From the Departments of Pharmacology and
§ Chemistry, Cornell University,
Ithaca, New York 14853
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ABSTRACT |
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Aluminum tetrafluoride
(AlF4)
activation of heterotrimeric G-protein
-subunits is a well
established aspect of the biochemistry of these proteins; however,
until recently it has been thought that
AlF4
does not mediate effects on the Ras superfamily of low molecular weight
GTP-binding proteins. Recent work demonstrating aluminum fluoride-induced complex formation between Ras and its
GTPase-activating proteins (RasGAP and NF1) has provided important
insights into the mechanism of GAP-stimulated GTP hydrolysis. We have
characterized the
AlF4
-induced
complex formation between the GDP-bound form of the Rho subfamily
G-protein Cdc42Hs and a limit functional domain of the Cdc42-GAP using
a variety of biochemical techniques. Our results indicate that the
apparent affinity of GAP for the
AlF4
-mediated
complex is similar to the affinity observed for the activated
(GTP-bound) form of Cdc42 and that beryllium (Be) can replace aluminum
in mediating fluoride-induced complex formation. Additionally, the
AlF4
-induced
interaction is weakened significantly by the catalytically compromised GAP(R305A) mutant, indicating that this arginine is critical in transition state stabilization. Unlike Ras, we find that
AlF4
and
BeF3
mediate complex formation between Cdc42Hs·GDP and downstream target/effector molecules, indicating that there are important differences in the mechanism of effector binding between the Ras and
Rho subfamily G-proteins.
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INTRODUCTION |
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The interaction between members of the Ras superfamily of GTPases and GTPase-activating proteins (GAPs)1 is a critical step in the signaling pathways of these proteins. GTP-binding proteins exhibit intrinsic hydrolytic activity, allowing them to cycle between an active GTP-bound state and an inactive GDP-bound state (1). The binding of a GAP significantly increases this hydrolytic rate, down-regulating the signal carried by the GTPase (2-4).
The -subunits of heterotrimeric G-proteins are comprised of two
domains: a Ras-like GTP binding domain and a large helical domain
(5-7). Heterotrimeric G-proteins exhibit a much higher intrinsic
hydrolytic rate than GTPases in the Ras superfamily (1, 8, 9). A large
body of evidence suggests that the helical domain of the
-subunits
serves as an internal GAP by contributing a critical arginine residue
to the GTP binding site (Arg-174 in
-transducin or Arg-178 in
Gi
). Covalent modification of this arginine by cholera
toxin or mutations at this site lead to a constitutively active
-subunit that is incapable of hydrolyzing GTP (10-12). The x-ray
crystallographic structures of
-subunits bound to GTP
S show that
this arginine is coordinated with the
-phosphate (
-transducin) or
positioned nearby (Gi
), indicating that this may be an
essential catalytic residue involved in GTP hydrolysis (6, 7). The
analogy between the helical domain of
-subunits and GAPs for the
small G-proteins is supported by mutational analyses of the GAPs
identifying catalytically important arginine residues similar to the
catalytic arginine of the heterotrimers (13,
14).2
An interesting aspect of the biochemistry of heterotrimeric G-protein
-subunits is the ability of AlF4
to
stimulate their activation (15). Upon binding of
AlF4
to the GDP-bound
-subunit, the
G-protein adopts an activated conformation capable of signaling to
downstream effectors. Crystallographic and biochemical studies indicate
that AlF4
binds in the position of the
-phosphate to induce this active conformation (7, 16, 17). Crystal
structures of AlF4
-bound
-transducin and Gi
show important structural
differences from the corresponding GTP
S-bound forms. The unique
coordination of the aluminum suggests that the
AlF4
structure represents a transition
state analog of the GTP-hydrolytic pathway. GTP hydrolysis proceeds
through a penta-covalent intermediate in which the
-phosphate acts
as the general base, de-protonating the attacking water (18). The
planar structure of AlF4
approximates
the transition state structure of the
-phosphate (7, 17).
Importantly, the catalytic arginine has a critical role in coordinating
and stabilizing this transition state structure.
Recently, Mittal et al. (14) made an important connection
between the high and low molecular weight GTP-binding proteins by
demonstrating AlF4-mediated effects on
Ras (14).2 Two GAPs, NF1 and RasGAP, form stable complexes
with the GDP-bound form of Ras in the presence of aluminum fluoride,
indicating that it also mimics the transition state of GTP hydrolysis
in low molecular weight GTP-binding proteins. This observation provides
an important opening for investigation of the GTPase mechanism in Ras
and other low molecular weight GTPases.
