From the Max-Planck Institut für Molekulare Physiologie,
Abteilung Strukturelle Biologie and Physikalische
Biochemie, Rheinlanddamm 201, 44139 Dortmund, Germany and
¶ Onyx Pharmaceuticals,
Richmond, California 94608
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ABSTRACT |
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Wiskott Aldrich syndrome is a rare hereditary
disease that affects cell morphology and signal transduction in
hematopoietic cells. Different size fragments of the Wiskott Aldrich
syndrome protein, W4, W7 and W13, were expressed in Escherichia
coli or obtained from proteolysis. All contain the GTPase binding
domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have
developed assays to measure the binding interaction between these
fragments and Cdc42 employing fluorescent
N-methylanthraniloyl-guanine nucleotide analogues. The
fragments bind with submicromolar affinities in a
GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not
sufficient for tight binding. Rate constants for the interaction with
W13 have been determined via surface plasmon resonance, and the
equilibrium dissociation constant obtained from their ratio agrees with
the value obtained by fluorescence measurements. Far UV circular
dichroism spectra show significant secondary structure only for W13,
supported by fluorescence studies using intrinsic protein fluorescence
and quenching by acrylamide. Proton and 15N NMR
measurements show that the GBD/CRIB motif has no apparent secondary
structure and that the region C-terminal to the GBD/CRIB region is
-helical. The binding of Cdc42 induces a structural rearrangement of
residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich
syndrome protein fragments have an ensemble of conformations, one of
which is stabilized by Cdc42 binding. Thus, in contrast to Ras
effectors, which have no conserved sequence elements but a defined
domain structure with ubiquitin topology, Rac/Cdc42 effectors have a
highly conserved binding region but no defined domain structure in the
absence of the GTP-binding protein. Deviating from common belief
GBD/CRIB is neither a structural domain nor sufficient for tight
binding as regions outside this motif are necessary for structure
formation and tight interaction with Rho/Rac proteins.
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INTRODUCTION |
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Members of the Rho subfamily of GTP-binding proteins, namely Rho, Rac, and Cdc42, are involved in the regulation of the actin cytoskeleton. They cycle between a biologically inactive GDP-bound and an active GTP-bound state. The ratio of these states is regulated by GEFs,1 guanine nucleotide exchange factors, and GAPs, GTPase-activating proteins. In the GTP-bound state they interact with so-called effectors or downstream targets that are defined as molecules that have a high affinity to the GTP-bound state as opposed to a low affinity to the GDP-bound state.
The Wiskott Aldrich syndrome is a genetically determined, X-linked recessive disorder that affects cells of the hematopoietic lineage and fibroblasts. Molecular symptoms include cytoskeletal abnormalities in T-cells and platelets as well as deficient chemotaxis in neutrophils. The three major phenotypic appearances are thrombocytopenia, eczema, and recurrent infections due to defects in the immune response toward polysaccharide antigens. The gene is allelic to X-linked thrombocytopenia, a rare recessive disorder with other clinical symptoms than WAS (1). Cloning of the gene (2) showed that the responsible protein WASP harbors several domains involved in signal transduction such as a proline-rich region binding to SH3 domains of adaptor proteins like Nck- and Src-type kinases. Other domains termed WH1 and WH2 (for WASP homology) are also found in other signaling molecules. A homologue called N-WASP has been cloned which shows 50% homology to WASP over the entire length (3).
WASP and N-WASP contain a sequence motif of approximately 14 residues, the GBD, or CRIB motif (for Cdc42/Rac interactive binding) which has been shown to specifically bind the GTP-bound form of Cdc42 or Rac, with a preference for Cdc42 (4, 5). The first proteins containing the CRIB motif were described by Lim and co-workers (6) as the non-receptor tyrosine kinase ACK and the serine/threonine kinase called PAK (7). It was found that the GBD/CRIB is conserved in other effectors of Cdc42/Rac such as the various isoforms of PAK and the yeast homologue STE20 (8-12). Data base searches using the CRIB motif turned up many more potential effector molecules with a more or less conserved motif (4, 13).
