From the Department of Cell Biology and Physiology, Washington
University School of Medicine, St. Louis, Missouri 63110
GTP hydrolysis by guanine nucleotide-binding
proteins, an essential step in many biological processes, is stimulated
by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs
stimulate GTP hydrolysis are unknown. We have used mutational,
biochemical, and structural data to investigate how RGS4, a GAP for
heterotrimeric G protein
subunits, stimulates GTP hydrolysis. Many
of the residues of RGS4 that interact with
Gi
1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-G
interface.
GAP-defective RGS4 mutants invariably were defective in binding
G
subunits in their transition state; furthermore, the
apparent strengths of GAP and binding defects were correlated. Thus,
none of these residues of RGS4, including asparagine 128, the only
residue positioned at the active site of Gi
1, is
required exclusively for catalyzing GTP hydrolysis. These results and
structural data (Tesmer, J. G. G., Berman, D. M.,
Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251-261) indicate that RGS4 stimulates GTP hydrolysis primarily by
stabilizing the transition state conformation of the switch regions of
the G protein, favoring the transition state of the reactants.
Therefore, although monomeric and heterotrimeric G proteins are
related, their GAPs have evolved distinct mechanisms of action.
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INTRODUCTION |
By cycling between active GTP-bound and inactive GDP-bound states,
guanine nucleotide-binding proteins control many biological processes,
including translation, vesicular trafficking, cytoskeletal organization, and signal transduction (1-3). GTP hydrolysis is required to deactivate guanine nucleotide-binding proteins, as shown by the effects of hydrolysis-resistant GTP analogs, or mutations and toxins that block this reaction. Because guanine nucleotide-binding proteins have slow intrinsic rates of GTP hydrolysis, they are acted
upon by GTPase-activating proteins
(GAPs),1 achieving rates of
GTP hydrolysis in the physiological range (4).
RGS (regulators of G protein
signaling) proteins are GAPs for
subunits of
heterotrimeric guanine nucleotide-binding proteins (G proteins),
principally Gq and members of the Gi family (5, 6). RGS proteins, which are unrelated in primary sequence to GAPs that
act on monomeric guanine nucleotide-binding proteins such as Ras, have
been identified in the yeast Saccharomyces cerevisiae (Sst2p) (7-9), Aspergillus nidulans (FlbA) (10),
Caenorhabditis elegans (Egl-10) (11) and in mammalian cells
(at least 16 family members) (reviewed in Refs. 5 and 6). In addition
to acting as GAPs, certain RGS family members are capable of inhibiting signaling by binding activated (GTP-bound) G
subunits,
antagonizing effector binding (12). Therefore, RGS family members are
thought to govern the strength and duration of physiological responses triggered by an array of G protein-dependent signaling
pathways.
The mechanisms whereby RGS proteins, or other GAPs, stimulate GTP
hydrolysis are unknown. They could introduce residues into the active
sites of guanine nucleotide-binding proteins, participating directly in
catalyzing GTP hydrolysis as has been suggested by mutational studies
of p120GAP (13-15); however, proof of this mechanism
requires determining the structure of Ras-p120GAP
complexes. Alternatively, RGS proteins or other GAPs could act allosterically by stabilizing the transition state structures of the
"switch" regions of guanine nucleotide-binding proteins that change
conformation during the GTPase cycle, stimulating intrinsic GTPase
activity. Consistent with this mechanism, p120GAP and
certain RGS family members bind preferentially to the transition state
conformations of Ras and G
subunits, respectively
(16-18).