In the study presented below, we demonstrate that
AlF4 induces complex formation between
the Rho family GTPase Cdc42Hs and the Cdc42-GAP, and we characterize
this interaction using spectroscopic methods. Unlike Ras, we observe
that AlF4
is able to induce GDP-bound
Cdc42 to form stable complexes with effectors as well as GAPs,
suggesting important differences in effector binding between the Ras
and Rho subfamilies. As shown for heterotrimeric G-proteins,
BeF3
behaves in a manner similar to
AlF4
and is also capable of mediating
complex formation; however, unlike
AlF4
, the
BeF3
clearly acts as a true GTP analog
and is even more effective than AlF4
at mediating complex formation between Cdc42 and its target/effector proteins.
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EXPERIMENTAL PROCEDURES |
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Proteins
Cdc42Hs and Cdc42Hs(Q61L)--
Cdc42Hs was prepared as described
previously with some modifications (19). The cDNA encoding Cdc42Hs
(or the Cdc42Hs(Q61L) GTPase-defective mutant) was inserted into the
pET15b expression vector immediately downstream of the hexa-histidine
tag and transformed into Escherichia coli BL21 cells. The
bacteria were grown in 4-liter fermentors, and protein expression was
induced by the addition of 100 µM
isopropyl-1-thio--D-galactopyranoside when the cultures reached an OD595 of 4.0.
Cdc42-GAP--
The carboxyl-terminal half of the Cdc42-GAP
(residues 239-443; designated below as GAP), which is fully functional
in stimulating the GTPase activity of Cdc42Hs (19), was purified using
essentially the same procedure described above for Cdc42Hs with a few
modifications. GDP was omitted from the lysis step. The GAP was
precipitated with 55% NH4(SO4)2
after elution from the Ni2+ column. The precipitated
protein was pelleted by ultracentrifugation (30 min, 10,000 × g) and resuspended in 50 ml of HMA. Proteolytic digestion
with thrombin and dialysis were performed as above. The GAP binds to
the Q-Sepharose column and was eluted with a 0-1 M NaCl
gradient (in HMA). The eluted protein was pooled and again subjected to
NH4(SO4)2 precipitation. The
pelleted protein was resuspended to a final concentration of ~5 mg/ml
in HMA and then dialyzed against HMA plus 40% glycerol for storage at
20 °C.
PBD--
The Cdc42Hs/Rac-binding domain of mPAK-3 was prepared
essentially as described previously (21, 22) and stored at 20 °C as a ~2 mg/ml stock.
Gel Filtration Analysis of Complex Formation
A Superdex 75 16/60 gel filtration column coupled to a Pharmacia fast performance liquid chromatography system was used to analyze complex formation between Cdc42Hs and the GAP. The column was run in buffer B (20 mM MOPS, 5 mM MgCl2, 1 mM NaN3, pH 7.5) at a flow rate of 0.5 ml/min. Purified samples of Cdc42 and GAP (0.5 ml at 5 mg/ml) were applied to the column, and elution profiles were monitored by UV absorption.
Complexes between GAP and the GTPase-defective Cdc42Hs(Q61L) mutant were prepared by combining 2.5 mg of each protein. The sample was concentrated to a final volume of 0.5 ml and incubated at 4 °C for 1 h. The sample was then analyzed as described above.
To detect AlF4-induced complex
formation, 2.5 mg each of Cdc42Hs·GDP and the GAP were combined, and
the mixture was brought to 60 µM AlCl3 and 25 mM NaF. Similar aluminum and fluoride mixtures contain a
high proportion of the aluminofluoride species thought to be
responsible for activating heterotrimeric G-proteins
(designated below as AlF4
)
(23). The sample was again concentrated to a final volume of 0.5 ml and
incubated at 4 °C for 1 h. The gel filtration chromatography was preformed as described above with 60 µM
AlCl3 and 25 mM NaF added to the column buffer.
The elution fractions (2 ml) were collected and subjected to SDS-PAGE
analysis.
19F NMR Spectroscopy
Samples for 19F NMR were prepared at a concentration of ~0.5 mM total protein in 0.5 ml of HMA buffer plus 0.75 mM AlCl3, 5 mM NaF, and 5% D2O. 19F NMR spectra were recorded at 25 °C on a Varian VXR-400 spectrophotometer using the fluorine probe (resonance frequency for 19F = 376 MHz).
