Conformational Changes at The Carboxyl Terminus of Galpha Occur during G Protein Activation*

Chii-Shen YangDagger §, Nikolai P. Skiba§, Maria R. Mazzoni, and Heidi E. Hamm§parallel

From the § Northwestern University Institute for Neuroscience, Department of Molecular Pharmacology and Biological Chemistry, and Department of Ophthalmology, Northwestern University, Chicago, Illinois 60611, the Dagger  Department of Physiology & Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, and the  Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, Neurobiology and Pharmacology Laboratory, University of Pisa, 56126 Pisa, Italy

    ABSTRACT
Top
Abstract
Introduction
References

To understand the dynamics of conformational changes during G protein activation, surface exposed cysteine residues on Galpha were fluorescently labeled. Limited trypsinolysis and mutational analysis of recombinant Galpha t/Galpha i1 determined that two cysteines are the major fluorescent labeling sites, Cys210, located in the switch II region, and Cys347 at the C terminus. Mutants with serines replacing Cys210 (Chi6a) and Cys347 (Chi6b) were single fluorescently labeled with lucifer yellow (LY), while a double mutant (Chi6ab) was no longer labeled. When Chi6b was labeled with LY on Cys210, AlF4- caused a 220% increase in LY fluorescence, indicating that the fluorescent group at Cys210 is a reporter of conformational change in the switch II region. Chi6a labeled at Cys347 also showed an AlF4--dependent increase in LY fluorescence (91%), indicating that Galpha activation leads to a conformational change at the COOH terminus. Preactivation of the protein with AlF4- before labeling led to a decreased incorporation of LY into Cys347 suggesting that Galpha activation buries Cys347. This COOH-terminal conformational change may provide the structural basis for communication between the GDP-binding site on Galpha and activated receptors, and may contribute to dissociation of activated Galpha subunit from activated receptor.

    INTRODUCTION
Top
Abstract
Introduction
References

Heterotrimeric G proteins are activated by seven-transmembrane-spanning receptors and relay signals to downstream effectors, including cellular enzymes and ion channels. Upon agonist binding, receptors become activated and in turn interact with G proteins and catalyze GDP release from G protein alpha  subunits. After the release of GDP, the Galpha subunit, together with Gbeta gamma subunits, remains in a tight complex with the receptor, which dissociates when GTP binds to the empty Galpha subunit. Both the GTP-bound Galpha subunit and the Gbeta gamma subunit complex are then capable of regulating a variety of effectors on the intracellular face of the plasma membrane. The binding of Galpha and Gbeta gamma subunits is restored when the intrinsic GTPase activity in the Galpha subunit hydrolyzes the bound GTP to GDP.

In order to have a reliable method for studying the conformational changes in G proteins, we developed a fluorescent monitor because fluorescence is easily detectable and responsive to local environmental change. Cysteines are highly reactive and can be labeled with a variety of Cys-directed fluorescent groups. We determined the cysteines that are accessible to sulfhydryl-specific fluorescence labeling on Galpha subunits and characterized the fluorescence changes on Galpha when different sites are labeled. We replaced those accessible cysteines with serine to make a functionally cysteineless mutant in which to place additional cysteines at sites where we would like to monitor conformational changes or interaction with other proteins.

To study structural changes in Galpha t upon its activation, we used the functional derivative of Galpha t, Galpha t/Galpha i1 chimera (Chi6) in which residues 216-294 of Galpha t were replaced with the corresponding residues 220-298 from Galpha i1. Chi6 can be conveniently expressed in Escherichia coli, and it was shown to have a similar rate of rhodopsin-catalyzed GDP/GTP exchange as Galpha t does, implying that its receptor and Gbeta gamma binding properties are Galpha t-like (1). The crystal structure of Chi6 in complex with Gbeta 1gamma 1 has been solved and revealed an identical geometry with wild type, native Galpha t (2). High expression levels of this protein in E. coli provided us milligram amounts of pure protein for biochemical and fluorescent studies as well as ease in constructing Galpha mutants.

Lucifer yellow, an environmentally sensitive fluorescent probe, was selected because it is a good reporter of local changes. We previously used this probe as a reporter of the binding of the inhibitory subunit of cGMP phosphodiesterase to Galpha t (3).

Ho and Fung (4) previously reported that by using 5,5'-dithiobis-(2-nitrobenzoic acid) titration and N-ethylmaleimide modification, a total of five reactive sulfhydryls in native Galpha t and nine reactive sulfhydryls in the SDS-denatured Galpha t protein were found. Eight cysteines were found by cDNA sequencing of alpha t (5). In 1988, by using 125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine (125I-ACTP),1 a cross-linking reagent, Dhanasekaran et al. (6) showed that Cys210 and Cys347 were the major reactive cysteines. Here, we show that the major labeling sites for LY are located on Cys210 and Cys347 in Galpha t/Galpha i chimeras. Also, we show that single labeling at either Cys210 or Cys347 can be used to report the local conformational changes around the labeled sites. As expected, Cys210 in the switch II region reports an AlF4--dependent activating conformational change. Unexpectedly, there is also an AlF4--dependent conformational change at Cys347, which may be important for allosteric communication between receptor binding and GDP-binding sites on the molecule.

