From the Medical College of Wisconsin, Departments of
Microbiology and Molecular Genetics and
§ Biochemistry, Milwaukee, Wisconsin 53226 and the
¶ Institute of Pharmacology and Toxicology, University of
Freiburg, D-79104 Freiburg, Germany
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ABSTRACT |
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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 -helix as Arg128.
Arg41 and Arg128 are located within two
different secondary structure motifs, 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 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 GTP 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 Nucleotide Loading of Ras Proteins--
20 µM Ras
was incubated alone or with 1 mM of either GTP 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
Ras Determination of the Sites of ADP-ribosylation within
Ras--
Ras 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.
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
Ras
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
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,
Ras 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 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).
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
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 -sheet and
-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
-sheet and
-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
S from Sigma. Recombinant
factor activating exoenzymes (FAS) and a c-Ha-Ras vector were gifts
from H. Fu (Emory University).
70 °C. These conditions were optimal for exchange of bound nucleotide from Ras proteins.
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]GTP
S.
CAAX, FAS, and
N222 (a catalytic, deletion peptide of ExoS).
FAS and
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. Ras
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.
CAAX or Ras
CAAX-R41K,R128K (20 µM) was
incubated with 0.4 µM
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).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 Ras CAAX
(panel A) or Ras
CAAX-R41K,R128K (panel B) was
ADP-ribosylated by
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).
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.
N-terminal amino acid sequencing of Ras peptides that have been
ADP-ribosylated by ExoS
CAAXR41K,R128K, was engineered by site-directed mutagenesis.
Under saturation conditions, ExoS ADP-ribosylated Ras
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 Ras
CAAX at a 4-fold greater velocity than Ras
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 Ras
CAAXR41K,R128K was determined as
described for wild type Ras
CAAX. rpHPLC analysis of peptides from V8
protease-digested ADP-ribosylated Ras
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.
ADP-ribosylation of Ras and Ras mutants by Exoenzyme S
CAAX, or Ras
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 GTP
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
N222 ExoS. Stoichiometries of the ADP-ribosylation of
wild type Ras was determined in reaction mixtures containing 5 µM Ras and 12 nM
N222 ExoS. Stoichiometry
of the ADP-ribosylation of Ras
CAAX (wild type or mutant) was
determined in reaction mixtures containing 5 µM Ras and 4 nM
N222 ExoS.
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Fig. 2.
ExoS ADP-ribosylates R41K, R128K
Ras CAAX at a lower rate than wild type
Ras
CAAX. Reaction mixtures contained: 200 mM Na acetate (pH 6.0), 50 µM [adenylate
phosphate-32P]NAD, 0.4 nM
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.
-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
-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
-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]GTP
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 Ras
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 -sheet as
indicated, whereas the second site and alternative site of
ADP-ribosylation, arginine 128 (R128) and arginine 135 (R135), respectively, are in an
-helix near the C
terminus.
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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
-helix containing Arg128 and Arg135 and that
ExoS can ADP-ribosylate Ras at Args on two distinct surfaces of Ras,
the
-sheet containing Arg41 and the
-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).
-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
-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 -strand of
Ras containing arginine 41, and the
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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The abbreviations used are:
ExoS, exoenzyme S;
rpHPLC, reversed-phase high performance liquid chromatography;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
FAS, factor activating
exoenzymes.
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