A Conserved Phenylalanine as a Relay between the α5 Helix and the GDP Binding Region of Heterotrimeric G_i Protein α Subunit

Background: GPCRs regulate heterotrimeric G protein activation. However, the intermediate steps regulating GDP release are still unknown.

Results: Energy analysis pinpoints information flow through Gα-receptor interaction and GDP release.

Conclusion: Hydrophobic interactions around α5 helix, β2-β3 strands, and α1 helix are key for GDP stability.

Significance: G protein activation defines regulation of high affinity receptor interactions and plays a role defining different cellular responses.

Abstract

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.

Introduction

Heterotrimeric G proteins play a critical role as molecular switch proteins that couple the activation of cell surface receptors, G protein-coupled receptors (GPCRs),² to different intracellular effector proteins mediating intracellular responses. Therefore, G proteins have a crucial role in defining the specificity and temporal characteristics of many different cellular responses (1–5).

Several structural and biophysical studies have proposed the conformation of the receptor in its active state and have identified potential receptor-mediated mechanisms for G protein activation and GDP release (6–16). Two well studied receptor-mediated G protein activation routes have been hypothesized. In the first, the binding of the GPCR to the C terminus of Gα is thought to trigger conformational changes that can be transmitted via rotation of the α5 helix of Gα to the β6-α5 turn on the purine ring of the GDP (Fig. 1) (3, 9, 17–19). In the second proposed mechanism, the GPCR is thought to take advantage of Gβγ as a nucleotide exchange factor to disrupt the phosphate interactions of the nucleotide binding pocket via destabilization of switch I-II regions through perturbing α5 interaction with the β2-β3 strands (Fig. 1) (20–25).

FIGURE 1.

Heterotrimeric G protein; localization and function α5 helix in G proteins. A, ribbon model of heterotrimeric G protein (G_iαβγ, Protein Data Bank entry 1GP2). The Gα subunit is composed of nucleotide binding (GTPase domain, light blue) and helical domains (green). The α5 helix and switch (SW) regions are colored yellow and purple, respectively. GDP is shown as sticks. B, receptor-mediated (orange) G protein activation routes. The binding of the GPCR to the C terminus of Gα is thought to trigger conformational changes that can be transmitted via rotation of the α5 helix (black, arrow 1) of Gα to the β6-α5 loop (purple, arrow 2) that binds the purine ring of the GDP. In the second route, disruption of the phosphate interactions with the nucleotide binding pocket via destabilization of switch I-II regions through perturbing α5 interaction with the β2-β3 strands (arrow 3). The rhodopsin-G_i complex model adapted from Alexander et al. (26). C and D, the α5 helix is one of the most critical regions for G protein stability and activation. A and B, the α5 helix (yellow) is protected by six β strands (β1–β6) and one α helix (α1) (green). The structure is adapted from the crystal structure of the G_i heterotrimer (Protein Data Bank entry 1GP2).

In 2011, Kobilka and co-workers (18) provided an important missing piece of the puzzle in the receptor-mediated G protein activation cycle by determining the structure of the β₂-adrenergic receptor-G_s heterotrimer complex structure. This groundbreaking study detailed the receptor-G protein interaction and G protein activation. This structure represents the end point in the signal transduction step. The signaling route by which an active receptor interacts with an inactive G protein and causes conformational changes that lead to the final high affinity complex of a receptor with its cognate G protein and GDP release is still unknown.

To address the conformational dynamics underlying nucleotide release from the Gα subunit, we recently generated a predictive computational model of the energy of receptor activation with the goal of understanding conformational changes and connections between potential key residues during G protein activation (26). In this model of the rhodopsin-G_iαβγ complex, it was suggested that the α5 helix is the most critical region for G protein stability and activation and is consistent with previous studies (9, 12, 14, 15, 27). The α5 helix is protected and surrounded with primarily hydrophobic interactions within six β strands (β1–β6) and one α helix (α1) (Fig. 1, C and D). Energetic analysis predicted that residues Phe-191 and Phe-196 in β2-β3, Ile-265 and Phe-267 in β5, Tyr-320 and His-322 in β6 strands, and Gln-52 and Met-53 in the α1 helix are making critical interactions with the α5 helix in both basal and receptor-mediated G protein activation (26). These key residues might either be important for the overall structural integrity of the GTPase domain during the activation process, or they may be directly involved in activation.

To identify the residue-residue interactions that are critical for activation as a part the signaling pathway, we systematically tested the effects of these residue-residue interactions on G protein activation. The residues were examined using biochemical, computational, and structural approaches in both basal and receptor-bound states. In this study, recombinant Gα_i1 was used for all experiments instead of visual G protein, given that Gα_i is a very close homolog of Gα_t yet much more easily expressed in Escherichia coli. Our data showed that single mutations in the β5 and β6 strands that face the α5 helix were not able to break hydrophobic interactions and trigger GDP release from G protein in both receptor-bound and unbound states. In the receptor-bound state, using pairwise coupling energy analysis, we predicted that the α5 rotation compensates the effect of β5-β6 mutations on protein activation.

However, the hydrophobic interactions on the opposite side of the α5 helix were predicted to directly affect G protein function. Energetic analysis predicted that Phe-336 is the most critical residue in the α5 helix; it creates a hydrophobic hot spot of G protein activation, consistent with previous studies (14, 28, 29). The amplitude of this effect was correlated with decreasing hydrophobicity of the side chain. Experimentally tracing the hydrophobic interactions around the Phe-336 residue together with computational analysis provided evidence for a dynamic interplay between Phe-336, the β2 and β3 strands, and the α1 helix on the G protein activation route.

