Báo cáo khoa học: The unique pharmacology of the scorpion a-like toxin Lqh3 is associated with its flexible C-tail pdf

The unique pharmacology of the scorpion a-like toxin Lqh3
is associated with its flexible C-tail
Izhar Karbat1, Roy Kahn1, Lior Cohen1, Nitza Ilan1, Nicolas Gilles2, Gerardo Corzo3, Oren Froy1,
Maya Gur1, Gudrun Albrecht4, Stefan H. Heinemann4, Dalia Gordon1 and Michael Gurevitz1
1
2
3
4

Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv, Israel
´
´ ´
´
CEA Saclay, Departement d’Ingenierie des Proteines, Gif-sur Yvette, France
´
´
´
´
Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Cuernavaca Morelos, Mexico
Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena, Germany

Keywords
pH-dependent toxin binding; scorpion a-like
toxin; structure–function relationships; toxin
effect on inactivation; toxin receptor site on
sodium channel
Correspondence
D. Gordon and M. Gurevitz, Department of
Plant Sciences, George S. Wise Faculty of
Life Sciences, Tel Aviv University, Ramat
Aviv, Tel Aviv 69978, Israel

Fax: +972 3 6406100
Tel: +972 3 6409844
E-mail: and

(Received 9 January 2007, revised 6
February 2007, accepted 12 February 2007)
doi:10.1111/j.1742-4658.2007.05737.x

The affinity of scorpion a-toxins for various voltage-gated sodium channels
(Navs) differs considerably despite similar structures and activities. It has
been proposed that key bioactive residues of the five-residue-turn (residues
8–12) and the C-tail form the NC domain, whose topology is dictated by a
cis or trans peptide-bond conformation between residues 9 and 10, which
correlates with the potency on insect or mammalian Navs. We examined
this hypothesis using Lqh3, an a-like toxin from Leiurus quinquestriatus
hebraeus that is highly active in insects and mammalian brain. Lqh3
exhibits slower association kinetics to Navs compared with other a-toxins
and its binding to insect Navs is pH-dependent. Mutagenesis of Lqh3
revealed a bi-partite bioactive surface, composed of the Core and NC
domains, as found in other a-toxins. Yet, substitutions at the five-residue
turn and stabilization of the 9–10 bond in the cis conformation did not
affect the activity. However, substitution of hydrogen-bond donors ⁄ acceptors at the NC domain reduced the pH-dependency of toxin binding, while
retaining its high potency at Drosophila Navs expressed in Xenopus
oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident
from the known solution structure, we suggest that acidic pH or specific
mutations at the NC domain favor toxin conformations with high affinity
for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and
suggests a novel mechanism of dynamic conformer selection during toxin
binding, enabling a-like toxins to affect a broad range of Navs.

Voltage-gated sodium channels (Navs) are responsible
for the depolarization phase of the action potential in
most excitable cells. Due to their pivotal role in excitability, Navs are targeted by a large variety of toxins
that modify their gating, such as long-chain scorpion
toxins. These toxins are 61–76 residue-long polypeptides that share a similar a ⁄ b scaffold and are divided

into two classes, a and b, according to their mode of
action and different receptor sites [1,2]. Scorpion
a-toxins prolong the action potential by slowing channel inactivation upon binding at a site that involves
extracellular regions of channel domains 1 and 4 [2–4].
Although different a-toxins are similarly toxic to
mice when injected subcutaneously and similarly affect

Abbreviations
Aah2, alpha toxin 2 from the scorpion Androctonus australis hector; BmK M1, alpha toxin from the scorpion Buthus martensii Karsch; CHO,
Chinese hamster ovary; Lqh2, Lqh3, LqhaIT, alpha toxins from the scorpion Leiurus quinquestriatus hebraeus; Nav, voltage-gated sodium
channel.

1918

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

a-Like toxin binding is linked to its flexibility

Fig. 1. Sequence alignment of scorpion a-toxins representing three pharmacological groups. Positions are numbered according to Aah2.
Dashes indicate gaps for best alignment. Residues of the five-residue turn and C-tail are shaded. Residues of the conserved Core domain
are in bold. Lqh2, Lqh3, LqhaIT [9], Lqh6, and Lqh7 [40] are from the scorpion L. quinquestriatus hebraeus; Aah2 is from Androctonus

australis hector; Lqq3 and Lqq5 are from L. quinquestriatus quinquestriatus; Bmk-M1, Bmk-M2, Bmk-M4, and Bmk-M8 are from Buthus
martensii Karch; Bom3 and Bom4 are from Buthus occitanus mardochei [9].

rat skeletal muscle Navs [5–7], they exhibit profound
differences in potency when injected into mice brain,
and in their affinity for insect and rat-brain neuronal
preparations [7,8]. Accordingly, scorpion a-toxins were
divided into three pharmacological groups (Fig. 1): (a)
Classical anti-mammalian toxins that bind with high
affinity to rat brain synaptosomes and are practically
nontoxic to insects [1]; (b) a-toxins highly active on
insects that bind with high affinity to insect Navs and
are weakly toxic in mammalian brain; and (c) a-like
toxins that are active in both mammalian brain and
insects (Fig. 1 [8,9]).
To correlate the selectivity of a-toxins with their
structure, the bioactive surface of the anti-insect
LqhaIT (from L. quinquestriatus hebraeus) and its
putative equivalent in the anti-mammalian Aah2 were
investigated and shown to consist of two domains [10].
Four residues located on short loops that connect the
conserved secondary structure elements of the molecule
core form the Core domain, while the five-residue-turn
(residues 8–12) and the C-terminal segment (residues
56–64) form the NC domain. The division of the bioactive surface into two domains is supported by muta-

genesis of the a-like toxin BmK M1 (from Buthus
martensii Karch) [11–13]. As the amino acid composition and spatial arrangement of the NC domain varies
among a-toxins, it was suggested to confer toxin preferential binding to various Navs. The high insecticidal
potency of LqhaIT was correlated with a protruding

conformation of the NC domain, a feature typifying
all scorpion a-toxins active on insects. This protrusion,
mediated by a nonproline cis peptide bond between
residues 9 and 10 of the five-residue turn, differs markedly from the flat conformation dictated by a trans
peptide bond conformation between residues 9 and 10,
which characterizes the NC domain in toxins highly
active in the rat brain [10,14]. In this respect, the high
potency of a-like toxins for both insect and various
mammalian Navs [7] cannot be readily explained and
was addressed here using Lqh3, the most pharmacologically characterized toxin with known structure of
the a-like group [15]. Lqh3 is highly toxic to insects
and competes with LqhaIT on binding to insect Navs.
Lqh3 differs from classical anti-mammalian a-toxins
as it inhibits Nav inactivation in cell bodies of hippocampus brain neurons, on which the anti-mammalian

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1919


a-Like toxin binding is linked to its flexibility

I. Karbat et al.

Lqh2 is inactive, and is unable to affect Nav1.2 in the
rat brain, on which Lqh2 is highly active [16]. Moreover, it has been shown that the pharmacological
properties of Lqh3 are unique in that its binding affinity for insect channels drops >30-fold at pH 8.5 versus
pH 6.5, and its rate of association with receptor site-3
on both insect and mammalian Navs is 4–15-fold
slower compared with LqhaIT and Lqh2 [6,17,18].

