The novel sex-specific potassium channel inhibitor IsTX, a41-residue peptide, was isolated from the venom of male Opisthacanthus madagascariensis.. Even though MTX has a different disulfid
Trang 1The novel sex-specific potassium channel inhibitor IsTX, a
41-residue peptide, was isolated from the venom of male
Opisthacanthus madagascariensis Two-dimensional NMR
techniques revealed that the structure of IsTX 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-helix and two b-strands connected by four disulfide
brid-ges and binds to voltage-gated K+channels and
apamin-sensitive Ca2+-activated K+ channels The
three-dimen-sional structure of IsTX is similar to that of Heterometrus
spinifertoxin (HsTX1) HsTX1 blocks the Kv1.3 channel at
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 clearly indicated that 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; potassium channels; scorpion toxin; sex-specific toxin
The venom of Opisthacanthus madagascariensis scorpions
from the Ischnuridae family collected 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 Ischnuridae
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 Furthermore, 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 been 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 Ca2+-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 structure of IsTX is composed of a single a-helix and triple b-strand connected by four cysteine-disulfide bridges The amino acid sequence of IsTX is 50% similar to HsTX1 and MTX and 43% similar to Pi1 These findings indicate that IsTX is highly similar to the a-KTx6 subfamily Although IsTX has a highly sequential and structural similarity to HsTX1, the activity of IsTX toward Kv1.3 is
10 000 times lower than that of HsTX1 The reason for this difference may be elucidated by comparing 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 Ca2+-activated K+
channel.
Database: The coordinate for the 20 lowest conformers IsTX has been
submitted to the RCSB Protein Data Bank database under the
accession number 1WMT.
(Received 14 June 2004, revised 26 July 2004, accepted 4 August 2004)
Trang 2of IsTX, (b) determination of the three-dimensional
structure of IsTX 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) were 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/H2O supplemented with
0.1% (v/v) trifluoroacetic acid in order to obtain a saturated
solution Samples mixed 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 Biosystems; Fragmingham,
MA, USA) in positive mode Crude venom was recorded in
linear mode
Peptide purification
The venom supernatant was loaded directly onto to an
RP-C18 HPLC column (Tosoh, TSK gel, ODS 120T,
250· 4.6 mm, 5 lm particle size) The temperature was
controlled at 40C The flow rate was set at 1 mLÆmin)1
and a linear gradient from 0–64% (v/v) acetonitrile/H2O
with 0.1% (v/v) trifluoroacetic acid was run for 60 min The
detector absorbance was set at 220 nm
Crude fraction 17, which contained the peptide IsTX, was
collected and lyophilized It was further purified by RP-C18
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) acetonitrile/HO with 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 purified peptide was collected and lyophilized
Primary structure analysis About 20 pmol of 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 C18HPLC (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/H2O with 0.1% (v/v) trifluoroacetic acid
at a flow rate of 1 mLÆmin)1 was run Half of the pyridylethylated peptide was applied to an automated Edman degradation gas-phase sequencer (PPSQ-10; Shim-adzu, Kyoto, Japan) and the remaining half was digested using V8 proteases at 37C for 12 h The digestion products were checked with MALDI MS and separated
by RP-C18 HPLC with the same conditions as described above Four fractions were collected and analyzed by a combination of MS ladder sequencing and automated Edman degradation sequencing
cDNA cloning The total RNA from one scorpion venom gland was 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: 5SP1, 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 sequencer (Perkin-Elmer, CA, USA)
Fig 1 Sequence alignment of IsTX with selected toxins froma-KTx6 subfamily and disulfide bridge patterns The amino acid sequences of HsTX1
(H spinnifer), Pi1 (P imperator) and MTX (S maurus) were aligned according to the eight half-cysteine residues Gaps (–) have been introduced to maximize the alignment *, C-terminal carboxyamidated extremity The two disulfide bridge patterns observed in HsTX1/Pi1 and MTX are shown below the sequences.