EXPERIMENTAL PROCEDURES

Materials

The TSKgel G2000SW column, GDP, and GTPγS were purchased from Sigma. All other reagents and chemicals were of the highest available purity.

Rosetta Interface Energy Calculations

Interface energies were computed following the Rosetta ΔΔG protocol previously described (26). Briefly, we leveraged the previously published ensembles of 10 structures of the G protein in the basal state and receptor-bound state. Residue-residue interactions across α1 helix/GTPase domain interface were evaluated by measuring energetic perturbations when computationally removing the α1 helices from the models. The α1 helix was defined as residues 45–58. For all analyses, GDP remained fixed within the nucleotide binding pocket. The ΔΔG value is reported as an average over the 10 structural models in Rosetta energy units (REU). Absolute values larger than 0.5 REU are considered to be significant. Using the standard deviation over the 10 structures, a Z-score was computed. The total ΔΔG value across the interface is calculated as the sum of individual residue contributions.

Rosetta Pairwise Binding Energy Calculations

Average energies between pairwise interacting residues were computed using Rosetta's per residue energy breakdown protocol. The energy between all possible pairs of interacting amino acid residues within the G protein were calculated across the previously published ensembles of 10 structures (26). These energies between all residues pairs were then averaged across the 10 models in both the receptor-bound and basal state. Predicted energy values are reported in REU and considered significant if greater than 0.5 REU.

Preparation of Urea Washed Rod Outer Segment Membranes and Gβ₁γ₁

Urea washed rod outer segment membranes and Gβ₁γ₁ were prepared from bovine retina as described previously (30, 31).

Construction, Expression, and Purification of Proteins

Briefly, the pSV277 expression vector encoding Gα_i1 with N-terminal His tag served as the template for introducing individual mutant substitutions using the QuikChange system (Stratagene). All mutations were confirmed by DNA sequencing (DNA Sequencing Facility, Vanderbilt University). The mutant constructs were then expressed and purified as previously described (32). The purified proteins were cleaved with thrombin (Sigma; final concentration, 0.5 unit/mg) for 16 h at 4 °C to remove the N-terminal His tag. The sample was then loaded onto a nickel-nitrilotriacetic acid column to separate the protein from the cleaved His tag and any uncleaved fraction. For further purification, the protein solution was loaded onto a size exclusion column (TSKgel G2000SW) that was equilibrated in buffer A (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm MgCl₂, 40 μm GDP (or 1 μm GTPγS), 2 mm DTT, and 100 μm PMSF). SDS-PAGE was used to test the purity of the proteins. Protein concentrations were determined by Bradford assay (33).

Nucleotide Exchange Assay

The basal rate of GTPγS binding was determined by monitoring the relative increase in the intrinsic tryptophan (Trp-211) fluorescence (λex 290 nm, λem 340 nm) of Gα_i1 (200 nm) in buffer containing 50 mm Tris (pH 7.2), 100 mm NaCl, and different amounts of MgCl₂ for 60 min at 25 °C after the addition of 10 mm GTPγS. Receptor-mediated nucleotide exchange was determined with Gβ1γ1 (400 nm) in the presence of 50 nm rhodopsin at 21 °C for 60 min after the addition of GTPγS. The data were normalized to the baseline and maximum fluorescence and then fit to the exponential association equation (Y = Y_max * (1 − e^−kt)), to calculate the rate constant (k) as previously described (9).

Intrinsic Trp Fluorescence Assay with AlF

Intrinsic tryptophan (Trp-211) fluorescence upon AlF₄⁻ activation, relative to emission in the GDP bound state of G protein α subunit, was monitored as previously described (34). The data represent the averages from six to eight experiments.

Trypsin Digestion and Analysis

2 μg of Gα_i1 were incubated in buffer containing 50 mm Tris (pH 7.5), 100 mm NaCl, 20 μm GDP, and different amounts of MgCl₂ (0.5, 1, and 2 mm). 10 mm NaF and 50 μm AlCl₃ were added to samples and then incubated for 2 min at 25 °C. One microliter of a 1 mg/ml TPCK trypsin solution was added and incubated on ice for 25 min. The reaction was stopped by adding 2.5 μl of termination solution (10 mg/ml aprotinin, 10 mm PMSF). Subsequently, samples were boiled with Laemmli sample buffer for 5 min and run on a 12.5% SDS-polyacrylamide gel, stained with Coomassie Blue, and quantified by densitometry (Multimager; Bio-Rad) (30, 35, 36).

Cross-linking

An expression vector encoding Gα_i1 with six amino acid substitutions at solvent-exposed cysteines (Gα_i1 HI) and an internal His₆ tag between residues Met-119 and Thr-120 served as the template for introducing individual cysteine substitutions using the QuikChange system (Stratagene) as describe above. The bifunctional cross-linking reagent bis-maleimidoethane (Pierce) was incubated in a 2:1 molar ratio with Gα_i1 HI as previously described (37). The concentrated, cross-linked monomeric protein was then purified by size exclusion chromatography on a calibrated G2000SW column. Calibration was performed under the same conditions as purification, using a broad range of molecular weight standards (Bio-Rad) (37).

Membrane Binding Assay

The ability of mutant Gα subunits to bind rhodopsin in urea-washed rod outer segment membranes was determined as previously described (9). Each sample was evaluated by comparison of the amount of Gα_i1 subunit within the pellet or supernatant to the total amount of Gα_i1 subunit in both treatments expressed as a percentage of the total Gα_i1 protein. The data represent the averages of three experiments.

