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INTRODUCTION |
Snake venom cardiotoxins are small molecular mass (~7 kDa),
highly basic proteins cross-linked by four disulfide bridges (1-3). These toxins exhibit a wide variety of biological activities such as
contraction of cardiac muscle cells, lysis of erythrocytes, and
selective toxicity to certain types of tumor cells (4, 5). More
recently, cardiotoxins have been demonstrated to selectively inhibit
the action of certain key enzymes such as
Na+,K+-ATPase and phospholipid-sensitive
protein kinase (2, 6-8). Although the general molecular mechanism
underlying the enzyme inhibitory action of cardiotoxins is still an
enigma, it is contemplated that cardiotoxin could effectively block the
enzymatic activities of Na+,K+-ATPase and
phospholipid-sensitive protein kinase by competitively binding (in the
present study) to adenosine triphosphate (ATP), which is a key
substrate for the functioning of these enzymes. However, to date, the
evidence for the binding of a nucleotide triphosphate to cardiotoxin
has not been reported.
In the present study, for the first time, we demonstrate that the
cardiotoxin analogue II (CTX
II)1 isolated from the Taiwan
cobra venom (Naja naja atra) bind to nucleotide
triphosphates. Herein, we also propose a molecular model of the CTX
II·dATP complex and suggest a reasonable molecular mechanism to
explain the reported inhibitory action of snake venom cardiotoxin on
phospholipid-sensitive protein kinase and
Na+,K+- ATPase.
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MATERIALS AND METHODS |
CTX II was purified as described by Yang et al. (9).
The concentration of the protein was estimated from its absorbance at
280 nm (
1 mM = 4.08). The nucleotides were
purchased from Sigma. The concentration of the nucleotides was
estimated (10) from their extinction coefficient values
(
259 = 15,400 for AMP, ADP, ATP, and dATP;
271 = 9,000 for CTP;
253 = 13,700 for
GTP; and
260 = 1,000 for UTP). All other chemicals used
were of high quality analytical grade. All the experiments were
performed at pH 3.0 and 25 °C unless otherwise mentioned.
Fluorescence Experiments--
Fluorescence experiments were
conducted on a Jasco HP 777 spectrofluorimeter. For tyrosine
fluorescence, the excitation wavelength was set at 275 nm and the
fluorescence was monitored at 310 nm. Both the excitation and emission
slit widths were set at 5 nm in all the experiments.
Circular Dichroism Experiments--
Circular dichroism
measurements were made on a Jasco 720 spectropolarimeter. Cylindrical
quartz cells of path length 0.1 and 1 mm were used for the measurements
in the far (190~240 nm) and near (250~320 nm) UV regions. For
thermal denaturation experiments, water-jacketed quartz cells were used
and the temperature of the sample was controlled using a Neslab water bath.
NMR Experiments--
The NMR experiments were performed on a
Bruker DMX 600 MHz spectrometer. For all 1H NMR titrations,
the protein concentration used was 0.5 mM and for
two-dimensional experiments 3 mM protein was used. The
protein solutions were made using 50 mM
glycine-d5 (pH 3.0). For the
purpose of assignment of individual proton resonances of the CTX
II·dATP complex, TOCSY (11) and water-gated NOESY (12) spectra were recorded for appropriate molar ratio (as indicated under "Results and
Discussion") of the CTX II-nucleotide in 95% H2O, 5%
D2O. All spectra were obtained with 2048 complex data
points in t2 (detection period) and 512 points
in t1 (evolution period) with a spectral width
of 7500 Hz. For hydrogen-deuterium exchange experiments, magnitude COSY
spectra (2048 × 256 points) were recorded for the sample in 100%
D2O. Chemical shifts were calibrated against
3-(trimethylsilyl)propionate, sodium salt. The data were processed
on an Indigo II workstation using the UXNMR software.
