Báo cáo khoa học: The equinatoxin N-terminus is transferred across planar lipid membranes and helps to stabilize the transmembrane pore pot

The equinatoxin N-terminus is transferred across planar
lipid membranes and helps to stabilize the transmembrane
pore
Katarina Kristan
1
, Gabriella Viero
2
, Peter Mac
˘
ek
1
, Mauro Dalla Serra
2
and Gregor Anderluh
1
1 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
2 ITC-CNR Institute of Biophysics, Unit at Trento, Trento, Italy
Pore-forming toxins (PFTs) comprise one of the most
widespread groups of natural toxins. They have the
unusual characteristic of existing in two different
states: they are synthesized as soluble monomers,
which spontaneously insert into cellular and model
membranes to form transmembrane pores that are per-
meable to various compounds [1,2]. The formation of
transmembrane pores disrupts ion gradients, which
leads to osmotic swelling and ultimately to cell death.
PFTs differ in sequence and structure, but the major
steps of pore formation are similar. The first step is
attachment to the membrane surface, which is a non-
specific or a specific process, with the receptor being a
membrane protein or a lipid molecule; for example,

cholesterol-dependent cytolysins from Gram-positive
bacteria only bind to membranes including cholesterol
[3]. Binding to the membrane is essential for oligomeri-
zation, as it enables a high concentration and correct
orientation of molecules on the host cell membrane.
After initial attachment, PFTs undergo a series of
Keywords
actinoporin; equinatoxin; planar lipid
membrane; pore-forming toxin; pore
structure
Correspondence
M. Dalla Serra, ITC-CNR Institute of
Biophysics, Unit at Trento, Via Sommarive
18, 38050 Povo, Trento, Italy
Fax: +39 0461 810628
Tel: +39 0461 314156
E-mail:
G. Anderluh, Department of Biology,
Biotechnical Faculty, University of Ljubljana,
Vec
˘
na pot 111, 1000 Ljubljana, Slovenia
Fax: +386 1 257 3390
Tel: +386 1 42 333 88
E-mail:
(Received 27 August 2006, revised 11
November 2006, accepted 21 November
2006)
doi:10.1111/j.1742-4658.2006.05608.x
Equinatoxin II is a cytolytic protein isolated from the sea anemone Acti-

nia equina. It is a member of the actinoporins, a family of eukaryotic pore-
forming toxins with a unique mechanism of pore formation. Equinatoxin II
is a 20 kDa cysteineless protein, with sphingomyelin-dependent activity.
Recent studies showed that the N-terminal region of the molecule requires
conformational flexibility during pore formation. An understanding of the
N-terminal position in the final pore and its role in membrane insertion
and pore stability is essential to define the precise molecular mechanism of
pore formation. The formation of pores and their electrophysiologic char-
acteristics were studied with planar lipid membranes. We show that amino
acids at positions 1 and 3 of equinatoxin II are exposed to the lumen of
the pore. Moreover, sulfhydryl reagents and a hexa-histidine tag attached
to the N-terminus revealed that the N-terminus of the toxin extends
through the pore to the other (trans) side of the membrane and that negat-
ively charged residues inside the pore are crucial to define the electrophysio-
logic characteristics of the channel. Finally, we detected a new, less stable,
state with a lower conductance by using a deletion mutant in which the
first five N-terminal amino acids were removed. We propose that the first
five amino acids help to anchor the amphipathic helix on the trans side of
the membrane and consequently stabilize the final transmembrane pore.
Abbreviations
EqtII, equinatoxin II; His
6
–EqtII, fusion protein with hexa-histidine tag attached to the N-terminus of equinatoxin II; MTS, methane-
thiosulfonate; MTSEA
+
, (2-aminoethyl)-methanethiosulfonate hydrobromide; MTSES
–
, sodium (2-sulfonato-ethyl)-methanethiosulfonate;
MTSET
+

, (2-trimethylamonium)-ethyl methanethiosulfonate; PFT, pore-forming toxin; PLM, planar lipid membrane; SM, sphingomyelin.
FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS 539
conformational changes that expose previously hidden
hydrophobic parts for the interaction with the
membrane lipids. PFTs are commonly divided into
two subgroups according to structural elements of
the transmembrane pore. The pores of b-PFTs are
transmembrane b-barrels formed by interconnected
b-hairpins. Examples include the well-known Staphylo-
coccus aureus a-toxin, cholesterol-dependent cytolysins
and anthrax toxin protective antigen [4,5]. On the
other hand, a-PFTs build pores from amphipathic
a-helices [6]. The most notable examples are colicins
[7] and actinoporins [8]. Transmembrane b-barrel pores
are structurally stable, whereas pores formed by
a-helices are unstable, and consequently there is less
structural information available. Because of the above-
mentioned properties, PFTs comprise a useful and
unique model with which to study protein unfolding
on the surface of the lipid bilayer [9], oligomerization
in a hydrophobic environment and membrane protein
structure [10], protein–protein interactions on the
surface of the membrane [11], etc. In addition, PFTs
are particularly interesting as potential tools in
biotechnology; that is, they can be used for selective
killing of cancer cells, with built-in biological ‘triggers’
that activate in response to specific biological stimuli
[12,13], or they can be used in biosensor technology [14].
Actinoporins, cytolytic toxins synthesized by sea
anemones, comprise a unique group of PFTs [8]. They