Protein Crystallization, Data Collection, and Structure Determination

Purified GDP bound Gα subunits were exchanged into crystallization buffer (50 mm EPPS, pH 8.0, 1 mm EDTA, 2 mm MgCl₂, 5 mm DTT, 1 mm GDP) using a size exclusion chromatography column. The appropriate fractions were pooled as described above, and SDS-PAGE was used to assess to test the purity of the proteins. Crystals were grown by the hanging drop vapor diffusion method at 18 °C by equilibration against a reservoir solution containing 2.0–2.3 m (NH₄)₂SO₃ and 100 mm sodium acetate (pH 5.9–6.4). Proteins (10 mg/ml) were mixed 1:2.5 ratio with reservoir solution and crystals appeared after 14–18 days with in the space group I4. A similar strategy was used to grow crystals in GTPγS bound form of Gα_i1 proteins. Proteins were incubated with 10 μm GTPγS for 30 min on ice and then storage buffer replaced the crystallization solution containing 50 μm GTPγS instead of GDP. Gα_i1-GTPγS samples crystallized in the space group P3₂21. Crystals were cryo-protected prior to data collection by briefly soaking in stabilization solution containing 18% glycerol and 2.4 m (NH₄)₂SO₃ for ∼30 s and cryo-cooled by immersion in liquid nitrogen.

Data sets were collected at the Life Sciences Collaborative Access Team (21-ID-G) of the Advanced Photon Source at Argonne National Laboratory at −180 °C using a wavelength of 0.98 Å on a MAR CCD detector. The data were processed and scaled using the HKL2000, CCP4, and Phenix suites (38–40). Crystallographic data processing and refinement statistics are reported in Table 1. Criteria for data cutoff were a combination of R_sym and I/σ, which both rose to unacceptable levels if the resolution were extended by G_iα. The structures of the Gα_i1-GDP and Gα_i1-GTPγS complexes were determined by molecular replacement using 1GDD (WT Gα_i1-GDP) (41) and 1GIA (Gα_i1-GTPγS·Mg²⁺) (42) as search models for Phaser-MR in the Phenix suite (40). Because 1GDD and 1GIA preceded the requirement for deposition of structural factors R_free reflections were randomly selected for F336C variant and was the same as F336Y. As a result, the free R is of limited utility. Model building was performed in Coot (43) using composite omit maps calculated in Phenix (40) to minimize model bias. Refinement conducted by both CNS (44) and Phenix, final refinements done by Phenix suite. In the final model, the regions corresponding to amino acids 1–8 and 203–211 in F336C-GDP and amino acids 1–8, 202–217, and 233–240 in F336Y-GDP are not included. Similarly, in the GTPγS-bound structures, amino acids 1–32 and 349–354 are not included because of lack of electron density. Structural superpositions were performed using Superpose for the Cα carbon backbone in the CCP4 suite (45, 46). All structural images were made with PyMOL (PyMOL Molecular Graphics System, version 1.5.0.4; Schrödinger) unless otherwise indicated.

TABLE 1.

Crystallographic data collection and refinement statistics

	F336C-GDP	F336C-GTPγS	F336Y-GDP	F336Y-GTPγS
Data Collection and Processinga
Beamline	21-ID-G	21-ID-G	21-ID-G	21-ID-G
Space groups	I4	P3221	I4	P3221
Cell dimensions
a, b, c (Å)	121.1, 121.1, 68.18	79.2, 79.2, 107.9	121.5, 121.5, 68.2	79.3, 79.3, 105.1
α, β, γ (degrees)	90, 90, 90	90, 90, 120	90, 90, 90	90, 90, 120
Resolution (Å)	34–2.1 (2.18–2.1)	31–2.0 (2.07–2.0)	20–2.4 (2.5–2.4)	42–2.0 (2.07–2.0)
Total reflections	255,402	307,412	177,466	437,402
Unique reflections	28,903	26,186	19,617	26,483
Rsym (%)b	5.3 (37.9)	10.1 (44.7)	6.2 (32)	10.2 (44.6)
Rpim (%)c	2.9 (23.2)	5.2 (23.5)	3.3 (18.4)	4.7 (20.7)
<I>/<σ>	19.9 (2.6)	13.5 (3.1)	19.3 (3.46)	17.5 (3.9)
Completeness (%)	99.6 (99.5)	100 (100)	99.3 (99)	100 (100)
Refinement statistics
Rwork (%)d	18.8	16.4	18.2	16.9
Rfree (%)	21.8	20.8	23.2	20.6
Root mean square deviations
Bond (Å)	0.008	0.007	0.008	0.007
Angle (°)	1.029	0.981	1.011	1.009
Ramachandran statisticse
Favored (%)	98.5	99.06	98.11	98.42
Allowed (%)	1.5	0.94	1.89	1.58
Outliers (%)	0.0	0.0	0.0	0.0

^a Numbers in parentheses indicate statistics for the highest shell.

^b R_sym = Σ|I_i − (I)|/Σ|I_i|, where I is intensity, I_i is the ith measurement, and (I) is the weighted mean of I.

^c R_pim = Σ_hkl√[1/(N − 1)]Σ_i|I_i(hkl) − I(hkl)|/Σ_hklΣ_iI_i(hkl), where I is running over the number of independent observations of reflection hkl, and N is representing the number of replicate observations.

^d R_work = Σ||F_o| − |F_c||/Σ|F_o|, where F_o and F_c are the observed and calculated structure factor amplitudes. R_free is the same as R_work for a set of data omitted from the refinement.

^e Ramachandran analysis from MOLPROBITY (53).

RESULTS

In this study, our strategy was to test residues around the α5 helix that were previously identified as critical for the function of this helix during G protein activation. Residues were examined using biochemical, computational, and structural approaches in both basal and receptor-bound states.

