Solutionstructureof IsTX
A malescorpiontoxin from
Opisthacanthus madagascariensis
(Ischnuridae)
Nahoko Yamaji
1
, Li Dai
1
, Kenji Sugase
1
, Marta Andriantsiferana
2
, Terumi Nakajima
1
and Takashi Iwashita
1
1
Suntory Institute for Bioorganic Research, Mishima-Gun, Osaka, Japan;
2
Faculty of Science, University of Antananarivo,
Madagascar
The novel sex-specific potassium channel inhibitor IsTX, a
41-residue peptide, was i solated from the venom of male
Opisthacanthus madagascariensis.Two-dimensionalNMR
techniques revealed t hat the structureofIsTX contains a
cysteine-stabilized a/b-fold. IsTX is classified, based on its
sequential and structural similarity, in the scorpion short
toxin family a-KTx6. The a-KTx6 family contains a single
a-he lix and t wo b-strands connected by four disulfide brid-
ges and binds to voltage-gated K
+
channels and apamin-
sensitive Ca
2+
-activated K
+
channels. The three-dimen-
sional structureofIsTX is similar to that of Heterometrus
spinifer toxin (HsTX1). HsTX1 blocks the Kv1.3 channel a t
picomolar concentrations, whereas IsTX has much lower
affinities (10 000-fold). To investigate the structure–activity
relationship, the geometry of sidechains and electrostatic
surface potential maps were compared with HsTX1. As a
result of the comparison of the primary structures, Lys27
of IsTX was conserved at the same position in HsTX1. The
analogous Lys23 of HsTX1, the most critical residue for
binding to potassium channels, binds to the channel pore.
However, IsTX has fewer basic residues to interact with
acidic channel surfaces than HsTX1. MALDI-TOF MS
analysis clear ly indicated t hat IsTX was found in male
scorpion venom, but not in female. This is the first report
that scorpion venom contains sex-specific compounds.
Keywords: cysteine-stabilized a/b-fold; NMR; potass ium
channels; s corpion toxin; sex-specific toxin.
The venom o f Opisthacanthus madagascariensis scorpions
from the I schnuridae fam ily collec ted in Isalo (Madagascar)
was investigated by MALDI-TOF MS. Many highly toxic
venom components from the Buthidae family of scorpions
have been reported [1–4]. However, there have been few
reports on the venom of scorpions from the Ischnu ridae
family, due to its lower toxicity [5,6]. The resultant mass
spectrum differed among males and females. The bioassay
of each HPLC fraction of venom revealed that a number of
peptides showed either weak or no toxic effects with the
exception of IsTX. Further more, male venom was shown to
contain IsTX, whereas female venom did not. This is the
first report of the sex-related scorpion venom component,
IsTX [7,8].
IsTX is a 41-residue toxin and a member of the a-K
scorpion toxin family that functions to block K
+
channels.
The a-K scorpion toxins, comprising 23–43 amino acids,
have a/b-fold structures stabilized with three or four
disulfide bridges. These toxins have b een divided into 12
subfamilies (a-KTx1–12) based on the alignments of
cysteine and highly conserved residues [9,10]. IsTX belongs
to the four disulfide-bridged a-KTx6 subfamily composed
of maurotoxin (MTX) [11–13], HsTX1 [14–16] and Pand-
inus imperator toxin 1 (Pi1) [ 17](Fig. 1). These a-KTx6
toxins selectively act on voltage-gated and Ca
2+
-dependent
K
+
channels [18–20]. All the members of a-KTx6 subfamily
contain four disulfide bridges. HsTX1 and Pi1 show the
same disulfide bridge pattern (C1-C5, C2-C6, C3-C7 and
C4-C8) [14,17], however, only MTX shows a nonstandard
pattern (C1-C5, C2-C6, C3-C4 and C7-C8) [11]. Even
though MTX has a different disulfide bridge pattern from
HsTX1 and Pi1, it possesses very similar global fold with
other toxins.
The structureofIsTX is composed ofa single a-helix and
triple b-strand connected by four cysteine-disulfide bridges.
The amino acid sequence ofIsTX is 50% similar to HsTX1
and MTX and 43% similar to Pi1. These findings indicate
that IsTX is highly similar to the a-KTx6 s ubfamily.
Although IsTX has a highly sequential and structural
similarity to HsTX1, the activity ofIsTX toward Kv1.3 is
10 000 times lower than that o f HsTX1. The reason for this
difference may be elucidated by c omparing the structures of
IsTX with HsTX1.