The structure of RGS4-Gi
1 complexes in the transition
state has recently been solved to a resolution of 2.8 Å (19). The
structure suggests that RGS4 could act allosterically and/or catalytically to stimulate GTP hydrolysis. An allosteric mechanism is
suggested because binding of RGS4 stabilizes the switch regions of
Gi
1. However, a catalytic role is possible because an asparagine residue (Asn-128) of RGS4 interacts with an active-site glutamine residue (Gln-204) of Gi
1 that is thought to
bind or polarize the attacking water molecule in the GTPase reaction. Furthermore, modeling studies suggest that asparagine 128 of RGS4 potentially binds a water molecule when it associates with
GTP-bound Gi
1, earlier in the GTPase reaction mechanism
(19). Therefore, determining whether RGS4 acts allosterically or
catalytically requires mutational and biochemical data that reveal
which residues of RGS4 are required specifically to bind
G
subunits and/or catalyze GTP hydrolysis.
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EXPERIMENTAL PROCEDURES |
Mutagenesis of RGS4--
RGS4 was expressed in yeast from the
ADH promoter using the polymerase chain reaction to clone
the coding region of an RGS4 cDNA (rat (20); rat RGS4 was used for
structural studies (19)) into pVT102U cut with BamHI and
XbaI, creating pADH-RGS4. A C-terminally Myc-tagged form of
RGS4 (which did not affect RGS4 function in yeast; data not shown) was
generated by ligating an XbaI fragment encoding three
in-frame copies of the c-Myc epitope (in pUC119; gift of D. Pellman,
Whitehead Institute) with XbaI-cut pADH-RGS4, creating
pADH-RGS4-3xMyc. N-terminally His-tagged RGS4 was expressed in
Escherichia coli using plasmids described previously (17). Point mutations in the RGS4 coding regions of yeast and E. coli expression plasmids were generated using the QuickChange
mutagenesis kit (Stratagene). N- and C-terminal truncation mutations of
RGS4 were generated by the polymerase chain reaction and cloned into pET15B (Novagen). The RGS4 coding regions of all constructs were sequenced to verify that only the desired mutations had been
introduced. Pheromone response of yeast cells (BC180, an
sst2 mutant) expressing wild-type and mutant forms of RGS4
was determined by performing quantitative growth arrest (halo) assays
(20). Expression of Myc-tagged RGS4 in yeast cells was examined by
performing immunoblot experiments using 9E10 monoclonal antibodies and
enhanced chemiluminescence detection (Amersham Corp.).
Purification of Proteins, GTPase Measurements, and G Protein
Binding Assays--
N-terminally His-tagged forms of RGS4 (rat) and
Go
were expressed in and purified from E. coli (BL21(DE3)) by Ni2+-nitrilotriacetic acid
chromatography as described previously (17). Recombinant
myristoylated Gi
2 (provided by M. Linder) was
purified from E. coli as described previously (21). The activities of G proteins were assessed by determining the stoichiometry of [35S]GTP
S binding, which was >70%.
GTP hydrolysis by G
subunits during a single catalytic
turnover was determined as described previously (17). Briefly, His-tagged Go
(100 nM in 400 µl) was
incubated with [
-32P]GTP (0.1 µM,
20,000-30,000 cpm/pmol) in the absence of Mg2+;
stoichiometries of nucleotide binding were >70%. Aliquots (50 µl)
were removed 15 s before and 45 s after hydrolysis of GTP was
initiated at 5 °C by adding MgSO4 (10 mM
final concentration), unlabeled GTP (100 µM final
concentration), and either a buffer control or various amounts of
wild-type or mutant forms of recombinant His-tagged RGS4. The amount of
32Pi released was determined by liquid
scintillation spectrometry.
Binding of RGS4 to Gi
2 subunits was determined as
described previously (17). Briefly, purified myristoylated
Gi
2 (5 µg) was incubated with GDP, GTP
S, or GDP and
AlF4
. Purified His-tagged wild-type or
mutant forms of RGS4 (10 µg) were added. After a 30-min incubation on
ice, Ni2+-nitrilotriacetic acid beads were added, incubated
for 30 min with agitation at 4 °C, washed three times, and boiled in
Laemmli sample buffer to elute bound proteins. Eluted proteins resolved by SDS-polyacrylamide gel electrophoresis were transferred to nitrocellulose; Gi
2 subunits were detected by
immunoblotting with antiserum P960 (G
-common antiserum) and
peroxidase-labeled secondary antibodies and by enhanced
chemiluminescence.