    EXPERIMENTAL PROCEDURES

Materials-- GTP, GTPgamma S, GDP, deoxyribonucleotides, and imidazole were purchased from Boehringer-Mannheim. All restriction and DNA modification enzymes were obtained from Boehringer-Mannheim and Pharmacia Biotech Inc. Lucifer yellow vinyl sulfone was purchased from Sigma. All other reagents were from sources described previously (1).

Construction of Mutants and Chimera Expression and Purification-- The construction of Chi6 was described previously (1). The construction of all mutants were based on Chi6 (1), composed of Galpha t cDNA except amino acids 216 to 294, which were from Galpha i cDNA. Mutants were all constructed by using the QuikChangeTM site-directed mutagenesis kit from Stratagene Ltd. Strategy of oligo design for polymerase chain reaction-based mutagenesis as suggested by the QuikChangeTM kit instruction manual. The NH2-terminal sequence of Chi6 is derived from Galpha t expression construct (1) and preceded by MA(His)6A. All chimera proteins were expressed and purified by nickel affinity chromatography followed by anion exchange chromatography using Protein Pak Q15 HR resin (Millipore) as described previously (1). The concentrations of chimeras in the eluates were determined spectrophotometrically and proteins were stored in buffer A (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM MgCl2, 50 µM GDP, 0.1 mM phenylmethylsulfonyl fluoride and 5 mM beta -mercaptoethanol). Samples were aliquoted and kept at -80 °C for several months with no loss of functional activity. The final yield of all chimeras was between 1 and 3 mg/liter bacterial culture with an average of 90% purity.

Expression of Pgamma in E. coli-- The Pgamma expression and purification was performed as described (7). The Pgamma was additionally purified by reversed-phase chromatography on HPLC Vydac C-4 column (size of 0.46 × 20 cm) using a gradient of acetonitrite. Acetronitrite was removed from Pgamma samples by Speed-Vac re-concentrating procedure. The final yield of the Pgamma ranged from 15 to 30 mg/liter of more than 95% pure protein.

Lucifer Yellow Labeling-- Labeling of Galpha with LY was performed immediately after the samples were purified. Protein samples (in buffer A without beta -mercaptoethanol and phenylmethylsulfonyl fluoride) and LY were mixed in a molar ratio of Galpha protein:LY (1:5) followed by incubation on ice for 30 min. Labeling reactions were stopped by addition of 4 mM beta -mercaptoethanol. The excess LY was removed by overnight dialysis and the labeled samples were again purified and resolved by HPLC using protein Pak Q15 HR column (capacity of 2 ml).

Stoichiometry Determination-- All samples were scanned from 260 to 550 nm with 8452A Diode Array Spectrophotometer (Hewlett Packard). The stoichiometry (n) of LY labeling was calculated as a ratio of the concentration of LY and the concentration of the labeled protein in the HPLC eluted samples. The relationship applied is,
n=C<SUB><UP>LY</UP></SUB>/C<SUB><UP>Chi6-LY</UP></SUB> (Eq. 1)
where CLY is the concentration of LY and CChi6-LY is the concentration of labeled Chi6 protein (or the other labeled Galpha chimeras). The concentration of LY (CLY) was determined using,
C<SUB><UP>LY</UP></SUB>(<UP>M</UP>)=A<SUB>430,<UP>LY</UP></SUB>/ϵ<SUB>430,<UP>LY</UP></SUB> (Eq. 2)
where A430,LY is the absorbance of LY at 430 nm and epsilon 430,LY is the molar extinction coefficient of LY at 430 nm which is 12,400. Since LY also absorbs at 280 nm (epsilon 280,LY = 24,000) the concentration of total labeled protein (CChi6-LY) was determined by a subtraction of LY absorbance at 280 nm from the total absorbance at 280 nm using the relationship,
C<SUB><UP>Chi6-LY</UP></SUB>=(A<SUB>280,<UP>Chi6-LY</UP></SUB>−(A<SUB>430,<UP>LY</UP></SUB>ϵ<SUB>280,<UP>LY</UP></SUB>/ϵ<SUB>430,<UP>LY</UP></SUB>))/ϵ<SUB>280,<UP>Chi6</UP></SUB> (Eq. 3)
where A280,Chi6 is the absorbance of Chi6-LY at 280 nm and epsilon 280,Chi6 is the molar extinction coefficient of Chi6 at 280 nm which is 43,100. The value of epsilon 280,Chi6 was calculated by applying the relationship,
ϵ<SUB>280,<UP>Chi6</UP></SUB>=2ϵ<SUB>280,<UP>Trp</UP></SUB>+13ϵ<SUB>280,<UP>Tyr</UP></SUB>+ϵ<SUB>280,<UP>GDP</UP></SUB> (Eq. 4)
where epsilon 280,Trp, is the molar extinction coefficient of Trp at 280 nm (5,300), epsilon 280,Tyr is the molar extinction coefficient of Tyr at 280 nm (1, 400) while the molar extinct coefficient of GDP at 280 nm, epsilon 280,GDP is 9,000. This relationship used to determine epsilon 280,Chi6 is based on the Trp, Tyr, and GDP content (Chi6 has 3 Trp, 13 Tyr, and 1 GDP). For the concentration of Chi6, the following relationship is used,
C<SUB><UP>Chi6</UP></SUB>(<UP>M</UP>)=A<SUB>280,<UP>Chi6</UP></SUB>/ϵ<SUB>280,<UP>Chi6</UP></SUB> (Eq. 5)