Preparation of mant-Nucleotides and Cdc42Hs·mant-Nucleotide Complexes
N-Methylanthraniloyl (mant)-labeled nucleotides
(mant-dGDP and mant-dGTP) were synthesized as described previously
(24). 5'-Deoxynucleotides were used to obtain a single isomer of the mant-labeled nucleotide (the mant-moiety is covalently attached to the
3'-OH of the ribose ring). Cdc42Hs (or Cdc42Hs(Q61L)) was preloaded
with mant-nucleotide by incubation with a 20-fold excess of purified
mant-nucleotide and 25 mM EDTA in HMA at 4 °C for 1 h. The exchange reaction was quenched with 50 mM
MgCl2. The reaction mixture was passed over a 10-ml
Sephadex G-25 column equilibrated against HMA to remove unbound
nucleotide. The protein was brought to a final concentration of ~100
µM by filtration through a Centricon-10 (Amicon) membrane
and then dialyzed against HMA plus 40% glycerol for storage at
20 °C. Spectroscopic analysis confirmed that more than 90% of the
protein was loaded with mant-nucleotide after this procedure.
Fluorescence Spectroscopy
Anisotropy measurements of complex formation were taken on an SLM 8000 spectrofluorometer operating in the T-format. Excitation of the mant-moiety was accomplished with a xenon-arc lamp monochromated to 350 nm with an 8-nm bandwidth. Excitation in the vertical orientation was used to obtain a correction value, and anisotropy measurements were taken with horizontally polarized excitation light. Vertically polarized emission was measured on channel A over a 16-nm band selected at 445 nm, using a momochromator, and horizontal emission was measured on channel B selected at 440 nm with a glass filter. An integration time of 10 s was used for all measurements, and data points represent the average of at least three measurements on the same sample.
All titrations were carried out in 1 ml of HMN buffer (20 mM HEPES, pH 8.0, 5 mM MgCl2, 100 mM NaCl) in a quartz cuvette. To observe
AlF4-induced complex formation, the
HMN buffer was brought to 60 µM AlCl3, and 25 mM NaF (for BeF3
experiments, 60 µM BeCl2 was used in place of
the AlCl3). Cdc42Hs·mant-dGDP (or
Cdc42Hs(Q61L)·mant-dGTP) (1 µM) was titrated with
either the GAP or PBD. Additions were made directly to the fluorescence
cuvette from a concentrated stock (100-200 µM) and
allowed to equilibrate for 2 min before anisotropy measurements were
taken.
Data Analysis
The fluorescence anisotropy data were fit using a simple equilibrium model for the bimolecular interaction between Cdc42Hs and GAP.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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RESULTS |
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Demonstration of Stable Complex Formation between Cdc42Hs and the
GAP in the Presence of
AlF4
by Gel Filtration Analysis--
Fig.
1 compares elution profiles from the gel
filtration analysis of AlF4
-induced
complex formation between GDP-bound Cdc42Hs and GAP. Panels
A and B show the elution profiles of native Cdc42Hs and GAP, respectively. The elution volume of these proteins was consistent with their monomeric molecular masses of 22 and 25 kDa. In the absence
of AlF4
, a mixture of Cdc42Hs·GDP
and GAP eluted as two peaks (Fig. 1C) corresponding to their
native molecular masses. When the same analysis was performed in the
presence of AlF4
(Fig. 1E),
a new peak corresponding to an approximate molecular mass of 47 kDa
appeared, which matched the expected size of a complex between Cdc42Hs
and GAP. This result indicated that
AlF4
induced stable complex formation
between the GDP-bound form of Cdc42Hs and the GAP and is in agreement
with similar results recently reported by Ahmadian et al.
(25).
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19F NMR Spectroscopy of the
AlF4-induced GAP
Complex--
19F NMR spectroscopy was performed to probe
fluoride binding directly. This method was used successfully to study
the interaction of AlF4
with
-transducin (16). Free AlF4
shows a
single peak at
10 ppm (Fig.
3A). No change in the spectrum was observed for the 19F NMR spectrum of Cdc42Hs·GDP in
the presence of AlF4
, suggesting that
free Cdc42Hs did not bind to AlF4
(Fig. 3B). The 19F NMR spectrum obtained for
Cdc42Hs·GDP in the presence of equimolar GAP is shown in Fig.