comprise a group of 20 kDa, cysteineless proteins,
whose activity depends on the presence of sphingomye-
lin (SM) in the membrane. The most studied represen-
tative actinoporins are equinatoxin II (EqtII), isolated
from the sea anemone Actinia equina, and sticho-
lysin II, from Stichodactyla helianthus. Actinoporins
form cation-selective pores approximately 2 nm in
diameter on the surface of the target cell [15,16], lead-
ing to cell lysis through colloid osmotic shock. The
three-dimensional structures of EqtII and sticholysin II
monomers were determined in solution [17–19]. The
molecule is composed of a tightly folded hydrophobic
b-sandwich core, flanked on two faces by a-helices
(Fig. 1A). The first 30 N-terminal amino acids, inclu-
ding an amphipathic a-helix, form the only part that
can detach from the core of the molecule without
disrupting the general fold of the protein [17,19].
Recently, it has been shown that this is the only part
of the molecule that undergoes major conformational
changes during pore formation, and that its flexibility
is essential for formation of the final pore [20,21]. Act-
inoporin pore formation proceeds in distinct steps. The
initial attachment to the membrane is achieved by a
cluster of exposed aromatic amino acids situated on
the broad loops at the bottom of the molecule and the
C-terminal a-helix [20,22,23], and by a recently defined
phosphorylcholine-binding site [19]. In the next step,
the N-terminal a-helix detaches from the core of the
molecule and inserts into the lipid–water interface,
where it lies flat on the membrane [19,20,24]. Finally,

four toxin monomers oligomerize and form the pore
by inserting the N-terminal a-helix through the mem-
brane [24]. The final functional pore is thus composed
of four amphipathic helices from four monomers
[15,16,25] and most probably also by membrane lipids
in a so-called toroidal pore arrangement [26,27].
The pores formed by actinoporins have not yet been
directly visualized. Most previous experiments were
focused on the N-terminal a-helix, which extends from
Ser15 to Leu26 in solution [17,18] and from Asp10 to
Asn28 in a hydrophobic membrane environment [24].
The question remains of how the region that comprises
residues 1–10 is organized in the final pore, and what
the nature of the contacts is between the monomers,
which should stabilize the toroidal pore [19]. Thus, the
purpose of this work was to gain further insight into
the structure of the EqtII pore, especially the topology
of the first five N-terminal amino acids. We demon-
strated that the N-terminus is positioned on the trans
side of the membrane. Furthermore, the amino acids
at positions 1 and 3 are exposed to the ion conductive
pathway, and the N-terminus helps to stabilize the
final pore.
Results
Characterization of transmembrane channels
formed by EqtII, single cysteine and deletion
mutants
In this study, we used planar lipid membranes (PLMs)
and three N-terminal EqtII mutants to study the topo-
logy of the N-terminus in the final transmembrane

pore (Fig. 1C). We chose to study the S1C mutant, as
the modification of a thiol group with methanethiosulf-
onate (MTS) reagents allows incorporation of a posit-
ive charge by using (2-aminoethyl)-MTS hydrobromide
(MTSEA
+
) and (2-trimethylamonium)-ethyl MTS
(MTSET
+
), or a negative charge by using sodium
(2-sulfonato-ethyl)-MTS (MTSES
–
). In order to clarify
the effect of the first five residues on the electrophysio-
logic properties and stability of the pore, the deletion
mutant D5 was used. This mutant lacks one negative
charge (Asp3) and three hydrophobic residues
(Fig. 1C,D). Finally, a His
6
–EqtII variant contains a
hexa-histidine tag and prolongs the N-terminus of
EqtII for 13 amino acid residues, adding a strong
positively charged region to the N-terminal helix at
Topology of equinatoxin II N-terminus K. Kristan et al.
540 FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS
pH 5.5; at this pH, the majority of the histidines
should be protonated [28] (Fig. 1C). All of the mutants
were produced in Escherichia coli and purified to
homogeneity as shown by SDS ⁄ PAGE gels (Fig. 1B).
The hemolytic activity of S1C and D5 was as observed

previously [24,29]. The addition of the His-tag to
the N-terminus of EqtII decreased the hemolytic
activity (c
wt
50
¼ 0.21 lgÆmL
)1
± 0.03, c
HisÀEqtII
50
¼ 0.98
lgÆmL
)1
± 0.04; n ¼ 3–5 ± SD) (c
50
is the concentra-
tion of a protein that produces 50% of the maximal
rate of hemolysis).
Deletion mutant D5, S1C, chemically modified ver-
sions of S1C (S1C-MTSEA
+
, S1C-MTSET
+
, S1C-
MTSES
–
, in which S1C was chemically modified with
MTSEA
+
, MTSET