The Effects of β5-β6 Strand Mutants on G Protein Activation

In our previous study, we proposed four residues that face the α5 helix in β5 (Ile-265 ands Phe-267) and β6 (Tyr-320 and His-322) (26). Any one of these might be critical for α5 helix stability and therefore the G protein activation (Fig. 2A and Ref. 26). To test the effect of these residues on G protein function, we evaluated nucleotide exchange rates after introduction of site-directed mutations. Basal and receptor-mediated nucleotide exchange rates of mutants were determined by monitoring the relative increase in the intrinsic tryptophan (Trp-211) fluorescence of Gα_i1. All of the mutants showed similar nucleotide exchange rates compare with WT Gα_i1 in both receptor-bound and unbound states (Fig. 2B). The simplest way to explain these data would be that those residues do not play a major role in G protein activation or that a single mutation is not enough to disturb the α5 helix for GDP release. However, when we computed pairwise residue interactions, we identified interesting details for receptor-mediated activation. In the basal state, Ile-265, Phe-267, Tyr-320, and His-322 were interacting hydrophobically with Val-339, Val-335, Val-342, and Val-335, respectively, within the α5 helix. After receptor interaction and α5 helix rotation, the same residues in β5 and β6 were predicted to hydrophobically interact with new sets of residues in the α5 helix that were previously pointing toward solvent and not involved in binding in the basal state. Specifically Ile-265, Phe-267, Tyr-320, and His-322 started to interact with Ala-338, Asn-331, Ala-338, and Phe-334, respectively (Fig. 2, C and D; see supplemental Table S1 and supplemental Movies S1–S3 for full data). The α5 helix can glide along this hydrophobic surface during its rotation. These calculations thus suggested how new interactions on the rotated the α5 helix can possibly compensate for the effect of single mutations in β5 and β6 strands during receptor-mediated G protein activation.

FIGURE 2.

The effects of β5-β6 strands mutations on G protein activation. A, Rosetta energy analysis of the interface between the α5 helix (black) and the GTPase domain in the receptor-bound state. Residues are colored by the interaction energy as reported in REU (dark blue, the most attractive). Calculations are adapted from Ref. 26. B, basal (black bars) and receptor-mediated (gray bars) nucleotide exchange rates for the β5 strand (I265A and F267A) and β6 strand (Y320C and H322A) mutations in Gα_i1 proteins. Nucleotide exchange was monitored by measuring the enhancement in intrinsic tryptophan (Trp-211) fluorescence (excitation, 290 nm; emission, 340 nm) as a function of time after addition of GTPγS (52). C, most favorable interactions between the α5 helix (Val-335, Val-339, and Val-342), β5 strand (Ile-265 and Phe-267), and β6 strand (Thr-320 and His-322) interface in the basal state. D, after receptor interaction and α5 helix rotation (arrow), the same residues in β5 and β6 were hydrophobically interacting with new residues in the α5 helix (red). Please see supplemental Table S1 and supplemental Movies S1–S3 for full interactions in both receptor-bound and unbound states.

The Effects of Phe-336 Mutants on G Protein Activation

To test the role of interactions with the opposite site of the α5 helix post-rotation, we focused on one specific residue in the α5 helix, Phe-336. Phe-336 is one of the highly conserved residues in the Gα protein family as well as the small GTPases. The side chain faces the β1, β2, and β3 strands as well as the α1 helix, which creates one of the conserved hydrophobic clusters in the Gα subunit. Our previous energetic study predicted that Phe-336 is the most critical residue for both basal and receptor-mediated G protein activation within the α5 helix (Fig. 3, A and B) (26). To test the effect of mutating this residue, we substituted Phe-336 with residues with decreasing hydrophobicity. All of the Phe-336 mutants displayed increased basal exchange rates compared with WT (Fig. 3C). Furthermore, a strong correlation was identified between the hydrophobicity of this residue and basal activity (Fig. 3E). The fastest nucleotide exchange rate was detected for F336Y. However, in receptor-mediated activation, the nucleotide exchange rates were decreased compared with WT without any correlation with hydrophobicity (Fig. 3, D and F; supplemental Movies S2 and S4). This result is consistent with a rotation of α5, leading to a new surface-exposed location of Phe-336 during α5 helix rotation and translation caused by interaction with the receptor (26). Overall, these data suggest that Phe-336 is one of the critical control points that regulate GDP release during G protein activation.

FIGURE 3.

The effect of Phe-336 residue on G protein activation. A and B, Rosetta energy analysis of the interface between the α5 helix and GTPase domain in the basal state (A) and receptor-bound state (B). Residues are colored by the interaction energy REU (dark blue, the most attractive). Calculations adapted from (26). C and D, basal (C) and receptor-mediated (D) nucleotide (Nuc.) exchange (Exc.) rates of Gα_i1 Phe-336 mutants. The data were normalized to the baseline and maximum fluorescence and then fit to the exponential association equation (Y = Y_max * (1 − e^−kt)) to calculate the rate constant (k). The data were collected at 21 °C for 60 min. The results represent the means ± S.E. values of at least three independent experiments. E and F, correlation between nucleotide exchange rates and hydrophobicity identity of the amino acids in basal (E) and receptor-bound (F) state. The Engelman scale was used during comparison and correlation coefficients were calculated with or without F336Y mutant data. The Pearson correlation in the basal state with F336Y is 0.9358; without F336Y, it is 0.9945. In the receptor-mediated state, the Pearson correlation with F336Y is 0.6992; without F336Y, it is 0.4861.