3D. The addition of GAP produced a new peak shifted up-field
by approximately 22.5 ppm, indicating that fluoride was bound to the
complex. Fig. 3C shows the 19F NMR spectrum of
an equimolar mixture of GAP and Cdc42Hs bound to the nonhydrolyzable
GTP analog GppNHp. The spectrum was identical to that of free
AlF4
, indicating that the bound peak
observed in the spectrum of the AlF4
-induced Cdc42Hs·GDP·GAP
complex was caused by the binding of fluoride in the position normally
occupied by the
-phosphate.
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Characterization of
AlF4-induced
Complex Formation Using Fluorescence Spectroscopy--
Fluorescence
anisotropy measurements can be used to monitor changes in the
rotational life time mass of a fluorescently labeled protein. This
technique provides a sensitive read-out for protein-protein interactions and was used to characterize the
AlF4
-induced Cdc42Hs-GAP interaction
in more detail. Addition of GAP to preparations of Cdc42Hs·mant-dGDP
in the presence of AlF4
caused
dramatic changes in the anisotropy which were indicative of complex
formation. Fig. 4A shows a
typical titration of 1 µM Cdc42 Hs ·mant-dGDP with
increasing amounts of GAP in the presence of 60 µM
AlCl3 and 25 mM NaF. The binding isotherm was
well fit by a simple bimolecular equilibrium model for Cdc42Hs·GAP
complex formation (see "Experimental Procedures") and yielded an
apparent Kd of 239 nM ± 79 (n = 10). Increasing the concentration of
AlF4
did not lead to a significant
change in the Kd value, suggesting that
AlF4
is saturating under these
conditions. Additionally, both aluminum and fluoride were required such
that the addition of either compound alone was not sufficient to
promote the interaction between Cdc42Hs·mant-dGDP and GAP (data not
shown). A significantly weaker affinity was observed between
Cdc42Hs·GDP and GAP in the absence of
AlF4
. Assuming an identical anisotropy
value at saturation, a lower limit Kd of ~50
µM can be assigned to this interaction.
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The Affinity of the
AlF4-induced
Complex Formation Is Reduced Significantly by a Catalytically Deficient
GAP Mutant--
To pursue the hypothesis that
AlF4
represents a transition state
analog of the GTP-hydrolytic pathway, fluorescence anisotropy experiments were performed with a GAP that was mutated at arginine 305 (according to the sequence in Refs. 13 and 26), a residue that appears
to be essential for catalysis. The R305A mutant of GAP, while
binding to Cdc42Hs with wild type affinity, shows a significantly
reduced ability to catalyze the GTPase reaction (i.e.
25-fold lower GAP activity).2
Similar results have also been shown for the analogous mutations in a
number of other GAPs (13).
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Characterization of
AlF4-
and BeF3
-induced Interactions
between the GDP-bound Form of Cdc42 and Effectors--
The interaction
between Ras and its effector Raf was shown to be unaffected by
AlF4
(14). To investigate this
possibility in the Rho subfamily, the effects of
AlF4
on the binding of PBD were
studied by fluorescence spectroscopy. PBD is the binding domain of
mPAK-3 (21) and contains the CRIB (Cdc42Hs-Rac-interactive binding)
motif, originally shown to be a limit domain found on a number of
proteins that bind Cdc42Hs or Rac (27). Fig.
6A shows the binding isotherm
for Cdc42Hs·mant-dGDP titrated with increasing amounts of PBD in the
presence and absence of AlF4
. An
apparent Kd value of 461 nM was
determined for the binding of PBD to Cdc42Hs·GDP in the presence of
AlF4
. The affinity of PBD for this
complex was significantly weaker than its apparent affinity for the
GTP-bound (activated) Cdc42Hs (i.e. Kd ~72
nM). Still, it is clear that the addition of
AlF4
caused a significant enhancement
in the affinity of PBD for the GDP-bound state of Cdc42Hs
(i.e. in the absence of
AlF4
, the Kd value
for the interaction of GDP-bound Cdc42Hs with PBD has a lower limit of
~70 µM). The addition of
AlF4
also enhanced the affinity of
GDP-bound Cdc42Hs for the limit binding domain of the IQGAP molecule,
another putative target for Cdc42 (data not shown) (28).
