From the
Institute of Microbiology, ETH
Zürich, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland,
¶Max-Planck-Institut für Molekulare
Physiologie, Abt. Strukturelle Biologie, Otto-Hahn-Str. 11, D-44227 Dortmund,
Germany, and the **European Molecular Biology
Laboratory, Structural and Computational Biology Programme, Meyerhofstrasse 1,
D-69117 Heidelberg, Germany
Received for publication, March 11, 2003 , and in revised form, April 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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Activation of RhoGTPase signaling requires the exchange of the bound GDP for GTP. Three regions of the RhoGTPases (switch I (Cdc422645), switch II (Cdc425974), P loop), and a Mg2+ ion mediate high affinity G nucleotide binding (57). Spontaneous dissociation of the RhoGTPase-GDP-Mg2+ complex is very slow in the absence of regulators. In vivo, the G nucleotide release is catalyzed by guanine nucleotide exchange factors (GEFs)1 (7, 8).
There are at least two families of GEFs for RhoGTPases. One family comprises the vast majority of the known eukaryotic GEFs specific for RhoGTPases (9, 10). These Dbl-like GEFs contain a catalytic Dbl homology (DH) and an adjacent pleckstrin homology (PH) domain (11) required for membrane association and sometimes also for catalysis (10, 12). The structures of GEF-RhoGTPase complexes have revealed that the DH (and sometimes also the PH) domains of Dbl-like GEFs engage and reshape the switch I/II regions of their cognate GTPases into a conformation that disrupts both magnesium and nucleotide binding (1214). Recently, several GEFs for RhoGTPases that lack DH and PH domains have been identified in eukaryotic cells (15, 16).
Another family of GEFs for RhoGTPases that lacks DH or PH domains has been discovered in pathogenic bacteria. So far, this "SopE family" includes the virulence factors SopE and SopE2 from Salmonella enterica (1720) and the putative virulence factor BopE from Burkholderia spp. (Fig. 1a) (21). During the course of an infection, these proteins are injected via type III secretion systems into cells of the host animal in order to manipulate signaling (22). The catalytic domains of SopE and SopE2 (aa 78240 of SopE) were shown to accelerate G nucleotide release from Cdc42 and several other RhoGTPases (but not from Ha-Ras) by up to 105-fold (20, 2325). X-ray crystallographic analysis of the SopE-Cdc42 complex confirmed that SopE has an entirely different tertiary structure and employs different amino acid residues to bind RhoGTPases than Dbl-like GEFs (26). However, the switch I and switch II regions of Cdc42 in the SopE-Cdc42 complex had conformations closely resembling the switch I and switch II conformations in Dbl-like GEF-RhoGTPase complexes (1214, 26). This confirmed that SopE uses the same catalytic principle as Dbl-like GEFs.
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Based on the mechanism of Dbl-like guanine nucleotide exchange
(13) and the structure of the
SopE-Cdc42 complex, we have proposed a mechanism for the GEF activity of SopE
(26). Most strikingly, SopE
inserts the 166GAGA169 loop between switch I/II regions
of Cdc42, and a SopEG168A mutant retains less than 1% of its
catalytic activity (26). In
the nucleotide-free complex (which contains a sulfate ion that might mimic a
bound -phosphate), the displacement of switch I/II of Cdc42 is
stabilized by SopE residues, including the side chains of
Asp-124SopE, Lys-198SopE, Asp-103SopE, and
Gln-109SopE (Fig.
1b). Many of the amino acids located at the SopE-Cdc42
interface are conserved between all members of the SopE family
(Fig. 1a).
SopE and Dbl-like GEFs employ different amino acids to stabilize the rearranged switch I/II regions. Nevertheless, two residues of SopE engage Cdc42 in a similar fashion as analogous residues of Dbl-like GEFs; Asp-124SopE and Glu-1047Tiam1/Glu-639Dbs/Glu-1244Intersectin form equivalent polar contacts with Tyr-32Cdc42/Rac1/RhoA, Thr-35Cdc42/Rac1/RhoA, and Val-36Cdc42/Rac1/RhoA (Cdc42 numbering; Tyr-34, Thr-37, and Val-38 in RhoA), whereas Gln-109SopE and Asn-1232Tiam1/Asn-810Dbs/Asn-1421Intersectin form equivalent polar contacts with Asp-65Cdc42/Rac1/RhoA and Arg-66Cdc42/Rac1/RhoA (Cdc42 numbering) (Table I) (12, 13, 26). The functional significance of these observations has remained unclear. Moreover, these proposed mechanistic similarities were based on the structure of the nucleotide-free Cdc42-SopE complex. We do not know how closely the determined structure of this complex resembles the transition state(s) of the rate-limiting step(s) of the G nucleotide release reaction. Therefore, the catalytic role of the SopE residues required further functional analysis.