To clarify the molecular basis of the unique pharmacological features of Lqh3, we analyzed its bioactive
surface seeking for residues involved with its slow
association kinetics and sensitivity to pH changes upon
interaction with insect Navs. Our data reveal that residues at the NC domain, which may serve as hydrogen
bond acceptors or donors, are specifically associated
with these features. Re-examination of the solution
structures of Lqh3 disclosed a high conformational
flexibility of its C-tail, which may interconvert between
two distinct conformers that differ in their intramolecular hydrogen-bonding pattern. Based on these observations we suggest that the unique pharmacological
features of scorpion a-like toxins are associated with
the flexibility of the C-tail.

Results
The bioactive surface of Lqh3
Twenty-four residues were substituted and the toxin
mutants were produced in Escherichia coli as a fusion
peptide (His-Apamin-Lqh3), folded in vitro, and purified by RP-HPLC (see Experimental procedures).
Changes in activity were monitored in toxicity assays
on blowfly larvae and binding assays using cockroach
neuronal membrane preparations. CD spectroscopy
was used as a measure of secondary structure signature
to discern effects that were due to structural perturbations from those associated directly with toxin activity.

From a total of 49 mutants, the CD spectrum of only
I59R altered (Fig. 2B). Of the 24 modified residues,
substitution of 15 had a weak (DDG ẳ 1.1 kcalặmol)1)
to moderate (DDG ẳ 1.5 kcal ⁄ mol) effect on activity
(Table 1).
Substitutions in the five-residue turn (residues 8–12)
had no significant effect on the activity to insects, even

when charges were neutralized or inverted (Table 1).
These results imply that the five-residue turn in Lqh3
is most likely not involved in direct interaction with
the channel receptor, and that it tolerates considerable
changes with no perturbation of toxin folding, in contrast to LqhaIT [19] and BmK M1 [14]. Substitutions
in the loop preceding the a-helix had large effects on
activity, as shown by the replacement of His15 by
bulky aliphatic or charged residues (H15F ⁄ L ⁄ R),
Phe17 by Ala, and Pro18 by Arg or Gly (Table 1).
Substitutions in the loop connecting the second and
the third b-strands highlighted the importance for
activity of Phe39 and Leu45, as was shown for their
equivalents in LqhaIT and Bmk-M1 [10,12]. His15,
Phe17, Pro18, Phe39 and Leu45 constitute a distinct
amino acid cluster on the molecule surface interconnected by hydrophobic–aromatic interactions resembling the Core domain reported for LqhaIT [10].
Substitutions I59A ⁄ R had a marked effect on the
binding affinity (Table 1). Ile59 is mostly buried in the
molecule and forms hydrophobic contacts with Gly4,
Tyr5, Ile6, and Ala7 of the N-terminal region [15].
While I59R altered the CD signature of the molecule,
the CD spectrum of I59A was similar to that of the
unmodified toxin, which suggested that Ile59 might
form contact with the receptor site, as was suggested
for the equivalent residue in other a-toxins [10,11,20].
Neutralization or inversion of the charge of Lys64
(K64A ⁄ D) and His66 (H66A ⁄ E) significantly affected the activity, while a conserved substitution had
a minor effect (Table 1), which suggested that a

Fig. 2. The bioactive surface of Lqh3. (A)
The toxin backbone is shown in ribbon. Residues, whose substitution affected the function (see Table 1) are space-filled and

colored according to their chemical nature
(aliphatic, green; aromatic, magenta; positive, blue). (B) CD spectra of the recombinant
HA-Lqh3 and representative mutants.

1920

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

a-Like toxin binding is linked to its flexibility

Table 1. Effects of mutations in Lqh3 on binding to cockroach neuronal membranes. The change in apparent binding affinity is presented as
the ratio of K mut over K wt . K wt and K mut were obtained in competition against 125I-LqhaIT binding at pH 7.2, as previously described [18].
i
i
i
i
The K wt value is 1.0 ± 0.1 nM, n ¼ 6. Ki determination is described in the Experimental procedures. The change in binding energy was calcui
lated as DDG ¼ -RT ln(K wt ⁄ K mut ).
i
i
HA-Lqh3 mutant

K mut ⁄ K wt
i
i

DDG (kcalỈmol)1)

Lqh3 mutant

K mut ⁄ K wt
i
i

DDG(kcalỈmol)1)

Q8A
Q8K
P9G
E10A
E10Y
E10R
E10P
Y14A
H15A
H15F
H15L
H15R
F17A
F17L
F17Y
P18A
P18R
P18G
S20A
S21A
D24A

H36A
F39A
K40A
V41A

9.5
3.5
9.0
2.5
0.4
6.6
1.0
15.6
3.9
45.0
109.0
577.0
91.0
3.5
10.0
92.0
210.0
258.0
6.0
1.9
1.5
4.9
28.0
9.0
13.0

1.33
0.74
1.30
0.54
)0.54
1.11
0.00
1.62
0.80
2.25
2.77
3.75
2.66
0.74
1.36
2.67
3.16
3.28
1.06
0.38
0.24
0.94
1.97
1.30
1.51

H43A
H43R
H43E

L45A
I58A
I59A
I59R
V60A
V60L
E61A
E61L
E61R
E63A
E63R
K64A
K64D
K64L
K64R
H66A
H66R
H66E
S67A
P9C-E10C
E10Y-E63R

4.0
0.2
117.0
70.0
1.2
103.0
25.0
1.0

13.0
0.2
1.4
0.5
2.0
0.8
16.0
22.0
18.0
4.0
23.0
4.0
10.0
1.4
1.0
0.4

0.82
)1.05
2.81
2.51
0.11
2.73
1.90
0.00
1.51
)0.95
0.20
)0.41
0.41

)0.13
1.64
1.82
1.71
0.82
1.85
0.82
1.36
0.20
0.00
)0.54

positively charged C-tail was important for activity.
Substitutions at the negatively charged patch composed of Glu10, Glu61 and Glu63, which is unique to
Lqh3 compared to other a-toxins, had no effect on the
activity (Table 1).
We further examined if the bioactive surface of
Lqh3 towards insect Navs coincided with that presented toward rat skeletal muscle Navs by analyzing
the effects of various substitutions on Nav1.4 and
Drosophila melanogaster DmNav1 Navs expressed in
Chinese hamster ovary (CHO) cells and in Xenopus
oocytes, respectively (Table 2). Most substitutions
that markedly reduced the binding affinity for cockroach neuronal membranes reduced the toxin potency
towards rNav1.4 and DmNav1 to a similar extent.
However, H15A, which had only a slight effect on
the toxicity and binding affinity for insects and on
potency at DmNav1, profoundly affected the potency
at rNav1.4. This analysis highlighted also substitution
H66E, which had a larger effect on the potency at
DmNav1 than at rNav1.4 (Table 2). Thus, the bioactive surface of Lqh3 towards insect and rat skeletalmuscle Navs is similar, but not identical, where

Table 2. Comparison of the effects of selected Lqh3 mutants on
rat skeletal muscle Navs (rNav1.4) expressed in CHO cells and on
the Drosophila DmNav1 channel expressed in Xenopus oocytes.
The apparent effective concentration 50% (EC50) of each mutant
on rNav1.4 and DmNav1 were determined in at least three independent experiments (see Experimental procedures) and normalmut
wt
ized to the potency of unmodified Lqh3 (EC50 ⁄ EC50 ). The effect
on DmNav1 was assayed at pH 7.1 (EC50 ¼ 10.5 ± 1.6 nM).