Trang 3spectrometer (Bruker Biospin, Germany) The temperature
was set to 298 K Chemical shifts were referenced to internal
3-(trimethylsilyl)[2,2,3,3-2H4] propionate (TSP) 2D
DQF-COSY [22], NOESY [23,24], TOCSY [25] and HSQC
experiments were conducted 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 A1H,13C HSQC spectrum was recorded
in natural abundance using the echo-antiecho scheme
[27,28] The time domain data were processed using
XWIN-NMR2.5 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 spin systems were then sequentially connected
using the NOESY spectra NOE volumes were converted
into distance restraints classified as strong (1.8–2.7 A˚),
medium (1.8–3.3 A˚), weak (1.8–5.0 A˚), and very 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 H2O sample, redissoloved in 250 lL of
99.96% (v/v) D2O, 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 The3JNH-Hacoupling 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 the3JNH-Ha
values were used as / angle constraints within the range of
)90 and )40 for3JNH-Ha< 5.5 Hz, between)160 and
)80 for3JNH-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 torsion-angle molecular dynamics at 50 000 K The
structures were then subjected to a 15 ps and 1500 steps
slow-cooling torsion angle molecular dynamics stage in which 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 D2O data (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 as 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 and experimental restraints and atom overlapping using AQUA3.2 andPROCHECK-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, which were assumed to be short-chain neurotoxins (Fig 2) However, when each C18 HPLC fraction was tested for toxicity to crickets, only fraction 17 showed an apparent paralysis effect on crickets (Fig 3) This fraction was further purified and two components were collected One was a peptide with an Mrof 3147 and no toxicity effects, and the other was a peptide with an Mrof 4819 that caused paralysis
to crickets This toxic peptide was isolated from a scorpion collected in Isalo (Madagascar) and was named IsTX Due to the limited amount of natural peptide, the primary structure was determined using several techniques including MS/MS analysis, ladder sequencing, Edman degradation sequencing, and subsequently confirmed by cDNA deduction
The pyridylethylated peptide was sequenced using an automated Edman degradation method and the N-terminal
15 step was determined as shown in Fig 4 The V8 digested products of the pyridylethylated peptide were monitored by 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 of IsTX was deduced from the cDNA sequence Based on the known amino acid sequences,
Trang 4specific 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 Over 10 clones 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 of IsTX encodes a precursor of 63 residues, contain-ing a signal peptide of 21 amino acid residues and a mature 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, Japan) using an automated solid-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 overlap with the water resonance signal The amide protons of Cys23 and Arg25 were shifted upfield relative to their positions in random coil structures, and the Ala24 amide proton was shifted downfield This observation suggests that these protons were in close proximity to an aromatic ring
The structure of IsTX was calculated using 679 NOE distance constraints The 20 accepted structures, which had
no NOE violations (£ 0.4 A˚) are shown in Fig 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 of scorpion 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)1and the absorbance was
monitored at 220 nm Fraction 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 sequence determination of IsTX The reduced and
pyridylethylated peptide was digested with V8 proteases and 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 that of a male scorpion and the bottom is of a female Peptide IsTX was only found in the male.
Trang 5Overall structure description The solution structure of IsTX 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 structure of 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 extended conformation The results are in reasonably good agreement with what is expected based on other NMR parameters
Secondary structure NOE correlations and Ca-chemical shift values were used to identify elements of secondary structure in IsTX The strong HN-HNi,i+1NOE correlations together with small3JNH-Ha coupling constants suggested the presence of an a-helix conformation extending from Thr10 to Lys21 A proline insertion in the middle of the helix was observed to cause distortion from the regular pattern Medium-range Ha-HN NOE correlations between Glu15 and Lys21
Fig 6 Solution structures of IsTX and HsTX1 (A) Stereoview of the 20 final structures of IsTX superimposed over the backbone atoms of the well-defined region (residues 2–39) (B) Ribbon diagram of the backbone peptide folding of IsTX 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 the 20 lowest energy structures.
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
Distance restraints
Intraresidue (|i–j| ¼ 0) 199
Medium-range (2 £ |i–j| £ 4) 113
Long-range (|i–j| > 4) 178
Deviations from idealized covalent geometry
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˚.
