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INTRODUCTION |
Pseudomonas aeruginosa is a ubiquitous, Gram-negative,
opportunistic pathogen that is commonly found in soil, water, sewage, and even in hospital environments (1). This bacterium is a leading
cause of infections in burn, cystic fibrosis, and postoperative patients and in other various immune-compromised hosts. P. aeruginosa synthesizes a number of extracellular toxic products
believed to be involved in the pathogenesis of these infections. The
most toxic factor secreted by P. aeruginosa is the 66-kDa
protein exotoxin A (ETA).1
ETA belongs to a family of toxins related in their mechanisms of
action, which include diphtheria toxin (DT), pertussis toxin, cholera
toxin, and Escherichia coli heat-labile toxin. These toxins exert their effects via ADP-ribosylation of specific target molecules within eukaryotic cells. More specifically, ETA catalyzes the transfer
of the ADP-ribose moiety of NAD+ onto elongation factor 2 (2, 3). This covalent transfer inactivates eukaryotic elongation factor
2, rendering it incapable of polypeptide chain elongation, inhibiting
protein synthesis, and eventually killing the target cell.
The three-dimensional crystallographic structure of the 613-residue ETA
protein has been solved to a resolution of 3.0 Å (4). Based on the
crystal structure of the 66-kDa ETA molecule, along with other
physicochemical data, molecular models were proposed that consist of
three distinct functional domains (5). Domain Ia (residues 1-252) is
involved in receptor binding, domain Ib (residues 365-404) has no
known function, and domain II (residues 253-364) aids in translocation
across the endoplasmic reticulum membrane into the host cell cytoplasm
after receptor-mediated endocytosis. Domain III (residues 405-613)
comprises the catalytic domain, which contains an extended cleft
postulated to be the active site of the enzyme.
From the resolved 2.3-Å crystal structure of DT (6), some active site
residues are located in essentially identical positions within ETA and
DT. Previously, a model was proposed in which NAD+ fits
into the cleft with the adenine ring bound in the hydrophobic pocket
defined by the aromatic rings of Tyr-470 (Tyr-54, DT), Tyr-481 (Tyr-65,
DT), Trp-466 (Trp-50, DT), Trp-558 (Trp-153, DT), and His-440 (His-21,
DT) (7). The model suggests that either the nicotinamide or adenine
ring of NAD+ would stack on the indole ring of Trp-466.
More recently, the 2.5-Å crystal structure of domain III of ETA
complexed with NAD+ hydrolysis products (AMP and
nicotinamide) has been solved (8), and close inspection of this
structure revealed that NAD+ likely does not directly
interact with the Trp-466 side chain (9). Furthermore, a
three-dimensional structure of domain III of ETA complexed with the
NAD+ analogue,
-TAD+, has also been
determined (10), as has the structure of DT complexed with intact
NAD+ (11). Bell et al. (12) compared the
conformations of NAD(P+) bound to 23 distinct
NAD(P+)-binding oxidoreductases enzymes. The majority of
the oxidoreductase-bound NAD(P+) conformations were found
to be similar, but the conformation of NAD+ bound to DT
(and ETA) was found to be unusual. This difference was seen in the
highly folded conformation of the nicotinamide mononucleotide portion
of NAD+ and a strained N-glycosidic bond, which places the
nicotinamide ring outside the preferred anti and
syn conformations. In DT and ETA, the
NAD+-binding site is formed at the junction of the two
anti-parallel
-sheets that are orthogonal to one another. This is
again in sharp contrast to other NAD(P+) enzymes that bind
NAD(P+) at the C-terminal end of a parallel
-sheet.
Within domain III of ETA are three tryptophans (Trp-417, Trp-466, and
Trp-558) that could potentially participate in hydrophobic stacking
interactions as described previously. Previously, we demonstrated that
upon NAD+ binding to the catalytic domain, the intrinsic
protein fluorescence of ETA is significantly quenched (9). This effect
was attributed to the fluorescence quenching of one or more tryptophans
located within domain III. However, at that time, a catalytically
active fragment of ETA that consisted of domain III only was not
available. In order to elucidate the roles of these tryptophans in
substrate binding and catalysis, we made conservative substitutions
(phenylalanine for tryptophan) within PE24, a recombinant protein that
is a catalytically active fragment of ETA consisting primarily of
domain III, using a site-directed mutagenesis technique. The effects of
these mutations were determined by accessibility of the Trp residues to
acrylamide, CD spectroscopy, NAD+-glycohydrolase (GH)
activity, NAD+ binding affinity, protein folded stability,
fluorescence lifetimes, anisotropies, and quantum yields
(QF). The results suggest that Trp-558 is protected from
the solvent when NAD+ fills the active site and that a
major structural change occurs within the catalytic domain within the
vicinity of Trp-417.
