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INTRODUCTION |
Scorpion venom contains a diversity of protein toxins, which
modify the properties of neural ion channels. The excitatory toxins are
highly specific to insects, have a characteristic structure and mode of
action, and define a unique receptor-binding site on the insect sodium
channel (1, 2). Unlike all other "long" scorpion toxins with 60-65
amino acid residues and a highly conserved pattern of four disulfide
bridges, the excitatory toxins reported thus far are composed of 70 amino acid residues, and one of their disulfide bonds is shifted (3,
4). Excitatory toxins such as AaHIT, LqqIT1, and AmmIT
induce spastic paralysis caused by repetitive activity of motor nerves
(5-7) resulting from increased sodium current and slowed inactivation
(8-10). AaHIT and other excitatory toxins define a high affinity
(Kd = 1-3 nM), low capacity (1-2
pmol/mg of protein) binding site in insect neural membrane (1, 11).
Their high potency makes them as an attractive model for development of
anti-insect-selective biopesticides. Indeed, AaHIT played a prominent
role in early attempts to engineer entomopathogenic baculoviruses
(12-17).
It has become apparent that detailed structure-activity analysis of
these toxins depends on their efficient overproduction in heterologous
systems. However, many attempts to establish a genetic system for high
level production of AaHIT in COS cells (18), insect cells (12-14),
yeast (19), or bacteria (20) have been very limited, and toxin
detection could be achieved only with antibodies or by neurotoxic
symptoms. The difficulty in obtaining reasonable amounts of pure
protein also hampered three-dimensional structure determination of this
toxin group. Indeed, only one report describing the secondary structure
and overall fold of AaHIT has been published (21). Other obstacles limiting a genetic study of excitatory toxins are the small number of
toxins identified thus far and their prominent sequence similarity. Only two, highly homologous excitatory toxins (AaHIT and
LqqIT1) have been sequenced during the last 3 decades (5,
6), and two additional excitatory toxins have been isolated (AmmIT (7) and BjIT1 (9)).
Recently, we have determined the three-dimensional structure of a novel
excitatory toxin, Bj-xtrIT (3), and have shown that, despite the unique
architecture of its C-terminal module, the core consisting of an
-helix and a three-stranded antiparallel
-sheet was similar to
those found in various pharmacologically distinct scorpion toxins,
e.g. AaH II and CsE v3 (22). Evidently, the difference in
bioactivity of the various toxins is dictated by their non-similar
exteriors composed of various amino acid side chains, loops, and turns
connecting the conserved secondary structure elements and the C
termini. Herein we report the purification, cloning, and functional
expression of Bj-xtrIT and its utilization in a genetic modification
study. By combining site-directed mutagenesis with functional and
structural analyses, we delineated specific amino acid residues
clustered on a molecular surface forming the putative bioactive domain
of scorpion excitatory neurotoxins.
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EXPERIMENTAL PROCEDURES |
Biological Material
Buthotus judaicus scorpions were collected at the
Carmel mountains of Israel, and their venom was collected upon stinging a Parafilm membrane. Sarcophaga falculata blowfly larvae,
Spodoptera littoralis noctuid larvae, and Periplaneta
americana cockroaches were bred in the laboratory. Albino
laboratory ICR mice were purchased from the Levenstein farm (Yokneam,
Israel). Escherichia coli strains DH5
and BL21 were used
for plasmid construction and expression, respectively. The
translational vector used, pET-11cK, is a pET-11c derivative (23)
bearing a gene for kanamycin resistance.
Purification and N-terminal Sequence of Native Bj-xtrIT
Crude venom (7.7 mg) from 35 scorpions was lyophilized,
dissolved in 3 ml of 10 mM ammonium acetate (pH 6.7), and
subjected to three successive chromatographic steps. 1) Cation-exchange chromatography was carried out on a 2.5-ml CM52-cellulose column (Whatman) equilibrated in 10 mM ammonium acetate (pH 6.7).
After elution of 5 ml, a linear gradient of 10-400 mM
ammonium acetate (pH 6.7) was applied at room temperature (20 °C)
with a flow rate of 0.6 ml/min, and 1.2-ml fractions were collected. 2)
Anion-exchange chromatography was performed on a 0.8-ml DEAE-Sephadex
column (Sigma) equilibrated in 10 mM ammonium acetate (pH
8.0). The active fraction obtained in step 1 was chromatographed using
a linear gradient of 0.01-1.0 M ammonium acetate (pH 8.0)
at a flow rate of 0.5 ml/min at room temperature. Fractions were
collected in 1.5-ml aliquots. 3) Reverse-phase
HPLC1 was carried out on a
Vydac C18 column (250 × 10 mm) using 0.1% trifluoroacetic acid as solvent A and 0.1% trifluoroacetic acid and
acetonitrile as solvent B. Elution was performed with a linear gradient
of solvent B: 10 min with 0-23%, 40 min with 23-35%, 15 min with
35-40%, and a final 10 min with 40-100%. Amino acid sequence
analysis followed automated Edman degradation using an Applied
Biosystems gas-phase sequencer connected to its corresponding phenylthiohydantoin analyzer and data system.