Graphics--
Images were produced using RasMol and the
coordinates of the transition state structure of
RGS4-Gi
1 complexes (19).
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RESULTS AND DISCUSSION |
Identification of Mutations That Disrupt the GAP Activity of
RGS4--
To determine the mechanism whereby RGS4 stimulates GTP
hydrolysis by G protein
subunits, we analyzed the effects of
mutations affecting RGS4. If certain residues of RGS4 are required
exclusively for catalysis, they should be dispensable for binding
G
subunits, similar to what has been shown for
p120GAP (13-15). Alternatively, if RGS4 catalyzes GTP
hydrolysis strictly by binding and stabilizing the transition state of
G
subunits, then mutations that disrupt GAP activity
should invariably cause a corresponding defect in G
binding.
Because these experiments were initiated before the structure of
RGS4-Gi
1 complexes was reported, our first objective was
to define the minimal region of RGS4 (a 205-residue polypeptide) that
possesses normal GAP activity in vitro. This domain could then be subjected to extensive point mutagenesis.
Analysis of purified forms of several truncation mutants indicated that
the domain characteristic of RGS family members (RGS homology domain,
residues 58-177 of RGS4) had normal GAP activity toward
Go
(Table I), similar to
results obtained using ret-RGS-d (22). Truncations extending into the
RGS homology domain resulted in the production of RGS4 in an insoluble
form (Table I), indicating a defect in protein folding or stability.
The RGS homology domain of RGS4 therefore appears to be a single
functional domain.
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Table I
Effects of point mutations on GAP activity of RGS4
His-tagged forms of the indicated RGS4 mutants were assayed for GAP
activity as described under "Experimental Procedures." Data are
expressed relative to the values obtained using an excess (200 nM) of wild-type RGS4-(1-205) (0.6-0.9 pmol Pi
released). Each RGS4 mutant was analyzed at least three times; standard
errors are indicated in parentheses. Deletion mutants are indicated by the residues of RGS4 remaining. Point mutations were made in
RGS4-(13-205). Those mutations that did not affect GAP activity were
among those that did not affect RGS4 function in yeast.
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To identify amino acids in RGS4 that are functionally important, we
constructed a set of 43 point mutations (most of which were alanine
substitutions) that affect many of the charged or conserved residues of
its RGS homology domain (Fig. 1). As a
means of screening rapidly for loss-of-function mutations, C-terminally Myc-tagged forms of the RGS4 mutants were tested for their ability to
inhibit G protein-dependent signaling (mating pheromone
response) when expressed in yeast (20) (see "Experimental
Procedures"; results are summarized in Fig. 1). The results indicated
that many of the mutants were as functional as wild-type RGS4, whereas other mutants were nonfunctional. Mutants showing an intermediate activity in this in vivo assay were not obtained, even
though subsequent experiments (see below) revealed that many of the
nonfunctional mutants were partially defective in GAP activity in
vitro. This was expected because the in vitro assay is
much more sensitive. None of the loss-of-function mutations
significantly decreased expression of RGS4 in yeast, as indicated by
immunoblotting (data not shown).

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Fig. 1.
Effects of point mutations on the function of
RGS4. The sequence of the RGS homology domain of RGS4 is shown.
-Helices in the RGS homology domain of RGS4 are indicated. Positions
where alanine substitutions impaired (red) or preserved
(green) the function of RGS4 (ability to inhibit G protein
signaling in yeast) are indicated. Other amino acid substitutions also
preserved (E87D) or impaired (G72R, E87A,N88A double mutant, I114D,
R167K, and R167H) the function of RGS4. Conserved residues that were
not targeted for mutagenesis (black) are indicated. The
single letter amino acid code is used.