Fluorescence Assays-- To monitor AlF4--dependent conformational change of Galpha t and chimeras, tryptophan fluorescence, before and after addition of AlF4- (simultaneously addition of 10 mM sodium fluoride and 50 µM AlCl3 to the test sample), was determined with excitation at 280 nm and emission at 340 nm. For LY-labeled samples, an additional measurement for AlF4--dependent fluorescence change of LY was performed with excitation at 430 nm and emission at 520 nm. All the experiments were performed on an AMINCO Bowman® Series 2 Luminescence Spectrometer running with OS/2 Warp 4.0.

Limited Proteolytic Analysis-- Protein samples were treated with trypsin with a weight ratio of 16.6 (protein to trypsin) for 0, 5, 15, and 30 min before the reactions were stopped by TLCK (8, 9). After running in polyacrylamide gel electrophoresis gels, the LY-labeled fragments were observed by illuminating the gel with UV light.

Surface Exposed Area Calculation-- Solvent-accessible surface area was calculated using the method of Eisenberg et al. (10). The program used for this analysis was Access running in Silicon Graphic System IRIX 6.2.

Data Analysis-- All curve fitting and kinetic analysis were performed by using Prism version 2.01 for Windows 95 from GraphPad.

    RESULTS

Determination of Solvent-accessible Surface Areas of Cysteines in Galpha t-- There are eight cysteines in Galpha t and according to the crystal structure (11), only four of them are surface exposed: namely Cys62, Cys210, Cys321, and Cys347 (Fig. 1). To obtain a quantitative criterion of surface exposure, we calculated the solvent-accessible surface area by the method of Eisenberg and McLachlan (10) using the coordinates of Galpha t-GDP (11) and Galpha t-GTPgamma S (12). The solvent-accessible surface areas of all eight cysteines in Chi6 are summarized in Table I. Among these cysteines, Cys210 has greater surface exposed area in its GDP binding form than Cys62, Cys321, and Cys282. Three other cysteine residues (135, 216, and 250) are completely buried inside the protein according to this calculation. Cys210 becomes less accessible in the Galpha -GTPgamma S binding form. Since the COOH terminus of Galpha is disordered and not visible in the crystal structure, it is likely that Cys347 is highly accessible.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   Locations of all cysteines in Chi6 protein. Crystal structure of Galpha t-GDP (11) is shown. All cysteines (except Cys347) are space-filled (yellow), alpha  helixes are red, beta -sheets are cyan, while the loops are silver. GDP buried in the central part of the molecule is purple. Cys62, Cys210, and Cys321 (labeled with red text) are more surface-exposed than other cysteines. Cys347 is not shown because the last 7 amino acid residues are disordered in the crystal structure. Graphic was generated using "WebLab ViewerLite" V3.10 (Molecule Simulation Inc).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Area of surface exposure of all cysteines in Chi6 protein (Access, Eisenberg, Nature 1986)

Determination of LY Labeling Sites on Chi6-- To determine accessible cysteines in Chi6, we first labeled Chi6 with LY under mild conditions and identified the labeling sites. After the excess LY was removed, the labeled samples were purified by anion-exchange HPLC. The presence of LY adds negative charges to the protein and, thus, allows separation of variously labeled species by anion exchange chromatography (Fig. 2). Chi6 showed three major labeled peaks as well as one unlabeled peak which co-migrates with the original protein. Each peak in the HPLC elution was collected separately and the stoichiometry of labeling was determined. Proteins from labeled peaks 1 and 2 each contains a single fluorescence group per molecule, while protein in labeled peak 3 contains two fluorescent groups (Table II).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   HPLC purification of the LY-labeled Chi6 and molecular structure of LY bound to cysteine. A, labeled Galpha (Chi6) samples were purified by HPLC using protein Pak Q 15 column. The stoichiometry of LY to protein in labeled peak 1 as well as labeled peak 2 in Chi6 was 1:1, whereas it was 2:1 in labeled peak 3. B, molecular structure of LY bound to cysteine. Retention time for each fraction is indicated on the top of the peak.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Summary of percentage of LY labeling in various chimera proteins and AlF4--dependent fluorescence change for all the labeled peaks. The ratio indicated after protein is the stoichiometry of LY:protein labeling with LY