Lqh3 mutant

EC50 (rNav1.4)
(nM)

mut
wt
EC50 ⁄ EC50 rNav1.4
mut
wt
(EC50 ⁄ EC50 DmNav1)

Unmodified Lqh3
H15A
F17A
P18A
S20A
F39A
H43E
L45A

I58A
I59A
K64D
H66E

4.2
209
87.3
424
11.5
154
505
342
48.3
86.2
128
27.4

1
49.3 (10.7)
20.6
100
2.7
36
119
80
11.4
20.4
30.1 (2.2)
6.5 (117)

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

±
±
±
±
±
±
±
±
±
±
±
±

0.5
40
10.5
8.0
1.3
35
30
71
17.2
19.4
40
2.2

1921

a-Like toxin binding is linked to its flexibility

I. Karbat et al.

His15 and His66 seem to contribute to the differential
interaction of Lqh3 with channel receptors of various
origin.
In total, the bioactive surface of Lqh3 is composed
of two distinct domains, the Core and NC domains,
formed by residues of the loop preceding the a-helix,
the loop connecting the second and the third b-strands,
and the C-tail (Fig. 2A).

decrease in the dissociation rate constant at lower
pH [18].
To clarify the molecular basis of the pH-dependent
binding, we examined two mechanisms previously
suggested to affect toxin binding. It was suggested
that a cis–trans isomerization of the nonproline cispeptide bond between residues 9 and 10 of scorpion
a-like toxins might function as a molecular switch
that determines their preference for various Navs [14].
In Lqh3, the peptide-bond between Pro9 and Glu10
appears in solution as a mixed population of cis
and trans conformations, and a slow pH- and temperature-dependent interconversion between these two
isomeric forms was reported [15,21]. Thus, a
pH-dependent isomerization of the P9-E10 bond in
Lqh3 could underlie its pH-dependent binding. We
tested this hypothesis by constructing a toxin double

mutant, in which Cys substituted both residues.
Modeling of the double mutant (P9C-E10C) predicted
that the position of these two Cys residues on the
tight five-residue-turn would force their side chains
to adopt a solvent exposed conformation and create
a vicinal disulfide bond in a cis conformation
(Fig. 4A,B). The toxin mutant was successfully
expressed and folded in vitro, and exhibited identical
toxicity (EC50 ¼ 75 ± 5 ng ⁄ 100 mg blowfly larvae)
and binding affinity for cockroach neuronal membranes (Ki ¼ 1.06 ± 0.07 nm, n ¼ 3) as those of the
unmodified toxin (Table 1). The molecular mass of
the P9C-E10C toxin mutant was determined to be
7040 ± 0.1 Da, which corresponded exactly to the
theoretical mass calculated, assuming that the newly
introduced Cys residues were both oxidized. This

Effect of substitutions in Lqh3 on its
pH-dependent binding
The binding affinity of Lqh3 for cockroach neuronal
membranes decreased 32-fold when assayed at pH 8.5
compared to pH 7.2 (Table 3). We have further tested
the effect of pH transitions on Lqh3 interaction with
DmNav1 channels expressed in Xenopus oocytes.
Under control conditions, pH alterations of the bath
solution in the range 7.0–8.5 had no effect on the
sodium current amplitude, and a slight reduction of the
peak current was observed at pH 6.5 (not shown). The
effect of Lqh3 on DmNav1 increased markedly upon
transition from basic to more acidic pH with an estimated half saturation at pH 7.2 (Fig. 3A–C). This increase
was slow and typically saturated after 10–15 min

(Fig. 3D). The slow kinetics was also evident when the
toxin was pre-equilibrated at the tested pH prior to
application onto the oocyte, suggesting that Lqh3
sensitivity to pH is associated with some later stage in
the binding process to the channel. This is corroborated by previous binding studies, which demonstrated
that Lqh3 association rate did not change between
pH 7.5 and 6.5, and the increased affinity was due to

Table 3. Effect of mutations on the pH dependence of Lqh3 binding to cockroach neuronal membranes. All binding experiments were performed using 125I-LqhaIT, a pH-independent marker of receptor site-3 [18], and the data represent mean ± SE of 2–4 independent experiments; ND, not determined. Ki (pH 8.5) ⁄ Ki (pH 7.2) represents the change in ratio when the analysis was performed at pH 8.5 versus 7.2.

Mutant

Ki, pH 6.5
(nM)

Ki, pH 7.2
(nM)

Ki, pH 8.5
(nM)

Ki (pH 8.5) ⁄
Ki (pH 7.2)

Unmodified Lqh3
Q8A
E10Y
H15A
H15L
H36A

H43A
H43R
H43E
E63R
H66A
H66R
P9C-E10C
E10Y-E63R

0.49
0.56
0.16
3.86
20
0.59
ND
0.13
64.5
ND
6.05
1.14
0.55
0.09

1.0
9.5
0.42
3.85
109
4.9

3.95
0.17
117
0.83
22.7
4.1
1.0
0.44

32.67
5.4
5.9
1633
7207
81.2
170
3.2
3112
6.8
141
14.4
34
2.93

32.7
0.56
14
424
66.3
16.6

43
18.8
26.6
8.19
6.2
3.5
34
6.7

1922

±
±
±
±
±
±

0.11
0.05
0.08
0.03
1.00
0.11

± 0.04
± 2.40
±
±
±

±

1.55
0.27
0.10
0.03

±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.1
2.5
0.13
0.65
19
1.9
0.05
0.04

12
0.18
1.3
0.3
0.14
0.14

±
±
±
±
±
±
±
±
±
±
±
±
±
±

6.67
1.6
1.7
497
1927
10.9
10
0.06

1589
0.8
34
1.25
4.5
0.14

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

a-Like toxin binding is linked to its flexibility

B

A

C o n tr ol
8.0
7.5

Normalized effect

1.0
0.8

7.0

0.6

0.4

6.5
1 μΑ

0.2
pH 7.1
pH 7.85

5 ms

0.0
0

10

1

10

2

10

3

10

4

10

[Toxin] (nM)

D

1.0

0.7
0.6

0.8

Normalized effect

Normalized effect

C

0.6

0.4

0.2

0.5
0.4
0.3
0.2

6.5

7.5

7.0

0

8.0

200

400

pH

600

800

Time (s)

Fig. 3. pH-dependent effect of Lqh3 on DmNav1 channels expressed in Xenopus oocytes. (A) Concentration-response relationship of Lqh3 at
pH 7.85 (s) and pH 7.1 (d). Data were fit using the Hill equation (Eqn. 1, Experimental procedures) and the EC50 values obtained were
86.6 ± 15.1 nM (n ¼ 3; pH 7.85) and 10.5 ± 1.6 nM (n ¼ 3; pH 7.1). (B) Effect of Lqh3 at various pH values. Oocytes were incubated with
50 nM Lqh3 dissolved in buffer at pH 8.0, and the toxin effect was continuously monitored by step depolarizations to )10 mV from a holding
potential of )80 mV. The toxin effect was allowed to saturate for 10 min and the external solution was then replaced by 50 nM Lqh3 in
pH 7.5 buffer. This procedure was repeated stepwise down to pH 6.5. Current traces from a representative oocyte are shown. (C) Toxin
effect (Iss ⁄ Ipeak) at each pH in the range 6.5–8.0 was normalized to the maximal effect obtained at pH 6.5 and plotted as a function of the
pH. Each point represents mean ± SEM from three oocytes. (D) Kinetics of the effect developed upon transition from pH 7.5 to pH 7.0 for

the cell presented in B. The steady-state to peak current ratio was determined at intervals of 15 s from the transition to pH 7.0 and is plotted against the incubation time. The kinetics was fit by a single exponential with s ¼ 496 s.