+
and MTSES
–
, respectively), and
His
6
–EqtII were able to form pores in PLMs at final
concentrations of 1–5 nm (Figs 2 and 4).
EqtII and its mutants formed pores in PLMs with a
broad conductance distribution: 308 pS (wild-type),
329 pS (S1C), 349 pS (S1C-MTSEA
+
), 358 pS (S1C-
MTSET
+
), 247 pS (S1C-MTSES
–
) and 256 pS (D5)
(Fig. 3A). D5 frequently showed two different types of
pore. One was similar to the wild-type, and the other
had a lower conductance and was less stable (Fig. 2A,
bottom trace, arrows). This behavior was not seen in
the wild-type or other mutants used in this study, sug-
gesting that it was a peculiarity of the deletion mutant.
These lower-conductance pores typically had conduct-
ances of 100–150 pS (Fig. 2B) and remained open for
a few seconds to 60 s (Fig. 2A). After 5–10 min, when
pores with higher conductance opened, such events
were no longer observed, probably due to the typical
total noise of EqtII multichannel recordings.

All mutants showed cation selectivity, but to different
extents (Fig. 3B). Mutation of Ser1 to Cys did not
affect the selectivity significantly (P
+
⁄ P
À
WT
¼ 9.08;
P
+
⁄ P
À
S1C
¼ 10.14; p ¼ 0.321) (P
+
and P
)
are the
permeability of cation and anion, here K
+
and Cl
)
respectively). The addition of a negative charge (S1C-
MTSES
–
) leads to a significant increase in cationic selec-
tivity (P
+
⁄ P
À

MTSESÀ
¼ 15.1; p ¼ 0.021). On the other
hand, the addition of positive charge (S1C-MTSEA
+
,
S1C-MTSET
+
) or the deletion of a negative charge
in D5 caused a shift to less cationic selectivity
AD
B
C
Fig. 1. Structure of EqtII and alignments of
actinoporin N-terminal sequences, EqtII and
mutants used in the study. (A) Three-dimen-
sional structure of EqtII (Protein Data Bank
code 1IAZ) with its N-terminal amphipathic
a-helix shown in black. (B) SDS ⁄ PAGE gel
of proteins used in this study. Approxi-
mately 1 lg of each protein was resolved
on 12% SDS ⁄ PAGE gel and stained with
Coomassie Blue. (C) Alignment of the wild-
type EqtII, mutants and fusion protein used.
Negatively charged amino acids are in black.
The position of the N-terminal amphipathic
a-helix (amino acids 15–26) is shown above
the alignment by letters h [17]. The region
that was shown to be in an a-helical
arrangement in the final pore is underlined
[24]. An arrow denotes the thrombin clea-

vage site. (D) The alignment of known N-ter-
minal sequences of actinoporins compiled
from the literature [8,45–49]. Hydrophobic
amino acids (AGVLIFWYP) are shown in
black, polar amino acids (TSMCNQ) are
shown in dark gray, and charged amino
acids (DEKHR) are shown in light gray. The
numbering is according to EqtII.
K. Kristan et al. Topology of equinatoxin II N-terminus
FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS 541
(P
+
⁄ P
À
MTSEAþ
¼ 5.07, p ¼ 0.0003; P
+
⁄ P
À
MTSETþ
¼
4.03, p ¼ 0.0002; P
+
⁄ P
À
d5
¼ 2.58, p < 0.0001)
(Fig. 3B).
To further evaluate the charge distribution through
the pore and possibly the position of the N-terminus,

the current–voltage characteristic (I ⁄ V) was studied
(Fig. 3C). The symmetry of the I ⁄ V curve for S1C, i.e.
I
+
⁄ I
–
% 1 (Fig. 3C, inset), suggests that the distribu-
tion of the charges along the lumen of the mutated
pore was not significantly different from that of the
wild-type pore. The addition of a negative charge on
S1 (S1C-MTSES
–
) slightly decreased the I
+
⁄ I
–
ratio
(Fig. 3C, inset). On the other hand, the addition of a
positive charge (S1C-MTSET
+
) strongly increased the
slope of the I
+
⁄ I
–
ratio vs. applied voltage curve
(Fig. 3C, inset), which is consistent with the location
of that charge on the trans side. A similar strong
asymmetry in the I ⁄ V curve was also observed for D5,
which lacks the negative charge at position 3.