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EXPERIMENTAL PROCEDURES |
Chemicals and Enzymes--
Restriction endonucleases, T7 DNA
polymerase, and T4 DNA ligase were purchased from Amersham Pharmacia
Biotech (Baie D'Urfe, Quebec, Canada) and New England Biolabs
(Mississauga, Ontario, Canada). The BL21(
DE3) cells were obtained
from Novagen (Madison, WI); isopropyl
-D-thiogalactopyranoside was from Alexis Corp. (San
Diego, CA); Q-Sepharose Fast-Flow anion exchange and chelate-agarose resins were from Amersham Pharmacia Biotech. Dithiothreitol was from
Promega Corp. (Madison, WI). The following chemicals were obtained from
Sigma:
-NAD+, TRIZMA base, bovine serum albumin,
1,N6-etheno-AMP (
-AMP), and
1,N6-etheno-
-nicotinamide adenine
dinucleotide (
-NAD+). SequanalTM grade of
both guanidine hydrochloride (Gn-HCl) and urea were obtained from
Pierce, and imidazole was obtained from Fluka (Buchs, Switzerland).
Purification of PE24--
PE24, the C-terminal catalytic
fragment of P. aeruginosa exotoxin A, was overexpressed in
E. coli using the plasmid pPE
5-399, in which a poly-His
sequence was introduced into the protein at the C terminus by
recombinant DNA methods. The His6-tagged protein was then
purified from BL21 (
DE3) cells as described previously for PE40 (9)
except that the osmotic shock lysate solution from 500 ml of induced
cell culture in 20 mM Tris-HCl, pH 7.9, was passed through
a 2-ml chelate-agarose affinity column charged with 50 mM
NiSO4. The column was washed with 20 mM
Tris-HCl, pH 7.9, buffer containing 500 mM NaCl (Buffer A)
and 5 mM imidazole (5 ml) followed by washing with 10 ml of
Buffer A at a higher imidazole concentration (60 mM). PE24
protein possessing a poly-His sequence was eluted from the column with
8 ml of Buffer A containing 250 mM imidazole, the samples
were collected in 1-ml fractions, and a yield of 5 mg of protein was
routinely obtained. The appropriate fractions were pooled and
exhaustively dialyzed against 20 mM Tris-HCl buffer, pH
7.4, containing 50 mM NaCl.
Oligonucleotide-directed Mutagenesis--
Mutagenesis was
performed using the Kunkel method as described previously (13). Dideoxy
sequencing was performed using an ABI Prism model 377 DNA sequencer
using dye termination and cycle sequencing. DNA samples were analyzed
in 4.5% acrylamide gels that were 36 cm in length.
Spectroscopic Measurements--
Steady-state fluorescence
measurements were performed using a PTI Alphascan spectrophotometer
(Photon Technology International, South Brunswick, NJ) equipped with a
water-jacketed sample chamber set to 25 °C.
NAD+-glycohydrolase Assay--
For the GH assay, the
excitation wavelength was 305 nm, the emission monochromator was set to
0 nm, and a 309 nm cutoff filter (Oriel Corporation, Stratford, CT) was
placed on the sample chamber side of the emission monochromator to
maximize signal detection (4 nm excitation and emission slit widths).
Buffer (20 mM Tris-HCl, pH 7.4, unless otherwise stated)
and
-NAD+ (Sigma N-2630; various concentrations were
used up to 600 µM from an
-NAD+ stock
solution, 30 mM prepared in distilled water,
M265 nm = 6000 M
1
cm
1) were combined in an ultramicro (3 mm path length)
cuvette (Helma Inc., Concord, ON). The cuvette was placed in the sample
chamber for 5 min to allow temperature equilibration of the sample. The reaction was started by the rapid addition and mixing of 2.0 µM PE24 (final concentration), and the reaction progress
monitored by the increase in the fluorescence intensity of the
fluorescent substrate,
-NAD+. For each concentration of
-NAD+, a corresponding control sample was prepared, in
which buffer was added to the cuvette in place of PE24. This control
was used to confirm that the increase in fluorescence intensity
observed in the presence of PE24 was due to the presence of the enzyme. The water-catalyzed GH activity was determined to be less than 10% of
the enzyme-catalyzed reaction (reactions performed in the absence of enzyme).