A

B

Q8

C

Q8

P9
C9
C12

C12
C10

E10

N11

N11

4.0Å

3.9Å

E10

H66

E63

Fig. 4. Conformations of the five-residue turn and the C-terminal segment of Lqh3. (A,B) Fixation of the peptide bond between residues 9
and 10 in Lqh3 in a cis conformation by an engineered vicinal disulfide bond. The five-residue turn of Lqh3 (A) is compared with its modeled
equivalent in the P9C-E10C mutant (B). The modeling was based on the structure of Lqh3, and energy minimized in vacuo using the
GROMOS96 implementation of Swiss-pdbViewer [39]. The arrows point to the cis peptide bond between residues 9 and 10. (C) Hydrogen
bond network that involves the sidechains of Glu10, Glu63 and His66.

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1923


a-Like toxin binding is linked to its flexibility

I. Karbat et al.

finding suggested that the two Cys residues were
indeed linked by a vicinal disulfide bond (Fig. 4B).
Still, the binding affinity of the double mutant
remained highly dependent on pH, similar to the
unmodified Lqh3 (Table 3). Therefore, we concluded
that the cis–trans isomerization of the P9-E10 peptide-bond was most likely unrelated to the pH
dependence of Lqh3.
To test the possibility that the pH-dependent binding of Lqh3 is associated with protonation of surface
histidines [18], we examined the effects of toxin
mutants H15A ⁄ L, H36A, H43A ⁄ R, and H66A ⁄ R ⁄ E

on the binding affinity for cockroach synaptosomes at
various pH values (Table 3). Whereas substitutions of
His15, His36 and His43 did not reduce Lqh3 sensitivity to pH, substitutions of His66 had a clear impact
with the utmost decrease obtained with H66R
(Table 3). Unexpectedly, substitutions of neutral or
negatively charged residues of the five-residue turn
(Q8A and E10Y) and C-tail (E63R), which were not
assigned to the bioactive surface, reduced markedly
the dependence of binding affinity on pH (Table 3).
Combined with the slow build up of Lqh3 effect on
DmNav1 upon pH transitions, these results indicate
that the dependence of Lqh3 binding on pH is not dictated by the protonation of His residues per se. These

A

LqhαIT

findings prompted us to examine structural features of
the NC domain that could explain its relatedness with
the pH dependency.
Lqh3 pH-dependent binding is associated with
the conformational flexibility of the C-tail
Inspection of Lqh3 solution structure reveals that the
C-terminal segment is by far more flexible than its
equivalent in LqhaIT (Fig. 5A,B). The conformational
heterogeneity focuses on a short loop spanning residues 60–64 (Fig. 5B), and is mediated by alternations
in a hydrogen bond network among the negatively
charged carboxyl groups of Glu10 and Glu63, and the
guanidinium moiety of His66 (Fig. 4C), substitution of
which clearly affected the pH-dependent binding of

Lqh3 (Table 3).
To examine whether changes in this hydrogen bond
network alter Lqh3 sensitivity to pH, we constructed a
double mutant, in which Tyr and Arg substituted
Glu10 and Glu63 to eliminate the intramolecular polar
interactions of His66 with these two Glu residues.
The binding affinity of the E10Y-E63R mutant to
cockroach neuronal membranes (Table 3), as well as
its potency at DmNav1 channels at neutral pH, was
similar to that of the unmodified toxin (Fig. 6A).

Lqh 3

B

r.m.s.d. ( )

3.0
2.5

Lqh3
Lqh IT

2.0
1.5
1.0
0.5
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66

Residue

1924

Fig. 5. Conformational heterogeneity in
LqhaIT and Lqh3. (A) Solution structures of
LqhaIT (PDB ID: 1LQI) and Lqh3 (PDB ID:
1FH3) in a ‘sausage’ representation. The Ca
carbon trace is depicted as a tube with a
radius proportional to the mean rmsd
observed within the various conformers in
the NMR ensemble. a-Helices are highlighted in red; b-strands are colored in cyan.
The arrows point to the C-terminal segment
of the molecule. (B) The rmsd of the Ca
atoms in the solution structures of LqhaIT
and Lqh3. For each model in the NMR
ensemble (LqhaIT )29 structures [41];
Lqh3–30 structures [15]) the rmsd of each
Ca atom was calculated using the mean
structure as reference. The rmsd of the individual models were averaged and presented
for each toxin residue.

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

a-Like toxin binding is linked to its flexibility

which suggested that structural flexibility rather than
rigidity had an important role on its function.

A 1.0

Normalized effect

0.8

Comparison of the bioactive surface of Lqh3
to those of other a-toxins

0.6
0.4
0.2

Lqh3
E10Y-E63R

0.0
10 -2

10 -1

10 0
10 1
[Toxin] nM

10 2

10 3

B 1.0

Normalized effect

0.8
0.6
0.4
0.2

Lqh3
E10Y-E63R

0.0
6.5

7.0

7.5

8.0

pH
Fig. 6. Effects of mutant E10Y-E63R on the properties of interaction with DmNav1 channels. (A) Concentration–response relations
of the unmodified Lqh3 (s) and mutant E10Y-E63R (n) at pH 7.0.
Data were fitted using Hill equation (Eqn 1, Experimental procedures) and the EC50 values obtained are: Lqh3–10.5 ± 1.6 nM (n ¼
4), E10Y-E63R )6.5 ± 1.2 nM (n ¼ 3). (B) pH-dependent effect of
E10Y-E63R (n) compared with the unmodified toxin (s). Data were
collected and analyzed as in Fig. 3.

Surprisingly, the pH-dependence of E10Y-E63R

mutant binding to cockroach sodium channels
decreased markedly (Table 3), and it was highly potent
at DmNav1 channels even at basic pH (Fig. 6B).

Discussion
Insight into the molecular basis of preferential interactions of scorpion a-toxins with insect or mammalian
Navs was thus far obtained mainly from mutagenesis
and comparison of bioactive surfaces and overall structures of pharmacologically distinct toxins. These analyses were based on available crystal structures of
a-toxins and their mutants and highlighted the NC
domain as a rigid structural entity, whose precise
topology dictates toxin specificity for various Nav subtypes [10,14,20]. Here we focused on the a-like toxin
Lqh3 because of its unique pharmacological features,

Molecular dissection of Lqh3 highlighted a bi-partite
functional surface composed of a Core domain and an
NC domain (Fig. 2), as was previously shown for the
anti-insect toxin LqhaIT [10]. The chemical nature of
the Core domain is highly conserved among various
scorpion a-toxins, and is predominated by positively
charged and aromatic ⁄ hydrophobic residues. In Lqh3,
substitution of Core-domain residues (His15, Phe17,
Pro18, Phe39 and Leu45) had a profound effect on the
binding energy (Table 1). Residue 15 (His or Glu in
a-like toxins) is especially peculiar: It was not assigned
to the bioactive surface of LqhaIT or BmK M1
[10–13,19], but in Lqh3 it seems to be within atomic
proximity of the channel receptor, because substitutions which increased its side chain volume
(H15F ⁄ L ⁄ R) reduced profoundly the binding affinity
(Table 1). In addition, residue 15 is involved in toxin
selectivity, as implied from the different effects of

mutant H15A on insect versus rat skeletal muscle Navs
(Table 2). Thus, the Core domain of Lqh3 plays an
important role in both, interaction with the receptor
site and toxin selectivity.
The NC domain, composed of the five-residue turn
and the C-terminal segment, varies in amino acid composition and conformation among a-toxins (Fig. 1),
and was therefore suggested to play a role in toxin
selectivity [10,13,14,19,22,23]. In scorpion a-toxins (e.g.
LqhaIT, Aah2, BmK M1 and Lqh3), residue 58 (59 in
Lqh3) is involved in an intricate network of intramolecular contacts, which contribute to C-tail stabilization relative to the molecule core. Therefore, chemical
modifications or substitutions at this region resulted in
a number of instances in marked alterations in structure and function [11,22–25]. Although the residue in
position 58 is conserved in most scorpion a-toxins
(Arg or Lys), its equivalent in a number of a-like toxins is hydrophobic ⁄ aliphatic (e.g. Ile59 in Lqh3;
Fig. 1), and is highly important for activity, as shown
in Lqh3 (Table 1). The mutagenic dissection highlighted the importance of the C-tail residues Ile59,
Lys64 and His66 for activity and selectivity, but not of
residues at the five-residue turn (Tables 2 and 3).
Whereas substitutions at the five-residue turn of
LqhaIT and BmK M1 were shown to greatly affect
the activity [10,11,19], mutagenesis at this region in
Lqh3 had no effect (Table 1), suggesting that this
structural motif was not involved in direct contact with