The position of the N-terminus in the final pore
His
6
–EqtII was used to analyze in a more direct way
the position of the N-terminus in the final pore, i.e. to
which side of the membrane the N-terminus extends.
His
6
–EqtII pores are slightly less conductive than
the wild-type [G ¼ 204 ± 36.6 pS (p ¼ 0.004; n ¼ 21
average ± SD) (G ¼ conductance), compared to
308 pS for the wild-type]. The selectivity was not chan-
ged (P
+
⁄ P
À
His6ÀEqtII
¼ 9.37, p ¼ 0.807), suggesting
that the positively charged His-tag is positioned far
enough from the pore entrance. Interestingly, when
positive voltages (from + 40 to + 100 mV) were
applied across the membrane, the current increased
nonlinearly (Fig. 4B, inset). When the positive voltages
were switched to negative voltages, the channels rap-
idly closed and the current dropped close to zero
(Fig. 4A). The pores reversibly opened again when a
positive voltage was again established. This behavior
could be easily explained by the His-tag being on the
trans side. At this pH, histidines should not carry an
excess of positive charge; however, there is an addi-

tional arginine, which is part of a thrombin cleavage
site in the spacer (Fig. 1C) and possesses a positive
charge, which contributes to the observed behavior.
Therefore, an applied negative potential forces the
entire N-terminus with the His-tag and linker contain-
ing arginine to become closer to the trans entrance of
the pore, to enter the pore lumen and to clog it. The
rate of closures was voltage-dependent and increased
with the magnitude of the negative applied voltage.
Furthermore, when the pH of the buffer was lowered
to 5.5, i.e. below the pK
A
of His (pK
A
¼ 6.04), which
A
B
C
Fig. 2. Formation of pores in PLMs by the wild-type EqtII and
mutants. (A) PLMs were composed of 1,2-diphytanoyl-sn-glycero-
phosphocholine and 20% (w ⁄ w) SM. The wild-type EqtII and D5
were added at a final concentration of 1–5 n
M to the cis side,
where a constant voltage (+ 40 mV) was applied. The buffer was
10 m
M Tris ⁄ HCl and 100 mM KCl (pH 8.0) on both sides. Only the
initial few steps of pore formation are presented for the most rep-
resentative traces. Transient currents observed in D5 are indicated
by arrows. (B) Histograms show the distribution of pore sizes. The
numbers of events from eight and 10 independent experiments

were 24 and 83 for the wild-type and D5, respectively. (C) Pore for-
mation of S1C and its modified forms (S1C-MTSET
+
, S1C-MTSEA
+
,
S1C-MTSES
–
) at a final concentration of 1–5 nM, under the same
experimental conditions as in (A). The representative traces of at
least two independent experiments are shown.
Topology of equinatoxin II N-terminus K. Kristan et al.
542 FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS
drastically increases the positive charge of the His-tag
[28], the current at negative voltages was actually com-
pletely blocked (Fig. 4A).
To gain additional insights into the position of the
N-terminus across the membrane, MTS reagents were
applied to the cis or trans side of S1C preformed pores
(Table 1). The variation in selectivity, which has been
shown to be the most sensitive parameter, was meas-
ured after the addition of MTS reagents (see Fig. 3B
for details). When MTS reagents, which are mem-
brane-impermeable, were added to the cis side of the
membrane, small or no changes in reversal potential
(U
rev
) were observed (Table 1). Changes occurred only
when MTS reagents were added to the trans side of
the membrane, suggesting that the reagents had reac-

ted with the thiol group of S1C, and modified the pore
selectivity, confirming the trans position of the Ser1
residue.
Finally, heterobifunctional maleimeide-poly(ethylene
glycol)-N-hydroxysuccinimide was used to chemically
modify the thiol group of S1C after pores were
already formed. Maleimeide-poly(ethylene glycol)-N-
hydroxysuccinimide contains a maleimide group that
can react with the thiol group and could possibly
clog the channel when covalently attached close to
the pore entrance. In multichannel recordings, clo-
sures could be seen only when maleimeide-poly(ethy-
lene glycol)-N-hydroxysuccinimide was added to the
trans side. However, the extent of current reduction
did not exceed 15–20% of the total current
(Fig. 5A,B). When we performed a three-channel
recording, the addition of the maleimeide-poly(ethy-
lene glycol)-N-hydroxysuccinimide reagent caused
stepwise closures only after maleimeide-poly(ethylene
glycol)-N-hydroxysuccinimide was added to the trans
side of the membrane (Fig. 5C).
Discussion
The molecular mechanism of actinoporin pore forma-
tion has been unraveled in the last few years, with
Fig. 3. Properties of the channels formed by the wild-type and
mutants in PLMs. (A) Average conductance of the wild-type and
mutant pores. The dashed line shows the wild-type’s conductance
for comparison. *p < 0.05; n ¼ 16–60, average ± SD. The experi-
mental conditions were as in Fig. 2A. The bar corresponding to D5
refers to the normal channel, i.e. the one with higher conductivity