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DISCUSSION |
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We have demonstrated that AlF4
induces the formation of a stable complex between Cdc42Hs·GDP and GAP
using a variety of biochemical and spectroscopic techniques. The
Kd value measured for the
AlF4
-induced complex formation was
similar to that determined for the binding of activated (GTP-bound)
Cdc42Hs to GAP. We have also found that beryllium was able to replace
aluminum in mediating Cdc42Hs·GDP·GAP complex formation in the
presence of fluoride, consistent with results that have been obtained
for heterotrimeric G-proteins. Our results indicate that the aluminum
fluoride-mediated complex formation reported between Ras and RasGAP is
likely to be a general phenomenon observed between all low molecular
mass GTP-binding proteins and their GAP molecules.
The 19F NMR data are crucial to understanding the mechanism
of the AlF4-induced complex formation.
There are two potential mechanisms by which
AlF4
could promote the interactions
between the GDP-bound form of Cdc42Hs and its effectors. The first
mechanism is analogous to that seen with the heterotrimeric GTP-binding
proteins, where the AlF4
binds
directly to the GDP-bound Cdc42Hs, inducing an activated conformation
capable of interacting with the GAP. Mittal et al. (14),
however, proposed an alternative mechanism for the aluminum fluoride-induced complex formation between Ras and its GAPs. The first
step in this mechanism is the formation of a weakly interacting complex
between the Ras and RasGAP. The transient formation of this low
affinity complex creates a pocket for the aluminum fluoride which then
binds to stabilize the complex.
The results of the 19F NMR spectroscopy with Cdc42Hs
clearly implicate the second of these two mechanisms. The addition of
the GDP-bound form of Cdc42Hs to a solution containing
AlF4 did not give rise to additional
fluoride peaks in the NMR spectrum, indicating that
AlF4
did not bind to Cdc42Hs
uncomplexed. The addition of GAP produced an additional up-field peak
not observed with Cdc42Hs alone. Thus, the GAP must contribute
important residues to the active site, forming a binding pocket capable
of stabilizing the transition state and thereby allowing
AlF4
to bind. The chemical shift
observed for the binding of AlF4
to
the Cdc42Hs·GDP·GAP complex was similar to that observed for
-transducin, suggesting that the environment of the
AlF4
binding pocket is similar in
these two proteins. AlF4
did not bind
to the Cdc42Hs·GppNHp·GAP complex indicating that AlF4
occupies the position that
normally contains the
-phosphate, as expected from studies on
heterotrimeric G-proteins.
GAPs for the Rho family GTPases make up a diverse family with highly
divergent amino acid sequences. A few highly conserved residues have
been isolated which are critical to the catalytic activity of these
proteins; in particular, mutation of the universally conserved arginine
(Arg-305 in Cdc42-GAP) severely compromises the catalytic activity of
these proteins (13).2 The fluorescence titrations of
Cdc42Hs with the GAP(R305A) mutant show a significantly weaker affinity
for the AlF4-induced complex relative
to the affinity measured for the interactions of GAP(R305A) with the
activated form of Cdc42Hs. The 50-fold lower affinity exhibited by
GAP(R305A) for the Cdc42Hs·GDP complex in the presence of
AlF4
is consistent with the loss of a
salt bridge between Arg-305 and AlF4
,
suggesting that the markedly depressed catalytic activity observed with
this GAP mutant is caused by its inability to stabilize the transition
state. Taken together with the 19F NMR data, this supports
the conclusion drawn by Mittal et al. (14), arguing that
GAPs stimulate GTP hydrolysis by introducing critical catalytic
residues to the active site (14). The recent x-ray
crystallographic structures for the
Ras·GDP·AIF3·RasGAP complex, as well as
complexes between RhoGAP and Rho family GTPases in both the active
(GppNHp-bound) and transition state
(GDP·AlF4
-bound) conformations, show
that GAPs contribute an arginine residue to the nucleotide binding site
which specifically stabilizes the transition state for GTP hydrolysis
(29-31). The results presented here as well as the observations of
these crystallographic studies are summarized in Fig.
7A, showing the catalytically
important Arg-305 mediating the
AlF4
-induced complex formation between
Cdc42 and GAP.
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As stated in the Introduction, a great deal of biochemical and
structural data has implicated AlF4 as
a transition state analog for the GTPase reaction in heterotrimeric G-proteins. In the crystal structure of
-subunits complexed to AlF4
, the critical arginine residue,
which is located within the large helical domain, interacts with the
coordinating fluorides. Considering the data presented here in light of
the work performed on heterotrimeric G-proteins and Ras, it seems
likely that the GTP-hydrolytic mechanism is conserved across the
families of both large and small G-proteins and that Arg-305 in
Cdc42-GAP plays the critical role of stabilizing the transition state
in the GTP-hydrolytic pathway, analogous to arginine 174 in
-transducin, arginine 178 in Gi
, and arginine 789 in
the RasGAP.