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1925


a-Like toxin binding is linked to its flexibility

I. Karbat et al.

the channel receptor site. Still, the entire NC domain
is important for activity as indicated by the effect of
substitutions at the five-residue turn and C-tail on
toxin potency and its pH-dependent binding to insect
Navs (Table 2).
Dissociation of the toxin-receptor complex and
the slow association kinetics of Lqh3 are linked
to the flexibility of the C-tail
The substantial decrease in the sensitivity of binding to
alterations in pH of Lqh3 mutants modified at the NC
domain in residues other than His (Table 3), as well as
the slow onset of Lqh3 effect upon pH transitions
(Fig. 3D), have raised the possibility that the NC
domain undergoes a slow conformational change along
the toxin binding process with the channel. Close
inspection of the published Lqh3 solution structure

[15,21] has indicated a high degree of conformational
heterogeneity of the NC domain especially around the
short loop spanning residues 60–64. Detailed analysis
of the various backbone conformations of this loop
have suggested that the majority (26 out of 29) of
Lqh3 NMR models are divided between two main
populations (Figs 7A,B), in which the overall topology
of the NC domain varies greatly (Fig. 7B–F). They differ in the side chains of His66, Glu10 and Glu63,
which project to nearly opposite directions
(Figs 7C,D), and in the intramolecular contacts among

Gln8, Glu10 (of the five-residue turn), Glu63 and
His66 (Fig. 7C–F), whose substitution had profound
effects on Lqh3 pH-dependent binding (Table 3). As a
result, the five-residue turn adopts different conformations, although in both populations the backbone
torsion angles around the Pro9–Glu10 bond are
restricted to allow for a cis conformation. Exchange of

Fig. 7. Lqh3 solution structure exhibits two
distinct conformations at its C-terminal segment. (A) Lqh3 NMR ensemble (PDB ID:
1FH3) was divided into two separate populations, designated group A (16 structures)
and group B (10 structures), and for each
group, a geometric average structure was
calculated using MOLMOL [42]. The averaged
rmsd of the backbone atoms of residues
57–67 from the average structure is presented for each group, as well as for the complete NMR ensemble. Three structures,
which exhibited great structural variations
and could not be classified into these two
groups were omitted for clarity. (B) Ca trace
for residues 60–64 of NMR structures classified to group A (red) or group B (blue).
(C,D) The side chains of Gln8, Glu10, Glu63
and His66, whose substitution affected
Lqh3 pH-dependent binding, is presented
for two individual NMR structures that represents two extreme conformations typifying the group A (C) and group B (D)
structure populations. (E,F) Comparison of
the overall topology and disposition of the
NC domain relative to the molecule core in
group A (E) versus group B (F) model. NCdomain residues are colored as in (C, D); for
all other residues only backbone atoms are
displayed (gray).

1926

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

conformations between the two populations would
involve the formation and breakdown of hydrogen
bonds and changes in the tilt and twist angles of the
backbone, and should be sensitive to the pH of the
medium. As His residues contribute in part the hydrogen bonds that differ in the two molecule populations
(His66, His43; Fig. 7C,D), such a conformational
change may provide the basis for the dependence of
Lqh3 binding on pH. This hypothesis is supported by
the decreased sensitivity to pH of the E10Y-E63R
mutant, in which key Glu residues that participate in hydrogen bond formation were eliminated
(Fig. 4C).
On the basis of these structural observations and the
unchanged toxin association rate under various pH
values, as well as the slower toxin dissociation rate at
low pH [18], we speculate that upon toxin binding to
the channel, acidic pH favors toxin conformation with
high affinity for the receptor, and reduces the probability that the bound toxin spontaneously convert to
unfavorable conformations, hence stabilizing the
toxin-receptor complex. In the case of the E10Y-E63R
mutations, elimination of critical hydrogen bonds
(Fig. 7) allows it to assume conformation favourable
for the receptor at a wider pH range.
To explain the mechanism of slow association of

Lqh3 to various Navs, we propose that the rate-limiting step that governs Lqh3 binding is a slow transition
between the two conformational populations of the
toxin depicted in Fig. 7. In the course of Lqh3 binding
to its Nav receptor, specific toxin conformers are selected from a dynamic ensemble of structures with various C-tail conformations (Fig. 7). Such a mechanism
may also rationalize the broad-range potency of this
toxin on insect as well as mammalian peripheral and
brain Navs [5,16,18]. A similar explanation might hold
for the slow effect on toxicity and a broad range of
activity of the site-3 sea anemone toxin Av2 [26], in
which the bioactive surface involves a highly flexible
Arg14 loop [26–28]. The paradigm of dynamic conformer selection was recently demonstrated for the
interaction between the cleavage factor component
pcf11 and the C-terminal domain of RNA polymerase II
[29]. This C-terminal domain was found to exist in
solution as a dynamic disordered ensemble of conformers, and upon binding to pcf11 it assumed a structured conformation via induced fit. This adaptation
ability enables RNA polymerase II C-terminal domain
region to bind specifically a broad range of factors
involved in mRNA processing [29]. By analogy, the
ability of Lqh3 and possibly other members of the
a-like group to affect a wide range of Nav subtypes
may be attributed to their conformational flexibility.

a-Like toxin binding is linked to its flexibility

Experimental procedures
Bacterial strains and insects
Escherichia coli DH5a was used for plasmid constructions,
and the BL21 (DE3, pLys) strain was used for toxin expression using the pET-14b vector as was described previously
[30,31]. Sarcophaga falculata blowfly larvae were bred in
the laboratory.