in Fig. 2B. (B) Selectivity of pores formed by the wild-type EqtII
and mutants. The wild-type and mutants were added at a final con-
centration of 1–5 n
M to the cis side of the membrane. Initially, both
sides were bathed in a symmetric solution of 10 m
M Tris ⁄ HCl and
100 m
M KCl (pH 8.0). The KCl concentration was increased step-
wise only on the trans side, to reach a final KCl concentration of
1
M (10-fold gradient). U
rev
values were converted to the reported
permeability ratio (P
+
⁄ P
–
) with the Goldman–Hodgkin–Katz equation
(Eqn 2). The dashed line shows the wild-type’s selectivity for com-
parison. *p < 0.05; n ¼ 3–9, average ± SD. (C) The dependence of
the single-channel current on the applied voltage for the wild-type
EqtII and mutants. The I ⁄ V characteristics of the pores formed by
the wild-type EqtII (filled squares), S1C (filled diamonds), S1C-
MTSES
–
(open triangles), S1C-MTSET
+
(open inverted triangles)
and D5 (open circles) mutants are shown. They were derived from
the amplitude of the current steps caused by square voltage pulses

in experiments with membranes containing 5–50 channels. Current
values were then normalized at + 40 mV to the current flowing
through one single channel, as obtained from the histogram in
Fig. 3. All conditions were as described in Fig. 2A. Inset: the ratio
of currents (I
+
⁄ I
–
; in absolute values) when positive and negative
voltages were applied. n ¼ 3–9, average ± SD.
K. Kristan et al. Topology of equinatoxin II N-terminus
FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS 543
particular emphasis on the role of the N-terminal
region. It was shown that it needs to be flexible [20,21]
and that region 10–28 forms an amphipathic a-helix,
so far the only recognized structural element of the
final pore [23]. The present study provides additional
information about the structure and formation of the
EqtII pore. The following conclusions can be drawn
from the data presented here: (a) the first and third
amino acids of EqtII are exposed to the lumen of the
pore; (b) the N-terminal part of EqtII extends to the
trans side of the membrane in the final pore, i.e. to the
other side than the rest of the membrane-bound mole-
cule; and (c) the first five amino acids help to stabilize
the final pore.
The position of the first and third amino acids
in the final pore
The change in the fixed charge distribution through
the pore affects the pore conductance [30]. Therefore,

it can be used for determining the residues exposed to
the lumen and the structural organization of the pore
[24]. In our experiments, the effects on pore properties
were clearly shown for chemically modified S1C, for
which large changes in cation selectivity were observed
upon chemical modification with MTS reagents. In
Malovrh et al. [24], modifications with MTS reagents
and comparison of selectivity indices for chemically
labeled mutants proved to be very useful for the detec-
tion of sites exposed to the pore lumen, particularly
the ratio of selectivity indices of a mutant that was
chemically modified with MTSES
–
and MTSEA
+
[(P
+
⁄ P
À
MTSESÀ
) ⁄ (P
+
⁄ P
À
MTSEAþ
)]. This ratio is about
1 when the amino acid side chain is not facing the
lumen of the pore, as was observed for most of the
mutants in region 10–28 of EqtII [24]. For the mutants
exposed to the pore lumen, this ratio should be higher,

as selectivity increased with the negative charge
attached and decreased with the positive charge. This
was indeed observed for the residues from the polar
face of the amphipathic helix, with Asp10 showing the
Fig. 4. The effect of the N-terminal histidine tag on the voltage-gating
properties. (A) Proteins were added to the cis side at a final concentra-
tion of 2–20 n
M, and the current across the membrane was followed.
The buffer was 10 m
M Tris ⁄ HCl, 100 mM KCl, and 0.1 mM EDTA
(pH 8.0), on both sides, except in the lowest trace, where the buffer
was 10 m
M Mes, 100 mM KCl, and 0.1 mM EDTA (pH 5.5). Other con-
ditions used were the same as described in Fig. 2A. The currents
when positive and negative voltages were applied are shown. (B) Cur-
rent–voltage dependence of His
6
–EqtII. The inset shows the ratio of
currents (I
+
⁄ I
–
; in absolute values) when positive and negative vol-
tages were applied. The protein concentration was 5 n
M.
Table 1. MTS reagents were added to the cis or trans side in order
to study the accessibility of Cys1. Experiments were performed in
asymmetric conditions, and U
rev
was monitored from the first 15 s

after the addition of MTS reagents until U
rev
stabilized (see Fig. 3B
for experimental details). The typical changes in U
rev
of 1–3 experi-
ments are reported. The concentration of KCl was 100 m
M and 1 M
in the cis and trans chambers, respectively. The U
rev
values for the
S1C mutant before the addition of MTS reagents were 38.9 mV,
37.6 mV and 39.1 mV for MTSEA
+
, MTSET
+
and MTSES
–
, respect-
ively.
D(cis) (mV) D(trans) (mV)
MTSEA
+
+ 0.3 ) 3.9
MTSET
+
0 ) 2.4
MTSES
–
+ 0.3 + 2.0