The Effects of Phe-336 Mutations on β6-α5 Loop; Cross-linking α1 and α5 Helices

The most obvious connection between the α5 helix and the nucleotide binding pocket is the β6-α5 loop. Perturbation of the α5 helix during receptor-mediated activation would disturb the interaction between the β6-α5 loop and the guanine ring of the nucleotide, leading to destabilization of the GDP in its binding pocket and domain opening of the α subunit. To test the effect of Phe-336 mutations on this loop, we cross-linked α1 to α5 to minimize the disruption of its interactions with the guanine ring by translocation toward the receptor. Cross-linking was performed between I56C/T329C residues on a cysteine-depleted Gα_i1 (Gα_i1 HI) protein (Fig. 4A). Without cross-linking, Gα_i1 HI I56C/T329C showed higher basal nucleotide exchange rates compared with the Gα_i1 HI protein (Fig. 4B, black bars). Moreover, as expected, substitution of Phe-336 for Cys on Gα_i HI I56C/T329C further increased the protein activity. After cross-linking, the nucleotide exchange rate of cross-linked Gα_i1 cross-linking HI I56C/T329C was decreased as compared with un-cross-linked proteins, demonstrating the stabilizing effect of the cross-linking. Substitution of F336C on cross-linked Gα_i1 HI I56C/T329C increased basal protein activation as compared with the un-cross-linked Gα_i1 HI I56C/T329C-F336C mutant (Fig. 4B, black bars). This indicates that perturbation of Phe-336 can trigger the activation mechanism without translocation of α5 toward the receptor and disruption of β6-α5 loop region.

FIGURE 4.

Cross-linking of α1 and α5 helices of Gα1 HI. A, cartoon representation of cross-linking region. Cross-linking was performed between I56C (α1) and T329C (α5) (purple) residues on a cysteine depleted Gα_i1 (Gα_i1 HI) protein. The Phe-336 (α5) residue is colored red, and the Phe-189 (β2), Phe-191 (β2), Phe-196 (β3), and Met-53 (α1) residues are colored green. The α5 helix is colored yellow, and the β1–β6 strands and α1 helix are colored green. B, basal (black bars) and receptor (gray bars) mediated nucleotide exchange rates for cross-linked Gα_i1 HI proteins. C, basal nucleotide exchange rates in the presence of Gβγ subunit. Black bars, Gα; striped bars, Gαβγ. D, membrane binding of wild type and mutant Gα_i1 HI proteins. Assay was performed as described under “Experimental Procedures.” DS, supernatant from dark sample; DP, pellet fraction from dark sample; LS, supernatant from light sample; LP, pellet from light sample; GS, supernatant from light- and GTPγS-activated sample; GP, pellet from light- and GTPγS-activated sample; XL, cross-linked sample. E, densitometric quantification of supernatant from light samples. Each sample from SDS-PAGE (D) was evaluated by comparison of the amount of Gα_i1 subunits in pellet (P) or supernatant (S) to the total amount of Gα_i1 subunits (P + S) in both treatments and expressed as a percentage of the total Gα_i1 protein. The data represent the averages of three independent experiments.

Because receptor-mediated activation causes both a rotation of the α5 helix and an uncoiling of one turn of helix, we expected the cross-linked Gα would be resistant to receptor-mediated activation. This is indeed what was found in both cross-linked proteins (Fig. 4B, gray bars). This result might be caused by the reduced capability of cross-linked Gα to interact with either Gβγ subunits or the receptor. To test the first possibility, we measured the basal nucleotide exchange rates of Gα mutants in the presence or absence of Gβγ subunits (Fig. 4C). The results showed that basal nucleotide exchange rates decreased on both cross-linked and un-cross-linked mutant Gα proteins in the presence of the Gβγ subunit, like the WT protein. This suggested that cross-linked Gα subunits were still capable of interacting with Gβγ subunits. To test the receptor binding capability of mutant Gα_i1 subunits, we determined the effect of cross-linking on the membrane association of the G protein with light-activated rhodopsin, a measure of the formation of the high affinity receptor-G protein complex. As expected, cross-linking between α1 and α5 impaired this membrane binding (Fig. 4, D and E), consistent with a lack of ability of the cross-linked α5 helix to translocate toward the receptor and the decreased nucleotide exchange rates. Overall, the cross-linking data suggest that perturbation of Phe-336 triggers GDP release through destabilization of switch I-II regions via perturbing the α5 helix interactions along the α1 helix and β2-β3 strands rather than disrupting the β6-α5 loop region.

Hydrophobic Interactions around Phe-336: α1 Helix Interface Binding Energy and G Protein Activation

Previous data suggested that the interaction of Phe-336 with the α1 helix and β2-β3 strands might be crucial for domain opening because the α1 helix is positioned at the interface of the Gαi-GTPase domain and the helical domain (25). In addition, the α1 helix and β2-β3 strands interact with the phosphate binding loop and switch I-II, respectively. To probe the effects of hydrophobic interactions around Phe-336 with the α1 helix, we computed interaction energies for all residues within the α1 helix in both basal and receptor-bound states of the heterotrimeric Giαβγ using our established protocol (26). These ΔΔG values probed for a potential network of intramolecular interactions that could propagate the conformational changes necessary for G protein activation and nucleotide exchange. ΔΔG calculations predicted the importance of π-π interactions between the aromatic rings of Phe-189 and His-57 in the β2 strand and α1 helix, respectively (Fig. 5A and Table 2). This pairwise interaction couples with Phe-336 on the α5 helix. Other predicted stabilizing interactions between α1 (Gln-52 and Ile-56) keep the α5 helix (Thr-329) fixed in the receptor unbound state; receptor interaction triggers unwinding of a turn of the α5 helix, disturbing this interaction (Fig. 5B and Table 2). On the face of α1, in contact with the helical domain, residues (Lys-51, Lys-54, Ile-55, Tyr-61, and Leu-175) on both the α1 and αF helices assist to secure the helical domain in a “closed” GDP-bound conformation. The total interaction energy was ∼25.4 REU. In the basal state, the α1 helix was predicted to interact favorably with β2-β3 (Phe-189, Met-198, and Asp-200; 3.59 REU), α5 (Val-332 and Phe-336; REU 2.44), and helical domain (Glu-65 and Leu-175; 1.84 REU) (Table 2). In the receptor-bound state, the α1 helix was predicted to interact favorably with α5 (Asn-331 and Val-332; 2.1 REU), and as expected, the overall interaction was calculated as lower than the unbound state (Table 2).