Calculation of Dissociation Constants--
The dissociation
constants (kd) of the dATP and CTX II were
calculated from the fluorescence intensity changes using the linear
relation (13),
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(Eq. 1)
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where
F is the change in the tyrosine fluorescence
intensity upon addition of the nucleotide, Lo is the
nucleotide concentration, and Co is the
concentration of the protein. The slope of the plot, 1/
F
versus 1/(Lo
F(Co)), yielded the dissociation
constant value.
Molecular Modeling--
The modeling of the CTX II·dATP
complex was carried out in different steps. Minimizations were
performed using the CHARMm (14) energy function. The dATP molecule was
built using the CHEMNOTE model building facility that is available
within QUANTA (Molecular Simulations Inc.). The conformation of the
adenine ring with respect to the deoxy sugar was set to be "anti."
The molecule was then subjected to Powell energy minimization (500 steps) with the inclusion of the intramolecular NOE restraints that was
available between the deoxyribose unit and the adenine moiety of dATP.
The energy-minimized dATP molecule was then placed on the convex side
of the CTX II (average solution structure; PDB entry code 1CRE). Both
the dATP and the CTX II molecules were subjected to
Adopted-Basis-Newton-Raphson energy minimization with the inclusion of
the available intermolecular NOEs. Finally, with the inclusion of both
intermolecular and the intramolecular NOEs (for CTX II and dATP), the
entire complex was subjected to another few cycles of
Adopted-Basis-Newton-Raphson energy minimization so as to obtain the
lowest energy structure and avoid unnecessary bad contacts in the
molecule. During this process the atomic restraints that had been
imposed on the CTX II molecule were relaxed.
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RESULTS AND DISCUSSION |
Binding to NTPs--
CTX II is a three-finger shaped (15, 16)
protein (Fig. 1). The solution structure
of the protein shows that the secondary structural elements in this
toxin analogue include antiparallel double- and triple-stranded
-sheet segments (15, 16). In addition, CTX II is a class A protein
as it lacks tryptophan residues and the aromatic amino acids in the
toxin include three tyrosine residues (2). The binding affinity of the
various nucleotide triphosphates (NTPs) to CTX II was investigated by
monitoring the changes in the tyrosine fluorescence emission intensity
at 310 nm as a function of the increasing concentrations of the various nucleotide triphosphates. The data presented in Fig.
2 depicts that all four nucleotide
triphosphates (used in this study) cause a decrease in the emission
intensity of CTX II, implying that all the four NTPs used could bind to
protein (CTX II). The decrease in the 310 nm emission intensity might
be due to the quenching effects of the NTPs bound to CTX II, at site(s)
proximal to the tyrosine residues in the toxin molecule. The binding
constants estimated from the tyrosine fluorescence decay curves are
listed in Table I. Evaluation of the
binding affinities (of different NTPs) to the protein from the
calculated binding constant values (Table I) reveal that the binding
affinity of all the four NTPs are in a similar range (16-32
µM). Thus, these results show that CTX II is a general
NTP-binding protein.

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Fig. 1.
MOLSCRIPT representation of the backbone fold
of CTX II. Residues 2-5 and 11-14 form the antiparallel
double-stranded -sheet and residues 20-25, 34-39, and 50-55 form
the antiparallel triple-stranded -sheet segment.
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Fig. 2.
Changes in the tyrosine fluorescence
intensity at 310 nm [ ex = 275 nm] shown as a function of nucleotide triphosphate(s)
concentration. The concentration of CTX II was 20 µM. The fluorescence intensity was normalized with
respect to the native protein fluorescence.