Cloning of Bj-xtrIT
Fifteen venom gland segments (telsons) were ground for RNA
purification. Isolation of poly(A)+ RNA and cDNA
synthesis were performed as described previously (24). A library in
E. coli strain DH5
was obtained by cloning 50 ng of
cDNA into the SmaI polylinker site of pBluescript
phagemid (Stratagene). A degenerate oligonucleotide (primer 1),
5'-AA(A/G)AA(A/G)AA(T/C)GGNTA(T/C)CCN(C/T)T-3', designed according to
the N-terminal amino acid sequence, was PCR-amplified with a second
oligonucleotide (primer 2), 5'-ATTCCTGCAGCCCTTTTTTT-3', composed of a
pBluescript stretch at the SmaI site and additional deoxythymidines, using the cDNA library as a template. Reaction conditions were 30 cycles for 1 min at 94 °C, 1 min at 50 °C, and
1 min at 72 °C. The PCR product was blunt-ended, phosphorylated, cloned into the SmaI site of pBluescript, and subjected to
sequence analysis using Sequenase II (U. S. Biochemical Corp.).
Full-length cDNA clones were obtained using an oligonucleotide
(primer 3), 5'-CGGGATCCCTATGAGGGTATTATTTGG-3', designed
according to the sequence of the noncoding strand of the region
encoding the C terminus of Bj-xtrIT and with a terminal
BamHI site (underlined), and the KS primer (Stratagene).
Construction of Expression Vectors
Oligonucleotide primers were used to reconstruct via PCR the
cDNA termini of Bj-xtrIT.38E, Bj-xtrIT.38K, and Lqh-xtrIT (formerly termed LqhIT1) (25). For Bj-xtrIT, primer 4 (5'-GGAATTCCATATGAAGAAGAACGGATATCCTCTG-3' was designed (i)
to remove the putative leader sequence, (ii) to create an additional
codon for methionine at position
1 of the mature polypeptide, and
(iii) to provide an NdeI restriction site (underlined)
within the ATG start codon. Primer 3, designed beforehand to contain a
BamHI restriction site, was used for reconstruction of the
3'-end. Similarly, primers 5 (5'-AACATATGAAGAAGAATGGGTATGCTGTC-3') and
6 (5'-CCGGATCCCTAATTAATTATTGTGAAATC-3') were used to reconstruct Lqh-xtrIT. Primer 7 (5'-CGGGATCCCTACCCGCAATATTTTTTGGTG-3')
was designed to replace the last seven amino acid residues with glycine and to add a BamHI restriction site (underlined). This
primer was employed with primer 4 via PCR to construct the
Bj-xtrIT.
C-ter mutant. The PCR products were cloned into the
corresponding NdeI and BamHI sites of pET-11cK.
Expression and purification of recombinant proteins followed an
established method (26). Conditions for optimized folding of
recombinant proteins were previously described (26, 27).
Toxicity Assays
Four-day-old blowfly larvae (~100 mg of body weight) were
injected intersegmentally at the rear side. A positive result was scored when a characteristic paralysis (immobilization and contraction) was observed and lasted for at least 15 min. Ten larvae were injected with five concentrations of each toxin in three independent
experiments. S. littoralis third instar larvae (~70 mg)
were injected in their pseudopodia, and a positive effect was scored
upon paralytic immobilization. ED50 (effective dose 50%)
values for flies and PU50 (paralytic unit 50%) values for
moths were calculated according to the sampling and estimation method
of Reed and Muench (28). Twenty-gram female mice were injected
subcutaneously with toxin amounts up to 0.2 mg.
Competition Binding Experiments
Neural Membrane Preparations--
Insect synaptosomes were
prepared from heads of adult cockroaches (P. americana)
according to a procedure previously described for blowfly heads (29,
30). All buffers used contained a mixture of protease inhibitors (50 µg/ml phenylmethylsulfonyl fluoride, 1 µM pepstatin A,
1 mM iodoacetamide, and 1 mM
1,10-phenanthroline). Membrane protein concentration was determined
using a Bio-Rad protein assay with bovine serum albumin as a standard.
Radioiodination--
AaHIT and LqhIT2 were
radioiodinated with IODO-GEN (Pierce) using 5 mg of toxin and 0.5 mCi
of carrier-free Na125I as described previously (31). The
monoiodotoxins were purified on a Vydac RP C18 column using
a gradient of 5-90% solvent B at a flow rate of 1 ml/min (32). The
concentration of the radiolabeled toxins was determined according to
the specific activity of the monoiodotoxin ranging between 4200 and
3450 dpm/fmol.