To identify the labeling site(s) for each peak, limited tryptic digestion and mutagenesis studies were performed. The potential tryptic cleavage sites in Chi6 protein are lysine 18, arginine 204, and arginine 310 (8, 9) as illustrated at the bottom of Fig. 3. Protein from labeled peak 3 of Chi6 was treated with trypsin for 0, 5, 15, and 30 min before the reactions were stopped by TLCK. The LY-labeled fragments on the gel were observed with UV illumination. As shown in Fig. 3, the double labeled peak 3 showed fluorescent fragments of 38, 19, 15, and 5 kDa, which suggested the labeled cysteines were within the region composed of amino acids 205-350, which contain Cys210, Cys321, and Cys347. To identify the labeled site(s), a mutant (Chi6a) was constructed in which Cys210 was changed to serine. As shown in Table II, after been labeled with LY, Chi6a-LY (Cys347-LY) migrated on Mono-Q column as a single labeled peak with a retention time of 13 min similar to labeled peak 2 in Chi6-LY (Table II). After this peak was treated with trypsin, the size of the LY-labeled fragments were 38, 19, and 5 kDa, which suggested the labeled site was either Cys347 or Cys321 (Fig. 3).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Proteolytic analysis of labeled peak of Chi6 and Chi6a-LY. The proteins represented by labeled peak 3 of Chi6-LY (upper left panel) and labeled peak of Chi6a-LY (upper right panel) were treated with trypsin (weight ratio protein:trypsin = 16.6:1). Trypsin fragments were resolved by polyacrylamide gel electrophoresis and each gel contains protein samples with trypsin treatment time of 0, 5, 15, and 30 min before the reactions were stopped by TLCK. A UV transilluminator was used to observe the LY-labeled fragments on the gel. Simplified scheme for limited proteolytic digestion pattern of Chi6 is shown on the lower panel. The cleavage sites in Chi6 protein for trypsin are shown with a hollow arrow (under Lys18, Arg204, and Arg310). See "Experimental Procedures" for more details.

To further clarify the possible labeling sites suggested in tryptic digestion experiments, two more mutants were constructed: Chi6b, with Cys347 mutated to serine, and Chi6ab, which has both Cys210 and Cys347 mutated to serine. As shown in Table II, after labeling with LY, Chi6b showed a single labeled peak with a retention time of 12 min, which corresponded to peak 1 for Chi6, whereas in the double mutant (Chi6ab), all major labeled peaks disappeared. We therefore conclude that the protein eluted in labeled peak 1 was labeled at Cys210 while protein eluted in peak 2 was labeled at Cys347. Thus, cysteine 210 and cysteine 347 are the major accessible sites for LY labeling. A comparison of the areas of the peaks shows that the efficiency of labeling at Cys347 was 6.5 times higher than that of Cys210. However, for mutants with one cysteine available for labeling (Chi6a and Chi6b), the efficiency of LY labeling at Cys210 and Cys347 was comparable, which suggested that the labeling of Cys347 in Chi6 prevented the labeling of Cys210.

Functional Assays for LY-labeled and Unlabeled Chimera Proteins-- To determine whether the mutant proteins folded properly, had GDP bound, and could undergo GTP-dependent conformational changes, we measured the intrinsic fluorescence change of Trp207 after addition of AlF4-. All the mutant proteins were able to undergo conformational change upon binding to AlF4- (Table II). After LY labeling, the AlF4--dependent intrinsic fluorescence change of Cys347-LY in Chi6a protein was 50% but Cys210-LY in Chi6b protein showed significantly less (1.2%) fluorescence change (Fig. 4, Table II).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 4.   Kinetics of the AlF4--dependent fluorescence change monitored at 340 nm (Trp) and 520 nm (LY). The fluorescence of Trp (open circle) and LY (open triangle) in Chi6b-LY (Cys210-LY) (A) and Chi6a-LY (Cys347-LY) (B) was monitored (66 nM protein) during activation by AlF4- (10 mM sodium fluoride and 50 µM AlCl3) at the time indicated by arrow and expressed as the percent of initial fluorescence change. C, the fluorescence of LY at Cys347 was expressed as percentage of the maximum fluorescence change upon addition of AlF4-. The best fit obtained by using equation Ft = F0 + F1(1-expact-Kactt) for tryptophan (dashed line) and LY (solid line) are shown. Ft is fluorescent emission at time t, while F0 is the initial fluorescence before addition of AlF4-. F1 is maximal change in fluorescence, and Kact is the rate constant for Chi6 activation. Initial fluorescence was set to 0 and maximal fluorescent change is expressed as 100%. Both fluorescence change in LY and Trp can be reversed by addition of 10 mM of EDTA to chelate AlF4- and this reverse process is EDTA concentration dependent (data not shown).

LY Is a Fluorescent Reporter for Conformational Change in Switch II-- There was only a 1.2% AlF4--dependent intrinsic Trp fluorescence change in Cys210-LY Galpha (Table II). To determine whether there was an AlF4--dependent conformational change in Cys210-LY, we measured the LY fluorescence change in Cys210-LY (Chi6b), which is within the switch II region, after addition of AlF4-. The fluorescence of Cys210-LY increased 220% in response to AlF4- (Fig. 4, Table II). This fluorescence change can be reversed by 10 mM EDTA (data not shown).