Herein, the structure in solution and the interaction
between IsTX and the voltage-gated K
+
channel has been
investigated. The study is comprised of (a) characterization
Correspondence to N. Yamaji, Suntory Institute for Bioorganic
Research, 1-1-1 Wakayamadai, Shimamoto-Cho, Mishima-Gun,
Osaka 618–8503, Japan. Fax: +81 75 962 2115;
Tel.: +81 75 962 6142, E-mail: yamaji@sunbor.or.jp
Abbreviations: DQF-COSY, double-quantum-filtered COSY; HSQC,
heteronuclear single quantum coherence; TsTX-Ka, Tityus serrulatus
toxin; HsTX1, Heterometrus spinifer toxin 1; IsTX, Ischnuridae toxin;
MTX, maurotoxin; Pi1, Pandinus imperator toxin 1; RMSD, root
mean square deviation; Kv, voltage-gated potassium channel; ShB,
shaker K
+
channel; SKCa, small-conductance Ca
2+
-activated K
+
channel.
Database: The coordinate for the 20 lo west co nformers IsTX has been
submitted to the RCSB Protein Data Bank database under the
accession number 1WMT.
(Received 14 June 200 4, revised 26 July 2004, accepted 4 August 20 04)
Eur. J. Biochem. 271, 3855–3864 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04322.x
of IsTX, (b) determination of the three-dimensional
structure ofIsTX by NMR spectroscopy, (c) calculation
of the surface potential map of IsTX. A comparison of the
data with HsTX1 revealed the proposed functional sites for
scorpion toxin binding to potassium channels.
Materials and methods
Scorpions (O. madagascariensis) w ere collected in Isalo
(Madagascar) and maintained in our laboratory. The crude
venom was collected by electrical stimulation of the
scorpion telson and extracted using water. The supernatant
was stored in the freezer under )70 °C.
MALDI-TOF mass spectrometry
The matrix for the MALDI-TOF MS analysis was
prepared as follows: a-cyanohydroxycinnamic acid was
dissolved in 1 : 1 (v/v) acetonitrile/H
2
O supplemented with
0.1% (v/v) trifluoroacetic acid in order to obtain a saturated
solution. Samples m ixed with the matrix solution were
placed on a sample plate and allowed to dry for 5 min. MS
analysis was performed on a Voyager Elite MALDI-TOF
mass spectrometer (Applied B iosystems; Fragmingham,
MA, USA) in positive mode. Crud e venom was recorded in
linear mode.
Peptide purification
The venom supernatant was loaded directly onto to an
RP-C
18
HPLC column (Tosoh, TSK gel, O DS 120T,
250 · 4.6 mm, 5 lm particle size). The temperature was
controlled at 40 °C. The flow rate was set at 1 mLÆmin
)1
and a linear gradient f rom 0–64% (v/v) acetonitrile/H
2
O
with 0.1% (v/v) trifluoroacetic acid was run for 60 min. The
detector absorbance was set at 220 nm.
Crude fraction 17, which contain ed the peptide IsTX, was
collected and lyophilized. It was further purified by RP-C
18
HPLC (Tosoh, TSK gel, ODS 120T, 250 · 4.6 mm, 5 lm
particle size) using a 60 min linear gradient from 0–32%
(v/v) a cetonitrile/H
2
O w ith 0 .1% (v/v) trifluoroacetic acid at
a flow rate of 1 mLÆmin
)1
. The analysis was monitored at
220 nm and the fraction containing the p urified peptide w as
collected and lyophilized.
Primary structure analysis
About 20 pmol o f purified peptide was reduced and
alkylated using tributyl phosphine and 4-vinyl-pyridine
according to the standard protocol [21]. The pyridylethyl-
ated product was desalted by C
18
HPLC (Tosoh, TSK gel,
ODS 80T, 0.25 · 150 mm) and detected using a MALDI-
TOF MS detector. A 60 min linear gradient from 0–64%
(v/v) acetonitrile/H
2
O with 0.1% (v/v) trifluoroacetic acid
at a flow rate of 1 mLÆmin
)1
was run. Half of the
pyridylethylated peptide was app lied to an automated
Edman degradation gas-phase sequencer (PPSQ-10; Shim-
adzu, Kyoto, Japan) and the remaining half was digested
using V8 proteases at 37 °C for 12 h. The digestion
products were checked with MALDI MS and separated
by RP-C
18
HPLC with the same conditions as described
above. Four fractions were collected and analyzed by a
combination of M S ladder s equencing and automated
Edman degradation sequencing.
cDNA cloning
The total RNA from one scorpion venom gland w as
prepared using TRIzol Regent (Invitrogen) according to the
standard protocol. The cDNA sequences encoding the IsPT
precursor were determined using 3¢/5¢ RACE experiments.
The specific primers designed according to the known
amino acid sequence were: 3¢ RACE: 3SP1, 5¢-GTXCA
YACXAAYATHCCXTG-3¢;3SP2,5¢-AAYATHCCXT
GYMGXGGXAC-3¢;3SP3,5¢-GAYTGYTAYGARC
CXTGYGA-3¢.5¢ RACE: 5 SP1, 5¢-GACAGAAATTA
CATTTTCGTAGCGT; 5SP2, 5¢-CCATGGACAATTG
TTGTAGCAGTT-3¢;5SP3,5¢-TGCCTATTCATACAT
TTTGCCCT-3¢. The PCR products were cloned into the
pCR2.1 vector and the DNA sequence was analyzed using
an ABI Prism 310 automated sequen cer (Perkin-E lmer, CA,
USA).