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Table I
The dissociation constant of different nucleotides calculated from the
tyrosine fluorescence data, signifying the interaction between the
nucleotides and the CTX II molecule
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To examine the influence of phosphate groups in the binding of NTPs to
the CTX II molecule, we compared the binding affinities of ATP, ADP,
AMP, and inorganic phosphate to CTX II. Addition of inorganic phosphate
to the protein did not cause any significant change (data not shown) in
the 310-nm emission even up to a concentration of 70 mM (10 times the concentration of the protein), implying that the changes in
the emission intensity at 310 nm upon addition of the NTPs are due to
the binding to the nucleoside portion of the NTPs used. Interestingly,
the changes in the emission intensity at 310 nm upon addition of ATP,
ADP, and AMP to CTX II reveal that all nucleotide derivatives used
per se bind to the protein, but the binding avidity appears
to be dependent on the length of the phosphate tail attached to the
nucleoside. Thus, ATP with three phosphate groups shows the strongest
binding (kd = 15.69 µM, Fig. 2 and
Table I) and AMP which possess one phosphate group shows the weakest
(kd = 87.36 µM, Fig. 2 and Table I)
binding to CTX II. Hence, from the results of the experiments discussed
above it is clear that CTX II binds to NTPs and the strength of the
binding is strongly correlated to the length of the phosphate tail
tethered to the nucleoside moiety. It should be mentioned that although
CTX II appears to bind to all the four nucleotide triphosphates with
similar affinity, for reasons of stability, we have used dATP as a
model NTP to understand the NTP-CTX II structural interactions in
greater detail.
To probe if binding of NTP to CTX II is accompanied by conformational
changes in the protein, the far and near UV-CD experiments were
performed. Comparison of the far UV-CD spectra of the free CTX II and
CTX II bound to dATP (in the molar ratio of 3:1 for dATP:CTX II)
revealed no significant change in the 215 nm ellipticity values
(representing the
-sheet secondary structural elements in the
protein) was observed. These results thus indicate that binding of dATP
to CTX II does not bring about any major change in the backbone folding
of the protein but only causes minor perturbations in its side chain packing.
Effect of CTX II Stability upon Binding to dATP--
To
investigate the effect(s) of binding of dATP on the stability of the
protein (CTX II), we compared the thermal stabilities of the CTX II in
the free form and the dATP bound state by monitoring the change(s) in
the 275-nm ellipticity as a function of increasing temperature. It was
observed that the tertiary structural interactions in the protein
(represented by the 275 nm ellipticity) show no change up to 60 °C
but begins to melt rapidly beyond this temperature (Fig.
3). The unfolding process is complete at
about 80 °C. Interestingly, the CTX II sample bound to dATP is
resistant to denaturation even up to 75 °C (Fig. 3). It could be
observed that the unfolding process is not complete even at a
temperature of 95 °C (which is technically the maximum possible
temperature we could perform the experiment). It is amply clear from
the results of these experiments that binding of dATP to CTX II confers
conformational stability to the protein.

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Fig. 3.
Changes in the ellipticity values (at 275 nm)
of the dATP-bound ( ) and free CTX II ( ) as a function of
temperature. CTX II is found to unfold at 80 °C
(Tm = 70 °C) in the absence of dATP whereas the
unfolding of the protein is not complete even at 95 °C when it is
bound to dATP.
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Hydrogen-deuterium exchange detected by two-dimensional NMR technique
is a facile and a sensitive technique to detect even subtle change(s)
in the backbone of the protein, which sometimes could not be tracked by
circular dichroism. Hence, we performed the hydrogen-deuterium exchange
experiments to investigate for possible conformational alterations in
CTX II upon binding to dATP (molar ratio 1:1). Magnitude COSY spectrum
of free CTX II dissolved in D2O (pD 3.4) showed that 27 cross-peaks (NH, C
H) are protected from exchange in the native state
of the protein (data not shown). This probably implies that these 27 amide protons are either involved in hydrogen bonding or the
corresponding amide protons are located in the protein interior. In
comparison, magnitude COSY spectrum (data not shown) of CTX II bound to
dATP also showed the same number of 27 cross-peaks (NH, C
H) in the
fingerprint region. Thus, it appears that there is/are no drastic
conformational change(s) in the backbone of the protein upon binding to
dATP. Although the number of (NH, C
H) cross-peaks of the free and
the bound forms are the same, it is also important to compare the relative cross-peak (NH, C
H) intensities in the magnitude COSY spectra representing the free and the dATP bound form of CTX II (Fig.
