alpha Helix Content of G Protein alpha  Subunit Is Decreased upon Activation by Receptor Mimetics*

Takeshi TanakaDagger §, Toshiyuki KohnoDagger , Shun'ichi Kinoshita§, Hidehito Mukai, Hiroshi Itohpar , Masanao Ohya§, Tatsuo Miyazawa**dagger , Tsutomu HigashijimaDagger Dagger dagger , and Kaori Wakamatsu§§§¶¶

From the Dagger  Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194, Japan; the § Department of Biochemical Sciences, Faculty of Engineering, Gunma University, Kiryu, Gunma 376, Japan; the  Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan; the par  Faculty of Biosciences and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa 226, Japan; the ** Protein Engineering Research Institute, Suita, Osaka 565, Japan; the Dagger Dagger  Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9041; and the §§ Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

To elucidate the mechanism whereby liganded receptor molecules enhance nucleotide exchange of GTP-binding regulatory proteins (G proteins), changes in the secondary structure of the recombinant Gi1 alpha  subunit (Gi1alpha ) upon binding with receptor mimetics, compound 48/80 and mastoparan, were analyzed by circular dichroism spectroscopy. Compound 48/80 enhanced the initial rate of GTPgamma S binding to soluble Gi1alpha 2.6-fold with an EC50 of 30 µg/ml. With the same EC50, the mimetic decreased the magnitude of ellipticity, which is ascribed to a reduction in alpha  helix content of the Gi1alpha by 7%. Likewise, mastoparan also enhanced the rate of GTPgamma S binding by 3.0-fold and decreased the magnitude of ellipticity of Gi1alpha similar to compound 48/80. In corresponding experiments using a K349P-Gi1alpha , a Gi1alpha counterpart of the unc mutant in Gsalpha in which Pro was substituted for Lys349, enhancement of the GTPgamma S binding rate by both activators was quite small. In addition, compound 48/80 showed a negligible effect on the circular dichroism spectrum of the mutant. On the other hand, a proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon interaction with the compound, as did the wild-type Gi1alpha . Taken together, our results strongly suggest that the activator-induced unwinding of the alpha  helix of the G protein alpha  subunit is mechanically coupled to the enhanced release of bound GDP from the alpha  subunit.

    INTRODUCTION
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Procedures
Results
Discussion
References

The central role played by trimeric GTP-binding regulatory proteins (G proteins)1 in signal transduction in membranes has received considerable research attention (reviewed in Refs. 1-3). Upon ligand binding, a G protein-coupled receptor promotes the release of GDP from inactive trimeric Galpha beta gamma , which allows binding of cytosolic GTP to the remaining Galpha subunit, thereby resulting in dissociation of trimeric Galpha (GTP)beta gamma complex into active Galpha ·GTP and a beta gamma subunit complex. In this activation process of G protein, the release of bound GDP is of particular interest as it is the rate-limiting step (4). The analyses of x-ray crystallographic structures of the alpha  subunit of Gt and Gi1 have indicated the presence of two domains, i.e. a GTPase (or Ras-like) domain comprised of alpha helices and beta  strands and a highly alpha  helical domain. In addition, the conformational changes induced in the alpha  subunit by nucleotide exchange (GDP right-arrow GTPgamma S) and the mechanism of GTP hydrolysis have been determined (5-9). Conformational changes in the alpha  subunit upon binding with a beta gamma subunit complex have been determined as well (10, 11). However, the mechanism whereby liganded receptor molecules enhance the GDP release from the alpha  subunit remains unclear, as pointed out previously (3, 12). Likewise, the conformational change of the alpha  subunit upon receptor binding is unknown. The use of physicochemical methods to gain further insight into these key reactions presents difficulties due to the facts that (i) only small amounts of G protein-coupled receptor proteins are expressed in cells, and (ii) no method exists for suitably analyzing the structure of a protein complexed with a large membrane protein.

We considered that mastoparan (MP), a 14-residue peptide discovered in wasp venom as the agent that induces histamine release from mast cells (13), might provide some important clues because it activates Go and Gi in a similar manner; its activation is both Mg2+-dependent and blocked by ADP-ribosylation of G proteins (13, 14). In addition, it has an amphiphilic sequence, as do putative G protein-binding sites of many receptors, namely, second and third intracellular loops and C-terminal tail (15). In fact, peptide fragments corresponding to the third intracellular loop of beta  adrenergic receptors were found to activate Gs (Refs. 16-18, reviewed in Ref. 19). Also of interest, Go and Gi are known to be activated by another histamine releaser that is also amphiphilic, i.e. compound 48/80 (C48/80) (20).