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We subsequently determined whether these loss-of-function mutations
affected the stability or GAP activity of recombinant RGS4 expressed in
and purified from E. coli (Table I); as controls, several
mutations that did not affect RGS4 function in yeast were also
analyzed. Equivalent results were obtained using either
Go
or Gi
2 as a substrate (data not
shown). The results are interpreted below in light of the structure of
RGS4-Gi
1 complexes (19).
Mutations That Apparently Affect the Stability of the RGS
Domain--
The RGS homology domain of RGS4 forms a bundle of nine
-helices. The binding site for Gi
1 is a cleft
consisting of conserved amino acids at the ends of helices 4, 7, and 8 and loops between helices 3 and 4 and helices 5 and 6. However, many
other conserved amino acids in RGS4 are located distal to the
Gi
1-binding site. Some of these residues are important
for the folding and/or stability of the RGS domain. These include a
pair of phenylalanine residues (Phe-79 and
Phe-168) apposed at the interface of
helices 3 and 8; substituting either with alanine resulted in an
insoluble protein when expressed in E. coli (data not
shown). Other interacting pairs of residues may also maintain the
stability or rigidity of the RGS fold because mutations affecting them
resulted in soluble proteins with reduced GAP activity (Table I). These
interacting residues include an isoleucine-phenylalanine pair (Ile-114
and Phe-149) apposed between helices 5 and 7, a pair of phenylalanine residues (Phe-91 and Phe-118) apposed between helices 4 and 5, and an
isoleucine-tryptophan pair (Ile-67 and Trp-92) between helices 2 and 4. In contrast, two highly conserved serine residues (Ser-164 and
Ser-171), which were predicted to stabilize the RGS fold by acting as
helix breakers between helices 7, 8, and 9 (19), were not required for
RGS4 function in vivo (Fig. 1).

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Fig. 2.
Functional map of the surface of RGS4 that
binds Gi 1. A, effects of alanine
substitutions affecting residues of RGS4 that interact with
Gi 1. Part of the surface of RGS4 is shown. Residues of
RGS4 (spacefill) located within 4.5 Å of Gi 1 are
colored, each according to the magnitude of the defect in GAP activity
(see Table I and/or Fig. 1) caused by an alanine substitution: no
effect (green), moderate defect (yellow), severe defect (orange), nearly complete defect (red),
and not mutated (cyan) because the residue is not highly
conserved or because an alanine substitution would be a conservative
replacement. B, residues of Gi 1 that interact
with the functionally important surface of RGS4. The orientation of
RGS4 and color coding of its functionally important amino acids
(spacefill) are the same as in A. Residues of
Gi 1 (sticks) that interact with this surface of RGS4 are indicated (carbon atoms are gray; nitrogen,
blue; and oxygen, red). C, interaction
of leucine 159 of RGS4 with threonine 182 and lysine 180 of
Gi 1 (spacefill; color coding of atoms is as in
B). Switch I of Gi 1 (cyan ribbon)
and helix 7 of RGS4 (white ribbon) are indicated.
D, interaction of methionine 160 of RGS4 with asparagine 88 of RGS4, which interacts with threonine 182 of Gi 1.
Color coding of atoms (spacefill) is as in B; in addition,
sulfur atoms are yellow. Switch I of Gi 1
(cyan ribbon) and helices 4 and 7 of RGS4 (white
ribbons) are indicated. E, surface of
Gi 1 (spacefill; the indicated residues are color-coded
arbitrarily) that interacts with asparagine 128 of RGS4
(sticks; atoms are color-coded as in B).
F, position of asparagine 128 of RGS4 near active-site
residues of Gi 1. Atoms of asparagine 128 of RGS4
(spacefill) are color-coded as in B. Residues of
Gi 1, GDP, and AlF4 are
indicated (sticks; atoms are color-coded as in B,
except that phosphorus is orange, aluminum is
yellow, and fluorine is blue-gray). The backbones
of RGS4 (white ribbon) and Gi 1 (cyan
ribbon) are indicated. Nearly all the residues of
Gi 1 that interact with RGS4 are conserved in the
G subunits we have used for in vivo (Gpa1)
and in vitro (Go and Gi 2) assays of RGS4 function; an exception is that in Gpa1, a threonine residue occurs at the position equivalent to valine 185 of
Gi 1.