Binding Assays--
Equilibrium competition and saturation
assays were performed using increasing concentrations of unlabeled
toxin in the presence of a constant low concentration of radioactive
toxin. To obtain saturation curves ("cold" saturation), the
specific radioactivity and the amount of bound toxin were calculated
and determined for each toxin concentration. Equilibrium saturation or
competition experiments were analyzed by the iterative computer program
LIGAND (Elsevier Biosoft, Cambridge United Kingdom) using cold
saturation and "drug" analysis, respectively. Standard binding
medium composition was 135 mM choline-Cl, 1.8 mM CaCl2, 5.5 mM KCl, 0.8 mM MgSO4, 50 mM HEPES (pH 7.3), 10 mM glucose, and 2 mg/ml bovine serum albumin. Washing
buffer composition was similar, but with 5 mg/ml bovine serum albumin
and no glucose. Insect synaptosomes (10-14 µg/ml) were suspended in
0.2 ml of binding buffer containing 125I-AaHIT or
125I-LqhIT2. After incubation for 1 h at
22 °C, the reaction mixture was diluted with 2 ml of ice-cold
washing buffer and filtered through GF/C filters under vacuum. Filters
were rapidly washed with an additional 2 × 2 ml of buffer.
Nonspecific binding of toxin was determined in the presence of 2 µM LqhIT2 and yielded 20-30% of total
binding for 125I-AaHIT or 30-40% for
125I-LhqIT2. The curves were fit to the data
points by the empirical Hill equation (for Bj-xtrIT) and by nonlinear
regression (for LqhIT2). The best fit for
LqhIT2 was obtained using the two-site model
(p = 0.997 by LIGAND).
Electrophysiological Experiments
Recordings in current-clamp and voltage-clamp conditions were
performed on cockroach isolated giant axons using the double oil-gap
single fiber technique (33). Axon isolation and the recording technique
were previously described in detail (34). The isolated axon was
superfused by physiological saline (buffered to pH 7.2 with 1 mM HEPES) containing 210 mM NaCl, 3.1 mM KCl, 5.2 mM MgCl2, and 5.4 mM CaCl2. Potassium currents were blocked in
voltage-clamp conditions by superfusion with 0.5 mM
3,4-diaminopyridine (Sigma), and sodium currents with 0.5 µM tetrodotoxin (Sigma). Lyophilized recombinant and
native Bj-xtrIT toxins were dissolved in normal saline to a
concentration of 1 µM in the presence of 0.25 mg/ml
bovine serum albumin. Fibers, providing action potentials of at least
95 mV in amplitude, were used in current-clamp conditions. Action
potentials were evoked every 5 s by passing short (0.5 ms) 10-nA pulses.
Circular Dichroism Spectroscopy
Toxins were dissolved in 20 mM phosphate buffer (pH
7). Spectra were measured at 20 °C in 0.05-mm path length cells from
260 to 178 nm with a JOBIN-YVON UV CD spectrophotometer (Mark VI). Calibration was performed with (+)-10-camphorsulfonic acid. A ratio of
2.2 was found between the positive CD band at 290.5 nm and the negative
band at 192.5 nm. Data were collected at 0.5-nm intervals with a scan
rate of 1 nm/min. CD spectra are reported as 
per amide. Protein
concentration was in the range of 0.5-1 mg/ml as determined on a
Beckman amino acid analyzer. Secondary structure content was determined
according to the method of Manavalan and Johnson (35).
Modeling
The initial AaHIT model was constructed on the basis of the
structural alignment with Bj-xtrIT (3). Using the Insight Homology program (MSI, Cambridge United Kingdom), residues in the Bj-xtrIT model
were replaced by homologous residues in AaHIT according to the
alignment. As shown in Fig. 1, residues 17 and 26 of Bj-xtrIT are
aligned with residues 17 and 21 of AaHIT as a result of an insertion of
five residues in Bj-xtrIT. To accommodate the difference in length of
the polypeptide chain around this region, a minor manual adjustment was
performed using the program O (37). Further manual adjustments were
applied to side chains of several residues in order to alleviate severe
steric clash with close neighbors. This initial model was subjected to
2000 steps of energy minimization using the program X-PLOR (38) and the
energy parameters described by Engh and Huber (39). During the
minimization, protein atoms were allowed to move under the constraints
of known protein stereochemistry as applied in X-PLOR. However, since
the underlying assumption in modeling AaHIT was its structural
resemblance to Bj-xtrIT, the C-
atoms of AaHIT were harmonically
restrained (38) to their positions in Bj-xtrIT. The stereochemistry of
the final energy-minimized model of AaHIT was examined by the program
PROCHECK (40) and found to be in agreement with the stereochemical
parameters of proteins in the Protein Data Bank, Brookhaven National
Laboratory (Upton, NY).