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In this study, we have mutated specific amino acids of SopE to explore their role in G nucleotide exchange. The SopE mutants were analyzed in vitro using G nucleotide exchange assays and in tissue culture cell infection assays. The 166GAGA169 loop, Asp-124SopE, Asp-103SopE, and Gln-109SopE were found to be important for catalysis. These residues are highly conserved among all members of the SopE family. Interestingly, the residues Asp-124SopE and Gln-109SopE have functional equivalents in Dbl-like GEFs, whereas the 166GAGA169 loop is a unique feature of SopE-like GEFs. Therefore, despite all of the differences in structure, amino acid sequence, and catalytic core, both families of GEFs employ several equivalent amino acid interactions to stabilize the rearranged switch I and switch II region of RhoGTPases and accelerate G nucleotide release.
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MATERIALS AND METHODS |
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Site-directed Mutagenesis of SopE and Cdc42All SopE and Cdc42 proteins used in this study are derivatives of SopE from S. typhimurium SL1344 and of the human Cdc42Hs protein, respectively.
Mutations in sopE were introduced in pM136 (20), a pBAD24 derivative, which expresses M45-tagged SopE (SopE1240-M45) under the control of its native sopE promoter using the QuikChange site-directed mutagenesis kit (Stratagene) according to the protocol of the manufacturer. Briefly, we designed complementary primers carrying base changes to introduce the desired single amino acid changes or deletions (Table II). Mutant strand synthesis was achieved by thermal cycling (denaturation 95 °C/30 s, 1218 cycles; 95 °C/30 s, 55 °C/1 min, 68 °C/13 min). The parental DNA template was digested with DpnI (0.2 units/µl, 1 h/37 °C), and the mutated plasmid was transformed into XL-1-Blue E. coli cells. The transformed XL-1-Blue cells were selected for growth on ampicillin-containing agar plates. The mutated plasmids were isolated, and their sequence was verified by sequencing. Finally, pM136 and the mutated derivatives (coding for wild type SopE1240-M45 and variants thereof) were digested with Eco47III/SalI, and the inserts, including the native sopE promoter and the M45-tagged sopE allele, were cloned into the EcoRV/SalI sites of pACYC184 (29), yielding the low copy plasmids pM438 (wild type SopE1240-M45) and pM461472 (SopE1240-M45 variants) (Table III).
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To construct overexpression vectors for the purification of the catalytic domain of SopE (aa 78240, designated SopE') carrying the desired amino acid changes, the same mutations were introduced in pM164 (pGEX-KG, which expresses GST-SopE') using the protocol described above (plasmids pM441pM452) (Table III).
The mutation for Cdc42D65A (pM454) was introduced in Cdc42 using the QuikChange site-directed mutagenesis kit as described above using an expression plasmid for Cdc42 as a template (Table III).
Preparation of Recombinant ProteinsPreparation of the recombinant proteins was performed as described (24, 30). Briefly, all proteins used in this study were overexpressed in E. coli BL21 carrying derivatives of the pGEX expression vector (Amersham Biosciences) as GST fusion proteins, recovered from bacterial extracts by binding to glutathione-Sepharose 4B (Amersham Biosciences). The proteins were either eluted with 20 mM glutathione (GST-SopE' and variants thereof) or cleaved off of the column by digestion with thrombin protease (SopE' and variants thereof, Cdc42, Cdc42', and Cdc42D65A). All steps of the purification were performed in buffers containing 50 mM Tris-HCl, pH 7.6, 5 mM MgCl2, 100 mM NaCl, 2 mM dithiothreitol. Purified proteins were dialyzed against the same buffer containing only 20 mM Tris-HCl, pH 7.6, concentrated by ultrafiltration (Mr cut-off 8000), snap-frozen in liquid nitrogen, and stored at -80 °C. The purity of the protein preparations was assayed by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Blue (Fig. 2). Protein concentrations were estimated from Coomassie-stained gels and by the Bio-Rad protein assay system.
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Due to the design of the expression vectors, the proteins carry the following additional amino acids. SopE78240 and variants thereof carry the additional N-terminal amino acids GS; Cdc42 and Cdc42D65A carry the N-terminal amino acids GSRRASVGSKIISA. Cdc42' (residues 1178) carries two additional N-terminal residues (GS), and the 13 C-terminal amino acids (PPEPKKSRRCVLL) are absent.
To test the stability of the preparations of SopE' and GST-SopE' and variants thereof, a 60 µM solution of the proteins in buffer S (40 mM HEPES/NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2) was incubated at 25 °C, and at several time points between 45 min and 48 h, precipitated material was removed by centrifugation, and 1-µl aliquots of the soluble fractions were analyzed by SDS-PAGE and staining with Coomassie Blue.
Preparation of the Cdc42-mGDP ComplexCdc42, Cdc42', and Cdc42D65A were loaded with O-(N-methylanthraniloyl)-GDP (mGDP) essentially as described for Rac1-mGDP (25). Briefly, GST-SopE' was bound to a glutathione-Sepharose 4B column. Cdc42 in buffer A (50 mM Tris-HCl, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 2 mM dithiothreitol) was loaded to the column, and unbound Cdc42 was removed by extensive washing with buffer A. The Cdc42 bound to GST-SopE' was eluted as a Cdc42-mGDP complex with 180 µM mGDP in buffer A and separated from free mGDP by gel filtration chromatography. Fractions containing Cdc42-mGDP were identified by fluorescence spectroscopy, pooled, concentrated by ultrafiltration (Millipore Ultrafree-15; Mr cut-off 8000), snap-frozen, and stored at -80 °C.