Lqh3 expression
For expression in E. coli we used the cDNA encoding Lqh3
isolated from a cDNA library constructed from the RNA
of the scorpion L. quinquestriatus hebraeus. Because expression of Lqh3 using the pET-11c vector, as was described
for the toxin LqhaIT [19], was poor, we used a fusionpartner strategy, whereby the N-terminus of Lqh3 was
extended by fusion to a Histag-Apamin-linker (HA-Lqh3).
Two oligonucleotide primers were used to construct
HA-Lqh3 using the pET-14b vector as template DNA.
Primer 1, 5¢- GGCAGCCATATGTGTAATTGTAAGGCA
CCAGAAACTGCACTTTGCGC-3¢, was designed to add
a sequence encoding Apamin and a linker cleavable by
thrombin and Fx proteases at an NdeI site behind the
Histag. Apamin folds well in vitro [32] and was added with
the anticipation for improved folding of the Lqh3 sequence
behind. The 3¢ region of this primer included 11 bases
that overlapped the 5¢ region of Lqh3-cDNA. Primer 2,
5¢- GGATCCGGCTGCTAACAAAGCCCGAAAGG-3¢, was
designed for the opposite strand in reverse orientation at
the 3¢ side of the Lqh3 gene, and contained a BamHI
restriction site for insertion into pET-14b. The PCR conditions were 30 cycles of 1 min at 94 °C, 1 min at 45 °C, and
1 min at 72 °C. The final product was cleaved by NdeI and
BamHI and cloned into the corresponding restriction sites
in the polylinker of pET-14b. The recombinant Lqh3,
which accumulated in inclusion bodies, was folded in vitro
following denaturation (in 6 m guanidinium-HCl, 0.1 m
Tris ⁄ HCl pH 8.0, 1 mm EDTA, 30 mm reduced glutathione) and renaturation (by 100-fold dropwise dilution into a
0.2 m ammonium acetate pH 8.0, 0.2 mm oxidized glutathione) at 18 °C for 16–24 h. The soluble material was precipitated with 4 m ammonium sulfate at 4 °C for 16 h,
collected by filtration (GF ⁄ C Whatman paper) and suspended in H2O. Final purification of the recombinant toxin
was performed on a Vydac C18 reverse phase HPLC column, and HA-Lqh3 eluted as a single peak at 32% acetonitrile with a typical yield of 2 mg toxin per liter of E. coli

culture. The high yield of recombinant toxin seems to
involve both higher yield of inclusion bodies and improved
in vitro folding of the fusion polypeptide. The recombinant
HA-Lqh3 exhibited a very similar activity to that of the
native Lqh3 (purchased from Latoxan, Valence, France), in

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1927


a-Like toxin binding is linked to its flexibility

I. Karbat et al.

Table 4. Activity of recombinant HA-Lqh3 and native Lqh3.
Assay
Toxicity to blowfly larvae,
ED50 (ng ⁄ 100 mg)
Binding to cockroach synaptosomes,
Ki (nM)
Potency on Nav1.4 channels,
EC50 (nM)

HA-Lqh3

Native Lqh3

56 ± 7
1.93 ± 0.9

5.4 ± 1.2b

25 ± 3
1.24 ± 0.23a
4.23 ± 0.52c

[16]. b rNav1.4 expressed in CHO cells (this study).
expressed in human embryonic kidney (HEK) cells [6].

a

c

rNav1.4

toxicity assays to blowfly larvae, binding affinity to cockroach neuronal membranes, and potency to rat skeletal
muscle rNav1.4 channels expressed in mammalian cells
(Table 4). These results corroborated previous observations
that extension of the N-terminus of long-chain scorpion
toxins does not impair their activity [31]. Therefore, most
assays were conducted with HA-Lqh3 derivatives without
further cleavage and purification of the Lqh3 moiety.

analytical ResourceÒ RP-HPLC column (6.4 · 100 mm,
15 lm particle size; Amersham, Bjorkgatan, Sweden). The
ă
concentration of the radiolabeled toxin was determined
according to the specic activity of the 125I corresponding
to 2500–3000 dpmỈfmol)1 of monoiodotoxin, depending on
the age of the radiotoxin and by estimation of its biological

activity (usually 70–80%). Composition of media used in
the binding assays and termination of the reactions have
been previously described [18]. Non-specific toxin binding
was determined in the presence of 1 lm of the unlabeled
toxin. Equilibrium competition binding assays were
performed using increasing concentrations of unlabeled
toxin in the presence of a constant low concentration
of 125I-LqhaIT, and analyzed by the computer program
kaleidagraph (Synergy Software, Reading, PA, USA)
using a nonlinear Hill equation (for IC50 determination).
Ki values were calculated using the equation Ki ¼
IC50 ⁄ (1 + (L* ⁄ Kd)), where L* is the concentration of
radioiodinated toxin and Kd is its dissociation constant.
Each experiment was performed in duplicate and repeated
at least three times as indicated (n). Data are presented as
mean ± SE of the number of independent experiments.

Mutagenesis
Mutations in the cDNA encoding Lqh3 were introduced
via PCR using complementary oligonucleotide primers. All
toxin mutants were produced similarly to the unmodified
toxin and all sequences were verified before expression.

Toxicity assays
Four-day-old blowfly larvae (S. falculata; 150 ± 20 mg
body weight) were injected intersegmentally. A positive
result was scored when a characteristic contraction was
observed up to 5 min after injection. Five concentrations of
each toxin were injected to larvae (nine larvae in each
group) in three independent experiments. Effective dose

50% (ED50) values were calculated according to the sampling and estimation method of Reed and Muench [33].
Notably, even in doses exceeding the ED50, the toxin effect
was substantially delayed compared to the effect induced
by equivalent doses of the a-toxin LqhaIT, and fully developed 3–5 min post injection.

Competition binding experiments
Neuronal membranes were prepared from heads of adult
cockroaches Periplaneta americana [18]. Membrane protein
concentration was determined by a Bio-Rad (Hercules, CA,
USA) Protein Assay, using bovine serum albumin as standard. Radioiodinated LqhaIT was prepared by lactoperoxidase (Sigma, St Louis, MO, USA; 7 U per 60 lL reaction
mix) using 10 lg toxin and 0.5 mCi carrier-free Na125I
(Amersham, Chalfont St Giles, UK) following a published
protocol [34]. The monoiodotoxin was purified using an

1928

CD spectroscopy
CD spectra were recorded at 25 °C using a model 202 circular dichroism spectrometer (Aviv Instruments, Lakewood, NJ, USA). HA-Lqh3 and mutants thereof (150 lm)
were dissolved in 5 mm sodium phosphate buffer, pH 7.0
and their spectrum was determined using a quartz cell of
0.1-mm light path. Each spectrum was recorded three times
and averaged. Blank spectrum was subtracted from each
curve.

Expression of insect DmNav1 channels in oocytes
and two-electrode voltage clamp experiments
cRNAs encoding the D. melanogaster para (DmNav1) Nav
a-subunit, and the auxiliary TipE subunit (kindly provided
by J. Warmke, Merck, Whitehouse Station, NJ, USA, and
M. S. Williamson, IACR-Rothamsted, UK, respectively),

were transcribed in vitro using T7 RNA-polymerase and the
mmessage mmachineTM system (Ambion, Austin, TX,
USA [35,36]); and injected into Xenopus laevis oocytes as
was previously described [37]. Currents were measured
4–5 days after injection using a two-electrode voltage clamp
and a Gene Clamp 500 amplifier (Axon Instruments, Union
City, CA, USA). Data were sampled at 10 kHz and filtered
at 5 kHz. Data acquisition was controlled by a Macintosh
PPC 7100 ⁄ 80 computer, equipped with ITC-16 analog ⁄
digital converter (Instrutech Corp., Port Washington, NY,
USA), utilizing Synapse (Synergistic Systems, Sweden).
Capacitance transients and leak currents were removed by
subtracting a scaled control trace utilizing a P ⁄ 6 protocol
[36]. Bath solution contained (in mm): 96 NaCl, 2 KCl,

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

1 MgCl2, 2 CaCl2, 5 Hepes, pH 7.85. Oocytes were washed
with bath solution flowing from a BPS-8 perfusion system
(ALA Scientific Instruments, Westbury, NY, USA) with a
positive pressure of 4 psi. Toxins were diluted with bath
solution containing 1 mgỈmL)1 bovine serum albumin, and
applied directly to the bath to the final desired concentration. To discard any application artifacts, 1 mgỈmL)1
bovine serum albumin solution was applied before toxin
application.