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RESULTS |
Purification and Cloning of Bj-xtrIT--
Crude venom from 35 B. judaicus scorpions was separated into 19 fractions by
ion-exchange chromatography. Fraction IV, which was 19.5% of the crude
venom and contained 1.5 mg of protein, produced an excitatory effect on
blowfly larvae. Further fractionation of this component by
gel-filtration chromatography yielded a major peak constituting 15.6%
of the crude venom and containing 1.2 mg of protein. Final purification
of the excitatory fraction was achieved by reverse-phase HPLC, yielding
three major components, of which the 36-min fraction (31%
acetonitrile) contained the toxic activity. The pure toxin (60 µg)
constituted ~0.8% of the total crude venom protein, and its mass was
determined to be 8455 Da by mass spectrometry. The toxicity
(ED50) of the purified toxin (Bj-xtrIT) to blowfly larvae
was 4.1 ng/100 mg of body weight (Table
I).
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Table I
Bioactivity of Bj-xtrIT variants and mutants
ED50 values were determined for blowfly larvae (100 ± 20 mg). In the mutant Bj-xtrIT. C-ter, the last seven amino acid
residues have been replaced by a single glycine.
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The sequence of the 30 N-terminal amino acids of Bj-xtrIT was
determined (Fig. 1), enabling synthesis
of a degenerate oligonucleotide (primer 1) spanning the first seven
amino acid residues. This primer was reacted with an oligo(dT) primer
(primer 2) using the cDNA library of B. judaicus (see
"Experimental Procedures"), resulting in a PCR product encoding
most of Bj-xtrIT cDNA. Primer 3, designed according to the
3'-region of the PCR product, was reacted with the KS primer and the
cDNA library as a template to generate the full-length cDNA
encoding Bj-xtrIT. Two distinct cDNA sequences encoding toxins that
vary at position 38 of the mature polypeptide (Bj-xtrIT.38K and
Bj-xtrIT.38E) were identified in a multiple number of experiments. It
is noteworthy that, in previous experiments (25), four distinct genes
encoding the excitatory toxin of Leiurus quinquestriatus
hebraeus have been isolated. These results, together with the
report of Bougis et al. (18), indicate polymorphism among
the genes encoding excitatory toxins (Fig. 1).

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Fig. 1.
Comparison among scorpion excitatory
neurotoxins. The Bj-xtrIT sequences have been scanned against the
data base, and all sequences with significant relatedness are included.
Alignment is with the sequence of Bj-xtrIT-a (Bj-xtrIT.38E, EMBL
accession number AJ012312). Bj-xtrIT-b is a variant containing Lys at
position 38 (Bj-xtrIT.38K, EMBL accession number AJ012313). The AaHIT
variants (EMBL accession numbers as follows: AaHIT-a, M27705; AaHIT-b,
M27706; AaHIT-c, M27707; and AaHIT-d, X58376) are from
Androctonus australis Hector (18, 21, 36).
LqqIT1 is from Leiurus quinquestriatus
quinquestriatus (6). Lqh-xtrIT (formerly termed
LqhIT1) variants are from L. quinquestriatus
hebraeus (25). Identical residues are designated by
dots. Dashes indicate gaps in the aligned
sequences.
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Expression of Bj-xtrIT and Lqh-xtrIT Toxins--
An efficient
bacterial expression system, employed previously to express an
-toxin (26) and a depressant toxin (27), was utilized in an attempt
to produce an active recombinant excitatory toxin. Lqh-xtrIT-a cDNA
(Fig. 1), encoding the excitatory toxin of L. quinquestriatus
hebraeus (25), and Bj-xtrIT-a and Bj-xtrIT-b cDNAs (Fig. 1),
encoding the aforementioned variants of B. judaicus excitatory toxin, were engineered into a pET-11cK expression vector. Following isopropyl-
-D-thiogalactopyranoside induction,
the toxins, accumulating within E. coli inclusion bodies,
were subjected to a denaturation/renaturation procedure (26, 27).
Folding conditions to obtain a functional toxin conformation were
achieved by dissolving the denatured protein under conditions of
various ammonium acetate concentrations (0-0.25 M),
temperatures (4-37 °C), pH values (5.0-9.0),
-mercaptoethanol
concentrations (0-0.5 M), and incubation periods (0-96
h). Both Bj-xtrIT variants eluted under similar conditions from the
C18 column, resulting in a major active peak. However, negligible activity (as determined by the toxicity to blowfly larvae
and the ability to displace the depressant toxin LqhIT2 from its receptor-binding site on cockroach neural membranes) could be
detected (data not shown) among a large number of Lqh-xtrIT isoforms.
Rabbit anti-Lqh-xtrIT antibodies recognized Bj-xtrIT on Western blots,
indicating common immunoreactive epitopes (data not shown). The overall
yield of the recombinant Bj-xtrIT variants was 2.5 mg purified from 1 liter of an E. coli culture.