Topology of equinatoxin II N-terminus K. Kristan et al.
544 FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS
highest ratio of almost 10, suggesting it is located at
the constriction of the pore [24]. In our case, the ratio
(P
+
⁄ P
À
MTSESÀ
) ⁄ (P
+
⁄ P
À
MTSEAþ
) was about 3 for the
S1C mutant, thus clearly indicating that this side chain
faces the lumen of the pore. This value is approxi-
mately the same as the value observed for Asp17 [24],
and would place Ser1 at approximately the same posi-
tion with regard to the center of the pore.
The introduced charges along the ion conductive
pathway should also affect the conductance of pores.
Variation of the conductance upon addition of positive
charge (MTSEA
+
or MTSET
+
) or negative charge
(MTSES
–

) has been observed for the solution-exposed
amino acids of other PFTs [30,31]. The observed chan-
ges were explained primarily by the electrostatic nature
of these effects, on the assumption that the channel
Fig. 5. The effect of maleimeide-poly(ethylene glycol)-N-hydroxysuccinimide on currents of the wild-type and S1C pores. Multichannel
recordings of EqtII (left) and S1C (right) in PLMs. The lowest panel in S1C shows a three channel recording. The concentration of the protein
was 1 n
M, except for oligochannel recording, where it was 200 pM. Maleimeide-poly(ethylene glycol)-N-hydroxysuccinimide at a final concen-
tration of 50 l
M was added to the cis or trans compartment, as indicated by arrows. The voltage applied was + 40 mV in all experiments.
K. Kristan et al. Topology of equinatoxin II N-terminus
FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS 545
maintained a fixed structure. In the present study, only
slight differences were observed in the conductance of
S1C, modified or not with MTS reagents (Fig. 3A).
For a possible explanation of these small variations,
we must keep in mind that we modified the position at
the tip of a very flexible N-terminus [17,18,32] and that
EqtII forms toroidal pores [27] in which helices are not
rigidly arranged in the final pore. Accordingly, the
high standard deviations in conductance values, which
are characteristics of EqtII pores, demonstrate that the
helix can slightly change position according to circum-
stances [24]. As previously noted [24], we confirm here
that EqtII conductance is not the most sensitive
parameter with which to study the charge distribution
along the pore. We therefore propose that the effects
of charges on the conductance of EqtII pores observed
for MTS-modified S1C and the low-conductance state
of the D5 mutant could be ascribed to an electrostatic

effect and to a pore structural rearrangement, respect-
ively.
A strong effect on selectivity, as a result of the
change in the net charge, was observed for the D5
mutant. This mutant showed significantly lower cat-
ion selectivity than the wild-type, which is consistent
with removal of a negative charge from the ion
conductive pathway. The data obtained with D5
therefore suggest that Asp3 is exposed to the pore
lumen. Similarly, the addition of a positive charge at
position 1 decreased the cation selectivity, and the
addition of a negative charge increased it. According
to Malovrh et al. [24], the modulation of negative
charges is crucial for defining the electrophysiologic
characteristics of EqtII. In particular, selectivity is
the most sensitive electrophysiologic parameter and
the one that is most affected by charge modifications
in the pore lumen.
Furthermore, current dependence on the applied
voltage was studied for chemically modified S1C and
D5. Deletion of the first five amino acid residues (D5)
and positive charge addition (S1C-MTSET
+
) had the
greatest effect. The strong asymmetry of the I ⁄ V
curves of D5 and S1C-MTSET
+
provides the first indi-
cation that the N-terminus is exposed to the trans side
of the membrane. Furthermore, values of the I

+
⁄ I
–
ratio larger than 1, as measured for those mutants,
strongly suggest a trans position of Ser1 (Fig. 3C,
inset). In this case, the local trans concentration of cat-
ions is lower than the bulk concentration, due to the
repulsive effect of MTSET
+
,K
+
(and Cl
–
), so they
move from cis to trans (or trans to cis) according to
both electrical and concentration gradients, leading to
a higher current. The opposite happens when a negat-
ive voltage is applied.
The position of the N-terminus in the final pore
The asymmetry of the I ⁄ V curves indicates that the
first and third positions are exposed to the lumen of
the pore. Additional insights into the position of the
N-terminus were obtained with the His-tagged version
of EqtII. The data showed that His
6
–EqtII forms
channels of lower conductance, has an asymmetric I ⁄ V
curve, and exhibits rapid closures of pores at a negat-
ive applied potential voltage. As we did not observe
any changes in the selectivity of the pores formed by