Nothing is known about the structure of Rac effectors and their
complexes. In the case of Ras, small Ras binding domains (RBDs) in the
effectors Raf, Ral-GEF, (14-16) and Byr2 (17) have been identified
which fold into a stable domain. For Raf (18, 19) and Ral-GEF (20, 21),
it has been shown that these domains fold into the ubiquitin superfold
even though they are sequentially unrelated either to ubiquitin or to
each other. It has also been shown by x-ray structural analysis that
Ras/Rap interact with RafRBD by forming an intermolecular -sheet
close to the effector region of Ras (19). To study the interaction
between Cdc42 and proteins containing the CRIB motif and as a first
step toward understanding the molecular defects of WAS, we have
investigated their interaction using biophysical techniques such as
circular dichroism (CD), fluorescence spectroscopy, surface plasmon
resonance, and NMR.
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MATERIALS AND METHODS |
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Cloning and Purification of WASP Fragments-- The plasmid pFusion3 (4) coding for residues 48-321 of the human WASP was used as a template for polymerase chain reaction subcloning of fragments corresponding to residues 221-257 (W4) and 201-321 (W13). The oligonucleotides 5'-CGCGGATCCCCAGCTGATAAGAAACGCTCAGGG-3' and 5'-CAGAATTCATCCATTCTGGGGGTC-3' were used to amplify a 126-base pair DNA fragment coding for the 4-kDa peptide and the oligonucleotides 5'-CGCGGATCCGACATCCAGAACCCTGACATCACG-3' and 5'-GTGAATTCATCGAGATGGCGGTG-3' to amplify a 380-base pair DNA fragment coding for the 13-kDa peptide. After hydrolysis with BamHI and EcoRI, the fragments were cloned into the glutathione S-transferase (GST) fusion vector pGEX-2T (Amersham Pharmacia Biotech), and the identity of the constructs was verified by sequencing using the cycle sequencing procedure. Plasmids were transformed in Escherichia coli BL21(DE3) (22) for production of the protein.
An overnight culture of BL21(DE3) cells containing the appropriate plasmid was diluted 100-fold into LB medium supplemented with 200 µg/ml ampicillin. At an A600 = 0.8 synthesis of the GST fusion protein was induced by addition of 0.1 mM isopropyl-Purification of Cdc42·Nucleotide Complexes-- The plasmids coding for the GST fusions of the wild type (Cdc42wt) and G12V mutant (Cdc42V12) of the human Cdc42 protein were kindly provided by George Martin (Onyx Pharmaceuticals). After cleavage of the fusion proteins with thrombin, seven additional amino acid residues (GSKIISA) preceded the starting methionine at the N terminus of Cdc42wt, whereas the CdcV12 protein had only three additional amino acid residues (GSP) attached to its N terminus as determined by protein sequencing. Synthesis and initial steps of purification of Cdc42 were the same as described above for the WASP fragments except that the buffer used was buffer A with 5 mM MgCl2, 5 mM 1,4-dithioerythritol. The cleaved Cdc42 was in the GDP state as determined by high pressure liquid chromatography (see below). Exchange for both the fluorescent and non-fluorescent GTP analogue was achieved as described by John et al. (24). Complexation of Cdc42 with mGDP was possible by incubation with a 50-fold excess of mGDP in the presence of 10 mM EDTA for 2 h at ambient temperature. For all complexes further purification involved gel filtration on a G-75 Sephadex (Amersham Pharmacia Biotech) column in 20 mM HEPES/NaOH, pH 7.4, 1 mM MgCl2. Cdc42 concentrations were determined by the method of Bradford (25) or spectrophotometrically according to Hiratsuka (26) when complexed with mant nucleotides. Cdc42·nucleotide complexes were analyzed by isocratic high pressure liquid chromatography (27) on a C18 reversed phase column (Bischoff, Leonberg, Germany). Because of its higher stability in dilute solutions all measurements were done with the G12V mutant of Cdc42 (Cdc42V12), whereas for the NMR studies wild type Cdc42 (Cdc42wt) was used. Control experiments showed that the interactions between WASP fragments and Cdc42 are not influenced by the G12V mutation. However, due to the high intrinsic GTPase rate of Cdc42, it was assumed that the wild type protein might even hydrolyze Gpp-(NH)p to GppNH2 in long term measurements and that the G12V mutation would prevent this.