These compounds are particularly useful for analyzing G protein activation when employed as low molecular weight mimetics of receptors. As such, the present study uses circular dichroism (CD) spectra to analyze conformational changes in Gi1alpha upon interaction with these two compounds. Analysis of CD spectra is an ideal method for determining overall structural changes in proteins. For example, CD measurements of a DNA-binding domain of yeast transcription activator GCN4 estimated that its alpha  helix content increases from 70-73% to 95-100% upon interaction with DNA containing its binding site (AP-1 site) (21). This estimation was later confirmed by the NMR analysis of the structure in a DNA-free state (22) and the x-ray crystallographic analysis of the structure in a DNA-bound state (23). In the present study, the CD analysis of Gi1alpha allowed us to determine how the interaction affects the alpha  helicity of Gi1alpha with the resultant conformational changes leading to the enhancement of GDP release.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
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References

Materials-- The following materials were used: Ni2+-NTA agarose (Qiagen), GTPgamma S and sequencing grade endoproteinase Lys-C (Boehringer Mannheim), [alpha -33P]GTP and [35S]GTPgamma S (New England Nuclear), C48/802 (oligomer of p-methoxy-N-methylphenethylamine) (Sigma), Lubrol PX (Nacalai Tesque, Kyoto, Japan), 5'-GDP sodium salt (Seikagaku Corp., Tokyo, Japan), standard bovine serum albumin (2 mg/ml) solution (Pierce), and BA85 nitrocellulose filter (Schleicher & Schuell). GTPgamma S was purified by Mono Q (Pharmacia) anion exchange chromatography. MP was synthesized by standard solid-phase methodology and purified as described previously (24). All other reagents were of analytical grade (Wako Pure Chemicals, Osaka, Japan).

Preparation of G Protein alpha  Subunits-- Because histidine-tagged proteins can easily be purified by affinity purification on Ni2+-NTA agarose, we prepared Gi1alpha tagged with 10 histidine residues (His10-Gialpha ) as well as nontagged full-length Gi1alpha (FL-Gialpha ) as a control. We also prepared a Lys349 right-arrow Pro mutant in His10-Gialpha (K349P-Gialpha ), a mutant corresponding to the unc mutant in Gsalpha (25, 26) and is expected to be insensitive to activators, and a 325-amino acid proteolytic fragment of Gi1alpha lacking the N-terminal 29 residues (Delta N-Gialpha ), which allowed us to investigate whether the protein's N-terminal segment is involved in activation.

FL-Gialpha was expressed in Escherichia coli BL21(DE3) cells harboring the pQE60/Gi1alpha plasmid (27) and purified as described (28), and His10-Gialpha was prepared as follows. After cloning cDNA of Gi1alpha using polymerase chain reaction with appropriate primers and QUICK-Clone cDNA (CLONTECH) as a template, the product cDNA was ligated into the NdeI and BamHI sites of pET19b (Novagen), which we modified beforehand by adding an adapter sequence giving a sequence encoding Met-Gly-(His)10-(Ser)2-Gly-His-Ile-(Asp)4-Lys-His at the N terminus of Gi1alpha . Next, the complete coding sequence, the XbaI-BamHI fragment of pET19b-Gi1alpha , was ligated into the XbaI and BamHI sites of pET24a(+) (Novagen) to produce pET24a(+)/His10-Gialpha , which was transformed into E. coli BL21(DE3) cells. Finally, His10-Gialpha protein was expressed and purified as described (29). K349P-Gialpha was prepared by subcloning into the BamHI and SalI sites of pUC19 a BglII-SalI fragment of pET24a(+)/His10-Gialpha corresponding to the 3' terminal 490 bp of the entire Gi1alpha sequence. Then, to substitute Pro for Lys349, the generated plasmid was subjected to site-directed mutagenesis (27) using 5'-GAGACCACAGTCTGGTAGGTTATT-3' as a mutagenic primer; the mutations were confirmed by DNA sequencing. The entire coding sequence was subsequently obtained by ligating the DraI-SalI fragment of pUC19 possessing the mutated sequence of the 3' terminal of the Gi1alpha sequence into the DraI-SalI sites of pET24a(+)/His10-Gialpha . The mutant protein was correspondingly expressed and purified like wild-type His10-Gialpha .