Fluorescence SpectrometryAll fluorescence measurements were performed at 20 °C in buffer S (40 mM HEPES/NaOH, pH 7.4, 100 mM NaCl, 5 mM MgCl2) on a MOS-450/AF-CD kinetic spectropolarimeter (Bio-Logic). For all kinetic measurements, the time courses of mGDP release from Cdc42 or Cdc42' in the presence of 1 mM nonfluorescent GDP were monitored as decreased fluorescence (excitation at 366 nm, 8-nm bandwidth) using a 400-nm long pass filter. For analysis of Michaelis-Menten parameters kcat and Km in multiple turnover reactions, increasing concentrations of Cdc42'-mGDP (final concentration of 175 µM) were premixed with unlabeled GDP (1 mM final concentration). The reactions were started by the addition of SopE', GST-SopE', or variants thereof (final concentrations of 25 or 100 nM), and the decrease of mant fluorescence was recorded.
For measurement of the single turnover kinetics of SopE'-catalyzed nucleotide release, increasing concentrations of SopE', GST-SopE', or variants thereof (final concentration, 122.5 µM) in the presence of unlabeled GDP (final concentration, 1 mM) were mixed with Cdc42-mGDP or Cdc42'-mGDP (final concentration, 75 nM) at 20 °C in buffer S using an SFM-20 stopped flow device (Bio-Logic) attached to the MOS-450/AF-CD spectrometer, and release of mGDP from Cdc42 or Cdc42' was monitored. Single turnover kinetics with the "slow" variants of SopE' or GST-SopE' were measured by the addition of increasing concentrations of SopE (final concentration of 0.530 µM) to Cdc42-mGDP (final concentration of 75 or 750 nM) in the presence of 1 mM GDP and following the dissociation of mGDP from Cdc42 by fluorescence spectrometry. Single exponentials were fitted to all kinetic data using the program Biokine (Bio-Logic).
Far-UV CD SpectroscopyFar-UV CD spectra of the variants of SopE' and GST-SopE' (11.6 µM in 10 mM potassium phosphate, pH 7.0) were recorded at room temperature on the MOS-450/AF-CD in the spectropolarimeter mode. Sensitivity was set to 100 millidegrees, and spectra were taken with a scan speed of 12 nm/min and a bandwith of 1 nm in a 2-mm quartz cuvette. The spectra were accumulated twice, corrected for background, and converted to mean residue ellipticities according to Schmid (31).
Rearrangements of the Actin Cytoskeleton in Cultured COS7
Cells COS7 cells were grown on glass coverslips to 70% confluence
in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 5% fetal
calf serum as described (28).
Cells were infected for 20 min with S. typhimurium mutant M516 or
M516 transformed with expression vectors for wild type SopEM or
variants thereof (pM438 or pM461pM472; multiplicity of infection = 30
bacteria/cell). After infection, cells were fixed with 4% paraformaldehyde in
PBS and permeabilized with PBS containing 0.1% Triton X-100. Actin filaments
were stained with rhodamine-phalloidin (Sigma; 1:500 in PBS, 3% BSA), bacteria
with rabbit -Salmonella O-1,4,5,12
(8) antiserum (Difco, 1:400 in
PBS, 3% BSA), and a secondary goat
-rabbit fluorescein isothiocyanate
conjugate (Sigma; 1:250 in PBS, 3% BSA). DNA was stained with
4',6-diamidino-2-phenylindole (1:2000 in PBS, 3% BSA). The mounted
coverslips were analyzed by fluorescence microscopy. Cells associated with at
least three bacteria and not undergoing cell division (i.e. showing
intact nuclei and no chromosome condensation) were classified based on the
degree of actin cytoskeletal rearrangements (ruffling). Cells displaying
profound ruffling (++), weak ruffling (+), and no detectable cytoskeletal
rearrangements (-) (see Fig. 7) were enumerated by two different researchers in a "blinded"
manner. The average distribution (as a percentage) of the infected COS7 cells
among the groups ++, +, and - was calculated as follows: percentage of
"++ cells" = (number of "++ cells"/total number of
cells associated with
3 bacteria).
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RESULTS |
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In the complex, Asp-124SopE forms side chain contacts with Tyr-32Cdc42 and with main chain amides of Thr-35Cdc42 and Val-36Cdc42 (Fig. 1b; Table I). SopED124E was constructed to slightly distort and SopED124A to completely disrupt this contact.