Protein identities and purity were checked by SDS-polyacrylamide gel electrophoresis and Edman sequencing. Electrospray ionization mass spectroscopy on a Finnigan LCQ mass spectrometer was used to verify the molecular masses of the proteins and to determine the isotopic enrichment of 15N-labeled proteins.Spectroscopic Techniques-- Fluorescence measurements were done on a FluoromaxTM spectrofluorimeter (Spex Industries) equipped with a thermostat set to 25 °C using cuvettes with a 0.5 × 1-cm2 section. Proteins were diluted into 40 mM HEPES/NaOH, pH 7.4, 100 mM NaCl (standard buffer). In case of Cdc42, 5 mM MgCl2 was added. Emission spectra were recorded after excitation at 280, 295, or 366 nm (1-nm bandwidth) with time constants of 0.1 or 1 s and 2-nm bandwidth. All spectra were corrected for buffer contributions. Titrations of Cdc42·mGpp(NH)p with WASP fragments were performed with an automatic titrator using a Hamilton syringe as reservoir for the ligand. The decrease in fluorescence at 435 nm was followed. Equilibrium dissociation constants were obtained by fitting the solution of a quadratic equation describing a bimolecular association model assuming a 1:1 stoichiometry to the data. For measurement of the dissociation of fluorescent nucleotides from Cdc42 and its complexes with WASP fragments a 1000-fold excess of unlabeled nucleotide was added, and the decrease in fluorescence at 435 nm (8 nm bandwidth) was followed. Single exponentials were fitted to the data using the program Grafit (Erithacus Software).
Fluorescence quenching measurements using acrylamide as the quenching agent were carried out as follows. To 1.2-ml sample normalized to 5 µM tryptophan residues, aliquots of a 6 M acrylamide stock solution were added. After excitation at 295 nm (0.5-nm bandwidth) the fluorescence signal at 357 nm (2-nm bandwidth) was followed for 2 min and averaged. Buffer contributions and dilution effects were accounted for and the inner filter effect due to the absorption of acrylamide at 295 nm was corrected by multiplying the fluorescence intensity by 10Surface Plasmon Resonance-- Association and dissociation reactions involving Cdc42·Gpp(NH)p and Cdc42·GDP without a fluorescence label were studied by surface plasmon resonance in a BIAcoreTM system (BIAcore AB, Uppsala, Sweden). Since reaction of essential lysine residues can lead to partial inactivation of chemically coupled proteins, a sandwich assay (33) with anti-GST antibodies was used. These were coupled to the matrix following the protocol of the manufacturer (34). GST fusion proteins were incubated with the antibodies at a concentration of 0.2 mg/ml for 7 min, followed by incubation with free ligand. Regeneration with 20 mM glycine, pH 2.0, and 0.005% SDS resulted in complete dissociation of all noncovalently bound ligands, leaving the immobilized immunoglobulin at essentially full activity.
Binding of Cdc42 to binary complexes of antibody and GST-WASP fragments was analyzed in a concentration-dependent manner. To eliminate the contribution of nonspecific binding, equivalent controls with Cdc42·GDP were used to calculate specific signal changes. Experiments were performed at 20 °C in 10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.0005% (w/v) Igepal CA-630 (Sigma), a buffer system stabilizing the immobilized anti-GST antibodies. Binding and dissociation curves were fitted to the sum of a single exponential and a linear function to take into account the association and dissociation of GST complexes. ![]() |
RESULTS |
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Expression of Recombinant WASP Fragments-- It has been shown previously that small fragments of Rac/Cdc42 effectors containing the CRIB motif, also called the GTPase binding domain, GBD, fused to GST are able to bind to Cdc42 in an overlay assay and that the binding is dependent on Rac/Cdc42 being loaded with GTP (4, 5). In the case of human WASP the smallest fragment that retained this property is a peptide of 34 amino acids comprising residues 235-268 (4). Binding of an 81-residue human WASP fragment (amino acids 215-295) to Cdc42 has also been investigated by the ability of the fragment to inhibit the interaction of Cdc42-GTP and Cdc42GAP (35). In order to define a Cdc42 binding domain for structural studies, to see whether any such fragments are necessary and sufficient for high affinity binding and to develop methods for the direct measurement of these interactions, we have produced various fragments from the human WASP in E. coli as GST fusion proteins (Fig. 1A). SDS-polyacrylamide gel electrophoresis analysis of the purified fragments (Fig. 1B) and mass spectroscopy show that the WASP fragments of 4 kDa (residues 221-257), 7 kDa (201-268), and 13 kDa (201-321) molecular mass, termed W4, W7, and W13, respectively, can be isolated as soluble non-GST fusion proteins without degradation and in reasonable quantities.
