(Received for publication, October 3, 1994)
From the
Pseudomonas aeruginosa exotoxin A (ETA) is a member of
the family of bacterial ADP-ribosylating toxins which use
NAD as the ADP-ribose donor. By analogy to diphtheria
and pertussis toxins, the His
residue of ETA has been
proposed to be one of the critical residues within the active site of
the toxin. In this study the role of the His
residue was
explored through site-directed mutagenesis which resulted in the
production of ETA proteins containing Ala, Asn, and Phe substitutions
at the 440 position. The His
-substituted ETA proteins
were purified and analyzed. All substitutions at the 440 site displayed
severely reduced ADP-ribosylation activity (>1000-fold). However,
NAD glycohydrolase activity remained intact and in the case of ETAH440N
actually increased 10-fold. NAD
binding is not
affected by substitutions at the 440 site as indicated by similar K
values for the ETA variants tested.
Conformational integrity of the mutant toxins appears to be largely
unaffected as assessed by analysis with a conformation-sensitive
monoclonal antibody as well as sensitivity to proteinase digestion. In
view of the location of His
residue within or close to
the proposed NAD
-binding site, these results suggest
that His
may be a catalytic residue involved in the
transfer of the ADP-ribose moiety to the EF-2 substrate.
Pseudomonas aeruginosa exotoxin A (ETA) ()and diphtheria toxin (DT) are two well characterized
members of a group of bacterial protein toxins known as
ADP-ribosyltransferases, based upon their enzymatic mode of action.
Both toxins catalyze the transfer of the ADP-ribose moiety of
NAD
into covalent modification of the eucaryotic
protein elongation factor 2 (EF-2). The result is paralysis of the
function of the EF-2 protein and the resultant inhibition of protein
synthesis in the target cell which the toxin has
entered(1, 2) .
The three-dimensional structure of the 613-amino acid ETA protein has been resolved to the 3-Å level(3) . This study and others (3, 4, 5, 6) have led to an assignment of the functional domains within the ETA structure. According to this scheme, residues 405-613 (domain III) and 365-404 (domain Ib) form the catalytic domain responsible for the ADP-ribosyltransferase activity as well as associated NAD glycohydrolase activity. The N-terminal domains Ia (residues 1-252) and II (residues 253-364) are involved in binding to the ETA receptor and for translocation across the endosomal membrane of sensitive cells, respectively.
Recently the three-dimensional
structure of diphtheria toxin has been resolved(7) . Although
immunological cross-reactivity has not been demonstrated between the
intact ETA and DT molecules, considerable sequence homology exists
within the enzymatic domains of these two
toxins(8, 9) . The NAD-binding site in both ETA and DT
have been proposed on the basis of computer modeling
studies(9, 10) . Essentially, the NAD site consists of
a hydrophobic cavity composed of residues with aromatic side chains,
such as Tyr (Tyr
, DT), Tyr
(Tyr
, DT), Trp
(Trp
, DT),
Trp
(Trp
, DT), and His
(His
, DT). The hydrophobicity of the pocket favors
the binding of the nicotinamide and adenine portions of NAD
due to aromatic ring-stacking as well as hydrogen bonding
interactions. Two alternative NAD
binding arrangements
have been proposed. In one arrangement the nicotinamide ring of
NAD
faces Tyr
while the adenine ring
faces Trp
, or vice versa in the alternative arrangement.
The His
residue which is positioned at the bottom of the
cavity projects its imidazole ring into the cavity and rotates freely
in order to facilitate its involvement in hydrogen bonding. Several
hydrophilic residues such as Glu
(Glu
, DT),
Glu
(Glu
, DT), Glu
,
Asp
(Asn
, DT), and Asp
(Asp
, DT) form the edge of the NAD
binding cavity and participate in H-bonding with the two ribose
moieties of the NAD
molecule.
This inspection of
the ETA active site residues suggests an important role for the
His residue. The importance of His
is also
implicated by its strict conservation in the active sites of other
ADP-ribosylating toxins, e.g. DT (His
), pertussis
toxin (His
), cholera toxin (His
), and Escherichia coli heat-labile enterotoxin (His
).
