Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs*

Gregory R. HoffmanDagger , Nicolas NassarDagger §, Robert E. OswaldDagger , and Richard A. CerioneDagger

From the Departments of Dagger  Pharmacology and § Chemistry, Cornell University, Ithaca, New York 14853

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Aluminum tetrafluoride (AlF4-) activation of heterotrimeric G-protein alpha -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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 alpha -subunits serves as an internal GAP by contributing a critical arginine residue to the GTP binding site (Arg-174 in alpha -transducin or Arg-178 in Gialpha ). Covalent modification of this arginine by cholera toxin or mutations at this site lead to a constitutively active alpha -subunit that is incapable of hydrolyzing GTP (10-12). The x-ray crystallographic structures of alpha -subunits bound to GTPgamma S show that this arginine is coordinated with the gamma -phosphate (alpha -transducin) or positioned nearby (Gialpha ), indicating that this may be an essential catalytic residue involved in GTP hydrolysis (6, 7). The analogy between the helical domain of alpha -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 alpha -subunits is the ability of AlF4- to stimulate their activation (15). Upon binding of AlF4- to the GDP-bound alpha -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 gamma -phosphate to induce this active conformation (7, 16, 17). Crystal structures of AlF4--bound alpha -transducin and Gialpha show important structural differences from the corresponding GTPgamma 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 gamma -phosphate acts as the general base, de-protonating the attacking water (18). The planar structure of AlF4- approximates the transition state structure of the gamma -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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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-beta -D-galactopyranoside when the cultures reached an OD595 of 4.0.

The bacterial pellet was resuspended in 100 ml of lysis buffer (20 mM Tris-HCl, pH 8.0, 5 mM imidazole, 500 mM NaCl, 1 mM NaN3, 200 µM phenylmethylsulfonyl fluoride, 2 µg/ml aprotonin, 2 µg/ml leupeptin, 0.1 µM GDP, and 0.5 mg/ml lysozyme) and homogenized. 10 mg of DNase I and 5 ml of 1 M MgCl2 were added to degrade the chromosomal DNA. The lysate was subjected to ultracentrifugation (30 min, 10,000 × g) and affinity purified over a 25-ml imidodiacetic acid column (Sigma) charged with Ni2+. The column was washed with buffer A (20 mM Tris-HCl, pH 8.0, 20 mM imidazole, 500 mM NaCl, and 1 mM NaN3), and the protein was eluted with a gradient of 20-250 mM imidazole in buffer A.

The hexa-histidine tag was removed by incubating overnight with 100 µg of thrombin and dialyzed twice against 1 liter of HMA buffer (20 mM HEPES, pH 8.0, 5 mM MgCl2, 1 mM NaN3). The clipped protein was applied to a Q-Sepharose anion exchange column equilibrated against HMA. Cdc42Hs does not bind to the Q-Sepharose column under these conditions, and the protein-containing flow-through was pooled and concentrated to ~20 mg/ml using an ultrafiltration cell (Amicon). In the case of the constitutively active Cdc42Hs(Q61L) protein, reverse phase high performance liquid chromatographic analysis confirmed that more than 90% of the purified, E. coli expressed protein was bound to GTP (20). The concentrated protein was stored at -20 °C after dialysis into HMA plus 40% glycerol.

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.
<UP>Cdc42</UP>·<UP>GAP</UP>↔
<UP><SC>Reaction</SC> 1</UP>
In the case of the AlF4--induced complex formation, aluminum and fluoride were maintained in large excess so that the concentration of any intermediates could be ignored, and the binding equilibrium was assumed to follow Reaction 1. The dissociation constant (Kd) for this reaction is simply
K<SUB>d</SUB>=<FR><NU>[<UP>Cdc42</UP>][<UP>GAP</UP>]</NU><DE>[<UP>C</UP>]</DE></FR> (Eq. 1)
where [C] is the unknown concentration of the Cdc42Hs·GAP complex. Equation 1 can be used to solve for the concentration of the complex in terms of known parameters, which in turn yields the quadratic equation shown below.
[<UP>C</UP>]=<FR><NU>(K<SUB>d</SUB>+<UP>Cdc42<SUB>t</SUB></UP>+<UP>GAP<SUB>t</SUB></UP>)±<RAD><RCD>(K<SUB>d</SUB>+<UP>Cdc42<SUB>t</SUB></UP>+<UP>GAP<SUB>t</SUB></UP>)<SUP>2</SUP>−4(<UP>Cdc42<SUB>t</SUB></UP> · <UP>GAP<SUB>t</SUB></UP>)</RCD></RAD></NU><DE>2</DE></FR> (Eq. 2)
where Cdc42t and GAPt represent the total amount of each of these proteins (complexed and free in solution). Only the positive root from Equation 2 is used in subsequent calculations. The observed anisotropy (Aobs) is then a weighted average of the anisotropy of the free (Af) and complexed (Ac) mant-labeled Cdc42Hs.
A<SUB><UP>obs</UP></SUB>=<FENCE><FR><NU>[<UP>C</UP>]</NU><DE>Cdc42<SUB>t</SUB></DE></FR></FENCE>(A<SUB><UP>c</UP></SUB>−A<SUB><UP>f</UP></SUB>)+A<SUB><UP>f</UP></SUB> (Eq. 3)
Apparent Kd values for complex formation were obtained by fitting the titration data to Equation 3 using the iterative, nonlinear least squares fitting regime in the IGOR Pro software package (Wave Metrics).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

