From the 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
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
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
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ABSTRACT |
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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 subunit (Gi1
)
upon binding with receptor mimetics, compound 48/80 and mastoparan,
were analyzed by circular dichroism spectroscopy. Compound 48/80
enhanced the initial rate of GTP
S binding to soluble Gi1
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
helix content of
the Gi1
by 7%. Likewise, mastoparan also enhanced the
rate of GTP
S binding by 3.0-fold and decreased the magnitude of
ellipticity of Gi1
similar to compound 48/80. In
corresponding experiments using a K349P-Gi1
, a
Gi1
counterpart of the unc mutant in
Gs
in which Pro was substituted for Lys349,
enhancement of the GTP
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 Gi1
lacking the N-terminal 29 residues was activated and showed decreased ellipticity upon
interaction with the compound, as did the wild-type Gi1
.
Taken together, our results strongly suggest that the activator-induced
unwinding of the
helix of the G protein
subunit is mechanically
coupled to the enhanced release of bound GDP from the
subunit.
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INTRODUCTION |
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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 G, which allows binding of cytosolic GTP to the
remaining G
subunit, thereby resulting in dissociation of trimeric
G
(GTP)
complex into active G
·GTP and a
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
subunit of
Gt and Gi1 have indicated the presence of two
domains, i.e. a GTPase (or Ras-like) domain comprised of
helices and
strands and a highly
helical domain. In addition, the conformational changes induced in the
subunit by nucleotide exchange (GDP
GTP
S) and the mechanism of GTP hydrolysis have been determined (5-9). Conformational changes in the
subunit upon
binding with a
subunit complex have been determined as well (10,
11). However, the mechanism whereby liganded receptor molecules enhance
the GDP release from the
subunit remains unclear, as pointed out
previously (3, 12). Likewise, the conformational change of the
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 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 Gi1 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
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 Gi1
allowed us to determine
how the interaction affects the
helicity of Gi1
with
the resultant conformational changes leading to the enhancement of GDP
release.
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EXPERIMENTAL PROCEDURES |
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Materials--
The following materials were used:
Ni2+-NTA agarose (Qiagen), GTPS and sequencing grade
endoproteinase Lys-C (Boehringer Mannheim), [
-33P]GTP
and [35S]GTP
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).
GTP
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 Subunits--
Because
histidine-tagged proteins can easily be purified by affinity
purification on Ni2+-NTA agarose, we prepared
Gi1
tagged with 10 histidine residues (His10-Gi
) as well as nontagged
full-length Gi1
(FL-Gi
) as a control. We
also prepared a Lys349
Pro mutant in
His10-Gi
(K349P-Gi
), a mutant
corresponding to the unc mutant in Gs
(25,
26) and is expected to be insensitive to activators, and a 325-amino
acid proteolytic fragment of Gi1
lacking the N-terminal
29 residues (
N-Gi
), which allowed us to investigate
whether the protein's N-terminal segment is involved in
activation.
GTPS 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 Gi1
were
compared against those determined by CD spectra analysis.
Determination of GDP Bound to Gi1--
To examine
whether His10-Gi
is denatured in the
presence of C48/80, the amount of GDP bound to Gi1
under
the CD measurement conditions was determined according to Refs. 32 and
33. His10-Gi
(0.3 mM) was
incubated in 50 mM sodium Hepes (pH 8.0), 1 mM
EDTA, 10 mM DTT, 10 mM MgCl2, 2 mM [
-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.
[
-33P]GDP-bound His10-Gi
(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
[]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."
Secondary Structure Estimation--
To predict which helix
is unwound upon interaction with C48/80, secondary structure preference
of rat Gi1
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
helical in the crystal structure of Gi1
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
N2
helix (residues 20-23).
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RESULTS |
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Enhancement of GDP Release from Gi1--
The
effects of MP and C48/80 on GTP
S binding to FL- and
K349P-Gi
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 GTP
S binding
rates in the absence of the activators; (ii) MP and C48/80 similarly
enhance the initial binding rate of FL-, His10-, and
N-Gi
by 2.5-3.0-fold; and (iii) enhancement is very
weak for K349P-Gi
. Accordingly, the affinity of a GDP molecule for Gi1
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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Intactness of His10-Gi during CD
Measurements--
To confirm that His10-Gi
is not denatured in the presence of C48/80, the amount of GDP bound to
His10-Gi
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). GTP
S binding
activity of His10-Gi
in the presence of 100 µg/ml C48/80 was also determined with different preincubation times
with C48/80. The GTP
S binding activity did not change significantly
(<5%) up to 50 min (data not shown).
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Helix Content of Gi1
in the Absence of
Activators and Effect of the Bound Nucleotide on the Secondary
Structure of Gi1
--
Fig.
3A illustrates the CD spectrum
of FL-Gi
in the GDP-bound form. CCA and SELCON analysis
of the spectrum gave
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 Gi1
·GDP structure
(9), which should be expected because both CCA and SELCON are known to
show high accuracy in estimating
helix content (38). Fig.
3A also illustrates the CD spectra of
His10-Gi
in the GDP-,
GDP·AlF4
-, and GTP
S-bound forms. The magnitude
of ellipticity is greater in FL-Gi
·GDP than in
His10-Gi
·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-Gi
, indicating that the secondary
structure of Gi1
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
Gi1
in the GDP-, GDP·AlF4
-, and
GTP
S-bound forms and among corresponding forms with Gt
(5-9).4
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Effects of Activators on the CD Spectrum of
FL-Gi--
The difference spectra of
FL-Gi
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
helical conformation
when bound to Gi1
(41), although taking no ordered
conformation in an aqueous solution (42). When it is considered that
the magnitude of negative ellipticity of the
helix is larger than that of random coil from 205 to 240 nm (38) and that the fraction of
Gi1
-bound MP molecules is only 3.5% at most, this
indicates that MP shows negative ellipticity slightly larger in
magnitude in the presence of Gi1
than in its absence;
accordingly, the magnitude of ellipticity contributed by
Gi1
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 Gi1
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.