Comparison of crystal structures of GDP and GTPgamma S bound Galpha t revealed decreased accessibility of Cys210 in the activated form. Therefore, the accessibility of cysteine residues to thiol-specific reagents (like LY) should decrease upon activation. Chi6b was labeled with LY in the presence or absence of AlF4- and a decrease of 77.8% of LY labeling efficiency for Chi6b was observed in AlF4- bound Chi6b (Fig. 5). Thus, in the active conformation, Cys210 is less accessible for LY labeling.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 5.   LY labeling efficiency of Cys210 and Cys347 in the presence or absence of AlF4-. Equal amounts of chimera proteins from the same preparation were taken and divided into control and AlF4- preactivated group. Before LY labeling, the latter group was activated by addition of AlF4- (10 mM sodium fluoride and 50 µM AlCl3) for 10 min and followed by measurements of AlF4--dependent Trp207 intrinsic fluorescence change in both groups. Labeling efficiency for two chimera proteins, Chi6a and Chi6b, were shown in the figure. Total amount of labeled protein in control group was set to 100%. The labeling efficiency of LY to Chi6a (Cys347-LY) was reduced by 58.6% in preactivation group while a 77.8% reduction was observed in preactivated Chi6b (Cys210-LY).

To further test the existence of a switch II conformational change in Cys210-LY, the ability of the mutant to interact with the downstream effector enzyme, cGMP phosphodiesterase, was tested. Galpha t-GTP binds to the inhibitory gamma  subunit (Pgamma ) with higher affinity than Galpha t-GDP (1). The interaction of Pgamma with Cys210-LY Galpha in the presence or absence of AlF4- was monitored by LY fluorescence. There was only a small fluorescent change upon addition of increasing concentrations of Pgamma in the absence of AlF4-, while in the presence of AlF4- there was a Pgamma concentration-dependent increase in fluorescence (Fig. 6). Thus, the mutant that fluorescently labeled Galpha undergoes an activating conformational change that causes an increased affinity for PDEgamma , and association with PDEgamma further increases the fluorescence of Cys210-LY.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6.   Binding of Chi6b-LY protein to Pgamma subunit in the presence or absence of AlF4-. The relative increase in LY fluorescence change (% of initial) was measured after addition of increasing concentrations of Pgamma to 50 nM Chi6b-LY (square) or AlF4--preactivated Chi6b-LY (triangle). The kinetic parameters calculated from the best fit to the four parameter logistic (sigmoidal) equation were: Kd = 100 ± 11 nM, Delta FMAX 99%, and Hill slope 1.2.

Since the switch II region of Chi6b-LY undergoes a conformational change upon binding to AlF4-, and this conformational change can be monitored by LY, we therefore concluded that LY labeled at Cys210 is a sensitive fluorescent reporter for the conformation of the switch II region.

The COOH Terminus of Galpha Undergoes a Conformational Change Upon AlF4--dependent Activation-- The COOH terminus of Galpha t is not ordered in crystal structure (2, 11-13). To assess its conformation and whether it changes conformation during activation, we labeled Cys347 with LY in Chi6a (Cys347-LY). As shown in Table II and Fig. 4B, an 91% LY fluorescence increase was observed upon addition of AlF4- in Cys347-LY protein. This observation suggests that upon binding of AlF4-, the COOH terminus of Galpha undergoes a conformational change. To understand and confirm the existence of this COOH-terminal conformational change, we determined the accessibility of Cys347 for LY labeling in the presence and absence of AlF4-. In the AlF4--activated Chi6a, a decrease of 58.6% of LY labeling efficiency to Chi6a was observed (Fig. 5) suggesting a decreased accessibility of Cys347 in the activated protein.

To determine whether this fluorescence change had a similar rate as the AlF4--dependent fluorescence change at Trp207, the kinetics of the AlF4--dependent Trp and LY fluorescence at Cys347-LY were measured. The rates of the intrinsic fluorescence change of Trp207 and LY fluorescence at Cys347-LY proteins were very similar, with a Kact of 0.462 and 0.364, respectively (Fig. 4C). This result suggested that upon binding of AlF4-, the COOH terminus of Galpha undergoes a fast conformational change that follows the slow AlF4--dependent conformational change in the switch II region. Alternatively, the change in conformation of the switch II region could lead to an increased contact with the COOH terminus that resulted in the increase of LY fluorescence.

    DISCUSSION

Knowledge of the functional structure of heterotrimeric G proteins has been greatly advanced with the solution of the crystal structure of all their subunits (2, 11-18). A comparison of the three-dimensional structure of the alpha  subunits in their GDP, GTPgamma S, and transition state analogue forms has revealed the molecular principles of nucleotide binding, hydrolysis, and the nature of the conformation changes upon protein activation. The COOH-terminal region of Galpha t, known to be important for receptor interaction (19-24), was not ordered in crystal structures (2, 11-13). Thus, other biochemical or biophysical methods are needed to further define the structural features of this region and understand its role in mediating receptor interaction and receptor induction of GDP release.