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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Fig. 1.   Gel filtration analysis of stable complex formation between GDP-bound Cdc42Hs and GAP. 0.5-ml samples were applied to a Superdex 75 16/60 gel filtration column, and elution profiles were monitored by UV absorbance. Elution profiles are of native Cdc42Hs (A), native GAP (B), an equimolar mixture of Cdc42Hs·GDP and GAP (C), an equimolar mixture of the GTPase-defective Cdc42Hs(Q61L) mutant and GAP (D), and an equimolar mixture of Cdc42Hs·GDP and GAP in the presence of 60 µM AlCl3 and 25 mM NaF (E). Arrows indicate the observed elution volume of the Cdc42Hs·GAP complex (~60 ml; first arrow), native GAP (~68 ml; second arrow), and native Cdc42Hs (~72 ml; third arrow).

It has been well established that the GAP normally interacts with the GTP-bound form of Cdc42Hs. However, stable complexes between Cdc42Hs·GTP and GAP are not formed because of the rapid hydrolysis of GTP to GDP and the subsequent loss of affinity between the two proteins. Stable complexes between Cdc42Hs and the GAP can be obtained using nonhydrolyzable GTP analogs or GTPase-deficient mutants of the protein. For comparison with the AlF4--induced complex, Fig. 1D shows a gel filtration experiment where stable complexes were formed between GAP and the GTPase-deficient Cdc42Hs(Q61L) mutant. As expected, the appearance of a 47-kDa peak is observed, consistent with the formation of a Cdc42Hs·GAP complex.

SDS-PAGE analysis of the column fractions confirmed that the high molecular mass peak induced by the addition of AlF4- was indeed a complex of Cdc42Hs·GDP and GAP. Fig. 2 shows SDS-PAGE analysis of the column fractions from the gel filtration experiment shown in Fig. 1E. The high molecular mass peak (fractions 60-62 in Fig. 2) clearly contained both Cdc42Hs and GAP, indicating that these proteins were eluted from the gel filtration column as a stable complex of approximately 47 kDa. The two proteins appeared to be present in equivalent amounts which is in agreement with the 1:1 stoichiometry expected for this interaction.


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Fig. 2.   SDS-PAGE analysis of gel filtration fractions. 2-ml fractions were collected from the gel filtration experiment shown in Fig. 1E. 20-µl samples corresponding to the indicated elution volume were resolved on 15% SDS-PAGE and visualized using Coomassie Blue staining.

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 alpha -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 gamma -phosphate.


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Fig. 3.   19F NMR spectroscopy of AlF4--induced complex formation. 19F NMR spectra (376 MHz) of protein samples in AlF4--containing NMR buffer (0.75 mM AlCl3, 5 mM NaF, and 5% D2O in HMA) are shown. The samples were: buffer alone (A), GDP-bound Cdc42Hs (B), GppNHp bound Cdc42Hs plus equimolar GAP (C), and GDP-bound Cdc42Hs containing aluminum fluoride plus equimolar GAP (D). Chemical shifts are measured relative to free fluoride (-10 ppm).

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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Fig. 4.   Fluorescence anisotropy titration of AlF4-- and BeF3--induced Cdc42Hs·GAP complex formation using Cdc42Hs·mant-dGDP. Panel A, Cdc42Hs·mant-dGDP (1 µM) was titrated with the indicated amounts of GAP. Titrations were carried out either in the absence (open circle ) or in the presence (bullet ) of AlF4-. The solid lines represent best fits of the data to the bimolecular binding models described under "Materials and Methods." The lower curve represents additions of equivalent volumes of GAP storage buffer (black-square). Panel B, Cdc42Hs·Mant-dGDP (1 µM) was titrated with the indicated amounts of GAP either in the presence of 60 µM AlCl3 and 25 mM NaF (bullet ) or 60 µM BeCl2 and 25 mM NaF (open circle ). Panel C, Cdc42Hs(Q61L)·mant-dGTP (1 µM) was titrated with the indicated amounts of Cdc42·GAP (open circle ). For comparison, the titration of Cdc42Hs·mant-dGDP (1 µM) in the presence of 60 µM AlCl3 and 25 mM NaF with the indicated amounts of GAP is also pictured (bullet ).

