Pseudomonas aeruginosa Exoenzyme S, a Double ADP-ribosyltransferase, Resembles Vertebrate Mono-ADP-ribosyltransferases*

Anand K. GanesanDagger , L. Mende-Mueller§, Jorg Selzer, and Joseph T. BarbieriDagger parallel

From the Medical College of Wisconsin, Departments of Dagger  Microbiology and Molecular Genetics and § Biochemistry, Milwaukee, Wisconsin 53226 and the  Institute of Pharmacology and Toxicology, University of Freiburg, D-79104 Freiburg, Germany

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous data indicated that Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras at multiple sites. One site appeared to be Arg41, but the second site could not be localized. In this study, the sites of ADP-ribosylation of c-Ha-Ras by ExoS were directly determined. Under saturating conditions, ExoS ADP-ribosylated Ras to a stoichiometry of 2 mol of ADP-ribose incorporated per mol of Ras. Nucleotide occupancy did not influence the stoichiometry or velocity of ADP-ribosylation of Ras by ExoS. Edman degradation and mass spectrometry of V8 protease generated peptides of ADP-ribosylated Ras identified the sites of ADP-ribosylation to be Arg41 and Arg128. ExoS ADP-ribosylated the double mutant, RasR41K,R128K, to a stoichiometry of 1 mol of ADP-ribose incorporated per mol of Ras, which indicated that Ras possessed an alternative site of ADP-ribosylation. The alternative site of ADP-ribosylation on Ras was identified as Arg135, which was on the same alpha -helix as Arg128.

Arg41 and Arg128 are located within two different secondary structure motifs, beta -sheet and alpha -helix, respectively, and are spatially separated within the three-dimensional structure of Ras. The fact that ExoS could ADP-ribosylate a target protein at multiple sites, along with earlier observations that ExoS could ADP-ribosylate numerous target proteins, were properties that have been attributed to several vertebrate ADP-ribosyltransferases. This prompted a detailed alignment study which showed that the catalytic domain of ExoS possessed considerably more primary amino acid homology with the vertebrate mono-ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases. These data are consistent with the hypothesis that ExoS may represent an evolutionary link between bacterial and vertebrate mono-ADP-ribosyltransferases.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystic fibrosis patients, burn wound victims, and the immunocompromised are particularly susceptible to infection by Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen (1). A number of virulence determinants contribute to the pathogenesis of P. aeruginosa, including two ADP-ribosyltransferases, Exotoxin A and Exoenzyme S (ExoS)1 (2). ExoS is a 49-kDa protein (3) that is secreted by the type III mechanism of P. aeruginosa (4), and ADP-ribosylates a number of target proteins in vitro including apolipoprotein A1, IgG3 (5), vimentin (6), and several members of the Ras superfamily (7). In vivo, ExoS is a cytotoxin (8) that ADP-ribosylates Ras during the course of infection in cultured cells (9), disrupting Ras-mediated signal transduction (10). Indirect methods were used to determine that Exoenzyme S ADP-ribosylates c-Ha-Ras at multiple sites (10).

Several vertebrate ADP-ribosyltransferases have been identified to date. The vertebrate ADP-ribosyltransferases possess specific properties that are distinct from the bacterial ADP-ribosyltransferases. Several of the vertebrate ADP-ribosyltransferases have the capacity to ADP-ribosylate multiple target proteins. For example, a murine lymphocyte transferase ADP-ribosylates a number of cell surface proteins (11), whereas rabbit skeletal muscle ADP-ribosyltransferase ADP-ribosylates several proteins in skeletal muscle T-tubules (12). In addition, several vertebrate ADP-ribosyltransferases modify target proteins at multiple sites. Both turkey erythrocyte type A ADP-ribosyltransferase (13) and chicken ADP-ribosyltransferase (14) ADP-ribosylate actin at two arginine residues. These sites of ADP-ribosylation are located in different regions of actin, which suggests that each ADP-ribosylation event is independent. Although the role of endogenous ADP-ribosylation in the regulation of eukaryotic cell physiology has not been defined in detail, the RT6 transferase appears to be involved in the regulation of T-lymphocyte function (15), and endogenous ADP-ribosylation may contribute to hippocampal long-term potentiation (16).