Fig. 1. Sequence alignment o f I sT X with selected toxins fro m a-KTx6 s ubfamily and d isulfide bridge patterns. The amino acid sequences of HsTX1
(H. spinnifer), Pi1 (P. imperator)andMTX(S. maurus) were aligned according to the eight half-cysteine resid ues. Gaps (–) have been int rodu ced to
maximize the alignment. *, C-term inal carboxyamidated e xtremity. The t wo disulfide bridge patterns observed in HsTX1/Pi1 and M TX are shown
below the sequences.
3856 N. Yamaji et al. (Eur. J. Biochem. 271) Ó FEBS 2004
NMR experiments and structural calculations
Synthetic IsTX was purchased from Peptide Institute, Inc.
(Osaka, J apan), and identified by HPLC and activity
measurements.
For NMR experiments, synthetic IsTX was dissolved in
0.5 mL of H
2
O/D
2
O [90/10 (v/v)] containing 50 l
M
NaN
2
,
the sample concentration was 4.7 m
M
, and the pH was 3.91
in order to detect amide signals.
All NMR spectra were recorded on a Bruker DMX-750
spectrometer (Bruker Biospin, Germany). The temperature
was s et to 298 K. Chemical shifts were r eferenced to internal
3-(trimethylsilyl)[2,2,3,3-
2
H
4
] propionate (TSP). 2D DQF-
COSY [22], NOESY [23,24], TOCSY [25] and HSQC
experiments were c onducted. The NOESY spectra were
acquired with 50, 100 and 200 ms mixing times and water
suppression was achieved using the WATERGATE
sequence [26]. The TOCSY spectrum was recorded using
a MLEV-17 pulse sequence with a 71 ms mixing time. The
2D spectra were recorded using time-proportional phase
Incrementation (TPPI) for quadrature detection in the F1
dimension. Spectra were recorded at 512 points for t1 and
2048 points for t2. A
1
H,
13
C HSQC spe ctrum was recorded
in natural abundan ce using the e cho-antiecho scheme
[27,28]. The time domain data w ere p rocessed u sing
XWIN
-
NMR
2.5 program (Bruker Biospin).
Proton signal assignments were achieved using the stand-
ard strategy described by Wu
¨
thrich [29] with the graphical
software
ANSIG
.3.3 [30]. The DQF-COSY and Clean-
TOCSY spectra gave the spin system fingerprint of the
peptide. The s pin systems were then sequentially connected
using the NOESY spectra. NOE volumes were converted
into distance restraints classified as s trong (1.8–2.7 A
˚
),
medium (1.8–3.3 A
˚
), weak (1.8–5.0 A
˚
), and v ery weak
(1.8–6.0 A
˚
).
Backbone amide proton temperature coefficients were
calculated from the NOESY spectra recorded at four
different temperatures from 288–318 K [31,32]. Hydrogen-
deuterium exchange experiments were carried out by
lyophilizing the H
2
O sample, redissoloved in 250 lLof
99.96% (v/v) D
2
O, and running 3 h TOCSY experiments.
Slowly exchanging amide protons were interpreted as
hydrogen-bond donors. Distance restraints for the identified
hydrogen bonds were included in the subsequent structure
calculations. The
3
J
NH-Ha
coupling constants were estima-
ted from the DQF-COSY spectra using either the
DECO
program (Bruker Biospin) or directly measured from a 1D
spectrum. The dihedral angles estimated from the
3
J
NH-Ha
values were used as / angle constraints within the range of
)90° and )40° for
3
J
NH-Ha
< 5.5 Hz, between )160° and
)80° for
3
J
NH-Ha
> 8 Hz [33].
Structural calculations were performed on IsTX using
X
-
PLOR
-
NIH
2.0.5 [34–36] with 679 NOE-based distance
restraints, which contain 199 sequential, 302 medium range
and 178 long-range restraints. Starting structures were an
extended strand conformation, and were performed using
conjugate-gradient minimization. The disulfide bonds were
included as pseudo-NOE restraints. In the first stage, the
extended strand structures were subjected to 10 ps and 1000
steps of t orsion-angle molecular dynamics at 50 000 K. The
structures were then s ubjected t o a 15 ps and 1500 steps slow-
cooling t orsion angle molecular dynamics stage in which the
temperature was reduced from 50 000 K to 298 K over 250
steps. Finally, the structures were subjected to 200 steps of
conjugated-gradient minimization. The following scale fac-
tors were used during the three steps: van der Waals, 0.1, 1
and 1; NOE, 150, 150 and 75; dihedral restraints, 100, 200
and 400 and R amachandran restraints, 0.002, 1.0 and 1.0.
