From the § Howard Hughes Medical Institute and the
Department of Biochemistry, The University of Texas
Southwestern Medical Center, Dallas, Texas 75235-9050
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ABSTRACT |
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The structure of the G protein
Gi The G Structures of the G Mutagenesis experiments demonstrate that two residues of
Gi The active site of the Gi Non-myristoylated, recombinant rat Gi1 complexed with the nonhydrolyzable GTP analog
guanosine-5'-(
-imino)triphosphate (GppNHp) has been determined at
a resolution of 1.5 Å. In the active site of
Gi
1·GppNHp, a water molecule is hydrogen bonded to the
side chain of Glu43 and to an oxygen atom of the
-phosphate group. The side chain of the essential catalytic residue
Gln204 assumes a conformation which is distinctly different
from that observed in complexes with either guanosine
5'-O-3-thiotriphosphate or the transition state analog
GDP·AlF4
. Hydrogen bonding and steric
interactions position Gln204 such that it interacts with a
presumptive nucleophilic water molecule, but cannot interact with the
pentacoordinate transition state. Gln204 must be released
from this auto-inhibited state to participate in catalysis. RGS
proteins may accelerate the rate of GTP hydrolysis by G protein
subunits, in part, by inserting an amino acid side chain into the site
occupied by Gln204, thereby destabilizing the
auto-inhibited state of G
.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunits of heterotrimeric G proteins are GTP
hydrolyases which, upon receptor activation, bind to GTP and regulate effector molecules (1, 2). G
-catalyzed hydrolysis of GTP
to GDP releases the G
·GDP product complex from
effector and allows its sequestration by inhibitory G
subunits. The rate of the hydrolytic reaction thus determines, in part, the length of time during which the effector is regulated by activated G proteins.
subunits Gt
(transducin), Gi
1, and Gs
complexed with
Mg2+ and the nonhydrolyzable GTP analog
GTP
S1 have provided views
of the active site in the ground state (7-9). In particular, the
positions of the presumptive nucleophilic water (Wnuc),
Mg2+, and the side chains of catalytically significant
residues have been determined. Structures of Gi
1 and
Gt
complexed with
GDP·AlF4
·Wnuc, a mimic of the
pentavalent transition state for GTP hydrolysis, have also been
determined (8, 10).
1, Arg178 and Gln204, are
required for catalysis (11, 12). In crystals of
Gi
1·GTP
S, which mimics the ground state E·S
complex, the side chains of these residues are partly disordered and do
not interact with the substrate analog. In contrast, in complexes of
Gi
1 and its homolog Gt
with
GDP·AlF4
, both residues are well ordered and
interact with AlF4
and its axial water ligand
(8, 10). It was proposed that Arg178 and Gln204
stabilize the pentavalent transition state through an analogous set of
interactions (8, 10). It was also suggested that the low catalytic rate
(kcat
2-4 min
1) exhibited by
Gi
1 (14) and its homologs might be attributed to a high
activation energy for the conformational rearrangement of
Arg178 and Gln204 from the ground state to the
transition state (2). However, the nature of this rearrangement is not
established, in part because the Gi
1·GTP
S complex
may not accurately mimic the true E·S complex. Specifically, the
thiol substituent of the
-phosphate in GTP
S may sterically
perturb the catalytic site. The sulfur atom is both more bulky than the
corresponding oxygen atom of GTP (van der Waals radii of 1.8 and 1.4 Å, respectively) and has a longer bond length with the
-phosphate
atom (P-S, 1.86 Å; P-O, 1.52 Å). Hence, to obtain a more accurate
view of the E·S ground state, we have determined a high resolution
x-ray crystal structure of Gi
1 complexed with an
alternative nonhydrolyzable GTP analog: GppNHp. In GppNHp all three
terminal substituents of the
-phosphate group are oxygen atoms.