Expression of Nav1.4 channels in CHO cells

and whole-cell patch clamp recording
CHO cells were maintained in F12 medium, supplemented
with 10% fetal calf serum, in a 5% CO2 incubator. Transient transfection was achieved using FuGENE 6 (Roche
Applied Science, Manheim, Germany) with a 1 : 0.3 ratio
of the pAlter expression vector encoding rNav1.4 and with
a vector encoding the CD8 antigen [38]. Individual transfected cells were visualized with Dynabeads (Deutsche
Dynal GmbH, Hamburg, Germany) binding to CD8. Currents were recorded 2–3 days after transfection. Whole-cell
voltage clamp experiments were conducted using an Axopatch 200B amplifier (Axon Instruments) at room temperature. Data were acquired with a Macintosh G4 computer
equipped with an ITC-16 analog-to-digital converter
(Instrutech Corp., Port Washington, NY, USA) using
synapse software (Synergistic Systems). Currents were lowpass filtered at 5 kHz and sampled at a rate of 10 kHz. Cell
and electrode capacitance and series resistance were compensated with an internal voltage clamp circuit. Residual
linear leak and capacitance were removed by subtracting
scaled control traces using P ⁄ 6 protocol [36]. The patch
pipette contained 35 mm NaCl, 105 mm CsF, 10 mm
EGTA, and 10 mm Hepes (adjusted to pH 7.4 with CsOH).
The bath solution contained 140 mm NaCl, 5 mm CsCl,
1.8 mm CaCl2, 1 mm MgCl2, 2 mm Na2ATP, and 10 mm
Hepes (adjusted to pH 7.4 with NaOH). Toxins were dissolved in the bath solution containing 1% bovine serum
albumin, and perfusion of the cells was conducted with a
flow pipe glass barrel (400 lm, outer diameter) positioned
100 lm from the cell.

Concentration–response curves of Lqh3 effect
on fast inactivation
For the construction of concentration-response curves, currents were elicited by a depolarization to )10 mV from a
holding potential of )80 mV in the presence of several toxin
concentrations. At each toxin concentration, the currents
were allowed to reach a steady-state level prior to the final
measurement. The concentration-dependence for toxininduced removal of fast inactivation was calculated by plotting the ratio of the steady-state current remaining 50 ms

a-Like toxin binding is linked to its flexibility

after depolarization (Iss) to the peak current (Ipeak) as a function of toxin concentration and fitting with the Hill equation
Iss
a1 a0
ẳ a0 ỵ
1ị

H
Ipeak
EC50
1 ỵ ẵToxin
where H is the Hill coefficient, [Toxin] is the toxin concentration, and a0 is the offset measured prior to toxin application. The amplitude a1 ) a0 provides the maximal effect
obtained at saturating toxin concentrations. EC50 is the
concentration of half maximal inhibition of fast inactivation. To reduce variability, H was set to 1 in all cases.

Mass spectrometry
The mass of unmodified Lqh3 and that of the Lqh3 P9CE10C mutant (after Fx cleavage) was determined by the
Mass Spectrometry Unit of the Weizmann Institute (Rehovot, Israel) using a MALDI mass spectrometer, Bruker
REFLEXTM reflector time-of-flight instrument with
SCOUTTM multiprobe (384) inlet and gridless delayed
extraction ion source, with accuracy of 0.001 Da.

Three-dimensional modeling and structural
analysis
Three-dimensional models were prepared using pymol
(Delano Scientific LLC, ). Conformational variability of Lqh3 solution structure was assessed
with deepview/pdb viewer version 3.7 [39] using custom script for the extraction and comparison of backbone
atom coordinates.

Acknowledgements
We are thankful to J. Warmke, Merck, Whitehouse
Station, NJ, USA, and M. S. Williamson, IACRRothamsted, UK, for the kind gift of DmNav1. and
TipE clones, respectively, and to Y. Moran, Tel Aviv
University, for fruitful discussions. This research was
supported by the United States–Israel Binational Agricultural Research and Development grant IS-3480–03
(to MG and DG); by the Israeli Science Foundation,
grants 733 ⁄ 01 (to MG) and 1008 ⁄ 05 (to DG); by a
grant from the GIF, the German–Israeli Foundation for Scientific Research and Development No.
G-770–242.1 ⁄ 2002 (to DG), and by the German
Research Association HE2993 ⁄ 5 (to SHH).

References
1 Gordon D, Savarin P, Gurevitz M & Zinn-Justin S
(1998) Functional anatomy of scorpion toxins affecting
sodium channels. J Toxicol Toxin Rev 17, 131–158.

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1929


a-Like toxin binding is linked to its flexibility

I. Karbat et al.

`
2 Cestele S & Catterall WA (2000) Molecular mechanisms
of neurotoxin action on voltage-gated sodium channels.

Biochimie 82, 883–892.
3 Rogers JC, Qu Y, Tanada TN, Scheuer T & Catterall
WA (1996) Molecular determinants of high affinity
binding of a-scorpion toxin and sea anemone toxin in
the S3–S4 extra-cellular loop in domain IV of the
sodium channel a subunit. J Biol Chem 271, 15950–
15962.
4 Leipold E, Lu S, Gordon D, Hansel A & Heinemann
SH (2004) Combinatorial interaction of scorpion toxins
Lqh2, Lqh3 and LqhaIT with sodium channel receptor
sites-3. Mol Pharmacol 65, 685–691.
5 Gilles N, Chen H, Wilson H, Le Gall F, Montoya G,
Molgo J, Schonherr R, Nicholson G, Heinemann SH &
Gordon D (2000) Scorpion a- and a-like toxins differentially interact with sodium channels in mammalian CNS
and periphery. Eur J Neurosci 12, 2823–2832.
6 Chen H, Gordon SH & Heinemann S (2000) Modulation of cloned skeletal muscle sodium channels by the
scorpion toxins Lqh II, Lqh III, and LqhaIT. Pflugers
Arch - Eur J Physiol 439, 423–432.
7 Gordon D, Gilles N, Bertrand D, Molgo J, Nicholson
GM, Sauviat MP, Benoit E, Shichor I, Lotan I,
Gurevitz M et al. (2002) Scorpion toxins differentiating
among neuronal sodium channel subtypes: Nature’s
guide for design of selective drugs. In Perspectives in
Molecular Toxinology (Menez, A, ed.), pp. 215–238.
Wiley & Sons, Chichester.
`
8 Gordon D, Martin-Eauclaire MF, Cestele S, Kopeyan
C, Carlier E, Ben Khalifa R, Pelhate M & Rochat H
(1996) Scorpion toxins affecting sodium current inactivation bind to distinct homologous receptor sites on rat
brain and insect sodium channels. J Biol Chem 271,

8034–8045.
9 Possani LD, Becerril B, Delepierre M & Tytgat J (1999)
Scorpion toxins specific for Na+-channels. Eur J Biochem 264, 287–300.
10 Karbat I, Frolow F, Froy O, Gilles N, Cohen L,
Turkov M, Gordon D & Gurevitz M (2004) Molecular
basis of the high insecticidal potency of scorpion alphatoxins. J Biol Chem 279, 31679–31686.
11 Wang CG, Gilles N, Hamon A, Gall FL, Stankiewicz
M, Pelhate M, Xiong YM, Wang DC & Chi CW (2003)
Exploration of the functional site of a scorpion a-like
toxin by site directed mutagenesis. Biochemistry 42,
4699–4708.
12 Sun YM, Bosmans F, Zhu RH, Goudet C, Xiong YN,
Tytgat J & Wang DC (2003) Importance of the conserved aromatic residues in the scorpion a-like toxin
BmK M1. J Biol Chem 278, 24125–24131.
13 Liu LH, Bosmans F, Maertens C, Zhu RH, Wang DC
& Tytgat J (2005) Molecular basis of the mammalian
potency of the scorpion alpha-like toxin, BmK M1.
FASEB J 19, 594–596.