Trang 6and HN-HNi,i+2 NOE correlations between Thr10 and
Glu15 clearly identify residues Thr10-Lys21 as a-helical
Strong Ha-HNi,i+1 connectivities from His2 to Pro6,
Cys27 to Met29 and His32 to Cys34 together with large
3JNH-Hacoupling constants indicate a b-strand
conforma-tion The last two stretches are connected by the Met29 to
His32 fragment, which shows a HN-HNi,i+1 connectivity
pattern typically assigned to a tight turn This turn was
defined as a type II b-turn An Ha-HNi,i+1 interaction
between Met29 and Asn30 was not observed, because a
Ha proton of Met29 overlapped with the water signal
A number of long-range HN-HNi,j [Thr3:Cys33,
Lys27:Asn34], Ha-HNi,j [His2:Cys35, Cys28:Asn34],
HN-Hai,j[Asn4:His32, Thr3:Asn34, Lys27:Cys35, Met29:Cys33]
and Ha-Hai,j[His2:Asn34] NOE connectivities suggest that
the structure is a triple-stranded b-sheet
Disulfide bridge pattern
The disulfide bridge pattern of IsTX was established by
NMR and modeling using data obtained from XPLOR
-NIH The disulfide bridge pattern of IsTX 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 type disulfide pattern (C1-C5, C2-C6, C3-C4
and C7-C8); and (c) without disulfide bridge restraints
The total energy of the best 20 structures was compared
after 100 structures 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 NOE
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 that IsTX has a standard disulfide bridge
pattern; however, there were no NOE correlations
between C1-C5 and C2-C6 Finally, calculations with a standard pattern restraint (a) and without disulfide bridges converged to the same structure
Hydrogen bonds The amide proton exchange rates and the value of the amide proton temperature coefficients were used for the identification of hydrogen bonds [40] Values of the coefficients (DrHN/DT) larger than )4.6 p.p.b.ÆK)1 are good indicators of hydrogen bonds The amide proton temperature coefficients are summarized in Fig 8 The backbone amide protons of IsTX 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)1and 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 show positive coefficients In order to determine hydrogen bonds with higher confidence than using the 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˚, a 60 angle with the donor atom in over 80% of the 20 structures was calculated without hydrogen-bond restraints
Fig 7 NMR data summary of IsTX 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 ms and classified as very weak, weak, medium and strong,
are represented by the thickness of the bars 3 J NHCa coupling constants
are indicated by › (> 8 Hz) and fl (< 5.5 Hz) In the secondary
structure, large unfilled arrows show b-sheets and the coil indicates an
a-helix.
Fig 8 Graph of the amide proton temperature coefficients in IsTX Symbols refer to the values of the temperature coefficients as follows: slowly exchangeable hydrogen bond-forming proton, j; rapidly exchangeable nonhydrogen bond-forming proton; h The continuous line corresponds to a value of )4.6 p.p.b.ÆK )1
Trang 7longer than those of HsTX1 and there is one more b-strand
at N-terminus and two bulky residues at C-terminus, Pro40
and Trp41
Electrostatic surface potential
The electrostatic potential distribution was calculated using
MOLMOL2K.1 [41] The atomic radius was a van der Waals
radius, and a full Coulombic calculation was performed
using at least a 10 A˚ 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 mM, and the salt
radius 2.0 A˚ Atomic charges and the protonation state of
the amino acid residues were derived using the pdb-charge
MOLMOLmacro program
Discussion
Most K+ channel toxins were isolated from Buthidae,
whereas MTX [11–13], HsTX1 [14–16] and Pi1 [17] were
from Scorpionidae and IsTX was from Ischnuridae An
evolutionary correlation among these peptides has been
suggested
When comparing the amino acid sequence of IsTX with
those of other scorpion toxins, IsTX showed a 50%
sequence similarity to HsTX1 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
IsTX was found to have a tertiary 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 lM; Kv1.1, 12.7 lM)
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 structure of the full-length voltage-gated K+ channel was 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 mutagenesis at Lys27 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 channel blocking activity and forms a hydrogen bond with the Tyr395 carbonyl oxygen atoms of the 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 HsTX1 on the Kv1.3 channel revealed that the Lys23, Met25 and Asn26 residues,
Fig 9 Electrostatic potential surfaces of IsTX (A) and HsTX1 (B) The charge was assigned to normally ionizable residues (Asp, Glut, Lys and Arg) Negatively charged regions are shown in red and positively charged regions in blue The figure was generated using
Trang 8found in most a-KTx6 functional sites, are critical for
binding [15]
Figure 9 shows the surface electrostatic potential
distri-bution of IsTX 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 charged residues in the
peptides interact electrostatically with the acidic residues 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 symmetry with the homotetramer [46]
The positively charged residues of 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 These
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 venom 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 male scorpion 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 structure of a newly discovered scorpion toxin from O madagascariensis was determined The sequence shows that it is a member of a-KTx6 subfamily, which is a voltage-gated potassium channel blocking 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 of IsTX may prevent binding
Mutation experiments of IsTX in which two bulky C-terminal residues are cleaved would demonstrate this proposed prevention
Acknowledgements
We are grateful to Drs Jan Tytgat and Hideki Nishio for binding assay and chemical synthesis of IsTX This work was supported by a grant from the Research for the Future Program from the Japan Society for the Promotion of Science (JSPS).