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Mutations Affecting Residues on the Surface of RGS4 That Interact
with Gi
1--
In remarkable agreement with structural
data, the remaining loss-of-function mutations affect residues on the
surface of RGS4 that interact with Gi
1 (Fig.
2A and Table I). As discussed below, this indicated that
many of the interactions between RGS4 and Gi
1 are
important for GAP activity (Fig. 2A and Table I).
Furthermore, this allowed us to investigate the relative functional
importance of specific protein-protein contacts and the mechanism
whereby RGS4 stimulates GTP hydrolysis. In describing these results, we
have used the convention of Tesmer et al. (19), in which
residues of RGS4 are preceded with "r" and those of
Gi
1 with "a."
Interactions occurring at the edge of the RGS4-Gi
1
interface are functionally important (Fig. 2B), as indicated
by the effects of various alanine substitutions in RGS4. These include hydrophobic interactions between the side chains of r-Tyr-84 and a-His-213 and a salt bridge between r-Glu-87 and a-Lys-210. r-Glu-83 is
also functionally important, probably because it interacts with the
side chain of r-Arg-167 (Fig. 2A). Potentially, this interaction pulls r-Glu-83 and the adjacent residue, r-Tyr-84, into
place such that r-Tyr-84 interacts effectively with a-His-213. Alternatively, the side chain of r-Glu-83 may be important for GAP
activity because it contacts the side chain of a-Val-185 (Fig. 2B).
In the center of the RGS4-Gi
1 interaction footprint,
a-Thr-182 in switch I of the G protein binds a pocket in RGS4
consisting of several conserved residues, including r-Ser-85, r-Asn-88,
r-Leu-159, r-Asp-163, r-Ser-164, and r-Arg-167 (Fig. 2B)
(19). With the exception of r-Ser-85 and r-Ser-164, the side chains of
residues forming the pocket are functionally important because an
alanine substitution at each site diminished the GAP activity of RGS4 (Table I).
Based on these results and the structural data of Tesmer et
al. (19), we propose the following roles for residues forming the
pocket that binds a-Thr-182. Three residues of the pocket are important
because they interact directly with a-Thr-182. The bulky side chain of
r-Leu-159 may determine the size or shape of the pocket because it
interacts extensively with the side chain of a-Thr-182 (Fig. 2,
B and C) and because the r-L159A substitution causes a severe defect in GAP activity. Furthermore, the side chain of
r-Leu-159 may be of critical importance because it also participates in
a hydrophobic interaction with the side chain of a-Lys-180 (Fig. 2,
B and C). The side chains of r-Asn-88 and r-Asp-163 appear to be important because they form hydrogen bonds with
the side chain hydroxyl and backbone nitrogen of a-Thr-182, respectively. Although r-Ser-85 and r-Ser-164 form part of the pocket
that binds a-Thr-182, their side chain hydroxyl groups are unimportant
because alanine substitutions at these sites did not affect RGS4
function in vivo (Fig. 1). However, this does not exclude
the possibility that the
-carbon atoms of r-Ser-85 and/or r-Ser-164
that do interact with a-Thr-182 are functionally important.
Residues forming the pocket, but which do not interact extensively with
a-Thr-182, are also functionally important, but probably for different
reasons. r-Met-160, which lies at the floor of the pocket but does not
contact a-Thr-182, may be important because its side chain interacts
with that of r-Asn-88 (Fig. 2D), potentially orienting it
toward a-Thr-182. Similarly, because r-Arg-167 does not contact
a-Thr-182 extensively (their side chains are a minimum of 3.9 Å apart), its principal function may be to stabilize two parts of the
RGS4 surface such that each can interact with Gi
1. This
is suggested because 1) the side chain of r-Arg-167 interacts with side
chain carboxyl groups of r-Glu-83 and r-Asp-163 (Fig. 2B),
both of which are important for GAP activity; 2) an alanine substitution of r-Arg-167 leads to a stronger defect than an alanine substitution affecting either r-Glu-83 or r-Asp-163 (Table I); and 3)
changing r-Arg-167 to alanine, lysine, or histidine disrupted GAP
activity (Table I).