In this study, the ADP-ribosylation of Ras by Exoenzyme S was characterized. Using both mass spectrometry and rpHPLC coupled with Edman degradation, it was determined that ExoS ADP-ribosylates Ras at arginines 41 and 128. These residues are located in a beta -sheet and alpha -helix, respectively, which are spatially separated in Ras. Thus, ExoS has functional similarities to the eukaryotic ADP-ribosyltransferases because ExoS ADP-ribosylates a number of target proteins in vitro and ADP-ribosylate Ras at two nonadjacent arginine residues. In addition, homology studies have identified more primary sequence homology between ExoS and the vertebrate ADP-ribosyltransferases than the bacterial ADP-ribosyltransferases.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The following reagents were purchased: [adenylate phosphate-32P]NAD from NEN Life Science Products, Sculptor in vitro mutagenesis kit from Amersham Pharmacia Biotech, bovine serum albumin from Pierce Biochemicals, DNA oligonucleotides from Operon, and GTPgamma S from Sigma. Recombinant factor activating exoenzymes (FAS) and a c-Ha-Ras vector were gifts from H. Fu (Emory University).

Purification of His-tagged Ras Proteins-- Recombinant His-tagged Ras proteins were expressed in Escherichia coli and purified by Ni2+ affinity chromatography as described previously (10) with several modifications. His-tagged Ras proteins were eluted from the Ni 2+ affinity resin in elution buffer containing 3 µM GTP and 10 mM MgCl2. Eluted proteins were dialyzed into buffer (20 mM Tris-HCl (pH 7.6), 10 mM EDTA, 5 mM MgCl2, 1 mM dithiothreitol, and 10% glycerol) and stored at -70 °C. These conditions were optimal for exchange of bound nucleotide from Ras proteins.

Nucleotide Loading of Ras Proteins-- 20 µM Ras was incubated alone or with 1 mM of either GTPgamma S or GDP for 30 min at 30 °C. Reactions were stopped by the addition of 20 mM MgCl2. The stoichiometry of nucleotide loading of Ras was monitored radioanalytically, using [35S]GTPgamma S.

ADP-ribosylation of Ras Proteins by ExoS-- Reaction mixtures contained (25 µl): 0.2 M sodium acetate (pH 6.0), 50 µM [adenylate phosphate-32P]NAD (specific activity 0.25 µCi per 1.25 nmol NAD), 5 µM Ras or RasDelta CAAX, FAS, and Delta N222 (a catalytic, deletion peptide of ExoS). FAS and Delta N222 were added in equivalent amounts. Reactions were stopped at the indicated times by spotting an aliquot of the reaction mixture onto trichloroacetic acid-saturated Whatman 3-mm paper. The paper was washed three times for 10 min each in 7.5% trichloroacetic acid, and radioactivity was quantitated by scintillation counting. Stoichiometry of ADP-ribosylation was determined as the moles of ADP-ribose incorporated/mole of Ras. Velocity of ADP-ribosylation was determined by linear regression analysis. RasDelta CAAX is a deletion peptide of Ras where the four C-terminal amino acids have been deleted, which eliminates its capacity to be acylated, making it more amenable to biochemical manipulation.

Determination of the Sites of ADP-ribosylation within Ras-- RasDelta CAAX or RasDelta CAAX-R41K,R128K (20 µM) was incubated with 0.4 µM Delta N222, 400 µM NAD, and 1.2 µM FAS for 2 h at room temperature. Stoichiometry of ADP-ribosylated Ras was determined by subjecting an aliquot of the reaction mixture to SDS-polyacrylamide gel electrophoresis and subjecting the protein band corresponding to Ras to scintillation counting. Following ADP-ribosylation, the reaction mixture was dialyzed overnight into 10 mM Tris-HCl (pH 7.6), containing 20 mM NaCl, to remove unreacted NAD. Dialyzed samples were distributed into aliquots and lyophilized. Lyophilized samples were resuspended in 50 mM NH4Ac (pH 4.0), and Staphylococcus aureus V8 protease was added at a w:w ratio of 1:10 (protease:Ras). Samples were digested overnight, lyophilized, and suspended in 0.1% trifluoroacetic acid. The digested material was subjected to rpHPLC (C18), using a 5-40% acetonitrile gradient in 0.1% trifluoroacetic acid, and 1-min fractions were collected. Absorbance of eluted material was measured at 215 nm. Fractions were subjected to scintillation counting to identify peptides that were radiolabeled. Peptides in fractions containing radioactivity were subjected N-terminal amino acid sequencing. Protease-digested samples of ADP-ribosylated Ras and non-ADP-ribosylated Ras were also subjected to mass spectrometry as described previously (13).