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WASP as a Nucleotide Dissociation Inhibitor of Cdc42--
The
different fragments were tested for their ability to bind to Cdc42 in a
nucleotide-dependent fashion. We have shown previously (36,
37) that the Ras binding domains of c-Raf1 and Ral-GEF act as a GDI
protein by inhibiting nucleotide dissociation from Ras in a saturable
manner and that this effect can be used to measure the dissociation
constant in solution for this interaction. When Cdc42 is loaded with
the fluorescent non-hydrolyzable GTP analogue mGpp(NH)p, the
dissociation of the fluorescent analogue, as measured by the decrease
in fluorescence in the presence of excess unlabeled Gpp(NH)p, is
0.4·103 s
1, 2.6-fold slower than the
dissociation of unlabeled Gpp(NH)p (not shown), similar to what has
been found for the dissociation of mGDP versus GDP for Cdc42
(38). In the presence of increasing amounts of W13, the dissociation of
nucleotide is inhibited (Fig. 2). With
different WASP fragments different degrees of guanine nucleotide
inhibition can be achieved, and it appears that the dissociation rate
of mGpp(NH)p from the different Cdc42·mGpp(NH)p·WASP complexes is
different (Table I). This indicates that
in principle the affinity between the WASP fragment and Cdc42 can be
measured by the GDI assay and that WASP affects nucleotide binding of
Cdc42 in a similar manner as Ras effectors have an effect on nucleotide binding on Ras (36, 37).
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Equilibrium Fluorescence Titration and Nucleotide-specific Binding-- Since, however, the difference between the dissociation rate constants in the presence and absence of WASP is very small as compared with the Ras system, and the amplitude of the kinetics diminishes with increasing concentration of W13, a more direct approach to measure the equilibrium dissociation constant was developed. As both the WASP fragments and Cdc42 contain a tryptophan residue, intrinsic fluorescence was not used as a spectrometric probe. In Fig. 3A it is shown that the fluorescence of the nucleotide analog mGpp(NH)p bound to Cdc42 decreases on addition of W13 by a factor of more than 2 under saturating conditions and almost approaches the fluorescence of the unbound nucleotide. This change in signal can thus be used to measure the interaction directly by equilibrium titration. Following the decrease of fluorescence (excitation and emission wavelength 366 and 435 nm, respectively) with increasing concentrations of W13 and fitting a binding equation with a 1:1 stoichiometry to the data, a dissociation constant of 77 nM was obtained (Fig. 3B). The stoichiometry was verified by an active site titration where increasing amounts of W13 were added to 1 µM Cdc42·mGpp(NH)p, a concentration much larger than the KD of the interaction (not shown). The smaller WASP fragments W4 and W7 also bind with high affinity. However, the dissociation constants for both are approximately 6-fold higher (Table II). Thus the GBD/CRIB region seems to be necessary but not sufficient for tight binding. An indication that the extra sequences of larger WASP fragments supply additional interaction sites can be seen from the maximal change of the mant fluorescence in the three complexes; the decrease induced by W4, W7, and W13 is very different (Fig. 3A).