In this report, we have developed an approach of sensitive real-time monitoring of the structural changes in Galpha using targeted fluorescent labeling of single surface-exposed cysteine residues. Our studies have identified the labeling sites of LY on Galpha t/Galpha i1 chimera (Chi6), characterized each labeled protein and, most importantly, shown the existence of a previously unsuspected conformational change in the COOH terminus of Galpha upon activation.

The existence of two negative charges in LY and the mild conditions used for labeling Chi6 with LY allowed us to resolve differently modified proteins by anion-exchange chromatography (Fig. 2). The labeling sites on Galpha identified in this report, Cys210 and Cys347, confirms the studies of Dhanasekaran et al. (6) who found that Cys347 is highly accessible and Cys210 is partially accessible to 125I-ACTP, when this cross-linking reagent was used to label Galpha t.

All mutants were properly folded and functional as judged by several criteria: first, all mutants had an increased Trp fluorescence upon addition of AlF4- indicating that they all have GDP bound and undergo conformational changes. Second, all mutants activated with AlF4- had similar affinity to Pgamma when compared with Chi6 (1), which was in the range of 1 µM. Third, the cleavage patterns of tryptic digestion were identical in Chi6 and Chi6a and therefore it can be concluded the mutant chimeras were properly folded during expression.

Our data clearly show that LY at Cys210 is a reporter of conformational change of the switch II region. The fluorescence of LY attached to Cys210 (Chi6a-LY) increases significantly (220%) in the presence of AlF4-. However, the intrinsic Trp fluorescence of Cys210-LY (Chi6b-LY) under similar conditions underwent only 1.2% change, which is much less than 50% change in Trp fluorescence for the unlabeled mutant. But Chi6b-LY protein does indeed undergo switch II region conformational change upon binding of AlF4- as judged by acquisition of high affinity for Pgamma . An explanation for a decreased Trp fluorescence change in Cys210-LY may be that the fluorescent group at Cys210 increases local hydrophobicity in the environment of Trp207 that causes an increase in Trp fluorescence in the GDP-bound state of Galpha . Upon addition of AlF4-, switch II moves and adopts the conformation of active Galpha subunit, as Trp207 does. However, the increased hydrophobicity of Trp207 in the new environment is not much more than that created by the LY group, so that it does not result in a regular change of intrinsic fluorescence.

The solvent-accessible surface area of Cys210 decreased 68.6% in the activated form of Galpha t. This calculation matches well with our finding that the labeling efficiency at Cys210 dropped 77.8% in the presence of AlF4-. Thus, Cys210 of Chi6b becomes partially protected from LY labeling in the presence of AlF4-, reflecting these changes in accessibility. The method is thus clearly useful for monitoring known conformational changes.

The conformational changes seen at the COOH terminus of Galpha are the first indications that the GTP-dependent or activation-dependent conformational switch may extend to this region of the molecule. Several facts support the existence of those environmental changes around the COOH terminus. First, the fluorescence of LY at Cys347-LY (Chi6a-LY) increases by more than 91% in the presence of AlF4- and can be reversed by 10 mM EDTA. There is no nonspecific interaction between LY and AlF4- since in the presence of free LY, no fluorescence change was observed by addition of sodium fluoride and AlCl3 (data not shown). Second, the efficiency of Cys347 labeling with LY for Chi6a is significantly reduced with addition of AlF4-, suggesting that surface exposure and reactivity of this residue is decreased as a result of protein activation. To test whether a fluorescent tag on a cysteine in a part of the molecule that is known to have no conformation change upon activation, Chi6ab, with both reactive cysteines changed to serines, was mutated to replace Val301 with cysteine. There was no AlF4--dependent fluorescent change in the resultant mutant, Chi6ab-Cys301-LY (data not shown). Thus the fluorescence change is not seen in regions known not to change conformation. Third, these effects are very similar to those we observed for Cys210 in Chi6b, which is an indicator of the known conformational change of switch II region. Thus, we can conclude that it is likely that a change in conformation does exist at or near the COOH terminus of Galpha upon activation or more precisely in the GDP-AlF4- form. The comparison of the kinetics of fluorescence changes monitored by LY to the fluorescent changes monitored by Trp207 for Chi6a-LY upon addition of AlF4- shows that they have similar time course (Fig. 4C). This analysis implies that the structural change at the COOH terminus is rate limited by the relatively slow movement of the switch II region upon binding of AlF4-. Dhanasekaran et al. (1988) also proposed that conformational changes could be transmitted between domains containing Cys347 and Cys210 in Galpha t because the photoactivation of the phenylazide moiety of 125I-ACTP labeled at Cys347 caused an insertion to the 12-kDa fragment (residues Arg204-Arg310), which contains Cys210. Our data suggest that LY-bound Cys347 is in a more hydrophobic environment in GDP-AlF4- than in the GDP-bound Galpha . Another observation is the Cys347 is the major labeling site for Chi6 and the labeling of Cys347 reduces the labeling efficiency of Cys210, which confirms the result suggested in 125I-ACTP labeling study (6). This leads us to speculate that the COOH terminus of Galpha does not interact with the alpha 2/beta 4 loop in Galpha -GDP form, but in the active conformation the alpha 2/beta 4 loop and COOH terminus of Galpha move closer.