Alignment of Protein Sequences-- Protein sequences were aligned using the PILEUP program of GCG. Using an algorithm similar to that described in Ref. 17, this program creates a multiple sequence alignment.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ExoS ADP-ribosylates Ras at Arg-41 and Arg-128-- Previous studies indicated that ExoS ADP-ribosylated Ras at multiple sites. Although indirect, these studies suggested that ExoS ADP-ribosylated Ras at Arg41 and at a second site. However, no specific second site of ADP-ribosylation could be resolved (10). In the present study, a direct approach was used to determine the sites of ADP-ribosylation within Ras by ExoS. Recombinant histidine-tagged RasDelta CAAX was ADP-ribosylated by ExoS to a stoichiometry approaching 2 mol of ADP-ribose incorporated per mol of Ras. ADP-ribosylated Ras was digested with S. aureus V8 protease, and peptides were fractionated by rpHPLC. The absorbance of eluted peptides was measured at 215 nm, and elution fractions were assayed for radioactivity (Fig. 1A). Analysis of the V8 peptides of ADP-ribosylated Ras revealed three radioactive fractions. The first radioactive fraction corresponded to the void volume of the column (6 min), where NAD and free ADP-ribose eluted (data not shown). The two additional fractions that contained radioactivity eluted at 24 and 48 min, within the resolving region of the chromatogram.


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Fig. 1.   Identification of arginine residues within Ras that are ADP-ribosylated by ExoS. Wild type RasDelta CAAX (panel A) or RasDelta CAAX-R41K,R128K (panel B) was ADP-ribosylated by Delta N222 ExoS to near completion. Following ADP-ribosylation, the protein was dialyzed to remove unreacted NAD and digested with S. aureus V8 protease. The resulting peptides were chromatographed on reversed-phase HPLC, and the absorbance of eluted material was measured at 215 nm (upper). Additionally, the absorbance of eluted material was measured at 259 nm to detect the presence of ADP-ribose (data not shown). Eluted fractions were assayed to detect radiolabeled peptides (lower).

The 24- and 48-min fractions were subjected to N-terminal amino acid sequencing. The first ten amino acids of the 24-min fraction contained a peptide corresponding to amino acids 38-47 of Ras, which included Arg41 (Table I). Amino acid yields at each cycle were similar (within about 2-fold), with the exception of cycle 4 where there was a reduction in the yield of the predicted amino acid, Arg41. Others have demonstrated that ADP-ribosylarginine is hydrolyzed by treatment with strong base (5). During the coupling of the free N terminus with phenyl isothiocyanate, the peptide was subjected to pH 9.0. When the products of cycle 4 of the Edman degradation were analyzed, no aberrant peak corresponding to ADP-ribosylarginine or hydrolyzed ADP-ribosylarginine was detected, and only minute amounts of free arginine were detected. The absence of hydrolytic products of ADP-ribosylarginine indicated that the ADP-ribose-arginine bond was not cleaved during the Edman degradation procedure. Hydrophilic phenyl isothiocyanate-modified amino acids will not be extracted with butyl acetate from the glass fiber filter during Edman degradation as shown in the case of cysteine.2 The fact that radioactivity was recovered on the glass fiber filter but not in the eluate of the Edman degradation is consistent with the idea that ADP-ribosylated arginine was not extracted from the glass fiber filter during cycle 4, and Arg 41 is one site of ADP-ribosylation. The first ten amino acids of the 48-min fraction contained a peptide corresponding to amino acids 127-136 of Ras, which included Arg128 and Arg135 (Table I). Amino acid yields at each cycle were similar (within about 2-fold), with the exception of cycle 2 where there was a reduction in the yield of the predicted amino acid, Arg128. This was consistent with Arg128 having been ADP-ribosylated. The high yield of Arg at cycle 9 indicated that Arg135 had not been ADP-ribosylated and indicated that the reduction in yield seen in cycle 2 of the 48-min fraction and cycle 4 of the 24-min fraction was not because of a generalized decrease in yield of arginine after Edman degradation.