Quenching of Intrinsic Protein Fluorescence--
The
NAD+-dependent quenching of intrinsic protein
fluorescence was monitored as a function of the concentration of
NAD+. Duplicate reactions were performed over a
concentration range of 0-750 µM NAD+ in the
presence of toxin at 25 °C in an initial volume of 600 µl of 50 mM Tris-HCl, pH 8.2. Samples were excited at 295 nm (4-nm band pass) and the fluorescence intensity was measured over the range
of 305-400 nm (4-nm band pass) in the computer-controlled PTI
Alphascan-2 spectrofluorometer. The final concentration of PE24 used in
the experiments was 2.5 µM. The fluorescence intensity of
N-acetyltryptophanamide (NATA) as a function of
NAD+ concentration was used to correct for the absorptive
screening by NAD+ at the excitation wavelength. The binding
constants were calculated using the Scatchard binding analysis, and the
data plots were linear corresponding to a single set of
NAD+ binding sites.
Unfolding Conditions and
G Measurements--
The PE24 protein
in 100 mM NaCl, 20 mM Tris-HCl buffer (pH 7.4)
was mixed with the appropriate volume of 8 M
SequanalTM grade Gn-HCl to provide solutions from 0 to 6 M Gn-HCl and 0.1 mg/ml protein concentration. Unfolding
experiments with urea necessitated the preparation of fresh solutions
of a 9 M urea SequanalTM grade stock that was
added to the protein solutions to make solutions from 0 to 8 M urea. In all cases, the samples were allowed to equilibrate for 30 min at 25 °C prior to spectroscopic analysis. Refolding of PE24 was examined by diluting the protein/denaturant solutions with buffer in 0.25 M increments with a 15 min
incubation time between dilutions. The spectroscopic
unfolding/refolding data were analyzed as described previously
(13).
Kinetic Data Collection--
All kinetic data were obtained at
25 °C and were collected using an Applied Photophysics stopped flow
fluorescence spectrometer equipped with both excitation and emission
monochromators (Applied Photophysics, Leatherhead, UK) as described
previously (14). Excitation slit widths were 4 nm, and emission slit
widths were 6 nm for all measurements. The kinetic traces shown are an
average of three or more experiments. Changes in Trp fluorescence upon denaturation were monitored by 10:1 mixing of 4.44 M
SequanalTM grade Gn-HCl with the PE24 protein (20 µM) in 20 mM Tris-HCl buffer, pH 7.4, to
provide a final Gn-HCl concentration of 4 M and a peptide
concentration of 2 µM. Tryptophan fluorescence was monitored by 295 nm excitation, and emission was detected at right angles at an emission wavelength of 334 nm for WT PE24.
Kinetic Data Analysis--
Rate constants were calculated by
fitting kinetic data by nonlinear least squares analysis (MicroCal
Origin; MicroCal Software Inc., Northhampton, MA). A fit was deemed
acceptable if it provided a fit with random residuals and had reached
minimum error in all fitting parameters (standard errors were typically
between 5 and 10%). Kinetic traces, which fit single exponential
kinetics, were fit with the following
equation,
|
(Eq. 1)
|
where F is the fluorescence at time t,
F0 is the initial fluorescence at time 0, F1 is the fluorescence change during the unfolding reaction, and k is the rate constant. Kinetic
traces, which showed double exponential kinetics, were fit with the
following equation,
|
(Eq. 2)
|
where F and F0 are as
described above, and F1 and
F2 are the fluores-cence changes for the double
exponential unfolding reaction, with rate constants
k1 and k2, respectively.
Fluorescence Quantum Yield and Lifetime
Measurements--
Steady-state fluorescence measurements were made
with a PTI spectrofluorometer operating in the ratio mode, with
unpolarized excitation and an emission polarizer (Glan-Thompson)
oriented at 35° to the vertical to eliminate any effects of
anisotropy on quantum yield or intensity measurements. The signal from
the buffer blank was subtracted from the sample fluorescence, and the
spectra were corrected for the wavelength dependence of the instrument
response. Fluorescence quantum yields (QF) for each of the
proteins were measured at 25 °C, using
N-acetyltryptophanamide (pH 7.0) as a standard as described
previously (15). The excitation wavelength was 295 nm, and the emission
was scanned from 305 to 450 nm in 0.5-nm increments (excitation and
emission bandpasses were 2 and 4 nm, respectively). The absorbance of
the samples at the excitation wavelength was typically less than 0.1. The values reported represent the mean of at least three determinations.
Time-resolved fluorescence measurements were performed using a PTI
LaserStrobe model C-72 lifetime fluorometer (Photon Technology International, South Brunswick, NJ). The excitation source was a pulsed
nitrogen laser pumping a dye laser with a frequency doubler. The laser
operated at 10 Hz, and the detection channel consisted of an emission
monochromator with a stroboscopic detector. The data analysis was
performed with a 1-to-4 exponential fitting program involving iterative
reconvolution and minimization of
2. The excitation
wavelength was 295 nm, and the emission was set at 340 nm with 1-nm bandpasses.