4). Comparison of the relative cross-peak
intensities of the 27 protected amide protons in the native and dATP
bound forms of CTX II (Fig. 4) reveal that several residues in the dATP
bound form show higher protection against exchange than when the
protein is not bound to dATP. The residues showing relatively higher
protection against exchange include Lys2, Cys3,
Lys18, Cys21, Tyr22,
Met24, Val34, Tyr51,
Val52, Arg58, and
Asn60. Interestingly, most of these residues depicting
higher protection (in the dATP bound state of CTX II) are located in
the
-strands III, IV, and V and in the N- and C-terminal ends of the
CTX II molecule. Thus, the results of the hydrogen-deuterium exchange experiments not only give useful clues regarding the dATP-binding site
on the protein but also unambiguously demonstrate that binding of dATP
to the protein (CTX II) stabilizes the structure of the protein.

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Fig. 4.
Difference in the
(C H-NH) cross-peak intensities as calculated
from the magnitude COSY spectra recorded for the CTX II sample in the
presence and absence of dATP (50 mM
glycine-d5 in 100%
D2O]. The intensities were normalized with respect to
the intensities of the non-exchangeable aromatic protons. The data
represents the difference(s) in the normalized intensity observed in
free CTX II minus the normalized intensities in the dATP-bound CTX
II.
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As the results of the experiments described thus far demonstrate that
dATP binds to CTX II, it would be interesting to probe the location of
the dATP-binding site on the three-dimensional structure of the
protein. In this context, we employed one- and two-dimensional NMR spectroscopy.
Location of dATP-binding Site--
One-dimensional NMR experiments
provide an idea of the site(s) of interaction(s) of the ligand on the
protein. These experiments provide clear information on the chemical
environment at the ligand interaction site(s) on the protein. In
general, protein-ligand interactions using one-dimensional NMR
spectroscopy could be discerned from the changes in the 1) broadening
effects in the line width of the individual proton resonance(s) and/or
from 2) the relative chemical shift difference(s) between the free and
bound form of the protein. In this context, a series of one-dimensional
NMR spectra (Fig. 5) were recorded (at
25 °C) at varying dATP:CTX II ratios (0-1.5). The chemical shift
values of many proton resonances, in the presence of dATP showed
significant differences as compared with the free form of the protein.
Only the changes accompanying a few selected non-overlapping resonances
could be unambiguously followed at increasing molar ratios of dATP:CTX
II. The titration curves evaluated as a function of the molar ratio(s)
of dATP:CTX II are hyperbolic and tend to saturate when the dATP:CTX II
molar ratio reaches a value close to one (Fig.
6). This aspect apparently suggests a 1:1
binding of the nucleotide triphosphate to the protein (Fig. 6). A close
examination of the chemical shift values of the various proton
resonances (which could be unambiguously monitored in one-dimensional
spectrum) reveals that the amide proton resonances of
Ile39, Asn4, and Arg58 undergo the
most pronounced drift in the chemical shift values (Fig. 6). It is
interesting to note that Asn4 and Arg58 are
located at the terminal ends of the cardiotoxin molecule. Incidentally,
the solution structure of the free-form of CTX II reveals that the N-
and C-terminal ends of the CTX II molecule are in close proximity
tethered together by hydrogen bonds among their backbone atoms (15,
16). In this context, it is probable that dATP binds to CTX II at
region(s) where the N- and C-terminal ends of the molecule are bridged
together. In addition to the amide proton of Ile39, the
side chain methyl proton
(Ile39C
H3) also exhibited
reasonably significant upfield shift (Fig. 6). Ile39, which
is a part of the triple-stranded
-sheet segment is located at a site
which is spatially 10 Å from the N and C termini of the CTX II
molecule. Although this residue (Ile39) is located at a
position well separated from the putative dATP-binding site, the
observed large magnitude drift could be possibly due to the small local
destabilization of the native structural interactions at the
dATP-binding site. Such a subtle molecular reorganization in the
protein molecule, upon binding to dATP, possibly brings the isoleucine
terminal methyl group spatially closer to the conserved phenolic ring
of Tyr22 (whose aromatic ring is spatially close to
Ile39 in the native three-dimensional structure of CTX II).