The initial runs for structure calculations were performed
without hydrogen bond restraints, and the obtained struc-
ture was examined. Fourteen protons from amide groups
were identified to form hydrogen bonds according to the
amide proton temperature coefficients and hydrogen-deu-
terium exchange experiments. If the hydrogen bond
restraint was in agreement with amide temperature coeffi-
cients (> )4.6 p.p.b.ÆK
)1
) and exchange in D
2
Odata
(signal present after 24 h of exchange at 298 K) and if an
oxygen atom was within 2.8 A
˚
of an amide proton, the
amide proton was identified a s the acceptor of the hydrogen
bond. These restraints were then used in the following stage
of structure calculations. The structures were checked for
violations of geometric a nd experimental restraints and
atom overlapping using
AQUA
3.2 and
PROCHECK
-
NMR
3.4
[37]. Finally, a set of 20 conformers was selected based on
the least distance and dihedral angle restraint violations
(deviations). The coordinate for the 20 lowest conformers of
IsTX has been deposited in the RCSB Protein Data Bank
(http://www.rcsb.org), accession code 1WMT.
Results
Purification and primary structure determination
Scorpion (O. madagascariensis) venom contained several
peptides with molecular masses between 4 and 5 kDa,
whichwereassumedtobeshort-chainneurotoxins(Fig.2).
However, when each C
18
HPLC fraction was tested for
toxicity to crickets, only fraction 17 showed an apparent
paralysis e ffect o n crickets (Fig. 3). This fraction was further
purified and two components were collected. One was a
peptide with an M
r
of 3147 and no toxicity effects, and the
other w as a peptide with an M
r
of 4819 that caused p aralysis
to crickets. This toxic peptide was isolated from a scorpion
collected in Isalo (Madagascar) and was named IsTX.
Due t o the limited amount o f natural peptide, the
primary structure was determined using several techniques
including MS/MS analysis, ladder sequencing, Edman
degradation s equencing, and subsequently confirmed b y
cDNA deduction.
The p yridylethylated peptide was sequenced using an
automated Edman degradation method and t he N-terminal
15 step was determined as shown in Fig. 4. The V8 digested
products of the pyridylethylated peptide were monitored b y
LC/MS. Four main fragments were detected in the LC/MS
spectra and further separated by RP-HPLC. The amino
acid sequences of the four fragments were determined using
Edman and ladder sequencing. The complete sequence of
IsTX was determined by combining the results obtained
above and further confirmed by cDNA sequencing.
Molecular biological analysis
The precursor ofIsTX was deduced from the c DNA
sequence. Based on the known amino acid sequences,
Ó FEBS 2004 SolutionstructureofIsTX (Eur. J. Biochem. 271) 3857
specific primers were designated as described above.
Molecular cloning of the cDNA from the venom gland
mRNA was performed using 5¢/3¢ RACE. The cDNA was
completed by overlapping two fragments amplified by 3¢
and 5 ¢ RACE. O ver 10 c lones were obtained with a
polyadenylation signal (AATAAA) in the 3¢ untranslated
region and a poly(A) tail at the 3¢ end. The open reading
frame ofIsTX encodes a precursor of 63 residues, contain-
ing a signal peptide of 21 amino acid residues and a m ature
peptide of 41 residues followed by an additional basic Arg
residue that was removed during carboxyl-processing. The
deduced mature peptide sequences were completely consis-
tent with results of amino acid sequencing (Fig. 5).
Chemical synthesis
The peptide was synthesized by Peptide Institute, Inc.
(Osaka, J apan) using an automated s olid-phase peptide
synthesizer 433-A (Applied Biosystems) based on the Fmoc-
strategy. Disulfide bridges were oxidized by exposure to air.
The identity of synthetic and natural peptides was con-
firmed by MS analysis, coinjection experiments on
RP-HPLC and capillary electrophoresis (CE).
Structure quality
NOE correlations of 50 ms 2D NOESY spectrum were used
for sequential assignments. The Ha protons of Met29 and
Asn38 were not observed due to the o verlap with the water
resonance signal. The amide protons of Cys23 and Arg25
were sh ifted upfield relative to their positions in random coil
structures, and the Ala24 amide proton w as shifted
downfield. T his observation suggests that t hese protons
were in close proximity to an aromatic ring.
The structureofIsTX was calculated using 679 NOE
distance constraints. The 20 accepted structures, which had
no NOE violations (£ 0.4 A
˚
)areshowninFig.6A.The
average RMSD was 0.385 A
˚
for backbone atoms and
0.975 A
˚
for all heavy atoms (without the C-terminal
residues), which were poorly determined (Table 1). A
percentage of 52.1% of all residues occupy the most
favorable regions of the Ramachandran plot and 41.7% lie
in additionally allowed regions.