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Surface Plasmon Resonance Measurements--
To measure
quantitatively the kinetics of the interaction between WASP and
Cdc42·Gpp(NH)p surface plasmon resonance was used. In a sandwich
assay described earlier for the interaction of Ran with its effector
RanBP1 (33), a monoclonal antibody against GST was coupled to the
sensor chip and used to bind the GST fusion proteins GST-W4 and
GST-W13. This system can be used to measure the interaction with Cdc42
in a GTP- and concentration-dependent manner in real time.
Fig. 4A shows traces of the
change in mass on the sensor chip upon binding of Cdc42 to immobilized
GST-W4. Only Cdc42·Gpp(NH)p but not Cdc42·GDP nor nucleotide free
Cdc42 binds tightly to the surface. An association rate constant of 1.9·105 M1 s
1
(Fig. 4, B and C, Table II) was obtained for the
W13/Cdc42·Gpp(NH)p system, which is fairly slow for a bimolecular
association reaction. The dissociation rate constant measured by
surface plasmon resonance (not shown) was found to be
11.9·10
3 s
1, the ratio of the rate
constants resulting in a kinetically determined dissociation
equilibrium constant of 63 nM. The reversed system with
GST-Cdc42 immobilized and W13 as the freely diffusible ligand yields a
value of 69 nM (data not shown). These results are in good
agreement with the value of 77 nM obtained by equilibrium fluorescence titration taking into account the small differences in
buffer composition and temperature (20 versus 25 °C, 150 mM NaCl versus 100 mM NaCl). It
supports observations made for the Ran interaction with RanBP1 using
the sandwich coupling technique where good correlation between
fluorescence and surface plasmon resonance data is found (33).
Dissociation rate constants were also measured for the W4 and W7
fragments using this technique and are about 7-fold faster than those
for the W13 fragment. Association rate constants for W7 and W4 were not
evaluated quantitatively due to the small net signal change. Compared
with the affinities measured by fluorescence titration (Table II),
these data show that the difference in affinities between W13 on the
one side and W4/W7 on the other is almost completely due to a
difference in the dissociation rate constants.
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Structural Differences between WASP Fragments-- The difference in binding constants between W4, W7, and W13 could either be caused by the smaller fragment not being folded in a native-like conformation or due to the W13 supplying extra residues that are necessary for tight binding. From Fig. 5A it can be seen that the residue Trp-252 just outside the CRIB region in WASP exhibits different fluorescence emission properties in the W4, W7, and W13 fragments. W4 and W7 do not differ significantly in their fluorescence properties and show a fluorescence emission maximum at 357 nm. Thus, the tryptophan residue seems to be located in a polar environment. In contrast to this, a slight increase in fluorescence intensity and a blue-shift of 6 nm can be seen for W13 indicating a less polar environment for the tryptophan residue. Thus, not only the mant group in Cdc42·mGpp(NH)p bound to WASP fragments but also the tryptophan residue in uncomplexed WASP fragments is located in different structural environments.
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NMR, Sequence-specific Assignments--
In order to gain more
structural insight on WASP, two- and three-dimensional nuclear magnetic
resonance techniques were used. According to the data shown above, W13
is the most tightly binding fragment and shows significant secondary
structural content as assessed by circular dichroism. Yet, it tends to
aggregate under high concentrations as used for the NMR measurements.
Thus, both the W4 and W7 fragments were used for NMR investigations.
Here, the sequence-specific resonance assignment of W7 is reported. Fragment W13 was only partially assigned by comparing the chemical shifts from resonances of amino acids that were also present in W4 and
W7. Resonance assignment of W7 was achieved according to the standard
method developed by Wüthrich (39). Spin systems were obtained
from two-dimensional DQF-COSY and two-dimensional Clean-TOCSY spectra.
Three-dimensional 15N-TOCSY-HMQC spectra were recorded to
avoid ambiguous assignment of overlapping resonances. Sequential
connectivities were obtained from two-dimensional NOESY spectra with
mixing times of 200 ms and from a three-dimensional
15N-NOESY-HMQC with a mixing time of 100 ms. The strong
Arg-268 NH-H as well as the NH-H
signal of the unique Thr-208
were chosen as starting points for the assignment of the C and N
termini. The spin systems of the rings of Tyr-212 and Trp-252 were used to trace the chain to Lys-226 and Lys-230, respectively. The assignment of Arg-227 to Gly-229 was ambiguous in the case of W7 but not in W4.