In such an event, the terminal methyl group is expected to experience larger ring current effects (compared with native) leading to the
observed change in the chemical shift values for the methyl group
protons. In addition, to the amide protons and the side chain methyl
group(s), the
-proton (C
H) resonances of a few residues (which
could be monitored) also showed significant differences in the chemical
shift values upon binding to the nucleotide (Fig. 6). The changes in
the C
H chemical shift values of Tyr11,
Tyr22, and Lys2 are remarkable.
Lys2 is located in the double-stranded
-sheet segment
and as stated earlier plays a key role in bridging the N and C termini
(16). Tyrosine residues located at positions 11 and 22 are lodged in the double- and triple-stranded
-sheet segments, respectively. Tyr22 is one of the most well conserved residue found in
all the cardiotoxin isoforms (17-19). This residue (Tyr22)
is the locus of a prominent hydrophobic core that plays an important role in maintaining the structural integrity of the three-dimensional structure of cardiotoxins. Chemical modification of Tyr22
has been reported to result in drastic disruption of tertiary structural interactions, rendering the toxin molecule inactive (20). In
addition, a comparison of the three-dimensional solution structures of
various cardiotoxin isoforms isolated from Taiwan cobra (Naja
naja atra) venom shows that the aromatic rings of Tyr22 and Tyr51 lie parallel to one another
(2). It appears that local destabilization effects at the dATP-binding
site (upon binding to dATP) in the protein are effectively transmitted
to residues which are spatially far away from the putative binding site
accounting for the observed change(s) in the chemical shift values of
some of the protons of residues (spatially separated from the
dATP-binding site). The local perturbation occurring upon binding to
dATP (at the contemplated dATP binding site) appears to force the
Ile39 side chain atoms to move closer to the aromatic ring
of Tyr22. It appears that the mobility of a bulky
hydrophobic side chain (Ile39) further influences the
native molecular interactions stabilizing the phenolic ring of
Tyr22. Since the aromatic rings of Tyr22 and
Tyr51 are spatially juxtaposed, the subtle destabilization
effects are probably further transmitted to Tyr51. Our
proposal of this molecular "destabilization relay" is supported by
the observed chemical shift changes of the amide protons of the
residues involved in the above mentioned destabilization relay. The
slight decrease in the ellipticity value at 270 nm in the near UV-CD
experiment, upon protein binding to dATP supports our contention that
the native aromatic interactions (contributing to the CD signal) are
affected upon the protein molecule binding to dATP. Ubbink and Bendall
(21) recently characterized the plastocyanic-cytochrome c
complex. The authors observed changes in chemical shifts of those
protons, which are spatially far away from the binding site. This
phenomenon is attributed to arise from the "secondary effects"
resulting from the complex formation. Similarly, the chemical shifts
observed in the present study could be due to such secondary effects
(by destabilization relay). Estimation of the association constant
values from the proton chemical shifts reveal that dATP binds to the
CTX II molecule with a binding constant value in the micromolar range
which is in par with those obtained from the fluorescence
experiment(s). Thus, from the results obtained from the one-dimensional
NMR experiments, it appears that dATP binds specifically with CTX II
and the interacting site is contemplated to be located in the cleft
formed between the N- and C-terminal ends of the molecule.

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Fig. 5.
One-dimensional NMR spectra of CTX II in the
absence and presence of dATP. The numbers (near the water signal)
represent the molar ratio of dATP to CTX II. All spectra were recorded
in 50 mM glycine-d5 (pH 3.0) at
25 °C. The chemical shifts were referenced to
3-(trimethylsilyl)propionate, sodium salt. The asterisks on
selected proton resonance signals indicate those that arise from
dATP.
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Fig. 6.