References
1 Smith, C., Phillips, M & Miller, C (1986) Purification of char-ybdotoxin, a specific inhibitor of the high-conductance Ca 2+ -activated K + channel J Biol Chem 261, 14607–14613.
2 Park, C.-S & Miller, C (1992) Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin Biochemistry 31, 7749–7755.
3 Stampe, P., Kolmakova-Partensky, L & Miller, C (1994) Intimations of K + channel structure from a complete functional map of the molecular surface of charybdotoxin Biochemistry 33, 443–450.
4 Blanc, E., Romi-Lebrun, R., Bornet, O., Nakajima, T & Darbon,
H (1998) Solution structure of two new toxins from the venom of the chinese scorpion Buthus martensi karsch blockers of the potassium channels Biochemistry 37, 12412–12418.
5 Dai, L., Yasuda, A., Naoki, H., Corzo, G., Andriantsiferana, M.
& Nakajima, T (2001) IsCT, a novel cytotoxic linear peptide from scorpion Opisthacanthus madagascariensis Biochem Biophys Res Commun 286, 820–825.
6 Dai, L., Corzo, G., Naoki, H., Andriantsiferana, M & Nakajima,
T (2002) Purification, structure-function analysis, and molecular characterization of novel linear peptides from scorpion Opistha-canthus madagascariensis Biochem Biophys Res Commun 293, 1514–1522.
Fig 10 Comparison of structures of IsTX and HsTX1 Selected
side-chains are shown for IsTX (yellow) and HsTX1 (dark green) and
labeled for IsTX (black) and HsTX1 (green) Conserved residues
(Met29, Asn30, Arg31) surrounding Lys27 (A) and nonconserved
residues and bulky C-terminal residues of IsTX (B).
Trang 9Martin-Eauclaire, M.-F., van der Walt, J.J & Possani, L.D.
(1999) A unified nomenclature for short-chain peptides isolated
from scorpion venoms: a-KTx molecular subfamilies Trends
Pharmacol Sci 20, 444–447.
10 Rodriguez de la Vega, R.C., Merino, E., Becerril, B & Possani,
L.D (2003) Novel interactions between K+channels and
scor-pion toxins Trends Pharmacol Sci 24, 222–227.
11 Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Oughideni,
R., Van Rietschoten, J., Martin-Eauclaire, M.-F., Rochat, H & El
Ayeb, M (1997) Maurotoxin, a four disulfide bridge toxin from
Scorpio maurus venom: purification, structure and action on
po-tassium channels FEBS Lett 406, 284–290.
12 Blanc, E., Sabatier, J.-M., Kharrat, R., Meunier, S., El Ayeb, M.,
Van Rietschoten, J & Darbon, H (1997) Solution structure of
maurotoxin, a scorpion toxin from Scorpio maurus, with high
affinity for voltage-gated potassium channels Proteins 29, 321–
333.
13 Fajloun, Z., Mosbah, A., Carlier, E., Mansuelle, P., Sandoz, G.,
Fathallah, M., di Luccio, E., Devaux, C., Rochat, H., Darbon, H.,
De Waard, M & Sabatier, J.M (2000) Maurotoxin versus Pi1/
HsTX1 toxins: toward new insights in the understanding of their
distinct disulfide bridge patterns J Biol Chem 275, 39394–39402.
14 Lebrun, B., Romi-Lebrun, R., Martin-Eauclaire, M.-F., Yasuda,
A., Ishiguro, M., Oyama, Y., Pongs, O & Nakajima, T (1997) A
four-disulphide-bridged toxin, with high affinity towards
voltage-gated K+channels, isolated from Heterometrus spinnifer
(Scor-pionidae) venom Biochem J 328, 321–327.
15 Savarin, P., Romi-Lebrun, R., Zinn-Justin, S., Lebrun, B.,
Nakajima, T., Gilquin, B & Menez, A (1999) Structural and
functional consequences of the presence of a fourth disulfide
bridge in the scorpion short toxins: Solution structure of the
potassium channel inhibitor HsTX1 Protein Sci 8, 2672–2685.