Fluorescence Anisotropy Measurements--
Steady-state
fluorescence anisotropy values were determined at 25 °C in 20 mM Tris-HCl buffer, pH 7.4. The excitation and emission
wavelengths were 295 nm (4-nm bandpass) and 340 nm (6-nm bandpass),
respectively. The anisotropy measurements were conducted by using a
T-format configuration with Glan-Thompson prism polarizers. The
rave values were calculated from the following
equation: rave = IVV
G-factor · IVH/(IVV + 2G-factor · IVH). The G-factor was determined by measuring
both the vertical and horizontal fluorescence emission with horizontal excitation and was calculated using the equation G = IHV/IHH. These calculations were
performed using the built-in anisotropy function within the PTI
FELIXTM software.
Stern-Volmer Quenching--
Quenching experiments were performed
using excitation at 295 nm with fluorescence emission set at 340 nm
(4-nm bandpasses). Aliquots of 4 M acrylamide solution were
added to protein solution (25 °C) containing 20 mM
Tris-HCl buffer, pH 7.4. The fluorescence intensities were recorded for
30 s, and the trace was integrated to give more reproducible data.
The data were corrected for dilution effects and for absorptive
screening caused by acrylamide (
M295 nm,
0.25 M
1 cm
1 for acrylamide)
(16). Quenching data were plotted as the ratio of fluorescence in the
absence of quencher (F0) to the intensity in the presence of quencher (F) against quencher
concentration. The majority of the Stern-Volmer plots were linear, but
for those exceptions, only the linear region of the quenching curves
was fit by least squares linear regression analysis. The calculated slope was equated to dynamic parameters according to the modified Stern-Volmer equation: F0/F = 1 + KSV[Q], where KSV is equal to
kq
0 (17, 18). Protein
concentrations were adjusted to provide an optical density at the
excitation wavelength of less than 0.1.
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RESULTS |
X-ray Structure and Molecular Model of PE24--
Fig.
1A shows the primary sequence
of the PE24 protein. It contains the first three residues of domain I
from whole toxin followed by residues 399-613 from domain III. At the
C terminus is located a polyhistidine sequence (His6) added
to facilitate purification in a single step. This C-terminal domain III
fragment has been shown to be catalytically active (19). This domain III fragment possesses nine helices (H1-H9) and seven
-strands (B1-B7); the latter comprise two orthogonally positioned antiparallel
-sheets in the active site of the enzyme (10) (Fig. 1B).
Fig. 1B shows the complex of PE24 with
-TAD+
based on the x-ray resolved structure (10). The three naturally occurring Trp residues within domain III are rendered as space-fill structures, with
-TAD+ shown as a stick model. Trp-417
is remotely located from the active site cleft and is located in a loop
region that extends from the N terminus and runs to Val-419 at the
N-terminal end of Helix 1. Trp-417 is largely buried in the x-ray
structure but is mostly surrounded by residue backbone atoms as well as
a number of polar side chains (Thr-414, Gln-415, Thr-418, and Arg-421). In contrast, both Trp-466 and Trp-558 are located within the active site cleft of the toxin. Trp-466 is located in the most nonpolar environment of the three naturally occurring Trp-residues within PE24.
It is located in a nonpolar pocket in a coil region near the N terminus
of a three residue
-strand, B2 (Fig. 1A). The benzene
ring portion of this indole is embedded in a pocket lined with the
aliphatic side chains of Ala-464, Leu-518, Ala-523, Ala-524, and
Val-527. It is also adjacent to Arg-467 but only faces the nonpolar
aliphatic portion of this side chain because the guanido group points
away toward Trp-558. Trp-558 is located at the N terminus of a
6-residue
-helix, H-6 (Fig. 1A). It is only partly buried
in the x-ray structure, and its C2-C3 double
bond (known to give rise to the 1La band
involved in UV fluorescence emission) is located close (3.5 Å) to the
guanido group of the Arg-467 side chain. The NAD+-binding
site consists of a common core fold of approximately 100 residues that
exists for ETA, DT, pertussis toxin, cholera toxin, and E. coli heat-labile enterotoxin. This fold represents a new
structural motif for NAD+ binding that is unique among this
family of ADP-ribosyltransferase toxins (12). The NAD+
binding site is formed at the interface of two antiparallel
-sheets, and the dinucleotide is bound at the surface of each of the
-sheets, where they intersect at nearly a right angle (Fig. 1B).

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Fig. 1.
A, primary sequence of the PE24 protein
(10). The nine -helices (H1-H9) are indicated by
rectangles, the seven -strands (B1-B7) are underlined by
right-pointed arrows, and the coil structure is unmarked.