To prepare Delta N-Gialpha , His10-Gialpha protein was subjected to limited digestion with endoproteinase Lys-C as described (6). The cleavage site was confirmed by amino acid sequencing (Applied Biosystems 477A Protein Sequencer), and the integrity of the C terminus was determined by Western blot analysis using antiserum specific to the C-terminal 10 residues of Gi1alpha .3

In addition to these proteins, which are bound by GDP, we also prepared His10-Gialpha in GTPgamma S- and GDP·AlF4--bound forms to investigate the effect of bound nucleotide on the secondary structure of the protein. The GTPgamma S-bound form was prepared by incubating 10 mg/ml of His10-Gialpha in the GDP-form at 30 °C for 5 h in a buffer of 100 mM sodium Hepes (pH 8.0), 1 mM EDTA, 10 mM DTT, 10 mM MgSO4, and 2 mM GTPgamma S; the complete conversion was confirmed by demonstrating trypsin resistance (30). His10-Gialpha in the GDP·AlF4--form was correspondingly prepared using a buffer containing 30 µM AlCl3 and 10 mM NaF in place of GTPgamma S; the complete conversion was confirmed by verifying that this form did not bind [35S]GTPgamma S (7). The concentration of all proteins was determined by Amido Black staining using bovine serum albumin as the standard (31).

GTPgamma S Binding Assay-- Because the effects of MP and C48/80 have been studied mostly on trimeric G proteins reconstituted in phospholipid vesicles, their effects on soluble Gi1alpha were compared against those determined by CD spectra analysis.

Activator enhancement of the initial GTPgamma S binding rate was determined as follows. Binding buffer containing 50 mM sodium Hepes (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2, 10% glycerol, and 2 µM [35S]GTPgamma S (6000 cpm/pmol) with or without activators was preincubated at 30 °C for 5 min, G proteins (0.2 µM) were added, and the solution was incubated further at 30 °C as described (32). At the indicated times, 50-µl aliquots were diluted with 0.5 ml of ice-cold 20 mM sodium Hepes (pH 8.0), 1 mM EDTA, 160 mM NaCl, 0.2 mM GTP, filtered on BA85 nitrocellulose filter, washed with 25 mM Tris (pH 8.0), 100 mM NaCl, 25 mM MgCl2, and the amount of bound [35S]GTPgamma S was determined on a liquid scintillation counter. Glycerol (10%), which stabilizes His10-Gialpha , was added to the buffer to prevent precipitation. This addition led to approximately a 2-fold decrease in the GTPgamma S binding rate for all proteins, in both the absence and presence of the activators.

GTPgamma S binding activity of His10-Gialpha under the CD measurement conditions was determined as follows. His10-Gialpha (3.5 µM) was incubated in 20 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM DTT, and 10% glycerol in the absence or presence of 100 µg/ml C48/80 at 25 °C. At the indicated times, 50-µl aliquots were withdrawn and added to 450 µl of 50 mM sodium Hepes (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.1 mM MgCl2, and 2 µM [35S]GTPgamma S (1, 500 cpm/pmol), incubated at 30 °C for 30 min, and the amount of bound [35S]GTPgamma S was determined as described above. Assays were performed in duplicate at least twice using a fresh protein solution each time. Data consisted of average values of duplicate determinations and varied less than 5%.