Effect of C48/80 on CD of Modified Gi1--
Fig.
4, A-C, illustrates
difference spectra for His10-,
N-, and
K349P-Gi
with C48/80 (100 µg/ml). The CD spectra of
His10- and
N-Gi
show a marked and similar
decrease in the magnitude of ellipticity as for FL-Gi
.
This indicates that the His10 tag segment of
His10-Gi
and the N-terminal 29 residues of
FL-Gi
do not change their conformations upon interaction
with C48/80. This also suggests that
His10-Gi
can be conveniently used as a
substitute for FL-Gi
for conformation analyses, taking
advantage of the fact that the His10-Gi
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 Gi1
, the dependence of
CD spectrum of His10-Gi
on the preincubation time with 100 µg/ml C48/80 was examined. The [
]222
value did not change significantly up to 50 min (Fig.
4D).
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Secondary Structure Prediction of Gi1--
Table
II shows the predicted secondary
structure preferences of those sequences that are
helical in the
GTPase domain of Gi1
in the GDP form (9). Among six
helices, the
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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DISCUSSION |
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Enhancement of Initial GTPS Binding Rate of Soluble
Gi1
by MP and C48/80--
The enhancement of GTP
S
binding to soluble Gi1
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 Gi
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 Gs
(Fig. 1B).
The greater enhancement for reconstituted Gi
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 Go (45), the observation that the
N-Gi
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 Gi
was able to block
MP-stimulated GTPase activity (46), which indicates that MP interacts
with the C-terminal portion of Gi
. When it is considered
that receptors interact with the C-terminal portion of G protein
subunits (reviewed in Ref. 47) and that the presumed
receptor-binding domain (residues 314-354 in Gi1
) (48)
contains the C-terminal
helix (residues 329-350 in
Gi1
·GDP and 328-343 in
Gi1
·GTP
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
helical in the GTPase domain of
Gi1
·GDP (Table II) indicates that the C-terminal
5
helix possesses considerably lower helix-forming propensities than
other helices. Furthermore, the 11-amino acid peptide from the C
terminus of Gt
is known to adopt an
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 5 helix of Gi1
·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
2/
3 loop (9). This bidentate salt bridge seems to stabilize
the potentially unstable
5 helix structure and, by bridging the
5
helix and the
2/
3 loop, the whole tertiary structure as well.
Such a salt bridge is formed also in Gt
·GDP (6) and is
expected to occur in Gs
(50), Go
(51),
and Gz
(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
5 helix. Such an
interaction is compatible with the observation that
[Tyr3,Cys11]MP is cross-linked with
Cys3 of Go
(45). Although the N-terminal
eight residues are disordered and are not observed in the crystal
structure of Gi1
·GDP (9), Asp9 is located
very close to Asp341 and, therefore, to
Asp337.
Coupling of Helix Unwinding and Enhanced GDP Release--
C48/80
enhanced GTPS binding to Gi1
with nearly the same
EC50 as it decreased [
]222 of
Gi1
, 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 Gi1
,
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
helix content of
Gi1
is coupled to the enhanced GTP
S binding,
i.e. enhanced GDP release. In other words, the unwinding of
helical residues (presumably in the
5 helix) upon binding with
C48/80 (or MP) is considered to be propagated to the guanine nucleotide
binding sites of Gi1
such that the affinity of the protein to a bound GDP molecule is lowered. The disruption of the
bidentate salt bridge would release the
2 sheet (residues 185-191)
from the
5 helix and result in the dislocation of guanosine-binding residues (Leu175 and Arg176) positioned to the
N terminus of the
2 sheet; ultimately, this would decrease the
affinity to the bound GDP. In support of this postulation, the affinity
of GDP to Go
is known to decrease remarkably by the
removal of the C-terminal 14 residues of the protein (including Asp341) (53), which adopt an
helical conformation in
Gi1
·GDP (9).
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ACKNOWLEDGEMENTS |
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We are grateful to Prof. A. G. Gilman
(University of Texas Southwestern Medical Center) for the generous gift
of E. coli cells harboring pQE60/Gi1 plasmid,
Dr. A. Omori and S. Yoshida (Mitsubishi Kasei Institute of Life
Sciences) for amino acid sequencing of
N-Gi
, 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.
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FOOTNOTES |
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* 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.
¶¶ 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;
N-Gi
, Gi1
in which the N-terminal 29 residues are removed; DTT, dithiothreitol; FL-Gi
,
full-length Gi1
; GTP
S, guanosine
5
-O-(3-thiotriphosphate); His10-Gi
,
Gi1
with a tag of 10 histidine residues;
K349P-Gi
, Lys349
Pro mutant in
His10-Gi
; MP, mastoparan; SELCON,
self-consistent CD analysis; [
]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 2 helix found in Gi1
in the GTP
S form (7) is disordered in the GDP form (9). However, the
secondary structure contents of the two forms do not differ much
because Gi1
in the GDP form contains N1 and N2 helices
that are not found in the GTP
S form and its
5 helix is longer
than that of the GTP
S form.
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REFERENCES |
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