His
6
–EqtII, the changes in current must again be due
to changes in the pore structure and flexibility of the
His-tag. The above observations can be interpreted to
mean that the His-tag is translocated to the trans side
of the membrane and then blocks the channel when
negative voltages are applied. This is likely, because
the His-tag with the linker possesses at least one posit-
ive charge at the pH of the buffer used (pH 8), and
can therefore act as a voltage-dependent gate. At
pH 5.5 (Fig. 4), most of the histidines are protonated
and thus the N-terminus of His
6
–EqtII carries a large
excess of positive charge. Consistently, the rates of clo-
sure and opening of the pore, as well as the blocking
efficiency at negative voltages, are drastically increased.
The most likely mechanism by which it may close the
pore is by inserting into the pore lumen and thus
obstructing the ion permeability when a negative
potential is applied. This mechanism would be analog-
ous to the ‘ball and chain’ mechanism of channel inac-
tivation [33]. The voltage-dependent closures of His
6
–
EqtII pores are also extremely similar to those observed
for His-tagged diphtheria toxin [33]. In that study, it
was shown that diphtheria toxin is able to translocate
the His-tag at the N-terminal region across the lipid

membrane. The same occurs in the case of EqtII; how-
ever, the pore formation efficiency of His
6
–EqtII is
reduced, as this mutant is less hemolytically active. A
similar reduction in permeabilizing activity when the
His-tag was present was reported for the homologs
magnificalysin [34] and sticholysin II [35]. In fact, the
N-terminus is critical for the permeabilizing activity of
EqtII, and longer tags rendered it inactive [20].
Topological experiments with thiol-modifiable rea-
gents were also performed. MTS reagents were added
to either side of the membrane when S1C pores were
already formed, but changes were observed only upon
addition to the trans side of the membrane (Table 1).
Finally, maleimeide-poly(ethylene glycol)-N-hydroxy-
succinimide only had observable effects when added to
the trans side (Fig. 5C). Altogether, the data obtained
indicate that the N-terminal part of EqtII is exposed to
the trans side when the transmembrane pore is formed.
Topology of equinatoxin II N-terminus K. Kristan et al.
546 FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS
Stabilization of pores by first five amino acids
The most unusual behavior was observed with D5
(Fig. 2). Unstable pores with half the conductance of
the fully open D5 pores were observed. The current
data cannot exclude the possibility that the smaller
currents are due to the formation of channels by only
three or even fewer monomers. In this case, the
N-terminal end would help to assemble monomers in

the final pore. However, we believe that these smaller
channels represent an intermediate on the way to the
final pore, where the lack of five amino acids pre-
vents stabilization of final fully open pores. In this
model, the first five amino acids would act as an
anchor, which would help to restrict the N-terminus
to the trans side of the membrane. Region 1–5 is
highly hydrophobic in actinoporins (Fig. 1D). The
Asp present at position 3 in EqtII is an exception, as
most actinoporins possess a hydrophobic amino acid
at that position (Fig. 1D). The fourth amino acid is a
bulkier hydrophobic Val or Leu and could actually
have the most important role. The first five amino
acids are also highly flexible and were not resolved in
the crystal structure of EqtII [17]. Recently, an NMR
structure of a peptide corresponding to region 1–32
was determined in the presence of dodecylphospho-
choline micelles. It formed a continuous helix from
residues 6 to 28, but again the first five amino acids
showed the highest flexibility [32]. This region would
have a similar role as in aerolysin, a b-pore-forming
toxin from bacteria, where hydrophobic amino acids
from the tip of the b-loop anchor the b-barrel in the
membrane [36].
Experimental procedures
Materials
Bovine brain SM and 1,2-diphytanoyl-sn-glycerophospho-
choline were obtained from Avanti Polar Lipids (Alabaster,
AL). All other materials were from Sigma (Milan, Italy),
unless stated otherwise.

Cloning, expression and isolation of the mutants
The construction of expression vectors of mutants S1C and
D5 (deleted first five amino acids of EqtII) has been des-
cribed previously (Fig. 1C) [22,29]. The wild-type EqtII,
S1C and D5 were expressed in an E. coli BL21 (DE3) strain
and purified from the bacterial cytoplasm as described else-
where [37]. The wild-type EqtII was also constructed as a
His
6
fusion protein, which contains an N-terminal hexa-his-
tidine tag and the thrombin cleavage site (Fig. 1C). His
6
–
EqtII was expressed in an E. coli BL21 (DE3) pLysS strain
and purified from the bacterial cytoplasm by Ni-chelate
chromatography [38]. All mutants, fusion proteins and the
wild-type were purified to homogeneity on SDS ⁄ PAGE
gels.
Hemolytic activity
Hemolytic activity was measured by the use of a microplate
reader (MRX; Dynex Technologies, Deckendorf, Ger-
many). A suspension of bovine red blood cells was pre-
pared in hemolysis buffer (0.13 m NaCl, 0.02 m Tris ⁄ HCl,
pH 7.4) from well-washed erythrocytes. One hundred
microliters of erythrocyte suspension with A
630
¼ 0.5 was
added to 100 lL of two-fold serially diluted proteins.
Hemolysis was then monitored turbidimetrically by measur-
ing the absorbance at 630 nm for 20 min at room tempera-