Determination of GDP Bound to Gi1alpha -- To examine whether His10-Gialpha is denatured in the presence of C48/80, the amount of GDP bound to Gi1alpha under the CD measurement conditions was determined according to Refs. 32 and 33. His10-Gialpha (0.3 mM) was incubated in 50 mM sodium Hepes (pH 8.0), 1 mM EDTA, 10 mM DTT, 10 mM MgCl2, 2 mM [alpha -33P] GTP (87.6 cpm/pmol) at 30 °C for 3 h; bound GTP was hydrolyzed to GDP during this incubation. The binding reaction was quenched by adding 12 mM EDTA (pH 8.0) and the free nucleotide was removed by gel filtration. [alpha -33P]GDP-bound His10-Gialpha (3.5 µM, 82.3 cpm/pmol) was then incubated in 20 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM DTT, 10% glycerol in the absence or presence of 100 µg/ml C48/80 at 25 °C. At the indicated times, 50-µl aliquots were withdrawn, diluted with 0.5 ml of ice-cold 20 mM sodium Hepes (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.3 mM AlCl3, 10 mM MgCl2, 10 mM NaF and filtered on BA85 nitrocellulose filter, and the bound radioactivity was determined on a liquid scintillation counter.

Circular Dichroism-- G protein (3.5 µM) was incubated in a buffer of 20 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM DTT, 10% glycerol at 25 °C for 10 min unless otherwise indicated. CD spectra were then recorded at 25 °C on a J-720 spectropolarimeter (JASCO, Tokyo, Japan) using a cuvette with a path length of 2 mm. For each sample, four scans were accumulated in approximately 3 min. To eliminate contributions by the activator or buffer, the CD spectrum of a protein is shown as the difference spectrum: the spectrum recorded in the presence or absence of an activator minus the spectrum of an activator or buffer alone. Measurements were repeated at least twice using freshly prepared solutions, and the results were fully reproducible. The resultant difference spectra are representative of two or three measurements, which varied less than 1% at 210 nm. The dependence of [theta ']222 on C48/80 concentration was determined in duplicate twice, and an average value for a single run is presented. Corresponding analysis was not performed using MP as described under "Results."

The alpha  helix content of Gi1alpha was estimated by convex constraint analysis (CCA) (34) and a self-consistent method (SELCON) (35). Both techniques utilized CD data at wavelengths from 200 to 240 nm with the assumption of three independent basis curves. In contrast to methods that use ellipticities at a single wavelength (36, 37), these methods are known to estimate secondary structure contents accurately (38).

Secondary Structure Estimation-- To predict which alpha  helix is unwound upon interaction with C48/80, secondary structure preference of rat Gi1alpha was estimated by two independent methods: Garnier-Osguthorpe-Robson (GOR) (39) and neural network (NNPREDICT) (40). Those amino acid sequences in the GTPase-domain that are alpha  helical in the crystal structure of Gi1alpha in the GDP form (Protein Data Bank accession code, 1GDD) (9) with five residues extended to both the N- and C-terminal directions were analyzed by these methods. The prediction was not performed of the very short alpha N2 helix (residues 20-23).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Enhancement of GDP Release from Gi1alpha -- The effects of MP and C48/80 on GTPgamma S binding to FL- and K349P-Gialpha are illustrated in Fig. 1, A and B, respectively, and Table I summarizes the initial binding rates and fold enhancement of each G protein examined. Note that (i) all four proteins show similar initial GTPgamma S binding rates in the absence of the activators; (ii) MP and C48/80 similarly enhance the initial binding rate of FL-, His10-, and Delta N-Gialpha by 2.5-3.0-fold; and (iii) enhancement is very weak for K349P-Gialpha . Accordingly, the affinity of a GDP molecule for Gi1alpha was not altered by the addition of the His10 tag, substitution of Pro for Lys349, or the deletion of the N-terminal 29 residues. However, the substitution of Pro for Lys349 substantially weakened activation by MP and C48/80.


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Fig. 1.   The effects of MP and C48/80 on GTPgamma S binding to Gi1alpha . Gi1alpha proteins (0.2 µM) were incubated with 2 µM [35S]GTPgamma S (6000 cpm/pmol) at 30 °C in the absence or presence of MP or C48/80. At the indicated times, 50-µl aliquots of the reaction mixture were withdrawn and analyzed for bound [35S]GTPgamma S. A, time courses of GTPgamma S binding to FL-Gialpha in the absence or presence of MP (100 µM) or C48/80 (100 µg/ml). B, time courses of GTPgamma S binding to K349P-Gialpha in the absence or presence of MP or C48/80 (concentrations were the same as in A). The ordinates in these results indicate the fraction of GTPgamma S-bound Gi1alpha relative to the total GTPgamma S binding activity that had been determined by incubating Gi1alpha in a buffer containing 100 mM sodium Hepes (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.1% Lubrol PX, 0.1 mM MgCl2, and 2 µM [35S]GTPgamma S (3000 cpm/pmol) at 30 °C for 3 h (4). C, The initial rate of GTPgamma S binding to FL-Gialpha was dependent on C48/80 concentration.