The role of His
in DT has been analyzed recently in two
independent studies using site-specific substitution with a variety of
amino acids(11, 12) . Results of both studies indicate
that His
is involved in NAD
binding, NAD
glycohydrolysis, and ADP-ribosylation of EF-2. Likewise, the analogous
His
residue of pertussis toxin has been found to be
critical for NAD
binding and
glycohydrolysis(13) .
In order to further define and
establish the function of the ETA His residue, we
utilized site-directed mutagenesis to insert various amino acids in
place of the histidine at position 440. The mutated toxins (ETAH440A,
ETAH440N, and ETAH440F) exhibit dramatic reductions of
ADP-ribosyltransferase activity as well as a severe reduction of in
vitro cytotoxicity. However, NAD glycohydrolase activity appears
to be undiminished in the case of the His
substitutions
studied thus far.
In order to obtain suitable amounts of the
altered toxins for study, the toxA gene in each plasmid was
subcloned in the following manner for expression in the ToxA-negative P. aeruginosa host strain PA103A which carries an
insertionally inactivated toxA gene. ()A
1.3-kilobase pair EcoRI/BglII fragment from each
plasmid was subsequently cloned into pGW580 (16) by fragment
replacement, resulting in plasmids pXH202, pXH205, and pXH206 which
carry the toxA gene under control of its own promoter. In each
case, the toxA gene was subsequently extracted using a
2.7-kilobase pair EcoRI/HindIII fragment and inserted
into pRO1614, a broad-host range plasmid(18) . The resultant
plasmids, pXH302 (ETAH440A), pXH305 (ETAH440N), and pXH306 (ETAH440F)
were used to transform P. aeruginosa strain PA103A, a
non-toxigenic derivative of strain PA103. Transformation of PA103A was
carried out according to a previously described procedure (18) and transformants were selected on Pseudomonas Isolation Agar in the presence of 300 µg ml
carbenicillin (Sigma). Procedures such as plasmid isolation,
endonuclease digestion, transformation of E. coli cells, and
agarose gel electrophoresis of DNA fragments or plasmids, were carried
out according to the methods described by Sambrook et
al.(19) .
Wild-type ETA was purified from P.
aeruginosa strain PA103 transformed with a plasmid (pGW28)
containing the toxR gene which encodes for a transcriptional
activator known to enhance toxin production, as described
previously(20) . The point-mutated ETA proteins were purified
from the Tox PA103A strain transformed with the
appropriate plasmid carrying the mutated toxA gene. All toxins
were purified to homogeneity as indicated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and
Coomassie Blue staining(21) .
Figure 4:
Trypsin digestion profiles of purified ETA
and His-substituted ETA. Purified toxins (1.5
µg/µl) were digested in the presence of trypsin (0.08
µg/µl) for either 30 or 60 min at 25 °C. The reactions were
stopped by the addition of soybean trypsin inhibitor and the resulting
digests analyzed using 15% SDS-PAGE and Coomassie Blue staining. Lanes 2, 5, 8, and 11 represent undigested toxin; lanes 3, 6, 9, and 12 represent 30-min digests; and lanes 4, 7, 10, and 13 represent 60-min digests; lanes 14 and 15 represent trypsin (3 µg) and
soybean trypsin inhibitor (3 µg),
respectively.
An alternative method for the evaluation of
ADP-ribosyltransferase activity involves measuring the ability of toxin
to inhibit protein synthesis in toxin-sensitive cells. When functional
ETA is incorporated into susceptible mouse L-929 fibroblast cells,
ADP-ribosylation of EF-2 results in the inhibition of protein synthesis
which can be observed by a reduction in
[H]leucine incorporation into newly synthesized
protein. As an example, ETAH440A was analyzed in this fashion and it
can be seen by the results presented in Fig. 1that a
substantial reduction in the inhibition of protein synthesis occurs,
similar to that observed for the His
mutant (ETAH426L) as
described previously(20) . The His
residue has
previously been shown to occupy a critical site which may be involved
in the interaction with the EF-2 substrate(20, 25) .
Figure 1:
Inhibition of protein synthesis in
mouse L-929 fibroblasts. Mouse L-929 fibroblast cells (10 cells/well/24-well plate) were cultured in the presence of trace
amounts of [
H]leucine in RPMI medium followed by
a 1-h exposure to ETA or mutated ETA at the indicated concentrations.