The 166GAGA169 loop, which inserts between and forms
backbone contacts with residues of the switch I and switch II regions of Cdc42
is regarded as the key functional element of SopE-like GEFs. In a recent
study, we had shown that the catalytic efficiency of a
GST-SopEG168A fusion protein was at least 100-fold reduced
(26). To analyze the effect of
structural alterations of the 166GAGA169 loop in more
detail, we have now constructed two additional variants
(SopEG168 and SopEG168V). These
changes should allow to study the effect of steric hindrance in the insertion
of the 166GAGA169 loop between the switch I and II
regions of Cdc42.
Asp-103SopE is conserved between all members of the SopE family, and the side chain carboxylate of Asp-103SopE forms an ionic contact to the guanidinium group of Arg-66Cdc42 (Fig. 1b). SopED103E and SopED103A were constructed to analyze the effect of a slight distortion and the complete elimination of this contact.
Lys-198SopE was predicted to stabilize the rearranged switch I by forming a salt bridge with the Asp-38Cdc42 side chain (26). Interestingly, Lys-198 is replaced by an asparagine residue in SopE from other Salmonella strains with different virulence characteristics than S. typhimurium (i.e. Salmonella dublin) and is replaced by glutamine in BopE. We speculated that if this amino acid was involved in catalysis it might determine substrate specificity of SopE-like GEFs. SopEK198A was constructed to analyze the role of the Lys-198SopE-Asp-38Cdc42 contact in catalysis. Furthermore, we constructed SopEK198E and SopEK198R to study the effect of a negatively charged and a bulkier positively charged group at this position.
In order to analyze the effect of these mutations on the in vitro GEF activity of the catalytic fragment of SopE (aa 78240; called SopE' from here on), the GST-SopE' expression vector pM164 was mutated as described under "Materials and Methods" (Table III).
Preparation of Recombinant SopE' Variants
SopE' protein variants were purified as GST fusion proteins, and the
GST part was cleaved off by thrombin as described under "Materials and
Methods." The SopE' proteins were purified as described
(24), and the quality of the
protein preparations was analyzed by SDS-PAGE
(Fig. 2).
The SopE' variants carrying amino acid exchanges at Gln-109SopE, Asp-124SopE, and Lys-198SopE were stable at room temperature (data not shown; see "Materials and Methods") and showed far-UV CD spectra indistinguishable from that of wild type SopE' (data not shown). This indicated that the amino acid exchanges had no dramatic effects on the overall protein conformation. In contrast, variants with amino acid exchanges in Asp-103SopE and Gly-168SopE were found to precipitate after 45 min to 6 h of incubation at room temperature and showed altered far-UV CD spectra (data not shown; see "Materials and Methods"). Therefore, we could not use these latter SopE' variants for functional studies. However, the GST-SopE' fusion proteins altered at Asp-103SopE and Gly-168SopE were found to be stable at room temperature and showed far-UV CD spectra indistinguishable from that of wild type GST-SopE' (data not shown; see "Materials and Methods").
We found that wild type GST-SopE' fusion protein has a similar level
of activity as the "cleaved off" wild type SopE'
(Fig. 3). Therefore, we decided
to use GST-SopE'D103E, GST-SopE'D103A,
GST-SopE'D103E,
GST-SopE'G168,
GST-SopE'G168A, and GST-SopE'G168V to
characterize the roles of Asp-103SopE and Gly-168SopE in
G nucleotide exchange. A detailed kinetic analysis of these GST fusion
proteins is complicated due to the tendency of GST to form dimers.
Nevertheless, comparison of the observed rates of G nucleotide exchange of
wild type or variant GST-SopE' fusion proteins can still yield useful
information about the functional importance of Asp-103SopE and
Gly-168SopE.
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Kinetic Analysis of Guanine Nucleotide Exchange Activity of the SopE
Variants
Multiple Turnover CatalysisThe G nucleotide exchange
activity of the SopE' variants was analyzed by fluorescence spectrometry
using mGDP-loaded Cdc42 (aa 1179, designated Cdc42'-mGDP from
here on) as a substrate (30).
mGDP is a GDP derivative that is frequently used in kinetic studies, because
the fluorescence of the mant moiety can change dramatically upon binding to
GTPases. mGDP bound to Cdc42 has a 4-fold higher fluorescence intensity than
unbound mGDP (24,
32,
33). Generally, the presence
of the fluorophore has little effect on the kinetic parameters of G nucleotide
release or GTP hydrolysis (32,
3437).
The Michaelis-Menten parameters kcat and Km of SopE'K198A, SopE'K198R, SopE'K198E, and wild type SopE' were analyzed by following the decrease in mant fluorescence upon the dissociation of mGDP from the Cdc42'-mGDP complex. We determined the rate constants of the multiple turnover G nucleotide release reactions for increasing concentrations of Cdc42'-mGDP in the presence of 25 nM (or 100 nM in the case of SopE'K198E) SopE' protein and 1 mM GDP. The observed rates were plotted against the Cdc42'-mGDP concentration and the Michaelis-Menten parameters kcat and Km were determined by fitting hyperbolas to the data (Fig. 4a; Table IV). Removal of the Lys-198SopE side chain or replacement by another positively charged side chain (SopE'K198A, SopE'K198R) resulted in 24-fold increased kcat and Km values but did not affect the catalytic efficiency kcat/Km (Table IV). In contrast, the exchange of the Lys-198SopE side chain by a negatively charged residue (SopE'K198E) had a much more pronounced effect, and the catalytic efficiency of this SopE' variant was reduced by more than 20-fold (Fig. 4a, Table IV). The kcat and Km values for SopE'K198E could not be determined, since the multiple turnover reaction did not reach saturation up to a substrate concentration of 75 µM (Fig. 4a, data not shown).