1930

14 Guan RJ, Xiang Y, He XL, Wang CG, Wang M,
Zhang Y, Sundberg EJ & Wang DC (2004) Structural mechanism governing cis and trans isomeric
states and an intramolecular switch for cis ⁄ trans
isomerization of a non-proline peptide bond observed
in crystal structures of scorpion toxins. J Mol Biol
341, 1189–1204.
15 Krimm I, Gilles N, Sautiere P, Stankiewicz M, Pelhate
M, Gordon D & Lancelin JM (1999) NMR structures
and activity of a novel a-like toxin from the scorpion.

Leiurus Quinquestriatus Hebraeus J Mol Biol 285, 1749–
1763.
16 Gilles N, Blanchet B, Shichor I, Zaninetti M, Lotan I,
Bertrand D & Gordon D (1999) A scorpion a-like toxin
active on insects and mammals reveals an unexpected
specificity and distribution of sodium channel subtypes
in rat brain neurons. J Neurosci 19, 8730–8739.
17 Chen H & Heinemann SH (2001) Interaction of
scorpion alpha-toxins with cardiac sodium channels:
binding properties and enhancement of slow
inactivation. J General Physiol 117, 505–518.
18 Gilles N, Krimm I, Bouet F, Froy O, Gurevitz M,
Lancelin JM & Gordon D (2000) Structural implications on the interaction of scorpion a-like toxins with
the sodium channel receptor site inferred from toxin
iodination and pH-dependent binding. J Neurochem 75,
1735–1745.
`
19 Zilberberg N, Froy O, Loret E, Cestele S, Arad D,
Gordon D & Gurevitz M (1997) Identification of structural elements of a scorpion alpha-neurotoxin important
for receptor site recognition. J Biol Chem 272, 14810–
14816.
20 Ye X, Bosmans F, Li C, Zhang Y, Wang DC & Tytgat
J (2005) Structural basis for the voltage-gated Na channel selectivity of the scorpion a-like toxin BmK M1.
J Mol Biol 353, 788–803.
21 Krimm I, Volpon L & Lancelin JM (2002) Redundant
peptidic isomerisms revealed by NMR of animal toxins.
In Toxines et Recherches Biome´dicales (Goudey-Perriere
F Bon C Puiseux-Dao S & Sauviat MP, eds), pp. 183–
192. Elsevier, Paris.
´

22 Legros C, Ceard B, Vacher H, Marchot P, Bougis PE &
Martin-Eauclaire MF (2005) Expression of the standard
scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements.
Biochim Biophys Acta 1723, 91–99.
23 He XL, Li HM, Zeng ZH, Liu XQ, Wang M & Wang
DC (1999) Crystal structures of two alpha-like scorpion
toxins: non-proline cis peptide bonds and implications
for new binding site selectivity on the sodium channel.
J Mol Biol 292, 125–135.
24 Darbon H, Jover E, Couraud F & Rochat H (1983)
Alpha-scorpion neurotoxin derivatives suitable as potential markers of sodium channels. Preparation and characterization. Int J Pept Protein Res 22, 179–186.

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS


I. Karbat et al.

25 Fontecilla-Camps JC, Habersetzer-Rochat C & Rochat
H (1988) Orthorhombic crystals and three-dimensional
structure of the potent toxin II from the scorpion
Androctonus australis Hector. Proc Natl Acad Sci USA
85, 7443–7447.
26 Moran Y, Cohen L, Kahn R, Karbat I, Gordon D &
Gurevitz M (2006) Expression and mutagenesis of the
sea anemone toxin Av2 reveals key amino acid residues
important for activity on voltage-gated sodium channels. Biochemistry 45, 8864–8873.
27 Norton RS (1991) Structure and structure–function relationships of sea anemone proteins that interact with the
sodium channel. Toxicon 29, 1051–1084.
28 Blumenthal KM & Seibert AL (2003) Voltage-gated
sodium channel toxins: poison, probes, and future promise. Cell Biochem Biophys 38, 215–238.

29 Noble CG, Hollingworth D, Martin SR, EnnisAdeniran V, Smerdon SJ, Kelly G, Taylor IA & Ramos
A (2005) Key features of the interaction between Pcf11
CID and RNA polymerase II CTD. Nat Struct Mol
Biol 12, 144–151.
30 Froy O, Zilberberg N, Gordon D, Turkov D, Gilles N,
Stankiewicz M, Pelhate M, Loret E, Oren D, Shaanan
B et al. (1999) The putative bioactive surface of insectselective scorpion excitatory neurotoxins. J Biol Chem
274, 5769–5776.
31 Cohen L, Karbat I, Gilles N, Ilan N, Gordon D &
Gurevitz M (2005) Common features in the functional
surface of scorpion b-toxins and elements that confer
specificity for insect and mammalian voltage-gated Nachannels. J Biol Chem 280, 5045–5053.
32 Pease JH, Storrs RW & Wemmer DE (1990) Folding
and activity of hybrid sequence, disulfide-stabilized peptides. Proc Natl Acad Sci USA 87, 5643–5647.
33 Reed L & Muench H (1938) A simple method of estimating fifty-percent endpoint. Am J Hyg 27, 493–497.

a-Like toxin binding is linked to its flexibility

34 Rochat C, Tessier M, Miranda F & Lissitzky S (1977)
Radioiodination of scorpion and snake neurotoxins.
Anal Biochem 82, 532–548.
35 Gershon E, Weigl L, Lotan I, Schreibmayer W &
Dascal N (1992) Protein-kinase A reduces voltagedependent Na+ current in Xenopus oocytes. J Neurosci
12, 3743–3752.
36 Wallner M, Weigl L, Meera P & Lotan I (1993) Modulation of the skeletal muscle sodium channel a-subunit
by the b1-subunit. FEBS Lett 336, 535–539.
37 Shichor I, Zlotkin E, Ilan N, Chikashvili D, Stuhmer
W, Gordon D & Lotan I (2002) Domain 2 of Drosophila Para voltage-gated sodium channel confers insect
properties to a rat brain channel. J Neurosci 22, 4364–
4371.

38 Jurman ME, Boland LM & Yellen G (1994) Visual
identification of individual transfected cells for electrophysiology using antibody-coated beads. Biotechniques
17, 876–881.
39 Guex N & Peitsch MC (1997) swiss-model and the
swiss-pdbviewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723.
`
40 Hamon A, Gilles N, Sautiere P, Martinage A,
Kopeyan C, Ulens C, Tytgat J, Lancelin JM & Gordon D
(2002) Characterization of scorpion a-like toxin group
using two new toxins from the scorpion. Leiurus
Quinquestriatus Hebraeus Eur J Biochem 269, 3920–
3933.
41 Tugarinov V, Kustanovich I, Zilberberg N, Gurevitz M
& Anglister J (1997) Solution structure of a highly
insecticidal recombinant scorpion a-toxin and a mutant
of increased activity. Biochemistry 36, 2414–2424.
42 Koradi R, Billeter M & Wuthrich K (1996) molmol: a
program for display and analysis of macromolecular
structures. J Mol Graph 14, 29–32.

FEBS Journal 274 (2007) 1918–1931 ª 2007 The Authors Journal compilation ª 2007 FEBS

1931