The cells were placed in fresh medium to which 0.3 µCi of
[
H]leucine had been added and incubated an
additional hour. The cells were harvested, washed repeatedly, and lysed
in the presence of 0.1 N NaOH. Lysates were collected by
filtration and counted using liquid scintillation methodology. The inset indicates the concentration of ETA that resulted in 50%
inhibition of [
H]leucine incorporation into
protein.
Figure 2:
Comparative analysis of the NAD
glycohydrolase activities of wild-type and
His-substituted ETA. In each case 1 µg of purified
ETA was activated by treatment with urea/DTT and then assayed for
NAD
hydrolysis as described under ``Experimental
Procedures.'' Assays were carried out at 15 °C for a 4-h
period using the concentrations of
[
H]NAD
indicated (10-1800
µM).
Figure 3:
ELISA-based capture assay for
conformational analysis of activated and non-activated ETA and
His-substituted ETA. Individual wells of Immulon II
(Dynatech, Alexandria, VA) 96-well microtiter plates were coated with
purified IgG of the TC-1 mAb (2 µg/ml). Activated and non-activated
toxins were added and incubated for 2 h at 25 °C. TC-1-bound toxin
was subsequently detected using high-titered rabbit anti-ETA antibody
and donkey anti-rabbit IgG-horseradish peroxidase conjugate as
described under ``Experimental
Procedures.''
In a separate
analysis of conformational differences among the 440-substituted toxins
we used proteinase sensitivity as an indication of conformational
integrity. Our analysis of trypsin sensitivity (Fig. 4) of the
modified ETA proteins indicates that the His substitutions do not render ETA more sensitive to trypsin
digestion than the wild-type ETA. These results suggest that the
substitutions at the 440 site do not result in destabilization of the
ETA structure.
In this study we have demonstrated that the His residue of ETA plays a critical role in the ADP-ribosylation of
elongation factor 2. Previous studies have proposed that His
of exotoxin A, His
of DT, and His
of
pertussis toxin are functionally homologous residues within the
NAD
-binding sites of their respective
proteins(8, 9) . Analysis of the three-dimensional
structures of ETA and DT reveal that ETA His
and DT
His
occupy essentially identical positions within the
active sites of these toxins(3, 7) . Using a
site-directed mutagenesis approach in which the ETA His
residue is replaced by Ala, Asn, or Phe provides evidence that
substitution of the histidine at the 440 position reduces the
ADP-ribosyltransferase activity of the toxin more than 1000-fold (Table 2). This result is substantiated by the further
observation that a replacement of the histidine at the 440 position
substantially reduces the ability of the toxin to inhibit protein
synthesis in an in vitro assay (Fig. 1). In view of the
apparent analogy of ETA His
with His
of DT
these results are not surprising.
Significantly, and in contrast to
the findings recently reported for both DT and PT (11, 12, 13) alteration of the ETA
His residue does not appear to eliminate or reduce the
NAD glycohydrolase activity associated with the toxin. In fact,
substitution of an Asn residue at this position results in an apparent
10-fold increase in the NAD glycohydrolase activity (Fig. 2, Table 3). Analysis of the kinetics of the NAD glycohydrolase
reaction indicates that the catalytic efficiency (K
/K
) of this reaction is
similar for both the Ala
and Phe
substitutions, but increases 5-fold in the case of Asn
substitution (Table 3). However, similar K
values for both the wild-type and mutant toxins implies that the
NAD
binding function remains intact (Table 3).
These results are supported by fluoresence quenching experiments which
indicate that NAD
binding in the mutant toxins is
unaffected by substitutions at the 440 position (data not shown). The
increased NAD glycohydrolase activity associated with the Asn
substitution in ETA may result from the effects of the polar side
chain of the residue. It seems unlikely that the Asn side chain is
directly involved in the formation of hydrogen bonds with
NAD
during cleavage of the N-glycosidic bond,
as appears to be the case in DT, since by analogy with DT substitution
with Asn at this position should result in some retention of
ADP-ribosyltransferase activity. Similarly, the nonpolar Ala
and Phe
substitutions in ETA should result in
reduced NAD glycohydrolase activities as observed in the case of DT if
the same hydrogen bonding model applies. Our findings suggest that ETA
His
does not appear to follow the model proposed for DT
His
. Rather, Asn
substitution may facilitate
NAD glycohydrolysis in some indirect fashion. One possibility is that
the polar side chain may allow the ready access of water molecules in
order to accept the ADP-ribose moiety following cleavage of the N-glycosidic bond through the involvement of as yet
unidentified residues. Another possibility is regional conformational
changes which could allow more efficient NAD glycohydrolysis to occur.