Changes in the chemical shift of selected,
isolated proton resonances as detected from the one-dimensional proton
NMR spectra, as a function of molar ratio of dATP to CTX II. The
changes in the observed chemical shift values are found to saturate at
a dATP to CTX II molar ratio of 1:1.
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To obtain complete insight of the structural interactions stabilizing
the dATP·CTX II complex, two-dimensional NMR experiments (22) were
carried out (at 1:1 molar ratio of dATP:CTX II). Most of the proton
resonances pertaining to dATP, free CTX II, and dATP bound to CTX II
were assigned unambiguously using the TOCSY and NOESY spectra. Analysis
of the spectral data of the two-dimensional NMR experiments revealed
that the chemical shift values of many protons in the protein (in the
free form) had significantly changed upon interaction with dATP. Some
of these changes could be monitored only from the two-dimensional NMR
data because of the overlap of the resonances in the one-dimensional
1H NMR spectra discussed earlier. There are several protons
of the protein backbone which show significant differences (>0.02 ppm)
in the chemical shift upon complexation. These include the amide and/or
-protons of Lys2, Asn4, Lys5,
Val7, Tyr11, Tyr22,
Lys23, Met24, Arg36,
Cys38, Ile39, Val41,
Lys44, Ser46, Tyr51,
Cys53, and Arg58 (data not shown).
Interestingly, most of the residues showing appreciable change(s) in
their chemical shift values after binding to dATP are located in the N-
and C-terminal ends and
-strands III and IV of the CTX II molecule.
It should be mentioned that the differences observed in the amide and
the
-protons of few residues that are situated topologically at
site(s) far away from the contemplated nucleotide-binding site could
possibly be due to minor secondary conformational effects. It should be
of interest to note that the backbone amide protons (of tyrosine
residues) show significant differences in their chemical shift values
upon binding to dATP (data not shown). This feature strongly suggests that the three tyrosine residues are located near or in the
nucleotide-binding site on the protein. Similar conclusions were
reached from the results of the tyrosine fluorescence experiments and
the one-dimensional NMR experiments as discussed earlier. Clinching
evidence for protein-ligand interaction(s) based on two-dimensional NMR
experiments comes from the observation of intermolecular NOEs between
the protons of the ligand and that of the protein. Analysis of the NOEs
obtained in the NOESY spectrum of the dATP·CTX II complex shows that
there are at least 11 unambiguous intermolecular NOEs characterizing the complex between the nucleotide and the protein (Table
II). Several of the observed
intermolecular NOEs are with the sugar moiety of dATP. Prominent among
the intermolecular NOEs signifying the protein-dATP interaction include
Lys2NH-H2', Ile39C
H3-H2',
Ile39C
H3-H2'', and Asp57NH-H4'
(Fig. 7). The Leu1H
-H8
symbolizes the sole intermolecular NOE characterizing the protein-adenine ring interaction(s). In addition, several other NOEs
are observed for the protein-sugar interaction (Table II).
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Table II
List of intermolecular NOEs observed in the NOESY spectrum (250 ms) of
the CTX II·dATP (1:1) complex (50 mM glycine, pH 3.0, 25 °C)
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Fig. 7.
NOE cross-peaks that are observed between the
CTX II and the dATP molecule in the NOESY spectra recorded with a
mixing period of 250 ms. The NOEs in the NOESY spectra of free CTX
II is shown for comparison.
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It is important to address the question as to why only one
intermolecular NOE represents the protein-adenine ring interaction. In
this context, it is important to compare the amino acid sequences of
well known nucleotide-binding proteins and that of CTX II. Kabsch and
Holmes (23) upon detailed search for the consensus nucleotide binding
motif among the proteins in the data bank, reported that the amino acid
sequence,
(Ile/Leu/Val)-X-(Ile/Leu/Val/Cys)-Asp-X-Gly-(Thr/Ser/Gly)-(Thr/Ser/Gly)-X-X-(Arg/Lys/Cys), represents the nucleotide binding motif. The amino acid sequence of CTX
II interestingly revealed the presence of a nucleotide binding
sequence,
Val32-Pro33-Val34-Lys35-Arg36-Gly37-Cys38-Ile39-Asp40-Val41-Cys42.