There also was evidence that normal function of RGS4 requires the
additive effects of two sites that bind different regions of
Gi
1. r-Glu-87 and r-Asn-88, which are adjacent to one another, are likely to function cooperatively because they interact with a-Lys-210 of switch II and a-Thr-182 of switch I, respectively (Fig. 2B). Consistent with this hypothesis, a double mutant
in which r-Glu-87 and r-Asn-88 are changed to alanine had a much stronger defect in GAP activity than either single mutation (Table I).
Asparagine 128 of RGS4 Is Critical for GAP Activity--
A cluster
of residues in Gi
1 (including a-Lys-180 of switch I and
a-Gln-204, a-Ser-206, and a-Glu-207 of switch II) cradles r-Asn-128
(Fig. 2, E and F). This structural arrangement
has suggested that r-Asn-128 does the following: 1) facilitates GTP
hydrolysis by orienting and/or polarizing the side chain of a-Gln-204,
which interacts with the attacking water molecule in the transition state; 2) binds a water molecule early in the GTPase mechanism, before
the transition state is reached; and/or 3) binds and stabilizes switches I and II (19). Indeed, we found that r-Asn-128 has a pivotal
role because replacing it with alanine resulted in essentially a
complete loss of GAP activity (Table I).
Effects of Mutations on Binding of RGS4 and G
Subunits--
To investigate the mechanism of RGS4-stimulated GTPase
activity, we determined whether the panel of GAP-defective RGS4 mutants can bind G
subunits in their active (GTP-bound) or
transition state (GDP + AlF4
)
conformations. Strikingly, we found that all of the GAP-defective RGS4
mutants were impaired in their ability to bind G
subunits. RGS4 mutants lacking appreciable GAP activity, including
r-N128A, were unable to bind activated (GTP-bound) Go
subunits because at high concentration (3 µM), they
failed to block the ability of wild-type RGS4 (10 nM) to
stimulate GTP hydrolysis (Fig.
3A).

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Fig. 3.
Effects of mutations on binding of RGS4 to
G subunits. A, inability of GAP-defective
RGS4 mutants to bind GTP-bound G subunits. Measurements
of GAP activity (pmol of 32Pi released after
45 s of incubation at 5 °C, relative to that obtained with
wild-type RGS4 (0.2 µM)) were used to indicate whether mutant forms of RGS4 can bind GTP-bound Go subunits,
thereby inhibiting the ability of wild-type RGS4 to stimulate GTP
hydrolysis. Assays were performed using the indicated concentrations
(µM) of purified wild-type (WT) and mutant
RGS4 and Go subunits (0.1 µM). The results
shown are the average of three assays; standard errors are indicated
(bars). B, effects of mutations on the ability of
RGS4 to bind (GDP + AlF4 )-activated
Gi 2. Gi 2 (incubated with the indicated
nucleotides) was incubated with polyhistidine-tagged wild-type RGS4 and
the indicated mutant forms of RGS4. RGS4-Gi 2 complexes
bound to Ni2+-nitrilotriacetic acid beads were detected by
SDS-polyacrylamide gel electrophoresis and immunoblotting. The portion
of input (In) and bound fractions analyzed was as follows:
10% input, 10% bound (first row; wild-type and mutants
having weaker GAP defects); and 5% input, 20% bound
(second and third rows; mutants having moderate
and strong GAP defects, respectively). The experiment was performed
three times, with equivalent results.