Bioactivity of Bj-xtrIT Natural Variants--
The biological
activity of the native (Bj-xtrIT-n) and recombinant (Bj-xtrIT.38E and
Bj-xtrIT.38K) toxins was assessed by toxicity, binding, and
electrophysiological assays. Injection of blowfly larvae provided
ED50 values of 4.1, 8.6, and 8.6 ng/100 mg of body weight
(Table I) for the three toxins, respectively. Injection of Bj-xtrIT.38K
into S. littoralis lepidopteran larvae generated paralytic
symptoms, with a PU50 value of 1.4 µg/100 mg of larva.
Binding studies were performed using AaHIT and the depressant toxin
LqhIT2 as competitive ligands. Bj-xtrIT competed with
radioiodinated AaHIT for binding at an apparent affinity of ~1
nM (Fig. 2A),
which is similar to the value obtained with AaHIT itself (11, 41).
Displacement of radioiodinated AaHIT from its receptor-binding site on
the cockroach membrane preparation provided Ki
values of 1.0 ± 0.6 and 1.4 ± 0.2 nM for Bj-xtrIT.38K and Bj-xtrIT.38E, respectively (Fig. 2B). To
further establish the similarity in receptor recognition between
Bj-xtrIT and AaHIT, competition curves were generated using
125I-LqhIT2 (Fig. 2B). The
depressant toxin LqhIT2 was shown to bind to two
non-interacting binding sites on insect neural membranes, a high
affinity and low capacity receptor site and a low affinity site with
high capacity. Only the high affinity site, shown to be located on
insect sodium channels, is a target for binding competition with the
excitatory toxin AaHIT (31, 41). Bj-xtrIT competed for the high
affinity site of LqhIT2 with no influence on its binding to
the low affinity site (Fig. 2B). Thus, Bj-xtrIT and AaHIT
bind to the same receptor site on the cockroach sodium channel.

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Fig. 2.
Competition of Bj-xtrIT with
125I-AaHIT and 125I-LqhIT2 for
binding to cockroach neural membranes. Insect neural membranes (13 µg of protein/ml) were incubated with 0.18 nM
125I-AaHIT in the presence of increasing concentrations of
Bj-xtrIT.38K ( ) or Bj-xtrIT.38E ( ) (A) and 0.16 nM 125I-LqhIT2 in the presence of
increasing concentrations of unlabeled recombinant LqhIT2
( ) or Bj-xtrIT.38E ( ) (B). The amount of specifically
bound 125I-toxin is expressed as a percentage of the
maximum specific binding. Note that Bj-xtrIT competed only for the high
affinity site of LqhIT2. Equilibrium binding constants
(mean ± S.E.) are Ki(Bj-xtrIT.38K) = 1.02 ± 0.20 nM,
Ki(Bj-xtrIT.38E) = 1.40 ± 0.67 nM (for competition with AaHIT (A)), and
Ki(Bj-xtrIT.38E) = 2.67 ± 1.23 nM (for competition with LqhIT2
(B)). The binding constants for the high affinity site of
LqhIT2 are Kd1 = 1.7 ± 0.8 nM and Bmax1 = 3.7 ± 1.8 pmol/mg of protein, and those for the low affinity site are
Kd2 = 397 ± 238 nM and
Bmax2 = 33 ± 19.8 pmol/mg of protein.
Binding of Bj-xtrIT. C-ter was assessed in a similar procedure using
9.2 mg of protein/ml of insect neural membranes incubated with 0.12 nM 125I-LqhIT2 in the presence of
increasing concentrations of Bj-xtrIT.38E ( ) or Bj-xtrIT. C-ter
( ) (C). Note that even after the large decrease in
apparent affinity, Bj-xtrIT. C-ter still competed only for the high
affinity site as unmodified Bj-xtrIT.38E. The IC50 values
determined for Bj-xtrIT. C-ter and Bj-xtrIT.38E are 424 ± 44 and 3.5 ± 1.2 nM, respectively. The calculated
equilibrium inhibition constants (Ki) for
Bj-xtrIT. C-ter and Bj-xtrIT.38E are 395 ± 42 and 3.3 ± 1.1 nM, respectively.
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In current-clamp experiments, Bj-xtrIT depolarized the resting membrane
potential and generated spontaneous action potentials. Application of
the toxin (1 µM) caused an 8-15-mV depolarization in <6
min (Fig. 3A); this effect
could be blocked by 0.5 µM tetrodotoxin (data not shown).
Depolarization was accompanied by persistent repetitive firing of
action potentials (starting at
52 mV and observed 5-15 min after
toxin application) even in the absence of electrical stimulation (Fig.
3B). Artificial repolarization, by passing a constant
hyperpolarizing current, did not prevent a later depolarization
accompanied by repetitive activity (Fig. 3C). The repetitive
activity induced by the toxin was typified by action potentials with
slightly decreased amplitude (70-80 mV rather than 95-100 mV in
control action potentials). The frequency during the action potential
bursts varied between 80 and 200 Hz, and no "plateau potential" was
recorded. The native (Bj-xtrIT-n) and recombinant (Bj-xtrIT.38E) toxins
were equally potent on the axonal preparation.