In the case of the heterotrimeric G-proteins, beryllium was found to be the only cation capable of replacing aluminum in the fluoride-induced activation response (15). Fig. 4B shows the titration of Cdc42Hs·mant-dGDP with increasing amounts of GAP in the presence of BeF3-. The best fit to this binding isotherm yielded an apparent Kd value of 388 nM ± 47 (n = 2), which was similar to that determined for the AlF4--induced Cdc42Hs·GAP interaction.

To compare carefully the Kd values for Cdc42Hs·GAP complex formation induced by AlF4- and BeF3- versus the values for GAP interactions with activated (GTPase-defective) Cdc42Hs, experiments were performed with the Cdc42(Q61L) mutant. Fig. 4C shows the titration of 1 µM Cdc42Hs(Q61L)·mant-dGTP with increasing amounts of GAP. The titration profile was fit using the same bimolecular model as above and yielded an apparent Kd of 279 nM ± 134 (n = 6). This value is not significantly different from that determined for the AlF4-- or BeF3--induced complex formation.

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).

Fig. 5 shows a titration of 1 µM Cdc42Hs·mant-dGDP in the presence of AlF4- with increasing amounts of the GAP(R305A) mutant. The best fit to this titration profile yielded an apparent Kd of 2.15 µM, which represents a significantly weaker affinity than that measured for the interaction between GAP and the GTP-bound state of Cdc42Hs (see above). A similar loss of affinity for the BeF3--induced complex was also observed (data not shown). The titration of GTP-bound Cdc42Hs with the GAP(R305A) mutant gives an apparent Kd value of 245 nM, i.e. an affinity nearly identical to that observed for the wild type GAP.


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Fig. 5.   Fluorescence anisotropy titration of AlF4--induced complex formation between Cdc42Hs and the GAP(R305A) mutant. Cdc42·mant-dGDP (1 µM) was titrated with the indicated amounts of GAP(R305A) in the presence of 60 µM AlCl3 and 25 mM NaF (open circle ). A similar titration of Cdc42Hs(Q61L)·mant-dGTP (1 µM) with the indicated amounts of GAP(R305A) is also shown (bullet ).

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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Fig. 6.   Fluorescence anisotropy titration of AlF4-- and BeF3--induced complex formation between Cdc42Hs·mant-dGDP and PBD. Panel A, Cdc42Hs·mant-dGDP (1 µM) was titrated with the indicated amounts of the limit binding domain of PAK (PBD). Titrations were carried out in HMN buffer alone (black-square) or in HMN plus 60 µM AlCl3 and 25 mM NaF (open circle ). A similar titration of Cdc42Hs(Q61L)·mant-dGTP (1 µM) with the indicated amounts of PBD is also shown (bullet ). The solid lines represent best fits of the data to the bimolecular binding model described under "Experimental Procedures." Panel B, Cdc42Hs·mant-dGDP (1 µM) was titrated with the indicated amounts of PBD either in HMN plus 60 µM BeCl2 and 25 mM NaF (open circle ). A similar titration of Cdc42Hs(Q61L)·Mant-dGTP (1 µM) with the indicated amounts of PBD is again shown (bullet ).

Interesting differences arose when Cdc42Hs·mant-dGDP was titrated with PBD in the presence of BeF3- as shown in Fig. 6B. The affinity observed for the BeF3--induced complex formation was significantly higher than that measured for AlF4-, yielding an apparent Kd value of 45 nM. This value was similar to the Kd value determined for the binding of activated Cdc42Hs to PBD (~72 nM) and suggests that BeF3- induces an activated conformation within Cdc42Hs which mimics the conformational state induced by GTP.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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 alpha -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 gamma -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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Fig. 7.   Conceptual model of fluoride-induced complex formation. Panel A, the "arginine finger" model for the stabilization of the aluminum fluoride transition state analog in the Cdc42·GDP·AlF4-/Cdc42·GAP complex based on recently published crystallographic studies (29, 31). Panel B, by analogy, we have proposed a similar model for the complex between Cdc42·GDP·BeF3- and PBD. In this case BeF3- acts as a true GTP analog and is stabilized by the introduction of an "anti-arginine" residue into the nucleotide binding site by PBD.

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 alpha -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 alpha -transducin, arginine 178 in Gialpha , 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 gamma -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.

    ACKNOWLEDGEMENTS

We acknowledge Dr. Alfred Wittinghofer for generous support in the initial stages of this work and Cindy Westmiller for excellent assistance.

    Note Added in Proof

It has been reported that the transition-state complex for the GTP hydrolytic reaction for Ras contains AIF3 (29).

    FOOTNOTES

* 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); GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; PBD, PAK binding domain; PAGE, polyacrylamide gel electrophoresis; mant, N-methylanthraniloyl; GppNHp, guanosine 5'-(beta ,gamma -imido)triphosphate; GMP-PNP, guanosine 5'-(beta ,gamma -imino) triphosphate; GIP, GTPase inhibitory protein.

2 D. A. Leonard, R. A. Cerione, and D. Manor, manuscript in preparation.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References

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