Fig. 3. Purification of IsTX. (A) The crude venom ofscorpion was
separated using RP-C
18
HPLC (250 · 4.6 mm) with a 60 min linear
gradient from 0 to 64% acetonitrile/H
2
O with 0.1% trifluoroacetic
acid. The flow rate was set at 1 mLÆmin
)1
and the absorbance was
monitored at 220 nm. F raction 17 was collected, as it showed a
paralysis effect on crickets. (B) C
18
HPLC fraction 17 was further
separated with a 60 min linear gradient from 0 to 32% acetonitrile/
H
2
O with 0.1% trifluoroacetic acid. Two main components including
IsTX were collected.
Fig. 4. Amino acid seq uence determination of IsTX. The reduced and
pyridylethylated peptide was digested with V8 proteases a nd sequenced
using Edman and ladder sequencing. The amino acid sequences
determined by ladder sequencing are underlined.
Fig. 2. MALDI-TOF MS spectrum of crude
venom of two individual Scorpion (O. madaga-
scariensis) in the mass range m/z 500–10000.
The top spectrum is th at ofamale scorpion
and the bottom is ofa female. Peptide IsTX
was only found in the m ale.
3858 N. Yamaji et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Overall structure description
The s olution structureofIsTX comprises an a-helix running
from residue Thr10 to Lys21 and three b-strands from His2
to Pro6, Cys27 to Met29 and His32 to Asn34. The two
strands of the b-sheet are connected by a type II b-turn
formed by residues Asn30 and Arg31 (Fig. 7). In order to
further characterize the overall structureof the IsTX toxin,
deviations from the random coil position of the Ca chemical
shifts were analyzed using the chemical shift index method
[38,39]. The Ca chemical shifts exhibit upfield shifts with
respect to random coil values in the helical conformation
and downfield shifts in the b-strand e xtended conformation.
The results are in reasonably good agreement with what is
expected based on other NMR parameters.
Secondary structure
NOE c orrelations and Ca-chemical shift values were used to
identify elements of secondary structure in IsTX. Th e s trong
HN-HN
i,i+1
NOE correlations together with small
3
J
NH-Ha
coupling constants s uggested the presence o f an a-helix
conformation extending from Thr10 to Lys21. A proline
insertion i n t he middle of t he helix was observed t o
cause d istortion from t he regular pattern. Medium-range
Ha-HN
i,i+3
NOE correlations between Glu15 and Lys21
Fig. 5. The cDNA and deduced amino acid
sequences of I sTX. Amino acids are denoted
by the on e-letter symbols. The c DN A encodes
a 21 residue signal peptide (underlined), a
mature 41 residue peptide (bold) and an
additional Arg at the C-terminal end (boxed).
The stop codon is indicated by ÔendÕ.The
polyadenylation signal (aataaa) is italicized.
Fig. 6. Solution structures ofIsTX and HsTX1. (A) Stereoview of the 20 final stru ctures ofIsTX superimposed over th e bac kbone atom s of the well-
defined region (residues 2–39). (B) Ribbon diagram of the backbone peptide folding ofIsTX illustrating the single a-helix and triple-stranded
b-sheet. (C) Ribbon diagram of HsTX1 illustrating the single a-helix and double-stranded b-sheet (PDB code 1QUZ).
Table 1. Structural statistics for the20lowestenergystructures.
Measurement Value
Ramachandran analysis (residues 2–40)
a
Residues in most favoured regions (%) 52.1
Residues in additional allowed regions (%) 41.7
Residues in generously allowed regions (%) 6.0
Residues in disallowed regions (%) 0.1
RMSDs between 20 conformers
(residues 2–39)
b
Backbone (A
˚
) (N,Ca,C,O) 0.385
All heavy atoms (A
˚
) 0.975
Distance restraints
Intraresidue (|i–j| ¼ 0) 199
Sequential (|iu
ˆ
j| ¼ 1) 189
Medium-range (2 £ |i–j| £ 4) 113
Long-range (|i–j| > 4) 178
Total 679
Deviations from idealized covalent geometry
Bonds (A
˚
) 0.0065 (± 0.0007)
Angles (°) 0.719 (± 0.026)
Impropers (°) 0.619 (± 0.032)
Total energies (kcalÆmol
)1
) 322.32
a
PROCHECK
-
NMR
was used to calculate these values.
b
None of
these structures exhibited distance violations > 0.4 A
˚
.
Ó FEBS 2004 SolutionstructureofIsTX (Eur. J. Biochem. 271) 3859
and HN-HN
i,i+2
NOE correlations between Thr10 and
Glu15 clearly identify residues Thr10-Lys21 as a-helical.