                              
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Table I
Initial rates and fold enhancements of GTPgamma S binding of various Gi1alpha proteins in the presence of activators
GTPgamma S binding was measured as described in the legend to Fig. 1 by incubating for up to 50 min at 30 °C with or without 100 µM MP or 100 µg/ml C48/80. The initial binding rates were calculated by exponential fitting.

Intactness of His10-Gialpha during CD Measurements-- To confirm that His10-Gialpha is not denatured in the presence of C48/80, the amount of GDP bound to His10-Gialpha was determined under the CD measurement conditions. In the CD buffer that did not contain guanine nucleotides, dissociation of GDP did not occur in either the absence or the presence of 100 µg/ml C48/80 up to 50 min (Fig. 2). When free GDP was included in the buffer, marked release of GDP was observed, and its release rate was increased in the presence of C48/80 (data not shown). GTPgamma S binding activity of His10-Gialpha in the presence of 100 µg/ml C48/80 was also determined with different preincubation times with C48/80. The GTPgamma S binding activity did not change significantly (<5%) up to 50 min (data not shown).


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Fig. 2.   Absence of GDP release from His10-Gialpha in a buffer containing 100 µg/ml C48/80 under the CD measurement conditions. [alpha -33P]GDP-bound His10-Gialpha (3.5 µM, 82.3 cpm/pmol) was incubated in 20 mM Tris (pH 7.4), 0.1 mM EGTA, 0.1 mM DTT, 10% glycerol in the absence or presence of 100 µg/ml C48/80 at 25 °C. At the indicated times, 50-µl aliquots were withdrawn, diluted with 0.5 ml of ice-cold 20 mM sodium Hepes (pH 8.0), 1 mM EDTA, 100 mM NaCl, 0.3 mM AlCl3, 10 mM MgCl2, 10 mM NaF and filtered on BA85 nitrocellulose filter, and the bound radioactivity was determined on a liquid scintillation counter.

alpha Helix Content of Gi1alpha in the Absence of Activators and Effect of the Bound Nucleotide on the Secondary Structure of Gi1alpha -- Fig. 3A illustrates the CD spectrum of FL-Gialpha in the GDP-bound form. CCA and SELCON analysis of the spectrum gave alpha  helix values of 50.6 and 55.9%, respectively; these values were in good agreement with that obtained by x-ray crystallographic analysis of the Gi1alpha ·GDP structure (9), which should be expected because both CCA and SELCON are known to show high accuracy in estimating alpha  helix content (38). Fig. 3A also illustrates the CD spectra of His10-Gialpha in the GDP-, GDP·AlF4--, and GTPgamma S-bound forms. The magnitude of ellipticity is greater in FL-Gialpha ·GDP than in His10-Gialpha ·GDP. This is presumably due to the absence of an ordered structure in the His10 tag segment. There were few spectral differences among the three forms of His10-Gialpha , indicating that the secondary structure of Gi1alpha does not substantially change irrespective of the chemical structure of the phosphate moiety of the guanine nucleotides bound (see Fig. 3, B and C, for expansion around 210 and 220 nm, respectively). This is consistent with the x-ray analysis results indicating the presence of few differences among the secondary structure contents of Gi1alpha in the GDP-, GDP·AlF4--, and GTPgamma S-bound forms and among corresponding forms with Gtalpha (5-9).4


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Fig. 3.   CD spectra of FL-Gialpha in the absence and presence of MP or C48/80. Spectra were recorded as described under "Experimental Procedures." A, FL-Gialpha in the GDP-bound form (FL-GDP) and His10-Gialpha in the GDP-bound (His10-GDP), GDP·AlF4--bound (His10-GDP·AlF4-), or GTPgamma S-bound (His10-GTPgamma S) forms. B, and C, expansion of the A for His10-Gialpha around 210 and 220 nm, respectively. D, CD spectra of FL-Gialpha in the absence and presence of 100 µM MP (FL + MP) or 100 µg/ml C48/80 (FL + C48/80). E, C48/80 concentration dependence of the [theta ]222 of FL-Gialpha . Data points indicate the average of duplicate measurements, and bars indicate the range of duplicate values.