It could be discerned that the CTX II shares more than 80% amino acid
sequence homology with the reported consensus nucleotide binding motif.
A closer look at the nucleotide binding motif in CTX II shows the
absence of a crucial, non-variant aspartic acid present in the amino
acid sequences of all the nucleotide-binding proteins known so far
(23). The charged carboxylate group of aspartic acid is believed to
stabilize the adenine ring through a hydrogen bond with the amino group
located in the purine ring (23). A positively charged lysine residue in
CTX II replaces the conserved aspartic acid. It is possible that the
positive charge on lysine does not produce similar stabilization (due
to charge incompatibility or electrostatic repulsion(s)) of the dATP molecule. This renders greater mobility for the adenine ring accounting for the absence of the expected intermolecular NOEs with the protein molecule.
Conformation of dATP--
It is pertinent to address the question
of the spatial orientation of the adenine ring with respect to the
deoxyribose sugar in the dATP molecule bound to the protein. It is well
known that the nucleotides in the unbound or free form adopt a
syn conformation (24). Upon binding to macromolecules, the
nucleotides exist in either anti or syn
conformation (25). The conformation of the bound nucleotide is
considered to be syn, if the intramolecular NOEs (in the
dATP molecule) between the H2 proton in the adenine ring and H2', H2'',
H3', and H5' protons in sugar moiety (24, 26) are observed. On the
contrary, if one detects NOEs between the H8 proton (of the adenine
ring) and H1', H2', H4', and H5' of the sugar ring, the conformation of
the bound nucleotide could be termed to be anti. A critical
examination of the observed intramolecular NOEs between the sugar and
the adenine ring (in the dATP molecule bound to CTX II) shows that
intramolecular NOEs (between H8 proton of the adenine ring and the H1',
H2', H2'', H4' of the deoxy ribose sugar) are quite similar to that
expected for the anti conformation (of the nucleotide).
Therefore, dATP adopts anti conformation upon binding to the
CTX II molecule.
Molecular Modeling of CTX II·dATP Complex--
Molecular
modeling of the CTX II-dATP interaction was attempted to obtain a
visual concept of the topology of the dATP-binding site and the
structural interactions in operation at the nucleotide triphosphate-CTX
II interface. The proposed model of the CTX II·dATP complex is in
good agreement with the experimental NMR data. Interestingly, the total
CHARMm energy of the free form of CTX II (
675 kcal mol
1) was lower than that of the dATP bound form of the
protein (
781 kcal mol
1). This further implies that CTX
II gains extra stabilization upon binding to the nucleotide
triphosphate. This result is in conformity with the conclusions drawn
from the results of the thermal denaturation and hydrogen-deuterium
exchange experiments. The structure of the dATP·CTX II complex
depicts the dATP molecule lodged in the groove enclosed by the N- and
C-terminal ends of the molecule (Fig. 8).
The energy minimized structure reveals that the protein-dATP complex is
stabilized by a variety of electrostatic interactions. The negatively
charged triphosphate moiety of the dATP molecule is found to strongly
interact with the "cationic cluster" at the terminal ends (N and C
termini). The residues in the cationic cluster interacting with the
triphosphate moiety are Lys2, Asn4,
Arg36, and Arg58 (Fig.
9). It is interesting to note that among
the three phosphate groups in dATP, the oxygen atoms of the
- and
-phosphate groups are spatially closer to the residues comprising
the cationic cluster. The negatively charged oxygen atom of the
-phosphate group, interestingly, does not possess any significant
interaction(s) with the cationic residues in the protein. It appears
that the phosphate groups in the
and
positions of the
nucleotide triphosphate dictates the binding efficiency of the ligand
molecule to the cationic cluster spread on the cardiotoxin molecule.