We obtained interesting and rather unexpected results when examining
the effects of AlF4 on the
interactions between Cdc42Hs and its downstream target/effector molecules. Specifically, we found that
AlF4
enhanced the affinity of the
GDP-bound form of Cdc42Hs for its target/effector proteins. This was
unexpected in light of the finding that
AlF4
does not influence Ras-target
interactions and suggests that the Ras and Rho subfamily G-proteins
have distinctly different modes of effector binding. The
19F NMR data clearly demonstrate the inability of
AlF4
to bind to free Cdc42Hs, and thus
the AlF4
-induced complex formation
between Cdc42Hs and PBD must proceed in a manner similar to that
proposed for the GAP where PBD must first form a transient complex with
Cdc42Hs and thereby create a binding site for
AlF4
. As appears to be the case for
the GAP, the creation of the binding pocket for
AlF4
apparently relies on the
introduction of a specific residue by the effector molecule. The
crystal structure of the Ras-related G-protein Rap in complex with the
Ras-binding domain of Raf demonstrates that targets for Ras do not
introduce such a residue into the GTP-hydrolytic site (32). Unlike Raf,
the PAKs and other Rho family target/effectors must introduce a residue
into the active site, enabling AlF4
to
bind and mediate complex formation.
Another unique property of Rho family targets is their role as GTPase
inhibitory proteins (GIPs) (33). Such GIP activity is not observed for
effectors of Ras (34). One interesting possibility is that the GIP
activity of Rho family target/effectors is mediated by a residue
introduced into the -phosphate binding pocket of the G-protein which
stabilizes the GTP-bound state in a manner analogous to the
stabilization of the transition state for GTP hydrolysis by the GAP,
but with an opposite effect. This interpretation is supported further
by the complex formation observed between Cdc42·GDP and PBD in the
presence of BeF3
and is summarized in
Fig. 7B. Comparison of the crystal structures of myosin-ADP
bound with either AlF4
or
BeF3
has shown that although the
AlF4
complex represents a distinct
transition state conformation, BeF3
is
a true ATP analog, inducing a conformation indistinguishable from the
ATP-bound protein (35). The affinity of PBD for Cdc42Hs·GDP in the
presence of BeF3
is identical to that
observed for the activated form of Cdc42Hs, whereas the affinity in the
presence of AlF4
is significantly
weaker than that measured for the GTP-bound state. This is consistent
with the hypothesis that the fluoride-mediated interaction of Cdc42Hs
with effectors relies on the GIP activity of these proteins which
should stabilize a GTP analog (BeF3
)
more effectively than a transition state analog
(AlF4
). This implies a direct role for
target/effector molecules in modulating the GTPase activity of Rho
family G-proteins.
In conclusion, it is clear that aluminum fluoride represents a
transition state analog in the GTP-hydrolytic pathway of Cdc42Hs and is
capable of inducing complex formation with the Cdc42·GAP. Moreover,
we propose a role for BeF3 as a true
GTP analog, capable of inducing high affinity complex formation between
Cdc42Hs and target/effector molecules. Such fluoride-mediated complex
formation is singular to the Rho subfamily of GTPases and is a direct
result of the unique biochemistry of the G-protein-target interaction
in this important family of signaling molecules.
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ACKNOWLEDGEMENTS |
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We acknowledge Dr. Alfred Wittinghofer for generous support in the initial stages of this work and Cindy Westmiller for excellent assistance.
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Note Added in Proof |
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It has been reported that the transition-state complex for the GTP hydrolytic reaction for Ras contains AIF3 (29).
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FOOTNOTES |
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* These studies were supported by National Institutes of Health Grants EY06429 and GM47458 and by a grant from the Human Frontiers of Science Program.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.: 607-253-3650; Fax: 607-253-3659.
1
The abbreviations used are: GAP(s),
GTPase-activating protein(s); GTPS, guanosine
5'-3-O-(thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; PBD, PAK binding domain; PAGE, polyacrylamide gel
electrophoresis; mant, N-methylanthraniloyl; GppNHp,
guanosine 5'-(
,
-imido)triphosphate; GMP-PNP, guanosine
5'-(
,
-imino) triphosphate; GIP, GTPase inhibitory protein.
2 D. A. Leonard, R. A. Cerione, and D. Manor, manuscript in preparation.
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REFERENCES |
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