Effects of Activators on the CD Spectrum of FL-Gialpha -- The difference spectra of FL-Gialpha in the presence of MP (100 µM) or C48/80 (100 µg/ml) are illustrated in Fig. 3D. Their presence decreased the magnitude of the ellipticity at 205-235 nm, which indicates changes in its secondary structure. The difference spectrum in the presence of MP, however, is not considered to accurately reflect the structure of the protein for the following reason. The MP molecule is known to adopt an alpha  helical conformation when bound to Gi1alpha (41), although taking no ordered conformation in an aqueous solution (42). When it is considered that the magnitude of negative ellipticity of the alpha  helix is larger than that of random coil from 205 to 240 nm (38) and that the fraction of Gi1alpha -bound MP molecules is only 3.5% at most, this indicates that MP shows negative ellipticity slightly larger in magnitude in the presence of Gi1alpha than in its absence; accordingly, the magnitude of ellipticity contributed by Gi1alpha in the presence of MP should be smaller than that shown in Fig. 3D. The conclusion still holds, however, that the magnitude of ellipticity of Gi1alpha is decreased upon interaction with MP. On the other hand, the difference spectra in the presence of C48/80 are accurate because C48/80 shows no ellipticity from 200 to 250 nm (data not shown); only the difference spectra obtained with this activator were subsequently considered.

In agreement with the observation that GDP molecules are not released from His10-Gialpha in the absence of free guanine nucleotides even in the presence of 100 µg/ml C48/80 (Fig. 2), the addition of 50 µM GDP did not affect the CD spectra in either the absence or the presence of C48/80 (data not shown). These data confirm that the difference spectrum in the presence of this activator (Fig. 3D) reflects the secondary structure of Gi1alpha ·GDP in the activated state rather than the structure of guanine nucleotide-free Gi1alpha .

Fig. 3E illustrates the dependence of [theta ']222 on the concentration of C48/80 in FL-Gialpha . With an EC50 value of 31.8 ± 2.6 µg/ml, the spectral change reaches a plateau at about 100 µg/ml. At this saturation concentration, CCA and SELCON analyses gave alpha  helix values of 43.8 and 49.3%, respectively. Both of these values are 7% lower than the original values; this reduction corresponds to the unwinding of the alpha  helix by 20 of 354 amino acid residues.

Effect of C48/80 on CD of Modified Gi1alpha -- Fig. 4, A-C, illustrates difference spectra for His10-, Delta N-, and K349P-Gialpha with C48/80 (100 µg/ml). The CD spectra of His10- and Delta N-Gialpha show a marked and similar decrease in the magnitude of ellipticity as for FL-Gialpha . This indicates that the His10 tag segment of His10-Gialpha and the N-terminal 29 residues of FL-Gialpha do not change their conformations upon interaction with C48/80. This also suggests that His10-Gialpha can be conveniently used as a substitute for FL-Gialpha for conformation analyses, taking advantage of the fact that the His10-Gialpha can easily be purified in large amounts. To further confirm that the spectral change upon addition of C48/80 is not due to the release of GDP and resultant denaturation of Gi1alpha , the dependence of CD spectrum of His10-Gialpha on the preincubation time with 100 µg/ml C48/80 was examined. The [theta ']222 value did not change significantly up to 50 min (Fig. 4D).


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Fig. 4.   The effect of C48/80 on CD spectra of modified Gi1alpha proteins. Spectra were recorded as described under "Experimental Procedures." Shown are His10-Gialpha (A), Delta N-Gialpha (B), and K349P-Gialpha (C) in the absence (His10, K349P, and Delta N) or presence (His10 + C48/80, K349P + C48/80, and Delta N + C48/80) of 100 µg/ml C48/80. D, time courses of [theta ']222 of His10-Gialpha in the absence or presence of 100 µg/ml C48/80. Data points indicate the average of duplicate measurements; the difference of the duplicate values was less than 0.5%.

In marked contrast to these wild-type proteins, K349P-Gialpha shows only a small decrease upon addition of C48/80 (Fig. 4C). This observation indicates that the alpha  helical structure in K349P-Gialpha is not unwound upon interaction with C48/80.