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Fig. 3.
Effect of Bj-xtrIT on electrical activity and
sodium currents of a cockroach isolated axon. A: two
superimposed recordings of resting potentials and evoked action
potentials under control conditions. A 0.5-ms current pulse of 10 nA
evoked only one action potential of 95 mV in amplitude and 0.5 ms in
duration (~20 mV above the resting potential). Six minutes after
Bj-xtrIT application, the rest ing potential decreased by 6 mV; the action potential reached its
threshold more rapidly; and a second spontaneous action potential was
measured 6.5 ms later, giving an instantaneous frequency of 154 Hz.
B and C: spontaneous activity of the action
potential after 7 and 10 min, respectively, after Bj-xtrIT application.
Action potential frequencies higher than 200 Hz were obtained, and no
"plateau potentials" could be measured. After a 10-mV artificial
hyperpolarization generated by a constant current, a slow spontaneous
depolarization reached the threshold for a sustained repetitive firing
of short action potential (C). D: families of
Na+ currents recorded in the presence of 0.5 mM
3,4-diaminopyridine during 5-ms voltage pulses. Panel a,
control; panel b, after 6 min in the presence of Bj-xtrIT.
Note that after Bj-xtrIT application, Na+ currents
developed at more negative potential values than in the control. The
horizontal lines indicate the zero current level.
E: voltage dependence of the sodium conductance (expressed
as
gNa+/gNa+.max.control)
calculated from current families as illustrated in D. Note
the shift of the activation curve by 12.5 mV toward more negative
potentials after addition of Bj-xtrIT. F: panel
a, Na+ current during a 300-ms voltage pulse to
Em = 30 mV showing a sustained current, which did
not inactivate during the pulse, and the existence of an inward holding
current before and after the pulse (dotted line indicates
zero current level); panel b, superimposed recordings before
and after application of 0.5 µM tetrodotoxin, which
suppressed the Na+ peak and maintained the holding
current.
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In voltage-clamp experiments, Bj-xtrIT changed neither the amplitude
nor the kinetics of the K+ current recorded in the presence
of 0.5 µM tetrodotoxin (data not shown). During the first
5-8 min after toxin application, Na+ currents recorded at
voltage pulses of
20 or
10 mV (from a holding potential of
60 mV)
increased slightly, and an inward component developed. At
50 or
40
mV, inward Na+ currents could be measured, whereas no such
currents existed under control conditions. The maximum inward
Na+ current was obtained at
30 mV (Fig. 3, D
(panel b) and F). Furthermore, a small inward
constant current developed progressively up to 80-100 nA at holding
potential during the first 5-15 min (Fig. 3F, panel
b). These data imply that Bj-xtrIT-n as well as Bj-xtrIT.38E open
Na+ channels at very negative potential values. As
illustrated in Fig. 3D (panels a and
b), it was possible to calculate the relative sodium
conductance (gNa+ = INa+/(Em
ENa+)) under control conditions and in
the presence of toxin (Fig. 3E). Both native and recombinant
toxins caused a shift in the Na+ activation voltage curve
to more negative values by 12-15 mV, and the overall sodium
conductance of the membrane surface increased, especially between
60
and
20 mV. A decrease in this conductance could be observed 15-20
min after toxin application, when the holding current reached
80 nA.
At this stage, it became evident that the axoplasmic concentration of
sodium ions had increased, resulting in a decrease in the equilibrium
potential for Na+ ions
(ENa+). After the peak Na+
current, a maintained inward current (without inactivation) developed at
30 mV to a higher extent than at positive potential values (Fig.
3D (panel b) and F). This finding
indicates that the inactivation process slows down less drastically
than with
-type scorpion toxins, which favor plateau potentials. In
summary, Bj-xtrIT causes repetitive activity of short action
potentials, which explains the contractive response manifested by
excitatory toxins.
Modification of the C Terminus of Bj-xtrIT--
Comparison of the
three-dimensional structure of recombinant Bj-xtrIT (3) with the known
structures of other toxins (22) revealed a striking similarity of a
large portion of the molecule including the
-helix (residues 24-34)
(Fig. 1), the
-sheet, and the configuration of three of the four
disulfide bonds. However, an additional
-helix (residues 63-69)
(Fig. 1) and a long C-terminal tail are unique to Bj-xtrIT (3) and
therefore have become primary targets for modification to determine
their involvement in the characteristic neuropharmacology of scorpion
excitatory toxins. We used the E. coli expression and
in vitro reconstitution system to produce and analyze a
Bj-xtrIT.38E mutant (Bj-xtrIT.