1·GppNHp complex differs from
that with GTP
S in several respects, but most importantly in the conformation of the catalytic residue Gln204. In this
conformation, Gln204 interacts with Wnuc, but
neither contacts the nucleotide nor is positioned to interact with the
pentavalent transition state. Thus, Gln204 may participate
directly in the Gi
1·GTP ground state but must reorient
to stabilize the transition state. RGS4, a member of the RGS family of
G protein stimulatory factors (15, 16), may accelerate hydrolysis of
GTP by Gi
1, in part, by destabilizing the ground-state
conformation of Gln204.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 was
synthesized in E. coli and purified as described previously
(17). Crystals of Gi
1 complexed with GppNHp and
Mg2+ were grown and prepared for x-ray data collection as
described except that GppNHp was substituted for GTP
S (18). Crystals were transferred to a cryoprotection solution containing 15% (v/v) glycerol and frozen in liquid propane as described (19). Two x-ray
diffraction data sets were collected at 100 K, each from a single
crystal. The first was measured at the Cornell synchrotron source
(CHESS) A1 line (
= 0.91 Å) equipped with an ADSC Quantum 1 CCD
detector. The crystal diffracted beyond 1.5 Å, but data were measured
only to 1.7 Å. A second data set collected to 1.5 Å was collected at
the CHESS F1 line (
= 0.92 Å) equipped with an ADSC Quantum 4 CCD
detector. Data were processed using the DENZO/SCALEPACK programming
package (20). After simultaneous determination of the scale factors for
both data sets using all observations, data between 15-2.24 Å from
the first data set and between 2.24-1.50 Å from the second were
combined to obtain the final 15.00-1.50-Å data set (Table
I).
Data collection and final refinement
statistics
The structure of the Gi1·GppNHp complex was solved by
molecular replacement, using the Gi
1·GTP
S complex
(PDB accession code 1gia) with the nucleotide, Mg2+, and
with waters removed as the starting
model. SigmaA-weighted 2 Fo
Fc and
Fo
Fc difference maps
(21) indicated strong difference electron density for GppNHp, a
previously unobserved active site water molecule, and new side chain
positions for Glu43 and Gln204. Small changes
in the side chain conformations of other residues were also observed.
Simulated annealing omit maps (22) were used to confirm that the
observed differences were not caused by model bias. The protein model
was manually rebuilt and ligands were added using the interactive
graphics model building program O (23). Positional and atomic
temperature factor refinement was carried out on the rebuilt model
using XPLOR (24) followed by additional rounds of model building and
refinement. In the final rounds, a bulk solvent correction (25) was
applied using XPLOR. Electron density about the active site is shown in
Fig. 2. The free-R factor, computed using 5% of the data, was used to
monitor the refinement (26). The structure exhibits good stereochemistry, with 94% of the residues in the most favored regions
of the Ramachandran plot as analyzed by PROCHECK (27). Comparisons and
superposition of the model with other proteins was carried out using O. Data collection and refinement statistics are given in Table I.
The model of the RGS4·Gi1·GppNHp complex was
made by superimposing the C
atoms of residues 34-343 of
Gi
1 from the Gi
1·GppNHp complex onto
the corresponding atoms of the
RGS4·Gi
1·GDP·AlF4
complex (PDB accession code 1agr).
Gi
1·GDP·AlF4
was then
replaced by the superimposed Gi
1·GppNHp complex. Analysis and in silico mutations were then performed on the
resulting RGS4·Gi
1·GppNHp model using O.
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RESULTS |
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The structure of the Gi1·GppNHp complex (Fig.
2A) differs from that of Gi
1·GTP
S (Fig.
2B) in two ways. First, there are small changes in the
side-chain positions of several surface residues that are poorly
ordered in crystals of Gi
1·GTP
S. These
differences are most likely because of damping of thermal motions in
the frozen Gi
1·GppNHp crystals. In contrast, 2.0 Å data from Gi
1·GTP
S crystals were collected at
14 °C. Additionally, the Gi
1·GppNHp data set was
measured to 1.5 Å, permitting unambiguous assignment of side chain conformations.
The second, and more interesting, group of changes relative to
Gi1·GTP
S are observed in the active site and are
most likely attributable to GppNHp (Fig. 2A). For the most
part, the active site is similar to that observed in the
Gi
1·GTP
S complex (Fig. 2B). All of the
interactions between the guanine nucleotide, Mg2+, and the
protein observed in the latter complex are also present in
Gi
1·GppNHp. The presumptive water nucleophile
(Wnuc) is also clearly observed (Fig.