FIGURE 5.

The effects of hydrophobic residues around Phe-336 on nucleotide exchange rates. A and B, Rosetta energetic analysis of the interface between α1 helix and GTPase domain in the basal state (A) and receptor-bound state (B). Residues are colored by the interaction energy in REU (dark blue, the most attractive). C and D, basal (C) and receptor-mediated (D) nucleotide exchange rates of single, double, and triple mutants within the β2-β3 strands and α1 helix as determined by monitoring intrinsic tryptophan (Trp-211) fluorescence changes upon addition of GTPγS. The data were collected at 21 °C for 45 min. The results represent the means ± S.E. values of at least three independent experiments.

TABLE 2.

G protein α subunit α1 helix interface energetic prediction

Entity	Amino acid	Energy	Standard deviation	Z-score
		REU
Free Gα
β1	Leu-038	0.87	±0.04	22.75
α1	Lys-046	1.14	±0.28	4.01
α1	Ser-047	0.95	±0.04	21.55
α1	Thr-48	1.82	±0.05	38.27
α1	Ile-49	0.99	±0.09	11.42
α1	Lys-51	0.82	±0.1	8.10
α1	Gln-52	1.65	±0.05	34.32
α1	Met-53	1.33	±0.11	12.04
α1	Lys-54	2.49	±0.07	38.00
α1	Ile-5	1.03	±0.16	6.53
α1	Ile-56	1.07	±0.03	32.77
α1	His-57	1.73	±0.08	22.03
Helical	Glu-65	0.78	±0.11	6.96
Helical	Leu-175	1.06	±0.08	13.10
β2	Phe-189	1.41	±0.09	15.72
β3	Met-198	0.50	±0.12	4.28
β3	Asp-200	0.81	±0.33	2.45
β6-α5	Ala-326	1.62	±0.04	41.26
β6-α5	Thr-329	0.82	±0.02	41.22
α5	Val-332	0.85	±0.03	31.85
α5	Phe-336	0.72	±0.05	15.83
GDP		0.95	±0.13	7.32
GDP	Cumulative	0.95
α1	Cumulative	15.02
Helical	Cumulative	1.84
β6-α5	Cumulative	2.44
α5	Cumulative	1.57
β-strands	Cumulative	3.59
Overall	Cumulative	25.43
Receptor-Gα complex
β1	Leu-038	0.78	±0.16	4.85
β1	Gly-40	0.72	±0.34	2.12
α1	Lys-46	1.58	±0.41	3.88
α1	Ser-47	0.71	±0.14	5.01
α1	Ile-49	1.11	±0.1	10.77
α1	Val-50	0.75	±0.16	4.61
α1	Lys-51	0.52	±0.34	1.55
α1	Gln-52	1.08	±0.13	8.03
α1	Met-53	1.62	±0.15	10.72
α1	Lys-54	0.94	±0.45	2.11
α1	Ile-56	1.18	±0.2	5.85
α1	His-57	1.20	±0.57	2.12
β2	Phe-189	1.43	±0.17	8.56
β2	Phe-191	0.55	±0.08	6.60
α5	Asn-331	1.02	±0.04	23.43
α5	Val-332	0.68	±0.08	8.11
GDP		0.70	±0.19	3.79
GDP	Cumulative	0.70
α1	Cumulative	10.69
α5	Cumulative	1.70
β-strands	Cumulative	3.48
Overall	Cumulative	16.57

To test our computational results, we mutated two residues that are predicted to stabilize the α1-α5 interaction (Phe-189 and Phe-191). In the basal state, F189C increased nucleotide exchange 5-fold, whereas F191C showed no change relative to WT Gα_i1 (Fig. 5C). We prepared double and triple mutants with M53C and F196C mutants, which we had previously tested (26). Double mutants (M53C/F189C and F189C/F196C) exhibited similar basal activation and a triple mutant (M53C/F189C/F196C) showed an even higher basal exchange rate compared with the F336C Gα_i1 mutant protein (Fig. 5C). In receptor-mediated activation of exchange, there was again a pattern of only modest inhibition, with F191C showing the largest decrease (Fig. 5D) consistent with previously predicted α5 (26) and α1 interface binding energy calculations.

Perturbation of Phosphate Site of Nucleotide Binding Region with Phe-336 Mutants

To determine whether the hydrophobic pocket around Phe-336 was necessary to control the local order of the phosphate binding region of GDP, we used the sensitive monitor of Mg²⁺ binding into this region. Three different strategies were used to investigate the influence of Gα_i1 mutants on Mg²⁺ binding to this region: (a) [Mg²⁺] effects on the kinetics of nucleotide exchange, (b) AIF₄⁻ binding, and (c) trypsin digestion of Gα_i1 in the presence of different concentrations of Mg²⁺. The results showed that the high nucleotide exchange rates of the mutants could be decreased in elevated Mg²⁺ concentrations (Fig. 6, A and B), suggesting that these mutations had allosteric effects on the phosphate binding region that could be overcome with higher Mg²⁺ concentration. The highest decrease in the rate of exchange, as a function of increasing concentrations of Mg²⁺, was observed for the F336Y mutant, which showed the fastest exchange rate in the presence of low Mg²⁺ concentrations (Fig. 3C).