Two NH-H
resonances for residues 227-229 were found indicating existence of two conformations. Three additional signals left unassigned belong to spin systems of two alanine residues and one
glutamine residue. These signals had integrals of approximately 10% of
those peaks that could be assigned unequivocally. Proline assignments
were done by using the connectivities of their H
resonances and the
H
peaks of the preceding amino acid.
Secondary Structure of W4 and W7--
Short and medium range NOEs
were analyzed to characterize the secondary structure of W7.
Surprisingly, considering the tight binding of W7 to Cdc42, amino acid
regions in the N-terminal part of W7 including the GBD/CRIB motif
itself do not show any obvious secondary structure. Only the region
from Trp-252 to Asp-264 shows NOEs
(NHi-NHi;
Hi-NHii + 2; Hai-NHi;
H
i-NHi) typical of an
-helical
conformation (Fig. 6). Although only a few of these could be assigned unambiguously, calculation of the coupling constants confirms the existence of an
-helix: four JNH-H
couplings in the DQF-COSY
define
angles in this region that are significantly smaller than 6 Hz which are typical for
-helices. Thus the region of secondary
structure is limited to the C-terminal part of W7. Considering a
maximum length of 10 residues the helix content within W7 is
approximately 14%. This coincides well with the ~10% helical
content estimated from the CD spectrum using the procedure
developed by Holzwarth and Doty (40). The underestimation of
helical elements in peptides by CD spectroscopy is a well known
phenomenon since short or transient helices do not give rise to a
strong CD signal (41, 42). With exception of the NH-H
resonances of
the first and last amino acids of the chain, no coupling constants
higher than 7.5 Hz could be found thus pointing out that no
-sheet
structure occurs within the protein. Additionally, five slowly
exchanging NH protons belonging to residues Gln-255 to Asp-259 indicate
hydrogen bonding. Compared with the H
shifts of tripeptides
mimicking the random coil state of a protein (43), H
resonances in
the C-terminal part of W7 shift to lower ppm values, and together with
NOEs and 3JNH-Ha coupling measurements support
the
-helical assignment.
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Complexes of W4 and W7 with Cdc42·Gpp(NH)p--
To obtain
structural information on the interaction between Cdc42 and WASP, HSQC
spectra of free and Cdc42·Gpp(NH)p-bound W4 and W7 fragments were
recorded, using a 2-fold excess of the GTP-binding protein. Resonances
in the complex were either unperturbed or shifted. Some resonances were
broadened or could not be observed any longer. Fig. 6 and Table
IV (data not shown for W4) summarize the
15N-1H peaks that have shifted or that could
not be analyzed. Both in W4 and W7, the shifting resonances are
clustered in the CRIB region. No additional resonance shifts of NH-H
peaks in other regions were found.
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DISCUSSION |
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We have shown here that fragments of WASP ranging from 4 to 13-kDa molecular mass can be stably expressed in E. coli as fusion proteins and cleaved from the GST part without losing stability or posing problems like aggregation or diminished solubility at higher micromolar concentrations. The fact that these fragments are not proteolytically digested during expression in E. coli and chromatographic purification seems to suggest that they have some amount of tertiary structure.
The WASP fragments bind to Cdc42 in the triphosphate form with reasonable high affinity ranging from 77 to 490 nM (Table II), as determined both by fluorescence titration and surface plasmon resonance with GST fusion proteins bound to sensor chips via anti-GST antibodies. This confirms similar findings made on the interaction between Ran and its effector RanBP1, where also a good correlation between surface plasmon resonance and fluorescence techniques was found (33). Equilibrium measurements of the interaction between a 72-residue fragment of the protein kinase mPAK-3 (PBD) and Cdc42 (44), using as a readout the fluorescence of the mant group, gave a dissociation constant in the micromolar range, similar to that found for W4 and W7. Surprisingly, the fluorescence of Cdc42-bound mGpp(NH)p was increased on addition of PBD, whereas with WASP the fluorescence decreases. This may indicate that the interface between Cdc42 and the CRIB/GBD region is somewhat different between WASP and PAKs, reflecting a different sequence context between the different putative effectors of Cdc42. Zhang et al. (35) have assessed the affinity between an 81-amino acid fragment (residues 215-295) of WASP and Cdc42 by measuring its inhibition of the Cdc42-Cdc42GAP interaction. Surprisingly, the dissociation constant of such a fragment, the size and location of which is intermediate between W7 and W13, was found to be 4.1 µM, well above the 77-490 nM values found for W13, W7 and W4.