Secondary Structure Prediction of Gi1alpha -- Table II shows the predicted secondary structure preferences of those sequences that are alpha  helical in the GTPase domain of Gi1alpha in the GDP form (9). Among six helices, the alpha 5 helix is predicted by both the Garnier-Osguthorpe-Robson and NNPREDICT methods to possess the lowest propensity to form helices.

                              
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Table II
Secondary structure prediction of the sequences that are alpha -helical in the crystal structure of Gi1alpha · GDP
Those sequences that are alpha -helical in the crystal structure of the GTPase domain of rat Gi1alpha in the GDP form (9) with five amino acid residues extended both to the N- and C-terminal directions were subjected to secondary structure predictions by GOR and NNPREDICT methods (see "Experimental Procedures").

    DISCUSSION
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Procedures
Results
Discussion
References

Enhancement of Initial GTPgamma S Binding Rate of Soluble Gi1alpha by MP and C48/80-- The enhancement of GTPgamma S binding to soluble Gi1alpha by MP and C48/80 was 3.0-fold and 2.6-fold, respectively, being markedly lower in comparison with 11-fold (13) and 6-fold (20) enhancements of the release of bound GDP from trimeric Gialpha beta gamma reconstituted in phospholipid vesicles at the same activator concentrations. The observed values, however, are nevertheless significant because the enhancement was saturable (Fig. 1C) and quite small for the K349P, mutant which corresponds to the unc mutant in Gsalpha (Fig. 1B). The greater enhancement for reconstituted Gialpha beta gamma can be attributed to a higher concentration of activators, which have a high affinity for phospholipid membranes (42, 43) near the vesicle surface and to an appropriate conformation of the activators induced by lipidic environment (41, 44).

The Site of Unwinding-- Although MP cross-links to the Cys3 of Goalpha (45), the observation that the Delta N-Gialpha lacking the N-terminal 29 residues can be activated by MP, as well as by C48/80 (Table I), indicates that the N-terminal residues of the protein are not essential for activation or interaction. In fact, polyclonal antibody directed against the C-terminal nine residues of Gialpha was able to block MP-stimulated GTPase activity (46), which indicates that MP interacts with the C-terminal portion of Gialpha . When it is considered that receptors interact with the C-terminal portion of G protein alpha  subunits (reviewed in Ref. 47) and that the presumed receptor-binding domain (residues 314-354 in Gi1alpha ) (48) contains the C-terminal alpha  helix (residues 329-350 in Gi1alpha ·GDP and 328-343 in Gi1alpha ·GTPgamma S·Mg2+), our observations suggest that MP and C48/80 unwind some portion of the C-terminal helix. In agreement with this postulation, the secondary structure prediction of the sequences that are alpha  helical in the GTPase domain of Gi1alpha ·GDP (Table II) indicates that the C-terminal alpha 5 helix possesses considerably lower helix-forming propensities than other helices. Furthermore, the 11-amino acid peptide from the C terminus of Gtalpha is known to adopt an alpha  helical conformation when bound to unexcited rhodopsin, whereas it adopts an extended conformation when bound to photoexcited rhodopsin (49).

Mechanism of Helix Unwinding-- Although speculative, the following mechanism is conceivably that of the helix unwinding. On the same side of the alpha 5 helix of Gi1alpha ·GDP (9), Asp337 and Asp341 are adjacently located and are solvent accessible. The electrostatic repulsion between the two negative charges on these residues would destabilize the helix structure if they were not neutralized by some positive charges. Actually, the two carboxylate groups of these residues form a bidentate salt bridge with the side chain amino group of Lys192 in the beta 2/beta 3 loop (9). This bidentate salt bridge seems to stabilize the potentially unstable alpha 5 helix structure and, by bridging the alpha 5 helix and the beta 2/beta 3 loop, the whole tertiary structure as well. Such a salt bridge is formed also in Gtalpha ·GDP (6) and is expected to occur in Gsalpha (50), Goalpha (51), and Gzalpha (52), as well. Because both MP and C48/80 have multiple positive charges, interactions of each positive charge with either Asp337 or Asp341 may result in the destabilization and subsequent unwinding of the alpha 5 helix. Such an interaction is compatible with the observation that [Tyr3,Cys11]MP is cross-linked with Cys3 of Goalpha (45). Although the N-terminal eight residues are disordered and are not observed in the crystal structure of Gi1alpha ·GDP (9), Asp9 is located very close to Asp341 and, therefore, to Asp337.