FIGURE 6.

The effect of MgCl₂ on Gα_i1 basal activity. A, basal nucleotide exchange in the presence of 2 and 10 mm MgCl₂ concentrations. B, changes in the nucleotide exchange rate in the presence of different MgCl₂ concentrations. Fold change was calculated from A and normalized with Gα_i1 (WT) data. C, rates of intrinsic tryptophan fluorescence changes in Gα_i1 upon AIF₄⁻ addition in the presence of different MgCl₂ concentrations (0.1–2 mm). Intensity of tryptophan signal were monitored (excitation, 290 nm; emission, 340 nm) at 21 °C for 10 min before and after the addition of AIF₄⁻ (10 mm NaF and 50 μm AlCl₃). The data were calculated as described above and rate constants plotted against MgCl₂ concentrations. D, trypsin digestion and analysis of Gα_i1 protein subunit. The densitometric measurement of proteolytic fragments in the presence of GDP-AIF₄⁻ + 0.5 mm MgCl₂. The results were normalized with WT Gα_i1 data, and fragments were quantified by densitometry (Multimager; Bio-Rad). The results represent the means ± S.E. values of at least six to eight independent experiments.

To investigate the order of the Mg²⁺ binding region in the presence of GDP, the AIF₄⁻ binding assay was used. In this assay, changes in intrinsic tryptophan fluorescence rates of Gα_i1 were measured upon AIF₄⁻ addition in the presence of different MgCl₂ concentrations. Mg²⁺ is necessary for AIF₄⁻ binding and generation of the active or transition state. Thus, this assay reflects both AIF₄⁻ and Mg²⁺ coordination in that region without nucleotide exchange. All mutations showed destabilization effects that were overcome with increasing Mg²⁺ concentration. The EC₅₀ for Mg²⁺ stabilization of AIF₄⁻ binding for F336M, F336C, F336A, and F336Y was increased by 1.4-, 2.1-, 2.8-, and 3.1-fold, respectively, over the WT Gα_i1 under the same experimental conditions (Fig. 6C). In addition to the α5 helix mutants, the M53C/F189C/F196C mutant also exhibited statistically significant increased EC₅₀ (Fig. 6C).

The sensitivity of the Gα_i1 mutants to the trypsin digestion assay is a complementary assay to show the subtle changes in local order at the trypsin digestion site at Arg-208 in the presence of varying Mg²⁺ concentrations. After activation by either GDP-AIF₄⁻ or GTPγS, Gα_i1 yields a ∼34-kDa fragment following trypsin digestion. All high nucleotide exchange mutants had reduced stability as assayed by decreased 34-kDa fragment in the presence of low Mg²⁺ concentrations compared with the WT Gα_i1 subunit (Fig. 6D).

Structural Features of the X-ray Structures of the F336C and F336Y Mutants

To probe the structural basis for the increased rates of nucleotide exchange observed in the Phe-336 mutants, the crystal structures of the F336C and F336Y variants of the Gα_i1 subunit were determined in both the GDP- and the GTPγS-bound states. The data collection and refinement statistics are summarized in Table 1. The mutations in the protein were confirmed by the crystal structure, where electron density at position 336 corresponded to either cysteine or tyrosine (Fig. 7, A and B). The structures of the GDP-bound form of F336C and F336Y Gα_i1 were refined to 2.0 and 2.4 Å resolution, respectively. Both GTPγS-bound structures were refined to a resolution of 2.0 Å. The GDP- and GTPγS-bound structures for F336C and F336Y were determined in space groups identical to those of the WT Gα_i1 structures. Neither mutant showed significant structural differences compared with WT Gα_i1. Even with the Phe-336 mutations in the α5 helix, the crystal structures showed the same localization and similar average B (temperature) factors around Phe-336 region relative to those of WT Gα_i1 structures (Fig. 7C). The effects of Phe-336 mutations on the β2-β3 strands and β2-β3 loop were minimal (Fig. 7, D and E). Overall, the root mean square deviations between WT Gα_i1-GDP with F336C and F336Y Gα_i1-GDP were 0.42 and 0.36 Å (310 Cα atoms aligned of 324 total), respectively, whereas they were 0.31 and 0.29 Å (304 Cα atoms aligned of 315) for their GTPγS-bound structures.

FIGURE 7.

Structural features of GDP bound Phe-336 mutant structures. A and B, electron density for the F336C (A) and F336Y (B) side chains in the GDP bound state of Gα_i1. Corresponding regions in GDP-bound WT Gα_i1 (Protein Data Bank entry 1GDD (41); teal) are superposed. Difference electron density is from a |F_o| − |F_c| omit map calculated after the removal of residues 330–340 and contoured to 3σ around the omitted side chain. C, comparison of the α5 helix between F336C-GDP (white), F336Y-GDP (yellow) and WT Gα_i1-GDP (Protein Data Bank entry 1GDD, teal). D, overview of the β2-β3 strands and β2-β3 loop. E, comparison of relative localization of Phe-189 (β2), Phe-191 (β2), Phe-196 (β3), and Phe-336 (α5) residues between F336C-GDP (white), F336Y-GDP (yellow), and WT-Gα_i1 (Protein Data Bank entry 1GDD, teal) structures.

DISCUSSION

The α5 helix of the Gα subunit is a critical region for both the receptor-mediated and basal activity (1, 9, 14, 15, 18). It is encircled by hydrophobic interactions from six β strands (β1–β6) and the α1 helix (α1). In the current study, we tested residues around the α5 helix that we predicted as critical for the function of this helix during G protein activation in our previous studies. We highlight information flow within the G protein, starting from the α5 helix to the GDP binding site of Gα using biochemical, structural, and computational approaches.