The affinity of the W13 fragment is in the same range or even higher
than that found between Ras and its effectors Raf and Ral-GEF (15, 36,
37, 45), and lower than the affinities between Ran and its effectors
RanBP1, RanBP2, and importin which are in the nanomolar or subnanomolar
affinity range (33, 46, 47).3
The structure of RafRBD does not change appreciably on complex formation with Rap or Ras (15, 19). In contrast W13 seems to become
structured only on complex formation with Cdc42, and it is surprising
that the affinity of the latter complex is similar to that of
Ras/RafRBD which seems to indicate the formation of a tight interface,
the energy of which is used to induce a structural transition in the
GBD/CRIB motif. Although the affinity to the GDP-bound form could not
be determined due to the lack of a measurable spectroscopic signal (at
the concentration tested), we have estimated the affinity of W13 to
Cdc42·GDP to be at least 500-fold less tight, again in agreement with
observations for interactions between GTP-binding proteins and their
effectors, and with the assumption that WASP is a true effector for
Cdc42. The dissociation rate constants, measured with surface plasmon
resonance, are relatively fast, approximately 1 min1 for
W13. Assuming that full-length WASP has a similar dissociation rate,
one can conclude that the Cdc42·GTP·WASP complex has a short half-life in the cell, similar to the Ras-Raf complex
(45)4 which may allow fast
regulation of signaling via Ras or Rac/Cdc42.
We have shown that the region encompassing the GBD/CRIB motif is necessary but not sufficient for high affinity binding to Cdc42 and that other regions of WASP are necessary for the interaction. W4 and W7 bind with similar low affinity, whereas W13 which contains additional residues C-terminal to this region increases the affinity approximately 6-fold, which corresponds to a difference in free enthalpy of binding of only 4.4 kJ/mol at 25 °C. This small difference is nevertheless reflected in a different behavior of the corresponding Cdc42·Gpp(NH)p·WASP complexes, one indication being the stronger inhibition of nucleotide dissociation associated with the binding of W13 to Cdc42·Gpp(NH)p, as compared with the W4 and W7 complex.
Surprisingly, even though W4, W7, and W13 seemed to be stable protein
fragments which bound specifically and with high affinity to Cdc42 in
the triphosphate form, they did not show pronounced secondary structure
in either the CRIB/GBD motif itself or the region N-terminal to it, as
determined by proton NMR. Thus, although the GBD/CRIB region is
commonly called a domain, it is not a domain in a structural sense, as
an independent folding unit with a defined structure, in contrast to
the Ras binding domain of both Raf kinase (15, 19) and Ral-GEF (20,
21). The C-terminal part of W7, however, has a short and/or flexible
-helical structure as detected by NMR and CD, and it is tempting to
suggest that this structural feature is stabilized in W13 thereby
giving rise to the stronger CD signal of this fragment. The sequence
C-terminal to the CRIB region in WASP does not show conserved residues
in comparison to other proteins containing this motif. We could thus imagine that these residues do not bind directly but rather stabilize the CRIB region and thereby raise the affinity toward Cdc42.