The Trp residues are indicated with an asterisk, and the
polyhistidine C-terminal tag is underlined. B,
ribbon topology diagram of the PE24 protein with -TAD+
bound to the active site. The drawing was prepared using WebLab
ViewerPro 3.2TM based on the x-ray structure (10). The
-helices are shown in red, -strands in
blue, and coil in gray. The Trp side chains are
rendered as space filled structures, Trp-417 (green),
Trp-466 (magenta), and Trp-558 (yellow). The
-TAD+ substrate is shown in stick format, with the
nicotinamide-ribose-phosphate portion colored blue and the
adenine-ribose-phosphate colored pink.
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Fluorescence Quantum Yields and
emmax
Values--
The fluorescence emission spectra for the PE24 WT and
three single Trp mutant proteins are shown in Fig.
2. The WT protein possesses the highest
QF value (0.17) followed by W-466
(0.11),2 W-417 (0.10), and
finally W-558 (0.06, Table I). This
suggests that Trp-466 is located in the most nonpolar environment and
that Trp-558 is located in the most polar environment. The
emmax values also reflect these differences with W-466
possessing the most blue-shifted value (330 nm, Table I) followed by
W-417 (332 nm), and finally by W-558 (335 nm).

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Fig. 2.
Relative fluorescence emission spectra of
PE24 WT, single Trp mutant proteins, and NATA. The experimental
conditions were as follows: 20 mM Tris-HCl, pH 7.4, 25 °C; excitation was 295 nm with the emission wavelength
scanned from 305-450 nm and a 4 nm bandpass for both excitation and
emission. The spectra were corrected for
wavelength-dependent bias of the emission optical and
detection system. Each spectrum was normalized to the same absorbance
at 295 nm (A295 = 0.05) The various traces
correspond to the following samples:  , WT PE24;
· · · · ·, Trp-417; -----, Trp-466; - - - - -,
Trp-558; and - - - - -, NATA.
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The QF values of the three naturally occurring Trp residues
as measured in the corresponding mutant protein do not reflect their
weighted averages observed in the WT protein (sum-weighted average = 0.09; cf. 0.17 for the WT protein). This likely indicates that some structural perturbation caused by the conservative
substitution of Phe for Trp within this catalytic domain structure has
occurred. This perturbation was not global because the NAD+
binding properties and GH activities of the mutant proteins were similar to those of the WT protein (Table
II). Furthermore, the
emmax values for the mutants are consistent with those
observed for the WT protein, indicative of normally folded proteins
(13). Also, CD analysis showed that the percent
-helical content was similar for all the proteins (17 ± 3%; data not shown). It is known that the fluorescence quantum yields measured in proteins reflect
the average of a large number of interactions within proteins and is,
perhaps, a more sensitive measure of protein folded structure than
emmax values or
G (folding) measurements (15).
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Table II
NAD+ binding and GH activity of PE24 wild-type and Trp mutants
Parameters of NAD+ binding were derived from the data shown in
Fig. 3.
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Upon NAD+ binding to the PE24 protein, there was no
detectable shift in the
emmax values for either the WT
or the single Trp mutant proteins (Table I), indicating no large change
in the net polarity of the environment surrounding the three Trp
residues within the catalytic domain. However, the NAD+
substrate did induce a significant change in the quantum yields for all
the single Trp mutant proteins and the WT protein as well (Table
I).
Fluorescence Lifetimes--
The time-resolved decay components for
the WT and the three single Trp mutants of PE24 are shown in Table I.
The WT protein exhibited a short component (
= 1.55 ns, 40%) and a
long (6.56 ns, 60%) decay time component. Each of the single Trp
mutant proteins possessed a short decay component near 1 ns and a
longer component between 4.7 and 4.9 ns, with the relative amounts of
both decay times being similar. Given the differences in the
steady-state fluorescence parameters between these proteins, it is
surprising that the decay time components were nearly identical. This
is reflected in the
ave values, with WT (5.88 ns) and
the three mutant proteins possessing similar values ranging between 4.0 and 4.4 ns. Again, the differences exist between the WT protein and the
single Trp mutant proteins, perhaps reflecting subtle changes upon Trp
Phe replacement that are not evident in the folded conformation and
in the biochemical function of the catalytic domain.