Our previous study predicted that Phe-336 within the α5 helix is an important amino acid for both the active and inactive states (26), a finding consistent with other studies (14, 28). Mutation of this residue resulted in constitutive activity in both monomeric and heterotrimeric G proteins (14, 16, 29, 47). It is also known that in small GTPases, structural perturbation of that region through mutation causes increased guanine nucleotide turnover that can lead to several diseases, including Noonan, cardiofaciocutaneous, and Costello syndromes (47–49).

In contrast to strong constitutive G protein activation, in this study, we did not observe drastic differences in the crystal structures of either GDP- or GTPγS-bound Phe-336 mutants. Like another highly constitutively active G protein mutant, Gα_i1 A326S (50), Phe-336 mutants showed similar structural features compared with WT Gα_i1. The guanine nucleotide provides a number of stabilizing interactions to the protein, perhaps inhibiting our ability to visualize subtle allosteric changes in the protein. In addition, other residues in the α5 helix and β-strands may contribute in holding this region intact during the crystallization process.

How does the perturbation at Phe-336 connect to the GDP binding region that is ∼16 Å removed? Phe-336 is a part of a highly conserved hydrophobic core in the Gα subunit. The effect of Phe-336 mutations on basal G protein activation is correlated with the hydrophobicity of this region (Fig. 3, C and E). Once the receptor contacts the α5 helix and causes its rotation and displacement into the receptor binding site, this Phe-336 is now in a hydrophilic environment. We propose that breaking the hydrophobic core is a key event in perturbing GDP binding (26). Interestingly, we did not observe any effects of the hydrophilic mutants on receptor-mediated activation; this is likely due to the new solvent-exposed site, which prevents these side chains from contacting anything other than solvent upon receptor binding (Fig. 3, D and F).

To trace the hydrophobic interactions and to discern a possible interaction network from the Phe-336 residue to the GDP binding site, we computed binding energies of different regions in the Gα subunit by using different Rosetta algorithms. Adding to our previous calculations (α5 helix-G_iα interface binding energy (26)), we predicted that the Phe-336 side chain is mostly coupled with Met-53 (α1), Ile-56 (α1), Phe-189 (β2), Phe-191 (β2), Phe-196 (β3), Val-332 (α5), Gln-333 (α5), Val-339 (α5), and Thr-340 (α5). Thus, the effects of Phe-336 are not solely local and not coupled to a single residue but rather might be part of a distributed network of interactions in which the activation is coupled to changes in regions dispersed across both domains of the Gα subunit. Phe-336 is likely making direct hydrophobic contacts with Phe-191 and Met-53. It potentially communicates with Phe-189 via two paths.

The first is through residues Met-53, His-57, and Phe-191, which interact with Phe-189 through a π-π interaction between residues His-57 and Phe-189 (Fig. 5A). This is consistent with one of our previous studies (51) in which the constitutively active I56C(α1)/Q333C(α5) double mutant of Gα_i1 made a spontaneous disulfide bond between the α1 and α5 helices. This structure showed significant rearrangement of side chain residues His-57, Phe-189, Phe-191, and Phe-332 and disturbed π-π interaction between His-57 and Phe-189.

The second path begins from the direction of Phe-196, which interacts with Phe-336 via the Phe-191 and Thr-340 residues. These observations indicate that the perturbation effects of Phe-336 spread with complex interactions via the α1 helix and β2-β3 strands. These interactions also spread to the Mg²⁺ ion and the nucleotide binding region (Figs. 5 and 6) as evidenced by our nucleotide exchange data combined with the perturbations seen in the Mg²⁺ and AlF₄⁻ assays, which supports previous studies (8, 16).

We also tested the effects of residues within the β5-β6 strands (Ile-265 (β5), Phe-267 (β5), Tyr-320 (β6), and His-322 (β6)) interacting with the other side of the α5 helix on G protein activation. We observed no major effects from the mutations either in the basal or receptor-mediated exchange assays. These data suggest how new interactions on the rotated α5 helix can compensate for the effects of single mutations in the β5 and β6 strands during receptor-mediated G protein activation. It also strongly suggests that the activation route goes through the other side of the protein (β1-β3/α1 to Switch I, phosphate binding loop, Mg²⁺ binding site, and GDP binding site), consistent with previously published findings (28). In addition, after restricting the C-terminal rotation and translocation by cross-linking the α1 and α5 helices, Phe-336 mutants can still induce increased basal nucleotide exchange (Fig. 4). This observation indicates that G proteins do not need a large displacement of α5 for basal state activation; rather, perturbing the β2-β3 and α1 regions are sufficient.

In summary, our study used a predictive energetic analysis to pinpoint information flow through Gα from receptor interaction to triggering of GDP release. We highlighted the hydrophobic interactions around Phe-336 as a key for stability of GDP binding, as well as removal of these hydrophobic interactions by receptor-mediated helical rotation to trigger GDP release. We suggested the route of information triggers through the α5 helix, β2-β3 strands, and the α1 helix using energetic analysis and mutagenesis. We also showed that the dynamics of the Mg²⁺ and β-phosphate binding area of GDP are perturbed by mutagenesis of this conserved residue. The β5-β6 residues that face the α5 helix are likely important structurally rather than functionally according to our analysis. Thus, our data suggest that after the initial interaction of the G protein with the receptor and C-terminal rotation, disruption of a conserved hydrophobic network around Phe-336 engages both β1-β3 and α1 to Switch I and the phosphate binding loop, which decreases the stability of the GDP binding pocket and triggers nucleotide release.