Fluorescence studies on W4, W7, and W13 show differences in their
intrinsic fluorescence and also in accessibility of the tryptophan
residues as determined by quenching studies indicating different
environments of the Trp residue. This apparent difference in structure
is reflected in the affinities toward Cdc42, where W4 and W7 have
similar affinities as opposed to W13. The structural properties of the
Cdc42 effector are different to those of the Ras effectors c-Raf1 and
Ral-GEF and Rlf, which contain small Ras binding domains with a defined ubiquitin-like fold, but show no apparent sequence homology
(18-21)5 It is also
different from the Ran binding domain of RanBP1 and RanBP2 which have
conserved sequence elements and a defined
structure.6
The NMR data show that upon binding to Cdc42·Gpp(NH)p a chemical shift change of the 15N-1H resonances occurs only in the CRIB/GBD region of W4 and W7 indicating that the conserved sequence element experiences a reorganization of structure on binding to Cdc42·Gpp(NH)p, which is, however, not detectable via CD. In contrast, the structure of the Ras binding domain of c-Raf-1 is very stable and very similar between the bound and unbound conformation (18, 19).7 Whether or not the CRIB/GBD motif of WASP or PAK has defined structure cannot be decided conclusively at the current stage of investigation. However, for some 15N-labeled nuclei and their corresponding hydrogen atoms, which reflect the conformation of the main chain, two different chemical shifts are found. There are also some NH protons in the GBD/CRIB motif which are slowly exchanging within 24 h, indicating some degree of folding in this area. There are no large CD spectral changes on complex formation between WASP fragments and Cdc42. Taken together this could mean that the CRIB/GBD domain populates two or more conformations in solution, only one of which strongly interacts with Rac/Cdc42 which in turn stabilizes the bound conformation. It is also possible that the structure of the GBD/CRIB motif and the flanking regions depends strongly on the presence of the rest of the WAS protein, although we find it unlikely that the structure of the motif is very much different from the one it has in the W13 fragment. Nevertheless, to gain more insight into the structural requirements for the interactions between WASP and Cdc42, the structure of the complete WASP molecule in complex with the GTP-binding proteins may have to be solved by x-ray crystallography to learn about possible domain boundaries in the molecule and how WASP may contribute to the effect of Cdc42 on the cytoskeleton. Our studies show that the analysis of fragments of WASP and of other GBD/CRIB containing proteins such as the various PAK isoforms have to be treated with caution as the requirements for a productive interaction appear to be different between the fragments and the full-length proteins.
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ACKNOWLEDGEMENTS |
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We thank Dorothee Vogt for cloning of the WASP fragments; Bernhard Griewel for help with the NMR spectrometer; Christian Herrmann for stimulating discussions; and Rita Schebaum for secretarial help. P. B. thanks Roger Goody for continuous support.
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FOOTNOTES |
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* This work was supported in part by European Union Grant BIO4-CT96-1110.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.
§ Supported by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie/Deutsches Zentrum für Luft- und Raumfahrt.
To whom correspondence should be addressed.
1
The abbreviations used are: GEF, guanine
nucleotide exchange factors; Cdc42V12, G12V mutant of Cdc42; Cdc42wt,
wild type human Cdc42, placental isoform; NOESY, nuclear Overhauser
enhancement spectroscopy; ROESY, rotating frame enhancement
spectroscopy; TOCSY, total correlated spectroscopy; Clean-TOCSY, TOCSY
with suppression of ROESY-type cross-peaks; CRIB, Cdc42/Rac interactive binding; DQF-COSY, double quantum filtered correlated spectroscopy; GAP, GTPase-activating proteins; GBD, GTPase binding domain; GDI, guanine nucleotide dissociation inhibitor; Gpp(NH)p, guanosine 5'-(,
-imido)triphosphate; gsHSQC, gradient-selected HSQC; GST, glutathione S-transferase; HMQC, hetero multiple quantum
coherence; HSQC, hetero single quantum coherence; mant,
-O-(N-methylanthraniloyl); NOEs, nuclear
Overhauser effects; Wn, WASP fragment of approximate kDa
mass; WASP, Wiskott Aldrich syndrome protein.
2 J. Reinstein, personal communication.
3 C. Villa, unpublished data.
4 J. Sydor, M. Engelhard, A. Wittinghofer, R. S. Goody, and C. Herrmann, unpublished data.
5 P. Bayer, D. Esser, R. Cool, B. Bauer, and R. Wolthuis, unpublished data.
6 H. R. Kalbitzer, unpublished data.
7 A. Wittinghofer, unpublished data.
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