When NAD+ was bound to the active site, the
ave value for the WT PE24 protein decreased from 5.88 to
2.78 ns (Table I). The short decay time for the WT protein changed from
1.55 to 0.82 ns (39.7 to 84.6%, respectively), whereas the long decay
time decreased from 6.56 to 4.66 ns (the normalized pre-exponential also decreased to 15.4% from 60.3%). The
ave value for
the W-417 mutant protein increased slightly from 4.36 to 4.64 ns, and
the short and long lifetime components both increased (short component, 0.93 to 1.54 ns and 43.8 to 56.6%, respectively; long component, 4.88 to 5.72 ns and 56.2 to 43.4%, respectively). There was little or no
change in the
ave value for the W-466 mutant protein
upon substrate binding (4.12 to 3.96 ns) and there was also
little change in either decay time component for this protein
(Table I). The W-558 mutant protein showed a slight increase in
its
ave value upon substrate binding and both
decay time components increased (short component, 1.05 to 1.32 ns and
47.6% to 57.3%; long component, 4.71 to 5.29 ns and 52.4% to
42.7%).
Quenching of Trp Fluorescence and NAD+ Substrate
Binding--
The titration of the WT and single Trp mutant proteins
with NAD+ is shown in Fig. 3.
The WT PE24 exhibited similar behavior when titrated with
NAD+, as previously observed for whole toxin (20) and for
PE40 (9). The intrinsic fluorescence of the WT was quenched in a
dose-dependent manner to less than 20% of the original
fluorescence (Fig. 3, Table II). The origin of the fluorescence
quenching mechanism was unmasked through the deployment of the
Trp-deficient (Trp-) mutant (W417F/W466F/W558F) PE24 (Fig.
3). This mutant protein possesses normal GH activity, indicating that
the replacement of the three Trp residues within the catalytic domain
does not inhibit the ability of the protein to bind the
NAD+ substrate. The fluorescence quench data shown in Fig.
3 were surprising in that the W-417 mutant exhibited significant
quenching of its lone Trp-417 residue (63%). This indicates that
NAD+ must induce a significant structural change within the
catalytic domain that results in an altered chemical environment for
Trp-417. Also, this mutant was able to bind NAD+, similar
to the WT protein (Table II). The W-466 mutant protein also bound
NAD+ with affinity comparable to the wild-type protein
(Table II), but the fluorescence of its Trp residue was not quenched to
the same extent as for the WT or W-417 proteins (Fig. 3). The W-558 protein showed a reduction in its affinity for the NAD+
substrate (296 and 47 µM for the W-558 and WT proteins,
respectively; Table II). However, the level of fluorescence quenching
was higher than for W-466 but lower than either the W-417 or WT
proteins (Fig. 3 and Table II).

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Fig. 3.
Quenching of intrinsic protein fluorescence
by NAD+. Titrations were conducted as described under
"Experimental Procedures." Samples included WT ( ), W-417 ( ),
W-558 (+), W-466 ( ), and Trp- ( ). The percentage of
quenching at saturating NAD+ concentration was calculated
from the data and analyzed by Scatchard plots to determine the
dissociation constants, and the Kd values obtained
are summarized in Table II. The results shown are representative of
three independent experiments.
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The GH activity of the WT and single Trp mutant proteins is also shown
in Table II. The WT GH parameters were as follows: Vmax, 3.34 ± 1.0 µmol/liter·h;
Km, 49.4 ± 7.8 µM;
kcat, 1.67 ± 0.5 h
1; and
specificity constant, 0.0338 µmol/liter·h. Both W-417 and W-466
mutant proteins possessed the same activity as the WT protein, but the
W-558 and Trp-deficient mutant proteins were slightly less active.
These results compare favorably to an earlier report involving n-1 and
n-2 Trp mutant proteins of a larger catalytically active fragment of
ETA, PE40 (9).
Fluorescence Anisotropy and NAD+ Binding--
The
effect of NAD+ substrate binding to the PE24 active site
was also monitored by fluorescence anisotropy determinations (Table I).
The WT protein possessed a moderate anisotropy value in the absence of
bound substrate (0.118), which showed a significant increase upon
association with NAD+ (0.207, Table I). The single Trp
mutant proteins showed a range of rave values in
the absence of bound substrate, ranging from 0.095 (W-466) to 0.173 (W-417). W-417 showed the largest increase in its
rave value upon binding the NAD+
substrate (
rave = 0.046), followed in
decreasing order by W-466 (
rave = 0.034) and
finally by W-558 (
rave = 0.005). These data indicate that the largest structural change within the catalytic domain
upon NAD+ substrate binding occurs in the vicinity of
Trp-417.
Stern-Volmer Quenching--
The Stern-Volmer quenching profiles
are shown in Fig. 4. The WT protein
showed a protection of the Trp fluorescence quenching induced by
acrylamide in the presence to NAD+. This protection was
also observed for the W-558 mutant protein, but there was no
significant difference for either the W-417 or W-466 mutant proteins.