                              
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Table I
N-terminal amino acid sequencing of Ras peptides that have been ADP-ribosylated by ExoS
Peptide sequencing was performed with a ABI371 automated sequencer with the preparation of peptides as described under "Experimental Procedures."

The sites that ExoS ADP-ribosylated on wild-type Ras were also determined by mass spectrometry. Ras alone or following ADP-ribosylation by ExoS to a stoichiometry approaching 2 mol of ADP-ribose per mol of Ras was digested with trypsin and subjected to mass spectrometry. Mass spectra of peptides from ADP-ribosylated Ras possessed two unique peptides relative to peptides from non-ADP-ribosylated Ras. One peptide was identified which possessed a molecular mass of 3636 daltons, which corresponded to residues 17-42 of Ras plus 541 daltons, while a second peptide was identified which possessed a molecular mass of 1914 daltons, corresponding to residues 124-135 of Ras plus 541 daltons. Addition of 541 daltons to each peptide was consistent with the addition of 1 ADP-ribose moiety to the peptide. These data supported the determination that ExoS ADP-ribosylates Ras at Arg41 and Arg128.

Identification of Arg-135 as an Alternative Site of ADP-ribosylation of Ras by ExoS-- Earlier data indicated that ExoS ADP-ribosylated Ras at multiple sites and that Ras possessed undefined alternative sites of ADP-ribosylation (10). To identify a possible alternative site of ADP-ribosylation within Ras, a double Ras mutant, RasDelta CAAXR41K,R128K, was engineered by site-directed mutagenesis. Under saturation conditions, ExoS ADP-ribosylated RasDelta CAAXR41K,R128K to a stoichiometry of approximately 1 mol of ADP-ribose/mol of Ras (Table II). This was consistent with the presence of an alternative site of ADP-ribosylation within the double mutant. Under linear velocity conditions, ExoS ADP-ribosylated wild type RasDelta CAAX at a 4-fold greater velocity than RasDelta CAAXR41K,R128K (Fig. 2). These data suggested the alternative site of ADP-ribosylation was not accessible for ADP-ribosylation upon the ADP-ribosylation of Arg128, possibly because of steric limitations. The identity of the alternative site of ADP-ribosylation within RasDelta CAAXR41K,R128K was determined as described for wild type RasDelta CAAX. rpHPLC analysis of peptides from V8 protease-digested ADP-ribosylated RasDelta CAAXR41K,R128K showed the elution of two major fractions of radioactivity (Fig. 1B). The first fraction containing radioactivity eluted with the void volume of the column (6 min), corresponding to NAD or hydrolyzed ADP-ribose, whereas the second fraction containing radioactivity eluted at 46 min. N-terminal amino acid sequencing of the first eleven amino acids of the 46-min fraction identified a peptide which corresponded to amino acids 127 to 137 of Ras, which included Arg135 (note the presence of the R128K mutation at cycle 2) (Table I). Amino acid yields at each cycle were similar (within about 2-fold), with the exception of cycle 9 where there was a reduction of the yield of the predicted amino acid, Arg135. This was consistent with Arg135 having been ADP-ribosylated. It should be noted that Ser is often recovered at lower yield during automated Edman degradation of peptides.2 These data were consistent with Arg135 being the alternative site for ADP-ribosylation of Ras by ExoS.

                              
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Table II
ADP-ribosylation of Ras and Ras mutants by Exoenzyme S
Wild type Ras, RasDelta CAAX, or RasDelta CAAXR41K,R128K were prepared as described under "Experimental Procedures." For loading experiments, 20 µM Ras was incubated at 30 °C in 200 mM Na acetate, containing 1 µM GTPgamma S or GDP. The reaction was stopped by the addition of 20 mM MgCl2 after 30 min. Velocities of ADP-ribosylation of wild type Ras were determined in reaction mixtures containing 5 µM Ras and 1.2 nM Delta N222 ExoS. Stoichiometries of the ADP-ribosylation of wild type Ras was determined in reaction mixtures containing 5 µM Ras and 12 nM Delta N222 ExoS. Stoichiometry of the ADP-ribosylation of RasDelta CAAX (wild type or mutant) was determined in reaction mixtures containing 5 µM Ras and 4 nM Delta N222 ExoS.