What is the nature of this COOH-terminal conformational change? According to the crystal structure of Galpha -GTPgamma S (12), in two out of three molecules in the asymmetric unit, the final eight residues are disordered and not visible while in a third molecule, the COOH-terminal residues 343-349 of Galpha t can be seen. The residues 343-349 make van der Waals contacts with residues 212-215 of the alpha 2/beta 4 loop. It is not clear whether this is a crystal packing artifact or an indication of one possible orientation of the COOH terminus.

In Galpha t, Asn343 is the last residue that is consistently seen. It is ~10 Å from the alpha 2/beta 4 loop which is part of Switch II. Two possibilities exist: 1) the conformation of the COOH-terminal region changes upon activation, 2) the COOH terminus itself does not undergo a conformational change, but its environment changes. For example, the disordered COOH-terminal region including Cys347 is close to the alpha 2/beta 4 loop and in the active conformation a binding pocket for the COOH terminus opens up near the switch II region such that Cys347 becomes more buried. In either case, the protection from fluorescent labeling by preactivation, and the activation-dependent fluorescent change indicates an important communication between the GDP binding pocket and the COOH terminus of the protein.

The COOH-terminal conformational change that was detected in this study is of significant interest because of the implications for receptor interaction mechanisms. The COOH terminus of Galpha is known to be a key determinant of the fidelity of receptor activation (19, 23). Since known receptor-binding regions on Galpha are distant from the GDP-binding site, it is likely that an allosteric mechanism triggers GDP release (24). It appears from a number of studies that mutations at the COOH terminus of Galpha proteins can both regulate specific receptor interaction and affect GDP affinity (24-28). COOH-terminal peptides from Galpha can both block receptor-G protein interaction and stabilize the active conformation of G protein-coupled receptors (21, 29-32). These data suggest that the COOH terminus may be a key relay for communication between the activated receptor and the GDP binding pocket. The activation-dependent conformational change reported here thus suggests specific regulation of receptor affinity by the status of the guanine nucleotide bound in the binding pocket. It is known that when receptor, ligand, and G protein are bound, GDP release leads to a high affinity "ternary complex" which only slowly dissociates without guanine nucleotide binding (33-37). It is possible that an activation-dependent conformational change at the COOH terminus of Galpha might lead to a lowered receptor affinity and dissociation from the ternary complex. Currently, it is thought that dissociation of activated G protein from receptor is secondary to the GTP-dependent dissociation of Galpha from Gbeta gamma . This concept has not been rigorously tested, however. Future studies will test the notion that the COOH-terminal conformational change after GTP binding is a component of G protein dissociation from an activated receptor.

Site-specific Cys-directed fluorescent groups could be reporters for conformation changes in various regions of Galpha . For example, they could probe conformational changes in other regions of the Galpha subunit like the NH2 terminus, which was not resolved in crystal structures of either the free Galpha GDP form (11) or the GTPgamma S form (12, 14). Also, they could be used to monitor protein-protein interaction, such as Galpha interaction with receptors, Gbeta gamma , and effectors. Future studies will explore these possibilities.

    ACKNOWLEDGEMENTS

We thank Hyunsu Bae for helpful discussions, Carna Meyer and Jennifer Conner for technical help, and Theresa Vera and Tarita Thomas for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants EY06062 and EY10291 (to H. E. H).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Northwestern University, Institute for Neuroscience, Searle Building, Rm. 5-555, 320 Superior St., Chicago, IL 606011. Tel.: 312-503-1109; Fax: 312-503-7345; E-mail: h-hamm{at}nwu.edu

The abbreviations used are: 125I-ACTP, 125I-N-(3-indo-4-azidophenylpropionamido-S-(2-thiopyridyl)cysteine; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; LY, lucifer yellow vinyl sulfone; HPLC, high performance liquid chromatography; TLCK, tosyl-L-lysine chloromethyl ketone.
    REFERENCES
Top
Abstract
Introduction
References