16 Savarin, P., Zinn-Justin, S & Gilquin, B (2001) Variability in
automated assignment of NOESY spectra and three-dimensional
structure determination: a test case on three small
disulfide-bon-ded proteins J Biomol NMR 19, 49–62.
17 Delepierre, M., Prochnicka-Chalufour, A & Possani, L.D (1997)
A novel potassium channel blocking toxin from the scorpion
Pandinus imperator: a 1H NMR analysis using a nano-NMR
probe Biochemistry 36, 2649–2658.
18 Tenenholz, T.C., Rogowski, R.S., Collins, J.H., Blaustein, M.P &
Weber, D.J (1997) Solution structure for Pandinus toxin K-a
(PiTX-Ka), a selective blocker of A-type potassium channels.
Biochemistry 36, 2763–2771.
19 Delepierre, M., Prochnicka-Chalufour, A., Boisbouvier, J &
Possani, L.D (1999) Pi7, an orphan peptide from the scorpion
Pandinus imperator: a 1 H-NMR analysis using a nano-NMR
probe Biochemistry 38, 16756–16765.
20 Guijarro, J.I., M’Barek, S., Gomez-Lagunas, F., Garnier, D.,
Rochat, H., Sabatier, J.-M., Possani, L.D & Delepierre, M (2003)
Solution structure of Pi4, a short four-disulfide-bridged scorpion
toxin specific of potassium channels Prot Sci 12, 1844–1854.
21 Katsumi, A., Tuley, E.A., Bodo´., I & Sadler, J.E (2000)
Locali-zation of disulfide bonds in the cystine knot domain of human von
Willebrand Factor J Biol Chem 275, 25585–25594.
Reson 58, 370–388.
25 Bax, A & Davis, D.G (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy J Magn Reson 65, 355–360.
26 Piotto, M., Saudek, V & Sklenar, V (1992) Gradient-tailored excitation for single quantum spectroscopy of aqueous solutions.
J Biomol NMR 2, 661–665.
27 Palmer, A.G., Cavanagh, J., Wright, P.E & Rance, M (1991) Sensitivity improvement in proton-detected two-dimensional het-eronuclear correlation NMR spectroscopy J Magn Reson 93, 151–170.
28 Kay, L.E., Keifer, P & Saarinen, T (1992) Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity J Am Chem Soc 114, 10663–10665.
29 Wu¨thrich, K (1986) NMR of Protein and Nucleic Acids John Wiley & Sons, Inc, New York.
30 Kraulis, P.J (1989) ANSIG: a program for the assignment of protein 1 H 2D NMR spectra by interactive computer graphics.
J Magn Reson 84, 627–633.
31 Andersen, N.H., Neidigh, J.W., Harris, S.M., Lee, G.M., Liu,
Z & Tong, H (1997) Extracting information from the tem-perature gradients of polypeptide NH chemical shifts 1 The importance of conformational averaging J Am Chem Soc 119, 8547–8561.
32 Cierpicki, T., Zhoukov, I., Byrd, R.A & Otlewski, J (2002) Hydrogen bonds in human ubiquitin reflected in temperature coefficients of amide protons J Magn Reson 157, 178–180.
33 Pardi, A., Billeter, M & C¸thrich, K (1984) Calibration of the angular dependence of the amide proton-C a proton coupling constants, 3 J HN a, in a globular protein Use of 3 J HN a for identi-fication of helical secondary structure J Mol Biol 180, 741–751.
34 Clore, G.M & Gronenborn, A.M (1998) New methods of structure refinement for macromolecular structure determination
by NMR Proc Natl Acad Sci USA 95, 5891–5898.
35 Schwieters, C.D., Kuszewski, J.J., Tjandra, N & Clore, G.M (2003) The Xplor-NIH NMR molecular structure determination package J Magn Reson 160, 66–74.
36 Brunger, A.T (1992) X-PLOR, Version 3.1 Manual Yale University Press, New Haven, CT, USA.
37 Lakowski, R.A., Rullmann, J.A., MacArthur, M.W., Kaptein, R.
& Thornton, J.M (1996) AQUA and PROCHECK-NMR: pro-grams for checking the quality of protein structures solved by NMR J Biomol NMR 8, 477–486.
38 Spera, S & Bax, A (1991) Empirical correlation between protein backbone conformation and Ca and Cb13C nuclear magnetic resonance chemical shifts J Am Chem Soc 113, 5490–5492.