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Fig. 2.   ExoS ADP-ribosylates R41K, R128K RasDelta CAAX at a lower rate than wild type RasDelta CAAX. Reaction mixtures contained: 200 mM Na acetate (pH 6.0), 50 µM [adenylate phosphate-32P]NAD, 0.4 nM Delta N222 ExoS, 5 µM Ras, and 4 nM FAS. Reactions were stopped at the indicated times by spotting the reaction mixture on trichloroacetic acid paper. Data represent the results of three experiments performed in duplicate.

Influence of Nucleotide Occupancy on the Ability of ExoS to ADP-ribosylate Ras-- Exoenzyme S ADP-ribosylates Ras at two distinct locations within the Ras molecule. One site of ADP-ribosylation, Arg41, lies in a beta -sheet which is adjacent to the switch 1 domain of Ras, whereas the second and alternative sites of ADP-ribosylation, Arg128 and Arg135, respectively, lie within an alpha -helix located toward the C terminus of Ras. The crystal structure of Ras (18) predicts that Arg128 and Arg135 lie on the external face of the alpha -helix and are located on a different surface of the Ras molecule, relative to Arg41 (Fig. 3). Because both Arg41 and Arg128 are removed from the nucleotide binding site of Ras, nucleotide occupancy might not affect the ability of ExoS to ADP-ribosylate Ras. This was tested by loading Ras with GTP or GDP and measuring the stoichiometry and velocity of ADP-ribosylation relative to nucleotide-free Ras. Neither the stoichiometry nor the velocity of ADP-ribosylation of Ras was affected by the nucleotide occupancy (Table II). ExoS ADP-ribosylated either GTP-loaded, GDP-loaded, or nucleotide-free Ras to approximately 2 mol of ADP-ribose incorporated per mol of Ras. These results indicate that ExoS did not preferentially ADP-ribosylate either GTP- or GDP-bound Ras. Control experiments using [35S]GTPgamma S showed efficient nucleotide exchange conditions with the loading of nucleotide to a stoichiometry of approximately 1 mol of nucleotide per mol of Ras (data not shown). It should also be noted that Ras and RasDelta CAAX showed the same velocities and stoichiometry as targets for ADP-ribosylation by ExoS. Thus, the presence of an acylation site does not influence the ADP-ribosylation properties of Ras.


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Fig. 3.   Exoenzyme S ADP-ribosylates Ras at two distinct locations. The structure of GDP-bound Ras residues 1-169 (Protein Data Bank code 4Q21; Ref. 18) was visualized, and the sites of ADP-ribosylation were labeled using Bobscript software. One site of ADP-ribosylation, arginine 41 (R41), is in a beta -sheet as indicated, whereas the second site and alternative site of ADP-ribosylation, arginine 128 (R128) and arginine 135 (R135), respectively, are in an alpha -helix near the C terminus.

Identification of Primary Amino Acid Homology between the Catalytic Domain of ExoS and Vertebrate ADP-ribosyltransferases-- To date, several vertebrate ADP-ribosyltransferases have been identified, including rabbit skeletal muscle ADP-ribosyltransferase (RNART) (19), rat RT6 (20), a human ecto-ADP-ribosyltransferases (21), and chicken ADP-ribosyltransferase types I and II (22). Several of the vertebrate ADP-ribosyltransferases have the capacity to ADP-ribosylate several eukaryotic target proteins (11, 12) and to ADP-ribosylate at two independent sites (13, 14), which identified functional relationships with ExoS. Using the tFASTA algorithm, the vertebrate ADP-ribosyltransferases were observed to possess considerable primary amino acid homology with the catalytic portion of ExoS. ExoS possessed homologies of 26.6% with RNART, 28.3% with HNART, 31.9% with RT6, 35.4% with CHAT 2b, and 25.7% with CHAT 1a. This alignment included the Ser-Thr-Ser motif and extended through the catalytic glutamic acids. Fig. 4 shows a PILEUP of the catalytic portion of ExoS and the vertebrate ADP-ribosyltransferases. These data suggest the possibility that the catalytic domain of ExoS shares both structural and functional properties with the vertebrate ADP-ribosyltransferases. In contrast, the alignments of the catalytic domain of ExoS with prokaryotic ADP-ribosyltransferases showed a similar degree of homology: 26.9% with cholera toxin, 28.9% with heat-labile enterotoxin of E. coli, and 23.8% with pertussis toxin. However, the homology centered on the Ser-Thr-Ser motif, but did not extend to the catalytic glutamic acids (data not shown).