The quenching profiles were linear and were best fit by correlation
coefficients between 0.981 and 0.999. The Stern-Volmer constants for
these proteins are shown in Table III. In
the absence of substrate, the WT showed the highest value (8.28 M
1), followed in decreasing order by W-417,
W-558, and W-466. The bimolecular quenching constants
(kq) are also shown in Table III. The two single
Trp mutant proteins, W-417 and W-558, exhibited the highest collisional
constants (1.77 and 1.80 M
1ns
1,
respectively), followed by the WT protein (1.41 M
1ns
1) and finally by W-466
(1.32 M
1ns
1). NATA was included
as a control and under the same conditions as for the proteins showed a
value greater than 11 M
1ns
1
(Table III). Upon binding the NAD+ substrate the value for
the Stern-Volmer constant decreased for the WT protein (2.89 M
1). The collisional constant also showed a
significant decrease from 1.41 to 1.04 M
1ns
1. The W-417 protein showed
a small decrease in solvent exposure of Trp-417 upon substrate binding
(1.77 to 1.62 M
1ns
1) in
contrast to W-466, which showed an increase in the solvent exposure of
its Trp residue (1.32 to 1.58 M
1ns
1). The W-558 mutant
protein showed a significant decrease in both the Stern-Volmer constant
(7.35 to 4.70 M
1) and the collision constant
(1.80 to 1.12 M
1ns
1). This
indicates that the substrate docks near Trp-558 and provides some
solvent shielding for this residue. This effect seems to be intensified
within the WT protein, which may reflect some local perturbation of the
active site caused by the Trp
Phe substitutions.

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Fig. 4.
Stern-Volmer plots of Trp fluorescence
quenching by acrylamide. Quenching experiments were conducted as
described under "Experimental Procedures." In each panel, the
samples were investigated with ( ) or without ( ) NAD+.
The samples include wild-type PE24 (A), W-417
(B), W-466 (C), and W-558 (D). The
slopes of the best fit lines for each data set
(KSV values) are shown in Table III and were
used to calculate kq values for each
sample.
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Table III
Stern-Volmer quenching of wild-type and Trp mutants of PE24
Fluorescence parameters determined from the data shown in Fig. 4.
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Structural Integrity and Folding of PE24--
The titration
profiles for the denaturation of WT PE24 with urea and Gn-HCl are shown
in Fig. 5, A and B,
respectively. The
-helical content of the WT PE24 protein was
determined by CD spectroscopy to be 19%. Importantly, there was no
significant changes in the average secondary structure content for any
of the single Trp mutant proteins (data not shown). The unfolding of
PE24 was reversible in both types of denaturants (Fig. 5, A and B). The calculated
GU values for the WT
protein was dependent upon the method used to measure secondary
structure changes associated with denaturation. Values of 10.7 ± 1.7 and 13.8 ± 3.8 kJ/mol for the
GU (urea
denaturant) were obtained for the fluorescence-based and the CD-based
measurements, respectively (Table IV).
The difference was even more pronounced between the
GU
values for fluorescence and CD measurements in Gn-HCl (8.7 ± 2.9 and 19.6 ± 3.3 kJ/mol, respectively). The
GU
values for all the mutant proteins were identical to the WT protein as
assessed by Gn-HCl denaturation experiments using fluorescence
spectroscopy (8.7 ± 1.1). Interestingly, when NAD+
was bound to WT PE24 at saturating concentration, the enzyme was
stabilized by 7.9 kJ/mol (Table IV). The rates for the
unfolding/folding processes were determined by stopped flow
fluorescence spectroscopy, and a sample trace is shown in Fig.
5C. The rate constants for the unfolding reaction were much
slower than for the folding reaction (Table IV). All the rate processes
were faster in the Gn-HCl denaturant as compared with urea, and the
kf constant was too rapid for determination by
conventional stopped flow instrumentation (instrumental dead-time = 1.3 ms).

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Fig. 5.
Unfolding, refolding, and stopped flow
profiles for WT PE24. The conditions for the experiments were as
described under "Experimental Procedures." The samples include
unfolding ( ) and refolding ( ) profiles in urea (A),
unfolding ( ) and refolding ( ) profiles in Gn-HCl (B),
and stopped flow kinetic trace for the unfolding process (0-4
M Gn-HCl) (C).