1). A new feature of the active site is a
highly ordered (B = 21.9Å2) water molecule
(W600) bound to the O1G oxygen of the
-phosphate (Fig. 1
and Fig. 2A). Modeling
indicates that this water molecule cannot bind to the
Gi
1·GTP
S complex because it would be sterically
excluded by the
-thiophosphate sulfur atom of GTP
S. The
conformation of Glu43 is altered, and its carboxylate
moiety forms a hydrogen bond to W600 and consequently
cannot form the hydrogen bond to Arg242 that is present in
Gi
1·GTP
S (Fig. 2, A and B).
In Gi
1·GppNHp, Glu43 and
Arg178 form a doubly hydrogen-bonded ion pair (Fig.
2A), in contrast to the less intimate ion pair interaction
observed in the Gi
1·GTP
S complex (Fig.
2B). This enhanced salt-bridge may strengthen binding of GTP
in the ground state. Remarkably, the same interaction occurs in
crystals of Gi
1·GDP complexed with G protein
subunits (28). The conformations of Arg178 in
Gi
1·GppNHp and Gi
1·GTP
S differ
slightly, but in neither case does Arg178 interact with the
GTP substrate analog. In contrast, in the Gt
·GTP
S complex, the corresponding residue, Arg174, interacts
directly with the oxygen bridging the
- and
-phosphate atoms and
the
-thiophosphate sulfur atom. The bridging NH group of GppNHp
would not be capable of this interaction however.
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The orientation of Gln204 in the GppNHp complex is
particularly interesting. In the Gi1·GTP
S complex
the side chain of this residue, which assumes the favored
gauche +
1 conformation, is poorly ordered and directed
out of the active site, and does not appear to interact with any
other residues (Fig. 2B). Similar conformations for the
corresponding catalytic glutamine (Glncat) are observed in
the GTP
S-Gt
, -Gs
, and
-Gs
·adenylyl cyclase complexes (7, 9, 29). In
contrast, Gln204 exhibits strong density for the
gauche
1 conformation in the Gi
1·GppNHp complex and makes several contacts with
other moieties (Figs. 1 and 2A). Notably, while
Gln204 does not interact directly with the substrate
analog, its side chain amino group forms a hydrogen bond to
Wnuc. The orientation of the amide group is defined by the
functionality of the three other moieties to which Wnuc is
hydrogen bonded: the O1G oxygen of the
-phosphate group, which is
proposed to act as a catalytic base (30, 31), the main chain carbonyl
oxygen of Thr181, and the main chain NH group of
Gln204 (Fig. 2A). Gln204 is also
anchored by a hydrogen bond between its carboxamide oxygen atom and the
hydroxyl moiety of Ser206.
The thiophosphate moiety of GTPS does not prevent Gln204
from adopting the conformation observed in the GppNHp complex; however, it does block entry of W600 to the active site.
W600, in turn prevents Gln204 from assuming the
conformation that is partly populated in the GTP
S complex. In the
GppNHp complex, the hydrogen bond between the phosphate O1G oxygen and
Wnuc is shorter than the corresponding interaction in the
GTP
S complex (2.82 versus 3.27 Å). The position of
Wnuc closer to the
-phosphate permits a more favorable
interaction with the side chain of Gln204. The well ordered
conformation of Gln204 in the GppNHp complex may be
attributed to hydrogen bonds formed with Wnuc and
Ser206 but also to the conformational restriction imposed
by W600 as well as the cryogenic data measurement
conditions. In other G protein-GppNHp or -GppCp complexes,
Glncat also interacts with Wnuc (32, 33).
However, in these cases Glncat assumes the gauche +
1 conformation.