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Fig. 4.   Alignment of P. aeruginosa exoenzyme S with eukaryotic ADP-ribosyltransferases. A portion of the catalytic domain of Exoenzyme S was aligned with the eukaryotic ADP-ribosyltransferases, using Pileup from GCG. RNART is the rabbit skeletal muscle NAD ADP-ribosyltransferase, HNART is the human NAD ADP-ribosyltransferase, CHAT 1 is the chicken ADP-ribosyltransferase type 1, CHAT 2 is the chicken ADP-ribosyltransferase type 2, and RT6 is the rat RT6 ADP-ribosyltransferase. Residues conserved between the eukaryotic transferases and Exoenzyme S are shown in bold. Region 2 of the ADP-ribosyltransferases corresponds to the STS sequence, whereas region 3 of the transferases corresponds to the catalytic glutamic acids.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Previous data indicated that exoenzyme S ADP-ribosylated Ras at multiple sites, including Arg41 and a second site which could not be localized. The current study utilized direct biochemical and biophysical approaches to analyze how ExoS modified Ras. ADP-ribosylated Ras was digested with S. aureus V8 protease, and radiolabeled peptides were subjected to Edman degradation. During the Edman degradation reaction, radiolabeled ADP-ribosylated arginine was not recovered although a decrease in the yield of arginine at residues corresponding to Arg41 and Arg128 of Ras was detected. Others have demonstrated that radiolabeled ADP-ribosylarginine was not recovered when peptides ADP-ribosylated at arginine are subjected to Edman degradation although the site of ADP-ribosylation can be identified indirectly by a decrease in amino acid yield at a candidate arginine residue (14). The fact that arginines 41 and 128 were identified as the sites of ADP-ribosylation by two different methods, Edman degradation of ADP-ribosylated peptides and mass spectrometry, after digestion with two different proteases, V8 and trypsin, respectively, indicate that ExoS ADP-ribosylates Ras at arginines 41 and 128.

Analysis of the double mutant, RasR41K,R128K, revealed an alternative site for ADP-ribosylation by ExoS, Arg135. Because ExoS did not ADP-ribosylate Arg135 in Ras, it appears that the ADP-ribosylation of Arg128 sterically blocks the ADP-ribosylation of Arg135. Consistent with a steric mechanism of blockage, rather than an affinity effect, was the determination that ExoS ADP-ribosylated RasR41K,R128K at a rate that was within 4-fold of Ras. This indicates that ExoS can bind Ras in several orientations to facilitate ADP-ribosylation along that alpha -helix containing Arg128 and Arg135 and that ExoS can ADP-ribosylate Ras at Args on two distinct surfaces of Ras, the beta -sheet containing Arg41 and the alpha -helix containing Arg128 and Arg135. The plasticity of the ExoS-Ras interactions may explain the observed ability of ExoS to ADP-ribosylate numerous small molecular weight GTP binding proteins in vitro (7).

Coburn et al. (7) showed that ExoS ADP-ribosylates several members of the Ras superfamily in vitro. Only a limited subset of the family, Ras, Ral, and Rap, contained the Arg41 homologue. Alignment of the alpha -helix, which contains the second site ADP-ribosylated by ExoS, Arg128, identified numerous members of the Ras superfamily that contained an arginine residue as a potential site of ADP-ribosylation. However, no distinct recognition motif is apparent from examination of the primary amino acid sequences of members of the Ras superfamily (Fig. 5). Consistent with this prediction, we have observed that ExoS ADP-ribosylates RhoA in vitro.3 RhoA does not contain an Arg41 homolog but does contain several Args within the secondary alpha -helix. The potential of ADP-ribosylating Ras superfamily members at their Arg41 or Arg128 homologues may have different functional implications. Arginine 41 is a contact residue in the Ras-Rap co-crystal structure (23) and is in a region of close contact in the Ras-SOS crystal structure (24). Modification of Ras at Arg41 may inhibit Ras activation or Ras-Raf interactions. The secondary site helix has no ascribed function, although it is located adjacent to the Ras farnesylation site. Therefore, it is unclear how ADP-ribosylation of Ras at Arg128 or 135 would affect Ras function, or alternatively ADP-ribosylation could affect Ras posttranslational modification. While ExoS has been shown to ADP-ribosylate Ras in vivo (9), a more detailed analysis of the in vivo targets of ExoS is needed.