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Similarly, RGS4 mutants displaying various defects in GAP activity were
also defective in binding Gi
2 in its transition state
(GDP + AlF4
) (Fig. 3B). The
results of the binding assays, although somewhat qualitative, suggested
that the severity of the defects in G
binding and GAP
activity were correlated. Mutations that severely reduced or eliminated
GAP activity (E87A,N88A double mutant, I114D, N128A, L159A, and R167A)
appeared to cause severe defects in binding (binding was not detected)
(Fig. 3B, third row), whereas those that
partially disrupted GAP activity caused less severe binding defects
(binding was less efficient than with wild-type RGS4) (Fig.
3B, first and second rows; 10% of the
bound material was analyzed for each mutant in the first row
and 20% for those in the second and third rows).
Because all of these mutations disrupted or impaired the binding of
RGS4 to G
subunits, none of the residues they affect,
including r-Asn-128, is required exclusively to catalyze GTP
hydrolysis.
Proposed Mechanism of RGS4-stimulated GTP Hydrolysis--
Our
functional analysis of RGS4 mutants and the structural data of Tesmer
et al. (19) lead to the following conclusions regarding the
mechanism whereby RGS4 stimulates GTP hydrolysis by G
subunits. First, a principal function of asparagine 128 of RGS4 is to
bind and stabilize switches I and II of G
subunits,
contributing to the overall stability of the RGS4-G
transition state complex. High affinity binding of RGS4 and
Gi
1 may not require a hydrogen bond formed by the amide
nitrogen of r-Asn-128 and the carbonyl oxygen of the side chain of
a-Gln-204 because in several other RGS proteins, including GAIP,
r-Asn-128 is replaced by a serine residue. Therefore, interactions
between r-Asn-128 and a-Lys-180, a-Ser-206, or a-Glu-207 may be
important for stabilizing RGS4-G
complexes. Indeed,
a-Glu-207 of Gi
1, which is highly conserved in
G
subunits, is critical for RGS binding and
RGS4-stimulated GTP hydrolysis (18, 23), but it is dispensable for
intrinsic GTPase activity and effector recognition.
Second, our finding that GAP-defective RGS4 mutants are defective in
binding G
subunits provides direct evidence in support of the hypothesis of Tesmer et al. (19) that RGS4 stimulates GTP hydrolysis primarily, if not exclusively, by binding and
stabilizing the transition state conformation of G
subunits. Apparently, the rigidity or stability of RGS4 is important
for the mechanism because we found that pairs of interacting conserved
residues distal to the Gi
1-binding site of RGS4 are
important for GAP activity. Furthermore, the mechanism appears to
involve the additive effects of several binding interactions along the
RGS4-G
interface, as indicated by the analysis of double
mutants. Thus, structural and functional data indicate that RGS4
stimulates GTP hydrolysis by binding the switch regions of
G
subunits, creating a rigid environment that favors the
transition state of the reactants.
Finally, the mechanism used by RGS4 to stimulate GTP hydrolysis is
different in certain respects from that used by p120GAP.
Binding of p120GAP is believed to introduce one or two
invariant arginine residues into the active site of Ras (13-15). One
of these conserved arginine residues, which are required for GAP
activity but are dispensable for binding of p120GAP to Ras,
is thought to stabilize the developing negative charge of the
-phosphate during GTP hydrolysis, a role subserved by a-Arg-178 in
Gi
1 (24). Direct support of this conclusion has been
provided recently from the crystal structure of p120GAP
bound to Ras (25). However, a common feature of p120GAP and
RGS4 is that they both stabilize the structures of the switch regions
of their cognate GTP-binding proteins in the transition state (19, 25,
26). Therefore, although monomeric and heterotrimeric GTP-binding
proteins are evolutionarily related, their attendant GAPs are distinct
in terms of their sequences, structures, and mechanisms of stimulating
GTP hydrolysis.
We thank J. Cooper for providing comments; M. Linder for materials, advice, and comments; and S. Sprang for the
coordinates of the RGS4-Gi
1 structure.