39 Kuszewski, J., Qin, J., Gronenborn, A.M & Clore, G.M (1995) The impact of direct refinement against 13 C a and 13 C b chemical shifts on protein structure determination by NMR J Magn Reson Series B 106, 92–96.
40 Cierpicki, T & Otlewski, J (2000) Determination of a high pre-cision structure of a novel protein, Linum usitatissimum trypsin inhibitor (LUTI), using computer-aided assignment of NOESY.
J Mol Biol 302, 1179–1192.
Trang 1041 Koradi, R., Billeter, M & Wu¨thrich, K (1996) MOLMOL: a
program for display and analysis of macromolecular structures.
J Mol Graph 14, 51–55.
42 Kharrat, R., Mabrouk, K., Crest, M., Darbon, H., Oughideni, R.,
Martin-Eauclair, M.-F., Jacquet, G., El Ayeb, M., Van
Riet-schoten, J., Rochat, H & Sabatier, J.M (1996) Chemical synthesis
and charaterization of maurotoxin, a short scorpion toxin with
four disulfide bridges that acts on K + channels Eur J Biochem.
242, 491–498.
43 Kharrat, R., Mansuelle, P., Sampieri, F., Crest, M., Oughideni,
R., Van Rietschoten, J., Martin-Eauclair, M.-F., Rochat, H & El
Ayeb, M (1997) Maurotoxin, a four disulfide bridge toxin from
Scorpio maurus venom: purification, structure and action o
potassium channels FEBS Lett 406, 284–290.
44 Gomez-Langunas, F., Olamendi-Portugal, T & Possani, L.D.
(1997) Block of ShakerB K+channels by Pi1, a novel class of
scorpion toxin FEBS Lett 400, 197–200.
45 Fajloun, Z., Carlier, E., Lecomte, C., Geib, S., Di Luccio, E.,
Bichet, D., Mabrouk, K., Rochat, H., De Waard, M & Sabatier,
J.M (2000) Chemical synthesis and characterization of Pi1, a
scorpion toxin from Pandinus imperator active on K+channels.
Eur J Biochem 267, 5149–5155.
46 Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B.T &
Mackinnon, R (2003) X-ray structure of a voltage-dependent K +
channel Nature 423, 33–41.
47 Jiang, Y., Ruta, V., Chen, J., Lee, A & Mackinnon, R (2003) The
principle of gating charge movement in a voltage-dependent K +
channel Nature 423, 42–48.
48 Mackinnon, R., Cohen, S.L., Kuo, A., Lee, A & Chait, B.T.
(1998) Structural conservation in prokaryotic and eukaryotic
potassium channels Science 280, 106–109.
49 Fu, W., Cui, M., Briggs, J.M., Huang, X., Xiong, B., Zhang, Y.,
Luo, X., Shen, J., Ji, R., Jiang, H & Chen, K (2002) Brownian
dynamics simulations of the recognition of the scorpion toxin
maurotoxin with the voltage-gated potassium ion channels Biophys J 83, 2370–2385.
50 Zhang, M., Korolkova, Y.V., Liu, J., Jiang, M., Grishin, E.V & Tseng, G.-N (2003) BeKm-1 is a HERG-specific toxin that shares the structure with ChTx but the mechanism of action with ErgTx1 Biopys J 84, 3022–3036.
51 M’Barek, S., Mosbah, A., Sandoz, G., Fajloun, Z., Olamendi-Portugal, T., Rochat, H., Sampieri, F., Guijarro, J.I., Mansuelle, P., Delepierre, M., De Waard, M & Sabatier, J.-M (2003) Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K + channels Eur J Biochem.
270, 3583–3592.
52 Ellis, K.C., Tenenholz, T.C., Jerng, H., Hayhurst, M., Dudlak, C.S., gilly, W.F., Blaustein, M.P & Weber, D.J (2001) Interaction
of a toxin from the scorpion Tityus serrulatus with a cloned K+ channel from squid (sqKv1A) Biochemistry 40, 5942–5953.
53 Francke, O.F (1979) Observations on the reproductive biology and life history of Megacormus gertschi Diaz (Scorpiones: Chactidae: Megacorminae) J Arachnol 7, 223–230.
Supplementary material
The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/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 with 50 ms mixing time
at 298K
Fig S2 Co-injection of natural and synthetic IsTX in RP-HPLC
Fig S3 Induced shifts of Ca carbons of IsTX