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DISCUSSION |
The quantum yield of the WT PE24 protein is not the average of
that measured for the three single Trp mutant proteins (WT, 0.17;
mutant average, 0.09). This indicates that there must be some degree of
local perturbation of the protein environment upon Trp
Phe
substitution within PE24 or energy transfer between Trp residues of the
WT. However, the weighted average for the anisotropy of the three
single Trp mutant proteins is similar to the measured value for the WT
anisotropy, indicating little or no depolarization of the WT anisotropy
value. This effectively rules out the latter possibility. The origin of
the low quantum yield for Trp-558 is likely its close position to the
guanido side chain of Arg-467 in addition to its high degree of aqueous solvent exposure. It is well known that Arg will quench indole fluorescence, although the mechanism is not well understood (21). All
three Trp residues showed a significant degree of fluorescence quenching upon NAD+ binding. In the case of Trp-558,
because the adenine ring of NAD+ docks next to the benzene
portion of the indole, a ground state nucleic acid-indole complex could
be formed (9, 22). However, the origin of the fluorescence quenching of
Trp-417 and Trp-466 is less obvious. Previously it was suggested that a
conserved loop region within ETA (residues 456-470, and residues
39-46 in DT) that harbors two Asp residues, Asp-461 and Asp-463, could be the source of substrate-induced fluorescence quenching within the
active site of the toxin (9). Unfortunately, this loop is unresolved in
the x-ray structure of the PE24-
-TAD+ substrate complex
(10). The largest change in the fluorescence quantum yield upon
substrate binding was observed for Trp-417. Given that both bases of
the NAD+ substrate are no closer than 17 Å to the Trp-417
indole ring, the origin of the fluorescence quenching mechanism of this
Trp must lie in a structural change within the protein segment
surrounding it. A comparison of the protein environment of Trp-417 in
the substrate bound and substrate-free forms revealed that the likely candidates for the fluorescence quenching are a number of peptide bonds
from various residues including Thr-418 (23).
Upon NAD+ binding, the anisotropy of the WT increased
(Table I), and this increase was reflected in the anisotropies of the both single Trp mutant proteins, W-417 and W-466, the former showing the greatest change. This adds further credence to the idea that there
is a substrate-induced structural change within the protein near
Trp-417. Trp-558 was previously believe to dock against the adenine
ring of NAD+ (9). However, with the advent of the
-TAD+-PE24 structure (10), it can be seen that there is
3-4 Å between the Trp-558 and
-TAD+ rings. The
anisotropy results for W-558 binding to NAD+ confirm this
observation because the Trp-558 rave value does not change significantly upon substrate binding (Table I). If there was
direct van der Waals contact between the two ring structures, an
increase in Trp-558 anisotropy would be expected.
The kq value for the WT protein was similar to
the weighted average values for the three single Trp mutant proteins
(1.41 and 1.58 M
1 ns
1,
respectively). Trp-417 and Trp-558 were similar in their extent of
solvent accessibility, and both were considerably more exposed to
solvent than was Trp-466. These findings were consistent with earlier
suggestions based on x-ray data (10) and sensitivity to NBS oxidation
(9). Trp-417 remains solvent exposed upon substrate binding, whereas
Trp-466 becomes more exposed. In contrast, Trp-558 is protected
from solvent upon substrate complexation with the enzyme. This
latter observation is likely due to the close proximity of
NAD+ to the Trp-558 residue.
The GH activity of the various single Trp mutant proteins mirrored our
earlier report that none of the Trp residues were critical for GH
activity of the toxin-enzyme (9). The measured turnover number
(kcat value) was also nearly identical to the
reported value for WT PE40 (1.67 ± 0.5 and 1.84 ± 0.4 h
1 for PE24 and PE40, respectively) previously reported
by our laboratory. The GH activity was the lowest for the triple mutant
Trp-, which resulted in GH activity that was 16% of the
WT value. This difference is most probably due to the accumulated
perturbation of the active site of the enzyme and does not reflect a
specific role for the Trp residues within the PE24 active site.
However, the single Trp mutant W-558, which lacks Trp-417 and Trp-558, exhibited a lower affinity (albeit marginally) for the NAD+
substrate (Table II). This may indicate that when both Trp-417 and
Trp-466 are replaced by Phe, there is a cumulative structural change
within the active site of the protein that leads to a less effective
binding pocket. The replacement of Trp-558 by Phe does not seem to have
any such effect (Table II).
The enzyme domain of ETA alone (domain III) is less stable than the
whole toxin or PE40 (domains II and III) (Table IV) (24). Remarkably,
the stability of WT PE24 protein was increased by 2-fold upon
complexation of the NAD+ substrate (Table IV). The stopped
flow experiments using denaturants as the unfolding agents revealed
that PE24 (domain III) refolds extremely quickly
much more rapidly
than either whole toxin or PE40. This was proposed from our earlier
folding experiments involving whole toxin and PE40 (24). The biological
implications of the rapid refolding process of domain III may reflect
the need for this domain to refold upon translocation to the cytoplasm
upon crossing the endoplasmic reticulum membrane in order to avoid destruction by host endogeneous proteinases or other scavenging cellular machinery during the intoxication process.