  1. Skiba, N. P., Bae, H., and Hamm, H. E. (1996) J. Biol. Chem. 271, 413-424[Abstract/Free Full Text]
  2. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319[CrossRef][Medline] [Order article via Infotrieve]
  3. Artemyev, N. O., Rarick, H. M., Mills, J. S., Skiba, N. P., and Hamm, H. E. (1992) J. Biol. Chem. 267, 25067-25072[Abstract/Free Full Text]
  4. Ho, Y.-K., and Fung, B. K.-K. (1984) J. Biol. Chem. 259, 6694-6699[Abstract/Free Full Text]
  5. Yatsunami, K., and Khorana, H. G. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4316-4320[Abstract]
  6. Dhanasekaran, N., Wessling-Resnick, M., Kelleher, D. J., Johnson, G. L., and Ruoho, A. E. (1988) J. Biol. Chem. 263, 17942-17950[Abstract/Free Full Text]
  7. Skiba, N. P., Artemyev, N. O., and Hamm, H. E. (1995) J. Biol. Chem. 270, 13210-13215[Abstract/Free Full Text]
  8. Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510[Abstract/Free Full Text]
  9. Mazzoni, M. R., Malinski, J. A., and Hamm, H. E. (1991) J. Biol. Chem. 266, 14072-14081[Abstract/Free Full Text]
  10. Eisenberg, D., and McLachlan, A. D. (1986) Nature 319, 199-203[Medline] [Order article via Infotrieve]
  11. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628[CrossRef][Medline] [Order article via Infotrieve]
  12. Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) Nature 366, 654-663[CrossRef][Medline] [Order article via Infotrieve]
  13. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372, 276-279[CrossRef][Medline] [Order article via Infotrieve]
  14. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412[Medline] [Order article via Infotrieve]
  15. Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058[Medline] [Order article via Infotrieve]
  16. Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374[CrossRef][Medline] [Order article via Infotrieve]
  17. Sunahara, R. K., Tesmer, J. J., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1943-1947[Abstract/Free Full Text]
  18. Tesmer, J. J., Sunahara, R. K., Gilman, A. G., and Sprang, S. R. (1997) Science 278, 1907-1916[Abstract/Free Full Text]
  19. West, R. E., Jr., Moss, J., Vaughan, M., Lui, T., and Lin, T.-Y. (1985) J. Biol. Chem. 260, 14428-14430[Abstract/Free Full Text]
  20. Sullivan, K. A., Miller, R. T., Masters, S. B., Beiderman, B., Heideman, W., and Bourne, H. R. (1987) Nature 330, 758-760[CrossRef][Medline] [Order article via Infotrieve]
  21. Hamm, H. E., Deretic, D., Arendt, A., Hargrave, P. A., Koenig, B., and Hofmann, K. P. (1988) Science 241, 832-835[Medline] [Order article via Infotrieve]
  22. Simonds, W. F., Goldsmith, P. K., Woodard, C. J., Unson, C. G., and Spiegel, A. M. (1989) FEBS Lett. 249, 189-194[CrossRef][Medline] [Order article via Infotrieve]
  23. Gutowski, S., Smrcka, A., Nowak, L., Wu, D., Simon, M., and Sternweis, P. C. (1991) J. Biol. Chem. 266, 20519-20524[Abstract/Free Full Text]
  24. Onrust, R., Herzmark, P., Chi, P., Garcia, P. D., Lichtarge, O., Kingsley, C., and Bourne, H. R. (1997) Science 275, 381-384[Abstract/Free Full Text]
  25. Denker, B. M., Schmidt, C. J., and Neer, E. J. (1992) J. Biol. Chem. 267, 9998-10002[Abstract/Free Full Text]
  26. Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Nature 363, 274-276[CrossRef][Medline] [Order article via Infotrieve]
  27. Osawa, S., and Weiss, E. R. (1995) J. Biol. Chem. 270, 31052-31058[Abstract/Free Full Text]
  28. Conklin, B. R., Herzmark, P., Ishida, S., Voyno-Yasenetskaya, T. A., Sun, Y., Farfel, Z., and Bourne, H. R. (1996) Mol. Pharmacol. 50, 885-890[Abstract]
  29. Dratz, E. D., Fursteneau, J. E., Lambert, C. G., Thireault, D. L., Rarick, H., Schepers, T., Pakhlevaniants, S., and Hamm, H. E. (1993) Nature 363, 276-280[CrossRef][Medline] [Order article via Infotrieve]
  30. Rasenick, M. M., Watanabe, M., Lazarevic, M. B., Hatta, S., and Hamm, H. E. (1994) J. Biol. Chem. 269, 21519-21525[Abstract/Free Full Text]
  31. Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366[Abstract/Free Full Text]
  32. Gilchrist, A., Mazzoni, M. R., Dineen, B., Dice, A., Linden, J., Proctor, W. R., Lupica, C. R., Dunwiddie, T. V., and Hamm, H. E. (1998) J. Biol. Chem 273, 14912-14919[Abstract/Free Full Text]
  33. Iyengar, R., Abramowitz, J., Bordelon-Riser, M., Blume, A. J., and Birnbaumer, L. (1980) J. Biol. Chem. 255, 10312-10321[Abstract/Free Full Text]
  34. Pfister, C., Kuhn, H., and Chabre, M. (1983) Eur. J. Biochem. 136, 489-499[Abstract]
  35. Birnbaumer, L., Codina, J., Mattera, R., Cerione, R. A., Hildebrandt, J. D., Sunyer, T., Rojas, F. J., Caron, M. G., Lefkowitz, R. J., and Iyengar, R. (1985) Recent Prog. Horm. Res. 41, 41-99[Medline] [Order article via Infotrieve]
  36. Kahlert, M., Konig, B., and Hofmann, K. P. (1990) J. Biol. Chem. 265, 18928-18932[Abstract/Free Full Text]
  37. Panico, J., Parkes, J. H., and Liebman, P. A. (1990) J. Biol. Chem. 265, 18922-18927[Abstract/Free Full Text]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.