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SopE' variants SopE'Q109N, SopE'Q109A, SopE'D124A, and SopE'D124E have not been analyzed in multiple turnover assays, because the observed rates of G nucleotide release were just slightly above the rate of spontaneous mGDP release (5.0 ± 0.2 x 10-5 s-1; data not shown) from the Cdc42'-mGDP complex.
We also analyzed the rates of mGDP release from Cdc42'-mGDP catalyzed by GST-SopE'D103E, GST-SopE'D103A, and wild type GST-SopE' under multiple turnover conditions (1 mM GDP, 1.1 µg/ml GST-SopE' protein, 145 µM Cdc42'-mGDP) (Fig. 4b). The observed rates were plotted against the Cdc42'-mGDP concentration, and the data could be fitted by hyperbolas as described above. However, the analysis of the data obtained for the GST fusion proteins is complicated due to dimerization equilibria that are frequently observed between GST fusion proteins. Because of this phenomenon, only approximate values were known for the effective concentration (moles/volume) of the enzyme, and the effective concentration is likely to be lower (12-fold) than the total enzyme concentration. To account for this problem we have used the (weight/volume) enzyme concentration instead of the molar enzyme concentration. The catalytic constants (kcat* and Km*) calculated in this way should only be compared between different GST-SopE' fusion proteins. We found that GST-SopE'D103E had virtually the same and GST-SopE'D103A had a 2-fold lower catalytic efficiency (kcat*/Km*) than wild type GST-SopE'. The reduced catalytic efficiency of GST-SopE'D103A was mainly attributable to an increased Km* value and indicated that the negatively charged Asp-103SopE plays a small but detectable role in catalysis.
Multiple turnover catalysis by all GST-SopE' Gly-168 variants was so inefficient that the observed rates did not differ significantly from the rate of spontaneous mGDP dissociation from the Cdc42'-mGDP complex. For this reason, we did not determine their multiple turnover kinetic parameters.
Single Turnover CatalysisTo analyze the catalytic defects of SopE' variants altered at Gln-109SopE, Asp-124SopE, and Gly-168SopE in more detail, we have performed single turnover experiments. This type of experiment employs a large excess of enzyme and thus monitors the first part of the catalytic cycle, including formation of the enzyme-substrate complex, possible conformational rearrangements that might occur prior to mGDP release, and mGDP release. It is, however, "blind" for all later steps of the catalytic cycle. Cdc42'-mGDP (final concentration of 75 nM) was rapidly mixed with GDP (1 mM final concentration) and an excess of SopE' or GST-SopE' (final concentrations of 0.722.5 µM or 22860 µg/ml, respectively). The release of mGDP from the Cdc42'-mGDP complex was followed by fluorescence spectrometry (Fig. 5a; see "Materials and Methods"), and the rates of mGDP release were determined for all of the SopE' and GST-SopE' variants we had created. The observed rates (kobs) were plotted against the enzyme concentration (in µM or µg/ml; Fig. 5, b and c). In line with our earlier results (24), we observed a linear correlation between kobs and the SopE' and GST-SopE' concentrations over the entire range of enzyme concentrations tested (Fig. 5, b and c). From the slopes of the plots in Fig. 5, b and c, we have calculated the single turnover catalytic efficiency of SopE' (kreact/Kmsto in s-1 M-1), GST-SopE' (kreact*/Kmsto* in s-1 g-1 liter; Table V; see "Materials and Methods") and variants thereof.
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Replacement of the side chain of Lys-198SopE by another positively charged side chain (SopE'K198R) and its truncation to alanine (SopE'K198A) did not reduce the single turnover catalytic efficiency (kreact/Kmsto in s-1 M-1) by more than 2-fold (Table V). In contrast, a SopE' variant carrying a negatively charged side chain at this position (SopE'K198E) had a 30-fold reduced catalytic efficiency. This suggested that the Lys-198SopE interaction with the negatively charged side chain of Asp-38Cdc42 (Fig. 1b) did not contribute to catalysis, but that these positions of SopE and Cdc42 were located sufficiently close to allow repulsion between two negatively charged side chains (in SopE'K198E and Asp-38Cdc42) to destabilize the transition state.
SopE'Q109A, SopE'Q109N, SopE'D124A, and SopE'D124E had 160800-fold reduced catalytic efficiencies (Fig. 5b; Table V). Therefore, Gln-109SopE and Asp-124SopE seem to play key roles in the rate-limiting step(s) of the G nucleotide release reaction.