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Fig. 5.   Alignment of exoenzyme S target proteins. Small molecular weight GTP binding proteins of the Ras superfamily that have been identified as targets for Exoenzyme S ADP-ribosylation in vitro were aligned using Pileup from GCG. The switch 1 domain of Ras (residues 32-37), the beta -strand of Ras containing arginine 41, and the alpha -helix of Ras containing arginines 128 and 135 are depicted. Arginines within these regions in Ras and homologous proteins are shown in bold.

Neither nucleotide occupancy nor Mg2+ binding altered the ability of ExoS to ADP-ribosylate Ras, as the velocity and stoichiometry of the ADP-ribosylation of GTP-loaded, GDP-loaded, and nucleotide-free Ras were essentially identical. This indicated that the sites of ADP-ribosylation were not involved in nucleotide binding or chelation of Mg2+ and was consistent with structural data which predicted that Arg41 and Arg128 were distant from these binding sites. In contrast to ExoS, several bacterial toxins that modify Ras superfamily members preferentially target GDP bound forms of those proteins; Clostridium sordellii LT preferentially glucosylates GDP bound Ras (25) and Clostridium botulinum C3 ADP-ribosyltransferase preferentially ADP-ribosylates GDP-bound Rho (26). The fact that ExoS modifies both the GTP- and GDP-bound forms of Ras whereas other toxins described to date modify only the GDP-bound form indicates that ExoS modifies Ras signal transduction by a mechanism that is distinct from the Clostridial toxins.

The endogenous vertebrate ADP-ribosyltransferases share several properties with ExoS, including the ability to ADP-ribosylate multiple target proteins and the ability to ADP-ribosylate target proteins at multiple sites. Sequence alignment showed that the catalytic domain of ExoS aligns more extensively with the eukaryotic ADP-ribosyltransferases than bacterial ADP-ribosyltransferases. The primary amino acid alignments of mono-ADP-ribosyltransferases predicts the conservation of a basic amino acid, a Ser-Thr-Ser sequence, and a catalytic glutamic acid (27). Alignment between the catalytic domain of ExoS and the vertebrate ADP-ribosyltransferases extended from the Ser-Thr-Ser sequence through the active site glutamic acid. In contrast, tFASTA alignment between the catalytic domain of ExoS and the bacterial ADP-ribosyltransferases, CT, LT, and PT, identified alignments only within the Ser-Thr-Ser sequence. This suggests that with respect to both functional and sequence alignments, ExoS is more similar to the vertebrate ADP-ribosyltransferases than the prokaryotic ADP-ribosyltransferases. Thus, ExoS may have an evolutionary link with the vertebrate ADP-ribosyltransferases. A better understanding of the mechanism by which ExoS modifies eukaryotic physiology may provide insight into the mechanisms of action of the vertebrate ADP-ribosyltransferases.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Klaus Aktories, Dara Frank, and Kristin Pederson for helpful discussions. We thank Yuedi Wang and Michael Pereckas for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant AI-30162.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.

parallel To whom correspondence should be addressed: Medical College of Wisconsin, Microbiology and Molecular Genetics, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8412; Fax: 414-456-6535; E-mail: toxin{at}mcw.edu.

2 L. Mende-Mueller, personal communication.

3 A. K. Ganesan and J. T. Barbieri, unpublished data.

    ABBREVIATIONS

The abbreviations used are: ExoS, exoenzyme S; rpHPLC, reversed-phase high performance liquid chromatography; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; FAS, factor activating exoenzymes.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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