Strong Ha-HN
i,i+1
connectivities f rom His2 t o Pro6,
Cys27 to Met29 and His32 to Cys34 together with large
3
J
NH-Ha
coupling constants indicate a b-strand conforma-
tion. The last two stretches are connected by the Met29 to
His32 fragment, which shows a HN-HN
i,i+1
connectivity
pattern typically assigned to a t ight turn. This t urn was
defined as a type II b-turn. An Ha-HN
i,i+1
interaction
between Met29 and Asn30 was not observed, because a
Ha proton of Met29 overlapped with t he water signal.
A number of long-range HN-HN
i,j
[Thr3:Cys33,
Lys27:Asn34], Ha-HN
i,j
[His2:Cys35, Cys28:Asn34], HN-
Ha
i,j
[Asn4:His32, Thr3:Asn34, Lys27:Cys35, Met2 9:Cys33]
and Ha-Ha
i,j
[His2:Asn34] NOE connectivities suggest that
the structure is a triple-stranded b-sheet.
Disulfide bridge pattern
The disulfide bridge pattern ofIsTX was established by
NMR and modeling u sing data o btained from
XPLOR
-
NIH
. The disulfide bridge pattern ofIsTX was expected
to conserve those in a-KTX6 family. The structural
calculations were considered: (a) with a standard disulfide
bridge pattern (C1-C5, C2-C6, C3-C7 and C4-C8); (b)
with MTX t ype disulfide pattern (C1-C5, C2-C6, C3-C 4
and C 7-C8); and (c) without disulfide bridge restraints.
The total energy of the best 20 structures was compared
after 1 00 str uctures were calculated for each disulfide
bridge patterns. The total energy with MTX pattern
restraint (b) 8969.6 kcal was higher than the standard
pattern restraint (a) 6446.4 kcal. The number of N OE
violations in 100 structures was seven for the standard
pattern and 335 for the MTX pattern. In addition, the
NOE correlations of the Hb-Hb protons between C3-C7
and C4-C8 were less well resolved, but present. The fact
confirmed t hat IsTX has a s tandard disulfide bridge
pattern; however, there were no NOE correlations
between C1-C5 and C2-C6. Finally, calculations with a
standard pattern r estraint (a) and without disulfide
bridges converged to the same structure.
Hydrogen bonds
The amide proton exchange rates a nd the value of the
amide proton temperature coefficients were used for
the identification of hydrogen bonds [40]. Values of the
coefficients ( Dr
HN
/DT) larger than )4.6 p.p.b.ÆK
)1
are
good indicators of hydrogen b onds. T he amide proton
temperature coefficients are summarized in Fig. 8. The
backbone amide protons ofIsTX have highly variable
temperature coefficients, ranging from )10.2 to
3.6 p.p.b.ÆK
)1
. Temperature coefficient values become
more positive as NHs are shifted upfield relative to their
random coil value and more negative as NHs are shifted
downfield [31]. In this case, as the residues (e.g. Cys23:
2.5 p.p.b.ÆK
)1
and Arg25: 2.9 p.p.b.ÆK
)1
), which are close
to aromatic rings and their amide protons are shifted
upfield from random coil values (Cys: 8.23 p.p.m., Arg:
8.24 p.p.m), they s how positive coefficients. In o rder to
determine hyd rogen bon ds with higher confidence t han
using t he temperature coefficients alone, the following
criteria were used to identify them: ( a) the temperature
coefficient of the amide proton summarized in Fig. 8
was larger than )4.6 p.p.b.ÆK
)1
; (b) the amide proton was
slowly exchanging; (c) a single potential acceptor atom was
found within a radius of 2.8 A
˚
,a60° angle with the donor
atom in over 80% of the 20 structures w as calculated
without hydrogen-bond restraints.
Fig. 7. NMR data summary of Is TX. Secondary structure, a summary
of the NOE connectivities and
3
J
NHCa
coupling constants are shown.
The sequential NOEs, extracted from NOESY with mixing times of
200 m s and classified as very weak, weak, medium and strong,
are represented by the t hickness of the bars.
3
J
NHCa
coupling constants
are indicated by › (> 8 Hz) and fl (< 5.5 Hz). In the secondary
structure, large unfilled a rrows show b-sheets and the coil i ndicates an
a-helix.
Fig. 8. Graph of the amide proton temper ature coefficients in IsTX.
Symbols refer to the values of the temperature coefficients as follows:
slowly exchangeable hydrogen bond-forming proton, j; r apidly
exchangeable nonhydrogen bond-forming proton; h. The c ontinuo us
line corresponds to a value of )4.6 p.p.b.ÆK
)1
.