The variant GST-SopE'D103E had a 2-fold and GST-SopE'D103A a 4-fold lower catalytic efficiency than wild type GST-SopE' (see Table V). These data are in line with the multiple turnover experiments and suggest that Asp-103 plays a minor but detectable role in catalysis.
The most dramatic effects were observed with SopE variants altered at
Gly-168SopE. The catalytic efficiency was reduced more than
800-fold in the case of GST-SopE'G168, more
than 5000-fold in the case of GST-SopE'G168A, and more than
20,000-fold in the case of GST-SopE'G168V
(Fig. 5c;
Table V). Even at
concentrations of 430 µg/ml GST-SopE'G168V, the observed
rates of G nucleotide release were only 3.5-fold above the rate for
spontaneous dissociation of mGDP from Cdc42, whereas the same concentration of
wild type GST-SopE' accelerated G nucleotide release almost
105-fold. These data extend our earlier observations
(26) and demonstrate the
crucial role of the SopE 166GAGA169 loop for G
nucleotide exchange.
Mutagenesis of Asp-65Cdc42
The structure of the Cdc42-SopE' complex suggested that a network of
polar contacts of the Gln-109SopE side chain to backbone atoms of
Tyr-64Cdc42, Asp-65Cdc42, and Arg-66Cdc42 and
to the carboxylate group of Asp-65Cdc42 is important for catalysis
(Fig. 1b)
(26). In line with this
hypothesis, we found that SopE'E109A and
SopE'E109N had a strongly reduced catalytic efficiency (see
above). To analyze the role of the contacts mentioned above in more detail, we
have constructed an expression vector for GST-Cdc42D65A and
prepared the Cdc42D65A-mGDP complex as described under
"Materials and Methods." The intrinsic rate of mGDP dissociation
from Cdc42D65A (koff = 7.4 ± 0.7 x
10-5 s-1) (data not shown) was not significantly
different from that of wild type Cdc42 (koff = 7.2
± 2.5 x 10-5 s-1; data not shown). Single
turnover assays were performed to analyze the effect of the
Cdc42D65A amino acid exchange on G nucleotide release catalyzed by
wild type SopE', SopE'Q109N, and
SopE'Q109A.
The catalytic efficiency of wild type SopE' was about 7-fold lower with Cdc42D65A than with wild type Cdc42 (8.9 x 104 versus 6.9 x 105 s-1 M-1) (Fig. 6; Table VI). This indicated that the polar contact between the Gln-109SopE side chain and the carboxylate group of Asp-65Cdc42 is important for catalysis. In contrast to wild type SopE', the variants SopE'Q109N and SopE'Q109A were "blind" toward the Cdc42D65A amino acid exchange (5.0 x 103 versus 4.8 x 103 s-1 M-1 and 1.5 x 103 versus 1.1 x 103 s-1 M-1) (Fig. 6; Table VI). This demonstrated that the SopE' variants SopE'Q109N and SopE'Q109A are per se unable to form the contact to the Asp-65Cdc42 side chain during catalysis of G nucleotide release.
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In Vivo Studies: Actin Cytoskeletal Rearrangements
SopE is one of the Salmonella toxins (effector proteins) injected
into host cells via the SPI-1 type III secretion system during the course of
an infection (22). The
injected effector proteins lead to a variety of responses including profound
cytoskeletal rearrangements in epithelial cells and fibroblasts. In recent
years, SopE, SopE2, and SopB have been identified as the principal S.
typhimurium effector proteins involved in the induction of host cell
cytoskeletal rearrangements
(22,
38). SopE and SopE2 induce
these cytoskeletal rearrangements via activation of host cellular Cdc42 and/or
Rac1 (20,
23,
25), whereas the mechanism of
SopB-mediated cytoskeletal rearrangements is still a matter of debate
(39,
40). Nevertheless, it was
found that an S. typhimurium triple mutant (M516)
(28) lacking the three
effector proteins SopE, SopE2, and SopB is incapable of inducing these
profound cytoskeletal rearrangements in cultured cells
(25,
39). When the S.
typhimurium mutant M516 is transformed with a SopE expression plasmid, it
regains its capacity to induce actin cytoskeletal rearrangements in host cells
(25). Therefore, we could use
this complementation assay for in vivo analysis of the functional
defects of the SopE variants described above.
To express the SopE variants in M516, we have constructed a series of derivatives of pM438 (Table III). pM438 is a low copy pACYC184 derivative and encodes an M45 epitope-tagged version of SopE (SopEM; see "Materials and Methods") under the control of the native sopE promoter. The vectors for the expression of wild type SopEM (pM438) and the SopEM variants (pM461pM472) were transformed into M516.
First, we have determined the stability of the SopEM variants.