Coupling of Helix Unwinding and Enhanced GDP Release-- C48/80 enhanced GTPgamma S binding to Gi1alpha with nearly the same EC50 as it decreased [theta ']222 of Gi1alpha , i.e. 33.0 ± 1.5 µg/ml (Fig. 1C) and 31.8 ± 2.6 µg/ml (Fig. 3E), respectively. In addition, the K349P mutant in Gi1alpha , which is activated minimally by C48/80 and MP (Fig. 1B), demonstrated an insignificant decrease in the magnitude of ellipticity in the presence of C48/80 (Fig. 4C). Taken together, these results strongly suggest that the decrease in the alpha  helix content of Gi1alpha is coupled to the enhanced GTPgamma S binding, i.e. enhanced GDP release. In other words, the unwinding of alpha  helical residues (presumably in the alpha 5 helix) upon binding with C48/80 (or MP) is considered to be propagated to the guanine nucleotide binding sites of Gi1alpha such that the affinity of the protein to a bound GDP molecule is lowered. The disruption of the bidentate salt bridge would release the beta 2 sheet (residues 185-191) from the alpha 5 helix and result in the dislocation of guanosine-binding residues (Leu175 and Arg176) positioned to the N terminus of the beta 2 sheet; ultimately, this would decrease the affinity to the bound GDP. In support of this postulation, the affinity of GDP to Goalpha is known to decrease remarkably by the removal of the C-terminal 14 residues of the protein (including Asp341) (53), which adopt an alpha  helical conformation in Gi1alpha ·GDP (9).

In summary, the present study reports for the first time the conformational change of G protein alpha  subunit upon activation by receptor mimetics. Additional studies should prove MP and C48/80 useful in elucidating the interaction between receptor and G protein alpha  subunits in more detail.

    ACKNOWLEDGEMENTS

We are grateful to Prof. A. G. Gilman (University of Texas Southwestern Medical Center) for the generous gift of E. coli cells harboring pQE60/Gi1alpha plasmid, Dr. A. Omori and S. Yoshida (Mitsubishi Kasei Institute of Life Sciences) for amino acid sequencing of Delta N-Gialpha , Dr. G. D. Fasman (Brandeis University) for the use of CCA, Dr. R. W. Woody (Colorado State University) for the use of SELCON, and Dr. N. J. Greenfield (UMDNJ-Robert Wood Johnson Medical School) for providing us with these programs.

    FOOTNOTES

* This research was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan; the Protein Engineering Research Institute, Saneyoshi Foundation, New Energy and Industrial Technology Development Organization (NEDO), and The Institute of Physical and Chemical Research (RIKEN).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.

dagger Deceased.

¶¶ To whom correspondence should be addressed. Tel.: and Fax: 81-277-30-1439; E-mail: wakamats{at}bce.gunma-u.ac.jp.

1 The abbreviations used are: G protein, trimeric GTP-binding regulatory protein; C48/80, compound 48/80; CCA, convex constraint analysis; CD, circular dichroism; Delta N-Gialpha , Gi1alpha in which the N-terminal 29 residues are removed; DTT, dithiothreitol; FL-Gialpha , full-length Gi1alpha ; GTPgamma S, guanosine 5'-O-(3-thiotriphosphate); His10-Gialpha , Gi1alpha with a tag of 10 histidine residues; K349P-Gialpha , Lys349 right-arrow Pro mutant in His10-Gialpha ; MP, mastoparan; SELCON, self-consistent CD analysis; [theta ]222, molar ellipticity at 222 nm.

2 We found that some lots of C48/80 give noisy spectra in 200-210 nm; when large noises are observed in its CD spectrum, another lot should be tested.

3 H. Itoh, unpublished results.

4 The long alpha 2 helix found in Gi1alpha in the GTPgamma S form (7) is disordered in the GDP form (9). However, the secondary structure contents of the two forms do not differ much because Gi1alpha in the GDP form contains N1 and N2 helices that are not found in the GTPgamma S form and its alpha 5 helix is longer than that of the GTPgamma S form.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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