3860 N. Yamaji et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Structural comparisons with HsTX1
IsTX shows high sequence similarity with HsTX1. The
overall structureofIsTX i s similar to t hose of o ther
members of the a/b-fold scorpiontoxin family including
HsTX1 (Fig. 6B,C). All of these toxins contain a single
a-helix connected to an antiparallel b-sheet. The disulfide
bridge pattern of IsTX, C1-C5, C2-C6, C3-C7 and C4-C8, is
thesameasthatofHsTX1.IsTXhasdifferentC-terminal
and N-terminal regions. The terminal regions ofIsTX are
longer than those of HsTX1 and there is one more b-st rand
at N-terminus and two bulky residues at C-terminus, Pro40
and Trp41.
Electrostatic surface potential
The e lectrostatic potential distribution was calculated using
MOLMOL
2
K
.1 [41]. The atomic radius was a van der Waals
radius, and a full Coulombic calculation was performed
usingatleasta10A
˚
boundary extending beyond the longest
axis of the protein. The internal protein dielectric constant
was 2.0, the water solvent dielectric constant 80.0, the water
radius 1.4 A
˚
, the salt concentration 150 m
M
,andthesalt
radius 2.0 A
˚
. Atomic charges and the protonation state of
the amino acid residues were derived using the Ôpdb-chargeÕ
MOLMOL
macro prog ram.
Discussion
Most K
+
channel toxins were i solated from B uthidae,
whereas MTX [11–13], HsTX1 [14–16] and Pi1 [17] were
from Scorpionidae and IsTX was from Ischnuridae. An
evolutionary correlation among these peptides h as been
suggested.
When comparing the amino acid sequence ofIsTX with
those of o ther scorpion toxins, IsTX s howed a 50%
sequence s imilarity to H sTX1 and MTX and a 43%
similarity to Pi1. These toxins belong to a unique subfamily
(a-KTx6) of K
+
channel toxins. This subfamily consists of a
cysteine-stabilized a/b-fold that comprises an a-helix con-
nected to a double stranded b-sheet. Because of the high
similarity of the sequence with these toxins and similar a/b-
fold, IsTX is thus thought to belong to the same subfamily
as MTX, HsTX1 and Pi1.
The a-KTx6 subfamily has specific activity for the shaker
related volta ge gat ed K
+
channels [10]. MTX shows
activities on both voltage-gated K
+
channels and apamin-
sensitive Ca
2+
-activated K
+
channels (Kv1.1, 37 n
M
;
Kv1.2, 0.8 n
M
; Kv1.3, 150 n
M
;ShB,2n
M
;SKCa,5n
M
)
[42,43]. On the other hand, HsTX1 shows high activity on
voltage-gated K
+
channels (Kv1.3, 12 p
M
) but is inactive
on rat brain apamin-sensitive SK chan nels. Pi1, however, is
inactive on Kv1.1 a nd Kv1.3 c hannels at micromolar
concentrations (Kv1.2, 0.44 n
M
; ShB, 23 n
M
) [44,45].
IsTX w as found to have a t ertiary structure similar to that
of HsTX1. There are a number of structural similarities
between IsTX and HsTX1. However, the functional prop-
erties of these toxins are clearly different. HsTX1 blocks the
Kv1.3 channel with higher affinity, whereas IsTX binds to
this channel with lower affinity (Kv1.3, 1.59 l
M
; Kv1.1,
12.7 l
M
).
In order to elucidate the elements critical for binding to
Kv1.3 channel, it is important to compare the structural
features, critical residues, sidechain locations and the
electrostatic surface potential between IsTX and HsTX1
(Figs 9 and 10).
Recently, the crystal structureof the full-length voltage-
gated K
+
channel w as determined [46,47]. In order to
explain the affinity and selectivity for K
+
channels, several
binding models were proposed [15,48–51]. The mutagenesis
studies of TsTX-Ka (a- KTx family) indicate that Lys27 is
the most critical residue involved in blocking K
+
channels
[52]. The affinity measurement for the K
+
channel using
site-directed mutage nesis at L ys27 indicated that Arg
substitution reduced the affinity by about 25-fold, and Ala
and Glu substitutions reduced the affinity by > 1000-fold.
Several reports have shown that the Lys27 residue occludes
the pore of the K
+
channel [ 2]. Lys27 of IsTX, which inserts
into the channel pore, is located at the same position as
Lys23 of HsTX1. This position is considered critical for the
Kv1.3 c hannel blocking activity and forms a hydrogen bond
with the Tyr395 carbonyl oxygen atoms of t he human
Kv1.3 channel. Furthermore, several other residues (Met29,
Asn30 and Arg31) surrounding Lys27 for IsTX are also
conserved and located in similar positions to those of
HsTX1 (Met25, Asn26 and Arg27) surrounding Lys23
(Fig. 10A). Docking studies of HsT X1 on t he Kv1.3
channel revealed that the Lys23, Met25 and Asn26 residues,
Fig. 9. Electrostatic potential surfaces ofIsTX (A) and HsTX1 (B). The charge was assigned to normally ionizable residues (Asp, Glut, Lys and
Arg). Negatively charged regions are shown i n red and positively charged regions in blue. The figure was generated using
MOLMOL
.
