been shown in some instances to be a major determinant of GAP activity, and there are sequence signatures unique to each RGS subfamily that interact with this domain (18, 31). Although a selectivity switch was not achieved in our study, the GAP assay results are consistent with RGS activity being enhanced or inhibited by interactions with the α-helical domain. Interestingly, the RGS10 Y132K mutant, creating a Lys-Lys dyad in α7, did not retain activity for Gα_i1, but could be rescued by the addition of the K131E mutation. The disadvantage of having a Lys-Lys dyad may be due to electrostatic repulsion between the adjacent positions or with the α-helical domain. However, it seems clear that the α-helical domain is not a major Gα selectivity determinant because no substitution in this interface could promote activity on Gα_i/q-R183C by RGS10 (Table 2).

Instead, the evidence now points toward SwIII, which interacts with the N-terminal end of the RGS α6 helix, as being the primary determinant of selectivity, as suggested in Ref. 10. In SwIII, the side chain of Gα_q-Asp²⁴³ stacks with the side chain of RGS8-Phe¹²⁵. The analogous residue in Gα_i, Glu²³⁸, cannot make this interaction because the backbone of its SwIII is positioned differently. Phe is shared by several other R4 family members at this position, but not by any R7 or R12 members (Fig. 2A). R4 RGS domains typically also have a basic residue, e.g. RGS8-Arg¹²⁸, that is positioned to form a hydrogen bond with a backbone carbonyl of another SwIII residue in Gα_q, Glu²⁴¹. Members of the R7 and R12 subfamilies typically lack this basic residue. Notably, although the RGS region that comes in contact with SwIII is well ordered in complexes involving R4 family members (Fig. 2, B and C), the RGS10 α6 helix is disordered in its complex with Gα_i, and thus contacts with SwIII are nearly entirely lost (Fig. 2C). It is therefore quite possible that loss of productive interactions with SwIII mediated by the α6 region are responsible for the inability of some RGS subfamilies to recognize Gα_q. However, due to the poor sequence conservation and conformational heterogeneity of this region in R12 family members (R12 subfamily members also have a 1-residue deletion in the α6 helical region), it is not possible to easily test this hypothesis because conversion of SwIII contacts in RGS8 to those found in RGS10, and vice versa, is not possible by simple substitution.

Regardless, this model does not explain how RGS proteins in the R7 and R12 subfamilies retain activity for Gα_i if they fail to make productive interactions with SwIII. These subfamilies may have optimized interactions in other contact regions (e.g. αA and other regions in the Gα α-helical domain), as has been shown for some R4 family members in determining their relative activity on members of the Gα_i subfamily (26). This hypothesis is supported by the increased GAP activity observed for RGS8 variants that have RGS10 substitutions in α7, and decreased GAP activity for RGS10 variants with RGS8 substitutions (Table 2). This result is consistent with prior studies that have likewise probed positions in α7 and shown them to modulate RGS domain interactions and GAP activity (18, 26, 32). Another possible explanation might be found in the SwI interface. Gα_q has Pro¹⁸⁵, whereas Gα_i has Lys¹⁸⁰. The Gα_i-Lys¹⁸⁰ side chain buries more surface area with the RGS protein when compared with Pro¹⁸⁵ in Gα_q. Hence, if the interactions with SwIII are not strong, RGS proteins may be less active against Gα_q as a result of less buried surface area with SwI. Indeed the specific activity of RGS4 is ∼10 times lower than wild type when using the Gα_i-K180P variant as a substrate (33). Inversely, AlF₄⁻-activated Gα_q-P185K can be pulled down by RGS2 in significantly greater amounts than wild-type Gα_q (32).

In summary, the rules that dictate RGS domain selectivity for a given Gα subunit are complex. They involve leveraging beneficial versus negative interactions at different points of contact with the Gα subunits, as well as the ability of individual RGS proteins to undergo induced fit when required (18). However, the structure-function analysis reported here still points to the SwI and SwIII interactions as being the key determinants of selectivity for Gα_i versus Gα_q. Interactions with the α-helical domain can tune GAP activity (such as for RGS9 in the Gα_i/o subfamily) or can even rescue RGS2 from loss of activity when it binds to Gα_q (18). How is selectivity achieved for or against other Gα subfamilies? Gα_s is not a substrate for any known RGS protein due to presence of Asp²²⁹ in SwII (Ser in Gα_i and Gα_q) (34). Gα_z, a Gα_i/o subfamily member, is the preferred substrate for RZ subfamily member RGS20, but the mechanism for this selectivity is unknown (35). A full understanding of the intricacies of how RGS proteins interact with Gα subunits is required if one seeks to design an RGS domain specific to a particular Gα subunit, or, conversely, a Gα subunit that is a specific substrate for a select subgroup of RGS proteins. These would serve as useful tools to decipher the roles of individual RGS proteins in cellular signaling.

Author Contributions

V. G. T. and J. J. G. T. designed the experiments, determined the x-ray structure, and prepared the manuscript. V. G. T. and P. A. B. purified the complex. P. A. B. crystallized the complex. V. G. T. purified proteins for, and performed and analyzed the GAP assays. All authors approved the final paper.