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DISCUSSION |
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The most intriguing feature of the Gi1·GppNHp
complex is the conformation of the side chain of Gln204 and
its implications for the mechanism of both intrinsic and RGS-stimulated
GTP hydrolysis. As established by mutagenesis studies (11),
Gln204, and the corresponding residues in other G proteins
(12, 34, 35), is absolutely required for enzymatic activity. Studies of
substrate-assisted catalysis using GTPase-deficient Gs
have demonstrated that a hydrogen donor group near the
-phosphate can substitute for Glncat (36). However, the function of
Glncat during GTP hydrolysis by G proteins has been
debated. Hydrolysis of GTP in G proteins is believed to occur by a
direct, in-line attack on the
-phosphate atom by a nucleophilic
water (37-39). Crystal structures of Gi
1 and other G
proteins have identified, near the
-phosphate group, a well ordered
water molecule (Wnuc) that is positioned to carry out a
nucleophilic attack (7-9, 32, 40, 41). Although Gln204 is
near Wnuc in Gi
1·GTP
S, it does not
directly contact it. Further, Wnuc is observed in
Gln204
Leu Gi
1·GTP
S, and
Gln61
Leu Ras·GppNHp (Gln61 is
Glncat in Ras) (8, 42), indicating that Glncat
is not required for binding of Wnuc in the ground state. It
has been pointed out that the basicity of glutamine is low, and it is
therefore unlikely that Glncat acts as the general base
that deprotonates Wnuc (43). Rather, an oxygen of the
presumably dianionic
-phosphate is proposed to serve this function
in Ras (30, 31). Hence, Glncat may only polarize
Wnuc in the ground state.
X-ray crystallographic studies have indicated that Glncat
stabilizes the transition state (2), as originally proposed by Prive et al. (43). Structures of Gi1 and
Gt
complexed with the transition state analog
GDP·AlF4
, reveal Glncat
positioned within the active site and directly interacting with a
fluorine substituent and Wnuc of the
GDP·AlF4
·Wnuc complex. Fig.
2C shows these interactions in the
RGS4·Gi
1·GDP·AlF4
complex (44). It was proposed that the amino group of
Glncat stabilizes negative character on the equatorial
oxygen of the transition state and its carbamoyl oxygen stabilizes the
attacking nucleophilic water. A similar configuration is observed
in the Ras-GAP·Ras·GDP·AlF4
and the
Rho-GAP·Cdc42Hs·GDP·AlF4
complexes (45-47). In
addition, mutations that perturb the transition state conformation of
Glncat abolish GTPase activity (42, 48). These observations
indicate that Glncat stabilizes the transition state for
GTP hydrolysis.
The GppNHp complex provides novel insights into both the mechanism of
GTP hydrolysis as well as to the role of both Argcat and
Glncat in the ground state E·S complex. GppNHp, but not
GTPS, permits a water molecule, W600, to occupy a
position in which it could act as the ultimate proton acceptor from
Wnuc. Water molecules in similar, but not identical,
positions are present in the GppNHp- or GppCp-bound complexes of Ras
and Rac1 (32, 40). A proton could be relayed from Wnuc to
W600 via O1G of the GTP
-phosphate. This substituent
does not otherwise participate in hydrogen bonds with the protein and
corresponds to the thiol of GTP
S. The basicity of W600
may be enhanced by hydrogen bond formation with Glu43,
which is well conserved in G
proteins with the exception of Gz
where it is replaced with an asparagine residue.
Glu43 also forms a hydrogen-bonded ion pair with
Arg178. In this conformation, Arg178 is
restrained from interacting with the
-phosphate of GTP. Transfer of
a proton from Wnuc to W600 would tend to weaken
this ion pair, releasing Arg178 to stabilize the incipient
pentacoordinate phosphoryl transition state. W600 also
blocks the side chain of Gln204 from interacting with the
pentacoordinate phosphate. Thus, until it diffuses from the active
site, W600 impedes the reorganization of the catalytic site
that is required for transition state stabilization.