For this purpose, the SopEM protein pools present in the bacterial
cultures have been analyzed by Western blot using a monoclonal -M45
antibody (Fig. 7a). We
found that all SopEM variants were present in similar amounts, and
we did not detect significant levels of degradation with any of the proteins
(Fig. 7a). Then these
strains were used to infect COS7 tissue culture cells for 20 min at a
multiplicity of infection of 30 bacteria per cell. To stop the infection, the
cells were fixed and stained to visualize the polymerized actin
(Fig. 7b). The
capacity of each strain to rearrange the actin cytoskeleton was scored as
described under "Materials and Methods." As expected, we found
that M516 had a very low capacity and that M516 expressing wild type
SopEM (pM438) had a high capacity to induce actin cytoskeletal
rearrangements (Fig. 7, b and
c). In this assay, SopEMD103A,
SopEMD103E, SopEMK198A,
SopEMK198R, and SopEMK198E
displayed a similar level of activity than wild type SopEM. In
contrast, the expression of SopEMD124A,
SopEMD124E, and SopEMQ109N could
only partially restore the capacity of M516 to rearrange the host cellular
actin. It should be noted that a significant portion (1015%) of the
cells infected with M516 expressing SopEMQ109N and
SopEMD124A did not show any cytoskeletal rearrangements
(Fig. 7, b and
c). SopEMQ109A and
SopEM
G168 showed a low residual
activity, and SopEMG168A and
SopEMG168V were almost completely inactive
(Fig. 7c). Overall,
the results obtained in this in vivo assay are in line with our
kinetic analyses; SopE variants with a low G nucleotide exchange activity also
had a low capacity to mediate cytoskeletal rearrangements, confirming the
in vivo relevance of our structure-based and biochemical conclusions.
Interestingly, some residual in vivo activity was observed with SopE
variants that had a 160840-fold reduced catalytic efficiency. This is
explained by the fact that the experimental conditions for the tissue culture
cell infection experiments had been optimized to maximize the
Salmonella-induced responses. This also explains why the subtle
catalytic defects of SopE variants carrying amino acid exchanges at positions
Asp-103SopE and Lys-198SopE did not lead to detectable
defects in the in vivo assay.
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DISCUSSION |
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The insertion of the 166GAGA169 loop between the
switch I/II regions of Cdc42 was the most striking feature of the SopE-Cdc42
complex (Fig. 1b).
Kinetic analysis of SopE mutants demonstrated that the single turnover
catalytic efficiency decreased in the following order:
SopEG168 (800-fold reduced) >
SopEG168A (5000-fold reduced) > SopEG168V
(20,000-fold reduced). These data lend further experimental support to the
notion that the 166GAGA169 loop is a key functional
element of SopE and suggest that steric constraints are of great importance.
However, the functional importance of individual contacts could not be
analyzed further by mutagenesis studies, because contacts of the
166GAGA169 loop are made primarily via the peptide
backbone.
In the crystal structure, the side chain carboxylate of Asp-103SopE forms an ionic contact with the guanidinium group of Arg-66Cdc42 (26). A slight distortion (in SopED103E) resulted in a 2-fold reduced catalytic efficiency and the complete elimination of the contact (in SopED103A) in a 4-fold reduced catalytic efficiency of G nucleotide release. This indicates that the ionic contact made by Asp-103SopE makes only a small but detectable contribution to catalysis of the G nucleotide release reaction. It is interesting to note that a similar interaction is found in the Dbl-like GEF Tiam1 in complex with Rac1 between Glu-1239Tiam1 and Arg-66Rac1.
The functional roles of the SopE residues Gln-109SopE and Asp-124SopE were of special interest, because they have been suggested to serve equivalent functions as the residues Asn-810Dbs/Asn-1232Tiam1/Asn-1421Intersectin, and Glu-639Dbs/Glu-1047Tiam1/Glu-1244Intersectin in Dbl-like GEFs (12, 26). In the SopE-Cdc42 crystal, the Gln-109SopE side chain appears to stabilize the altered conformation of switch II via a network of polar contacts with backbone atoms of Tyr-64Cdc42, Asp-65Cdc42, and Arg-66Cdc42 and the carboxylate group of Asp-65Cdc42 (Fig. 1). Kinetic analysis of guanine nucleotide exchange of wild type SopE, SopEQ109N, and SopEQ109A with wild type Cdc42 and Cdc42D65A demonstrated that these contacts are of importance for catalysis. Disruption of the contact between Gln-109SopE and the Asp-65Cdc42 side chain (in Cdc42D65A) reduced SopE-mediated GEF activity by 7-fold. Disruption of all contacts made by Gln-109SopE (in SopEQ109A) reduced GEF activity even further (700-fold). Therefore, polar contacts to the backbone of Tyr-64Cdc42, Asp-65Cdc42, and/or Arg-66Cdc42 also contribute to catalysis.