C-ter) in which the entire C-terminal
tail following Cys69, namely,
Asp70-Val71-Gln72-Ile73-Ile74-Pro75-Ser76,
was replaced with a single Gly residue (primer 7; see "Experimental Procedures"). This deletion did not affect the folding capacity of
the mutant toxin as was suggested by the HPLC profile of the product
obtained under similar folding conditions used for renaturation of
Bj-xtrIT.38E. The HPLC-purified mutant polypeptide was analyzed for its
bioactivity and secondary structure content (Table
II). No toxicity could be detected upon
injection into blowfly larvae of quantities up to 16 µg/100 mg of
larva (Table I). In binding assays, Bj-xtrIT.
C-ter displaced
LqhIT2 from the high affinity binding site shared by
depressant and excitatory toxins (31) at concentrations above 400 nM compared with 1.4-3.0 nM unmodified toxin
(Fig. 2). The CD spectrum of the tailless mutant resembled the spectrum
of Bj-xtrIT.38K (Fig. 4), and its
secondary structure content was practically similar to those of the two
unmodified variants, Bj-xtrIT.38K and Bj-xtrIT.38E (Table II),
suggesting similarity in overall structure. To elucidate specific
residues at the C terminus that could participate in bioactivity, the C terminus was analyzed by sequential deletions. Deletion of the ultimate
Ser alone or together with the penultimate Pro revealed mutant toxins
with ~5-fold decreased activity. Any truncation beyond the last two
residues resulted in an inactive mutant toxin (Table I).
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Table II
Comparison of secondary structure content among scorpion neurotoxins
CD spectrum analysis was carried out according to the singular value
decomposition method (35).
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Fig. 4.
Circular dichroism spectra of scorpion
excitatory toxins. - - -, Bj-xtrIT.38E; ------, Bj-xtrIT.38K;
--- - ---, Bj-xtrIT. C-ter; ····, AaHIT.
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DISCUSSION |
In this study, we provide insight into the putative bioactive
surface of scorpion excitatory neurotoxins displaying anti-insect selectivity. The atypical features of Bj-xtrIT, a unique representative of this pharmacological group from the Israeli black scorpion (B. judaicus), enabled high yield expression, proper in
vitro folding, and crystallization (3). With these prerequisites, a genetic modification approach has become available for the
elucidation of the bioactive site of these important effectors of
insect sodium channels.
Uniqueness of Bj-xtrIT--
Bj-xtrIT is the longest scorpion
neurotoxin affecting sodium channels that has been described thus far
(76 residues; molecular mass of 8455 Da). The added length of Bj-xtrIT
is manifested by an additional pentapeptide (residues 21-25) preceding
the
-helical motif (residues 24-34) in the toxin core (3) and in
one additional C-terminal residue (Fig. 1). Its deduced amino acid
sequence differs substantially from all highly conserved sequences of
known excitatory toxins (49% similarity) (Fig. 1). Whereas all
excitatory toxins contain two prolines and four glycines, Bj-xtrIT is
relatively rich in these amino acids (five and seven, respectively),
which may suggest higher conformational flexibility. Despite the
differences, Bj-xtrIT displays, with high fidelity, the characteristic
features of excitatory toxins, e.g. poisoning symptoms,
displacement from the receptor-binding site (Fig. 2), sodium channel
modification (Fig. 3), CD spectrum (Fig. 4), and immunoreactivity with
antibodies raised against Lqh-xtrIT (data not shown). Although it has
been shown in quantitative radio immunoassays performed with scorpion
-toxins that cross-reactivity demanded at least 75% sequence homology (44), Bj-xtrIT and the other excitatory toxins, varying substantially in sequence, seem to share rather conserved immunogenic epitopes. The molecular mass, ED50 values for blowfly
larvae, amino acid composition, and overall length of Bj-xtrIT differ substantially from those determined for a B. judaicus
excitatory toxin, BjIT1, detected and partly characterized
by Lester et al. (9); yet it cannot be ruled out that
BjIT1 and Bj-xtrIT are identical.
The uniqueness of Bj-xtrIT, compared with other excitatory toxins,
seems to be of great importance and provides the following advantages:
1) amenability to genetic dissection and structural analysis due to the
easy production and ability to fold in vitro; 2) distinction
between conserved versus variable regions, which enables a
mutagenic approach to determine functionally important residues; and 3)
an applied potential arising from its prominent anti-lepidopteran
toxicity (PU50 = 1.4 µg/100 mg of body weight) compared
with the effect (PU50 = 2.5 µg/100 mg of body weight) induced by the classical excitatory toxin AaHIT (45). It is possible
that the metabolic fate of Bj-xtrIT in the hemolymph differs from that
of AaHIT with respect to accessibility barriers and to degradation
processes. These features place Bj-xtrIT as a preferred candidate among
scorpion toxins for improving the insecticidal efficacy of baculoviruses.
Further evidence for the structural uniqueness of Bj-xtrIT is provided
by its three-dimensional features (3). Unlike other known long toxin
structures (Ref. 22 and reviewed in Ref. 2), Bj-xtrIT consists of two
structural entities: a major entity (residues 1-59) encompassing the
/
-core found in all other toxins and a minor entity (residues
60-76) comprising the additional
-helix and the seven-residue
C-terminal tail. The pronounced ability of mutant Bj-xtrIT.