Gln204 is anchored in a noncatalytic conformation by
hydrogen bonds to both Wnuc and Ser206
(Ser206 is substituted by an Asp in Gs,). In
the ground state, Gln204 could orient and perhaps activate
Wnuc; however, to stabilize the transition state as
represented by G protein GDP·AlF4
complexes, Gln204 must sever its hydrogen bond with
Ser206 and Wnuc and rotate
120° about
1 and
90° about
2 and
3 (to gauche+ and gauche
,
respectively) such that its carbamoyl group donates a hydrogen bond to
the equatorial oxygen of the pentacoordinate
-phosphoryl group and
accepts a hydrogen bond from Wnuc. Such would incur a
substantial penalty in catalytic efficiency and perhaps account, at
least in part, for the low catalytic rate of GTP hydrolysis in
G
and perhaps in other G proteins.
We propose that the ground state Gi1·GTP complex is
"auto-inhibited" with Glncat locked into an
unproductive conformation. Active site residues in the EF-Tu·GppNHp
complex also assumes anti-catalytic positions; in this case
Hiscat, the residue corresponding to Glncat,
cannot interact with the substrates because of steric interference by
other active site residues (41). In Gi
1, catalysis could occur only if the bonds that hold Glncat in this position
are broken, and the side chain freed to interact with the
pentacoordinate transition state. This model predicts that changes that
disrupt the ground state conformation of Gln204, while not
otherwise compromising the active site, would increase kcat.
RGS proteins, which accelerate the rate of GTP hydrolysis by
Gi1 by 50-100-fold, may act in part by destabilizing
the ground state conformation of Glncat, as well as
stabilizing its productive conformation in the transition state (44).
The crystal structure of the
RGS4·Gi
1·GDP·AlF4
complex demonstrates that RGS4 stabilizes the active site of Gi
1 in the conformation corresponding to that of the
transition state complex (Fig. 2C) (44). No residues from
RGS4 are inserted into the active site except Asn128, which
could enhance catalysis by aiding in binding, orienting, and polarizing
Wnuc in the pre-transition state complex (44).
However, superposition of Gi1·GppNHp and
Gi
1·GDP·AlF4
from the
RGS4·Gi
1·GDP·AlF4
complex reveals that the carbamoyl groups of Gln204 and
Asn128 occupy nearly the same positions, although the side
chains approach from opposite directions (Fig. 2D). Further,
both residues are positioned such that they can bind the nucleophilic
water and Ser206. We suggest that Asn128 of
RGS4 displaces the side chain of Gln204 from its
"anti-catalytic" position in the ground state, freeing it to
participate in stabilization of the transition state.
Mutational analysis of RGS proteins supports this hypothesis. Mutation
of Asn131 in hRGSr (analogous to Asn128 of
RGS4) to either serine or glutamine resulted in a relatively small
decrease in the kcat of Gt (49).
In addition, hRGSr in which Asn131 was mutated to leucine
or alanine also retains substantial stimulatory activity, and the loss
of activity that was observed could be attributed to weakened binding
of these mutants to Gt
. Similar mutagenic studies have
been performed with RGS4 (50). Mutants of Asn128
analogous to those of hRGSr Asn131 were modeled in the
structure of the "RGS4·Gi
1·GppNHp" complex. In
all cases these residues were in steric conflict with
Gln204. These findings indicate that the bulk and binding
of the residue at position 128 is important to the stimulatory activity
of RGS4 although it is unlikely that it has a direct catalytic role in stimulation of GTPase activity (49).
The evidence presented is consistent with a self-inhibited or
anti-catalytic model of the ground state of G proteins, and a role for RGS proteins in stimulating GTPase activity by releasing G
subunits from this ground state
while stabilizing the transition state.
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FOOTNOTES |
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* 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.
The atomic coordinates and structure factors (code1cip) has been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.
¶ To whom correspondence should be addressed: Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235-9050. Tel.: 214-648-5008; Fax: 214-648-6336; E-mail: Sprang{at}howie.swmed.edu.
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ABBREVIATIONS |
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The abbreviations used are:
GTPS, guanosine
5'-O-3-thiotriphosphate;
RGS, regulator of G protein
signaling;
GppNHp, guanosine-5'-(
-imino)triphosphate;
GppCHp, guanosine-5'-(
-methylene)triphosphate;
Wnuc, nucleophilic water;
Glncat and Argcat, conserved catalytic residues in G
subunits.
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REFERENCES |
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