The side chain of SopEQ109N is merely shortened by a single CH2 group. Nevertheless, SopEQ109N was about 160-fold less active than wild type SopE. In addition, SopEQ109N was "blind" to the Cdc42D65A amino acid exchange, which indicated that SopEQ109N was unable to form the contact with the side chain of Asp-65Cdc42. From these observations, we conclude that the identity of functional groups of the Gln-109SopE side chain and their spatial arrangement are of critical importance. Only little experimental data is available about the functional role of the equivalent amino acid residue in Dbl-like GEFs (Asn-673Dbl, Asn-810Dbs, Asn-1232Tiam1). Nevertheless, a DblN673A,D674A mutant was virtually inactive (41). Despite caveats attributable to the double mutation, these data are in line with the structural analyses and indicate that Asn-673Dbl plays a similar role in Dbl-like GEFs as Gln-109SopE in SopE-like proteins.
Asp-124SopE was speculated to affect G nucleotide exchange by stabilizing the rearranged switch I via side chain contacts to Tyr-32Cdc42 and the main chain amides of Thr-35Cdc42 and Val-36Cdc42 (26). Indeed, complete disruption of these contacts (SopED124A) as well as small spatial changes (one additional CH2-group in SopED124E) dramatically reduced the catalytic efficiency and the in vivo activity. Mutation of the equivalent position of Dbl-like GEFs is also detrimental to GEF activity (DbsE639A (6); TrioE1240A (42)). This strongly supports the notion that Asp-124SopE is functionally equivalent to the analogous residue of Dbl-like GEFs.
Of the SopE residues analyzed in this study, only the side chain of Lys-198SopE, which was predicted to stabilize the rearranged switch I by forming a salt bridge with the Asp-38Cdc42 side chain (26), was found to be dispensable. Nevertheless, the 30-fold reduced catalytic efficiency of SopEK198E suggested that this amino acid is located close to the negatively charged Asp-38Cdc42 side chain in the transition state(s) of the rate-limiting step(s).
The catalytic domains (aa 78240 of SopE) of the three currently
known SopE-like GEFs (SopE, SopE2, and BopE) are predicted to share similar
folds (Fig. 1a) but
have only 23% identical amino acids. Lys-198SopE, which was not
found to be required for G nucleotide exchange, is present in SopE and SopE2
from S. typhimurium but not in BopE
(Fig. 1a). In
contrast, the catalytically important residue Gly-168SopE of the
166GAGA169 loop, Asp-124SopE,
Gln-109SopE, and Asp-103SopE are conserved in all
members of the SopE family. Based on these conserved functionally important
residues and on conserved elements of the secondary structure, we propose that
two motifs might be common to all members of the SopE family: (a)
DX5QX14D (where X
represents any amino acid, and underlined residues represent the
-helix; corresponding to aa 103124 of SopE; see
Fig. 1a) and
(b) a GAGXNP motif located further downstream (aa
166171 of SopE; see Fig.
1a) of the first motif. It might be possible to use these
motifs for identification of additional distantly related members of the SopE
family in genome databases. However, so far our attempts to screen for such
proteins in the current databases have only recovered SopE, SopE2, and BopE
sequences.
Our data demonstrate that SopE employs both elements unique to the SopE family (i.e. 166GAGA169 loop, Asp-103SopE) and residues (i.e. Gln-109SopE, Asp-124SopE) that are functionally equivalent to key residues of Dbl-like GEFs in order to catalyze G nucleotide exchange. Interestingly, the kinetic parameters of Dbl-like and SopE-like GEFs are also quite similar; the Michaelis-Menten constants (Km) for the multiple turnover G nucleotide exchange reaction are virtually identical (low micromolar range) between Dbl-like and SopE-like GEFs, and the catalytic rates also range in a similar order of magnitude (kcat = 0.10.3 s-1 for Dbl-like GEFs versus 120 s-1 for SopE or SopE2 (24, 25, 37, 43) (this study). However, despite all of these similarities, it should be kept in mind that the structure of the SopE-like GEFs differs completely from that of Dbl-like GEFs (26) and that both families of GEFs have probably evolved independently; the Dbl-like GEFs have evolved as highly regulated switches in eukaryotic cells, and the SopE-like family have evolved as bacterial toxins ("deregulated" GEFs), which allow the bacteria to efficiently manipulate signaling in eukaryotic cells.
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FOOTNOTES |
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These authors contributed equally to the experimental work.
|| Present address: Netherlands Cancer Institute, Molecular Carcinogenesis,
Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
To whom correspondence should be addressed: Institute of Microbiology, ETH
Zürich, Schmelzbergstr. 7, CH-8092 Zürich, Switzerland. Tel.:
41-1-632-5143; Fax: 41-1-632-1129; E-mail:
hardt{at}micro.biol.ethz.ch.
1 The abbreviations used are: GEF, guanine nucleotide exchange factor; DH,
Dbl homology; PH, pleckstrin homology; aa, amino acid; mant,
O-(N-methylanthraniloyl); mGDP, mant-GDP; GST, glutathione
S-transferase; PBS, phosphate-buffered saline; BSA, bovine serum
albumin; TRITC, tetramethylrhodamine isothiocyanate.
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ACKNOWLEDGMENTS |
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REFERENCES |
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