C-ter to
fold into a major isoform with secondary structure content (Fig. 4 and
Table II), similar to that of the unmodified toxin variants, suggests
that the C-terminal tail is devoid of typical secondary structure and
has no effect on the folding capability of the entire molecule.
Although the additional
-helix observed in Bj-xtrIT (residues
63-69) (3) is contiguous to the bioactive C-terminal tail, its actual
role remains to be determined.
Interestingly, the
-helix in the conserved
/
-core (residues
24-34) is preceded by an additional four-residue helical motif (residues 19-22), which is part of the five-residue insertion unique
to Bj-xtrIT (Fig. 1). The existence of additional helical elements
explains the relatively high content of
-helical secondary structure
observed in the singular value decomposition analysis of excitatory
toxins (Table II).
Putative Toxic Surface of Scorpion Excitatory Toxins--
The
ability of Bj-xtrIT to displace AaHIT from its receptor-binding site,
despite their moderate sequence homology, implies a common bioactive
surface. The effect of the modifications introduced at the C-terminal
tail of Bj-xtrIT on the bioactivity strongly suggests that the putative
active surface is located in the vicinity of the C-terminal region.
Comparison of the three-dimensional structure of Bj-xtrIT with the
AaHIT three-dimensional model, with particular emphasis on the
C-terminal module, could therefore highlight conserved and hence
functionally significant residues in this region. Taking the conserved
bioactive Ile73 and Ile74 as two common
reference points, another 11 residues comprising Lys1,
Lys2, Asn28, Thr32,
Lys33, Tyr36, Ala37,
Asp54, Asp55, Lys56, and
Asp70 (Bj-xtrIT sequence) (Fig.
5), centered around the aforementioned isoleucines, form a continuous patch in both Bj-xtrIT and AaHIT (Fig.
5). This patch might therefore be considered as part of the surface
presented to the ion channel in both toxins. The bioactive role of some
residues belonging to this molecular surface has also been provided by
chemical modifications introduced to AaHIT (36). Acetylation of
residues that belong to the putative bioactive surface, e.g.
Lys28 and Lys51 (Lys33 and
Lys56 in the Bj-xtrIT sequence), substantially decreased
the activity without perturbation of the CD spectrum of the modified
polypeptides. Conversely, acetylation of Lys34,
carbethoxylation of the imidazolyl ring of His30, and
modification of Arg60 by 1,2-cyclohexanedione had no effect
on the toxicity of AaHIT (36). These positions in Bj-xtrIT are occupied
by Ser39, Tyr35, and Thr65,
respectively, and in addition to the lack of conservation, they are
either buried or remote from the putative bioactive surface.

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Fig. 5.
Putative interaction surface of scorpion
excitatory toxins. A, a ribbon diagram of Bj-xtrIT
structure (3) with the side chain residues indicated in B.
B and C, the putative bioactive surfaces of
Bj-xtrIT and AaHIT, respectively, highlighted by the ellipsoid. The
model of AaHIT (C) was constructed according to the solved
structure (3) of Bj-xtrIT (B). Colors are according to the
side chain character: blue, positive; red,
negative; magenta, aromatic; green, hydrophobic;
yellow, polar; white, glycines. Note the position
of the essential Ile73, the last residue observed in the
x-ray structure (3), within the putative interaction surface. Labeled
in black are two nonconserved residues, i.e.
Glu38 and Glu53 of Bj-xtrIT (Asp33
and Asn48 in AaHIT), that may be shielded by the last three
C-terminal residues not detected in the x-ray structure. A
was produced in Setor (46) and B and C in Grasp
(47).
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On the basis of the structure and the putative active surface, it is
possible to interpret the similar activity of Bj-xtrIT.38K and
Bj-xtrIT.38E variants. Although the last three residues of the
C-terminal tail could not be observed in the crystal structure (3), it
is likely that they cover residue 38 and thus reduce the effect of its
charge on the bioactive surface (Fig. 5). Other residues that are
conserved in Bj-xtrIT and AaHIT are located farther away from the C
terminus and are less exposed to the solvent. As suggested by the
crystal structure of Bj-xtrIT, it is clear that Asp70 is
somewhat buried in the structure, and its conservation might be due to
its structural role in stabilizing the C-terminal module through
hydrogen bonds with Asn3 and Tyr48 and a salt
bridge with Lys66 (3). The residues in the conserved
surface lie, as was proposed by Fontecilla-Camps (22), on loops outside
the
/
-core, whereas the conserved residues not included in the
bioactive surface may play a structural role.
The putative toxic surface of Bj-xtrIT (and, most likely, of all other
excitatory toxins) is the first structural element described thus far
that is able to recognize exclusively sodium channels of insects. As
such, it becomes an important tool for the molecular characterization
of these channels and may be of practical value in the design of novel
insect-pest control agents.