Similar site-directed mutagenesis studies have recently been
reported for the analysis of the His residue of diphtheria
toxin (11, 12) . In these studies it was reported that
substitution of His
with Arg, Leu, Ala, and Gln
significantly reduces both ADP-ribosyltransferase and NAD
glycohydrolase activities. The reduction in these activities was shown
to be due to reduced NAD
binding as revealed by
fluoresence quenching analysis. Interestingly, an Asn
substitution only reduced the ADP-ribosyltransferase activity
3-fold. This conservation of function may be due to the ability of the
amide
-nitrogen to form hydrogen bonds. Consequently Blanke et
al.(11) proposed that the primary function of His
is to form a hydrogen bond with the nicotinamide carboxymide of
NAD
. This interaction would facilitate formation of
the initial DT
NAD complex, orient the N-glycosidic bond
of NAD
for nucleophilic attack by the incoming
diphthamide residue of EF-2, and stabilize the transition state
intermediate of this reaction. Similar mutagenesis studies of the
His
residue of pertussis toxin also indicate the
involvement of this residue in NAD
binding and
glycohydrolysis(13) .
However, the results of the present
study do not support a direct role for the ETA His residue in NAD
binding or hydrolysis. Our
results do indicate that His
is required for the
ADP-ribosyltransferase reaction and therefore suggest that the
His
residue must either be involved in the interaction
with EF-2 or participate in the catalysis of the reaction. It is
difficult to measure the interaction with EF-2 directly. However,
direct contact of His
with the incoming EF-2 substrate is
unlikely since His
is located on the floor of a
hydrophobic cavity within the active site of ETA(3) .
Furthermore, a role for the His
residue in maintaining an
appropriate conformation for interaction with EF-2 seems unlikely in
view of the fact that sterically conservative substitutions of Ala and
Asn do not maintain ADP-ribosyltransferase activity. In view of these
considerations it may be that His
plays a catalytic role
in the ADP-ribosylation reaction. This hypothesis is supported by the
location of the residue within the active site (30) and by its
close spatial relationship with Glu
, a crucial catalytic
residue(27) . Glu
was initially identified using
an NAD
-photolabeling technique (31) and upon
further analysis a catalytic function was suggested for this
residue(32, 33) . These studies demonstrated that
deletion of Glu
, or substitutions with Asp or Cys at this
site all abolished ADP-ribosyltransferase activity and cytotoxicity,
yet produced little or no change in NAD
binding or
glycohydrolysis. In conjunction with studies of Glu
of
DT, a functional homologue of Glu
, it was found that both
the negative charge and the chain length of the carboxyl group of ETA
Glu
(or DT Glu
) are crucial with respect to
catalytic function(17, 27) . Therefore it was proposed
that the carboxylate serves to deprotonate the incoming diphthamide on
EF-2, thereby activating the nitrogen at position 1 of the diphthamide
imidazole ring which then imposes a nucleophilic attack on the N-glycosidic bond of NAD
in a direct
displacement mechanism(30) . In some respects the functional
features of Glu
appear to be similar to those of
His
, as demonstrated in the present study. The fact that
the Asn
substitution severely reduces catalysis of the
ADP-ribosyltransferase reaction suggests that it is not the hydrogen
bond-forming ability of His
, but rather the acid-base
properties which may be important for catalysis. The aromatic nature of
the His
imidazole ring seem less important since
substitution of a Phe residue at this position does not maintain
function. Although the data presented in this study is not sufficient
to propose a detailed catalytic mechanism, we speculate that the
acid-base properties of His
may contribute to the
interaction with the incoming EF-2 diphthamide residue, stabilize the
transition state intermediate, and facilitate the transfer of the
ADP-ribose moiety to the diphthamide site.
The functional
differences between His of ETA and His
of DT
have encouraged us to search for additional active site residues which
may be critical for ADP-ribosyltransferase activity. However, an
inspection of the active site structure of ETA failed to indicate
potential residues which might serve a function homolgous to the
His
of DT (data not shown).