Ó FEBS 2004 SolutionstructureofIsTX (Eur. J. Biochem. 271) 3861
found in m ost a-KTx6 f unctional sites, a re critical fo r
binding [15].
Figure 9 shows the surface electrostatic potential distri-
bution ofIsTX and HsTX1. HsTX1 has large basic residues
(Arg4, Lys7, Arg14, Lys23, Lys28, Lys30 and Lys33) while
IsTX has fewer basic residues (Arg8, Arg25, Lys27 and
Arg31).
The extracellular surface of the entry pore of the Kv1.3
channel bears a large negative electrostatic potential, which
is centrosymmetric around the central pore [49]. On the
other hand, the surfaces of peptides have positive electro-
static potentials. The positively c harged residues i n the
peptides interact electrostatically with the acidic re sidues in
the channel. The electrostatic potential on the surface of the
peptides shows that charge anisotropy is the driving force
for the association of peptides to the Kv1.3 channel. The
channel has fourfold sy mmetry with the homotetramer [46].
The positively charged residues o f HsTX1, Arg4, Lys7,
Lys28 and Arg33, are uniformly distributed around Lys23
on the surface. IsTX has only four positively charged
residues, thus, the positive potential is biased. T hese
differences are indicative of the fact that IsTX is less potent
than HsTX1. Furthermore, IsTX has two bulky residues
(Pro40 and Trp41) at the C-terminus. These bulky residues
are unfavorable for the interaction with the Kv1.3 channel
pore region.
This is the first report that scorpion v enom contains
sexually linked toxins and that only male scorpions have
IsTX in their venom. In spider venoms, some male toxins
have been reported (e.g. d-atracotoxin-Ar1a [7], d-missu-
lenatoxin-Mb1a [8]). The MALDI-TOF MS clearly eluci-
dated the difference between the two genders (Fig. 1). A
sexually related sting in some species of scorpions was
reported previously [53]. The malescorpion uses its stinger
to puncture the female scorpion’s body for 3–20 min or
more. It is difficult to determine whether envenomation
occurs during this process. This behavior occurs early on
and then sporadically later in the mating promenade. If
envenomation occurs prior to mating, the sexual sting may
drug the female and thus function to subdue her normal
aggressive behavior. Whether the sexual sting occurs in
scorpion (O. madagascariensis) and whether IsTX plays a
role in this biological process is currently under investiga-
tion.
In summary, the three-dimensional structureofa newly
discovered scorpiontoxin from O. madagascariensis was
determined. The sequence shows that it is a member of
a-KTx6 subfamily, which is a voltage-gated potassium
channel b locking peptide. IsTX was suggested to have a
high affinity to the Kv1.3, as the tertiary structure is similar
to HsTX1. HsTX1 has a high sequence similarity to IsTX.
However, IsTX is 10 000-fold less potent than HsTX1 in
binding to the Kv1.3 channel. These results suggest that the
basic amino residues are crucial to binding to voltage-gated
potassium channels. Furthermore, two bulky residues at the
C-terminus ofIsTX may prevent binding.
Mutation experiments ofIsTX in w hich two bulky
C-terminal residues are cleaved w ould demonstrate this
proposed prevention.
Acknowledgements
We are grateful to Drs Jan Tytgat and Hideki Nishio for binding assay
and c hemical synthesis of IsTX. This work was supported by a grant
from the Research f or the F uture P rogram from the Japan Society for
the Promotion of Science (JSPS).
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Supplementary material
The following material is available from http://www.
blackwellpublishing.com/products/journals/sup pmat/EJB/
EJB4322/EJB4322sm.htm
Table S1. Proton chemical shifts of IsTX.
Table S2. Ca and Cb chemical shifts of IsTX.
Fig. S1. NOESY spectrum recorded w ith 50 m s mixing time
at 298K.
Fig. S2. Co-injection o f natural and s ynthetic IsTX in
RP-HPLC.
Fig. S3. Induced shifts of Ca carbons of IsTX.
3864 N. Yamaji et al. (Eur. J. Biochem. 271) Ó FEBS 2004
. Solution structure of IsTX A male scorpion toxin from Opisthacanthus madagascariensis (Ischnuridae) Nahoko Yamaji 1 , Li Dai 1 , Kenji Sugase 1 , Marta Andriantsiferana 2 , Terumi Nakajima 1 and. Nakajima 1 and Takashi Iwashita 1 1 Suntory Institute for Bioorganic Research, Mishima-Gun, Osaka, Japan; 2 Faculty of Science, University of Antananarivo, Madagascar The novel sex-specific potassium channel. obtained with a polyadenylation signal (AATAAA) in the 3¢ untranslated region and a poly (A) tail at the 3¢ end. The open reading frame of IsTX encodes a precursor of 63 residues, contain- ing a