The negatively charged phosphate groups of the dATP molecule are placed
across the middle of Loop II of the CTX II molecule (Fig. 9). Such a
topological location brings the negatively charged phosphate groups of
dATP spatially closer to some of the residues located in Loop I. For example, the aromatic residue, Tyr11, located in Loop I is
found to be located at a spatial distance of 6 Å from the
-phosphate group of dATP. Due to the proximity of the phenolic ring
(of Tyr11) to the negatively charged
-phosphate, it is
possible that the aromatic ring experiences an electrostatic repulsion
leading to minor perturbations in the native interactions in this
portion of the molecule. Interestingly, the change in the chemical
shift values of the amide protons of Tyr11 appears to
support our contention. In addition to the phosphate group(s)-mediated
interactions, the energy minimized CTX II·dATP complex structure also
reveals additional interactions between dATP and the protein. The
hydroxyl groups of the deoxy sugar are found to interact with the
protein through the amide groups (of the backbone) contributed by the
amino acid residues located at the N- and C-terminal ends such as
Lys2, Cys3, Asp57, and
Cys59.

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Fig. 8.
Graphical representation of the dATP molecule
interacting with CTX II. The dATP molecule could be seen to be
lodged in the groove between the N- and C-terminal ends of the CTX II
molecule.
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Fig. 9.
Molecular model of the CTX II·dATP
complex. The nucleotide could be seen to lodge in between the the
N- and C-terminal end of the CTX II molecule. It is also evident that
the negatively charged pyrophosphate group of the dATP segment is
stabilized by the electrostatic interactions with the positively
charged lysines and arginines of CTX II. The dATP molecule is shown in
green.
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Go and co-workers (27) recently analyzed the structural features of the
nucleotide-binding site in various proteins, which have strong
propensity to bind to nucleotides. Based on a thorough search, it was
found that the nucleotide-binding site is normally located in the
"charged sockets" comprising of cationic residues. In general these
charged sockets are found to be located at a topological site wherein
the N- and C-terminal ends of the protein come close to one another. In
addition, this study revealed that at the site(s) closer to the
nucleotide-binding site, the backbone of the protein to have strong
propensity to adopt
-sheet conformation. Interestingly, the
ATP-binding site in CTX II exhibits structural features quite similar
to that of the consensus nucleotide-binding site.
Implications of CTX II-dATP Interaction--
Cardiotoxins as
stated earlier are bestowed with a broad range of biological properties
(2). Recently, cardiotoxins isolated from various snake venom sources
have been demonstrated in vitro to competitively inhibit the
enzymatic activity of phospholipid-sensitive (Ca2+
dependent) protein kinase and activated
Na+,K+-ATPase bound to the erythrocyte membrane
and muscle cells (6-8). It is interesting to note that both these
enzymes are membrane bound and binding to ATP is obligatory for their
enzymatic activity. Cardiotoxins are membrane active proteins (2) and
are known to bind and penetrate through the membrane and also possess
the ability to strongly bind to nucleotide triphosphates (including ATP
as reported in the present study). Thus, it appears that CTX II due to
its nucleotide triphosphate binding ability, probably competes with
these ATP-dependent enzymes for binding to ATP leading to
the effective inhibition of the catalytic action of these enzymes. It
is interesting to note that the binding constant of dATP to CTX II is
in the same range as that of phospholipid-sensitive protein kinase
(~30 µM) (28) and Na+,K+-ATPase
(124 µM) (29). This supports our viewpoint that the snake
venom cardiotoxin block the enzymatic action of these two enzymes by
sequestering the ATP and inhibit the biological activities of protein
kinase and Na+,K+-ATPase.
The present study is the first report wherein the binding of nucleotide
triphosphates, including ATP, has been presented for a snake venom
cardiotoxin. This finding, in our opinion, implicates the role of
cardiotoxins in the control of many cellular processes. It is believed
that more detailed studies exploring the significance of binding of the
nucleotide triphosphates to snake venom cardiotoxins would be conducted
in the near future.