ture. The results are presented as c
50
, which is the
concentration of a protein that produces 50% of the max-
imal rate of hemolysis.
Chemical modification using MTS derivatives
Mutant S1C was chemically modified with MTS reagents
to introduce either a positive or a negative charge at the
thiol group [30,39]. MTSEA
+
and MTSET
+
were used
for the introduction of positive charges, and MTSES
–
was
used to introduce a negative charge (all from Biotium,
Inc., Fremont, CA). S1C at a concentration of 10–
50 lgÆmL
)1
(0.5–2 lm) in water was preincubated over-
night in a 200 molar excess of dithiothreitol (0.1–0.4 mm).
MTS reagents, freshly dissolved in water, were then added
at 1000 molar excess (0.5–2 mm). After 1 h of incubation
at room temperature, after which the majority of the rea-
gent had been hydrolyzed according to the manufacturer’s
specifications, the modified samples were used for PLM
experiments. The final concentrations of MTS reagents or
dithiothreitol in the cis chamber after addition of the
sample to the PLM were below 5 lm. The pore propert-

ies of the wild-type EqtII were not affected by the MTS
reagents [24].
PLM experiments
Solvent-free PLMs were composed of 1,2-diphytanoyl-
sn-glycerophosphocholine and 20% SM (w ⁄ w) [40]. The
chambers were made of Teflon, and the volume of the
chambers was 2 mL. The septum between the chambers
was also made of Teflon and contained a 100 lm hole. The
protein was added at nanomolar concentrations to stable,
preformed bilayers on the cis side only (the cis side is where
the electrical potential was applied, and the trans side was
grounded). All experiments were started in symmetric con-
ditions, using a buffer comprising 10 mm Tris ⁄ HCl and
100 mm KCl (pH 8.0) on both sides of the membrane. For
K. Kristan et al. Topology of equinatoxin II N-terminus
FEBS Journal 274 (2007) 539–550 ª 2006 The Authors Journal compilation ª 2006 FEBS 547
experiments with His
6
–EqtII fusion protein, 0.1 mm
EDTA was included in the buffer. A defined voltage,
generally + 40 mV, was applied across the membrane.
Miniature magnetic stir bars stirred the solutions on both
sides of the membrane. The currents across the bilayer were
measured, and the conductance (G) was determined as
follows [41]:
GðpSÞ¼IðpAÞ=U ðVÞð1Þ
where I is the current through the membrane, and U is the
applied transmembrane potential.
Macroscopic currents were recorded by a patch clamp
amplifier (Axopatch 200, Axon Instruments, Foster City,

CA). A PC equipped with a DigiData 1200 A ⁄ D converter
(Axon Instruments) was used for data acquisition. The
current traces were filtered at 100 Hz and acquired at
500 Hz by the computer using axoscope 8 software (Axon
Instruments). All measurements were performed at room
temperature.
For the selectivity measurement, KCl concentration was
increased stepwise on the trans side only to finally form a
10-fold gradient. At each concentration, the potential neces-
sary to zero the transmembrane current (i.e. the reversal
potential U
rev
), was determined. From the reversal poten-
tial, the ratio of the cation over anion permeability
(P
+
⁄ P
–
) was calculated using the Goldman–Hodgkin–Katz
equation [42–44]:
P
þ
=P
À
¼½ða
trans
=a
cis
Þ expðeU
rev

=kTÞÀ1=
½ða
trans
=a
cis
ÞÀexpðeU
rev
=kTÞ ð2Þ
where a
trans
and a
cis
are the activities of KCl on the trans
side and the cis side, respectively [a, thermodynamic
activity on trans or cis side of membrane; U
rev
, reversal
potential; e, elementary charge; k, Boltzmann constant; T,
absolute temperature (at 23 °C kT/e ¼ 25 mV)]. kT ⁄ e is
% 25 mV at room temperature. The P
+
⁄ P
–
values repor-
ted were measured at the same conditions, which were
100 mm KCl in the cis chamber and 1 m KCl in the
trans chamber.
For topological experiments with the S1C mutant, the
protein was preincubated for 30 min with 20 mm dithio-
threitol and added to PLMs to allow pore formation. MTS

reagents or heterobifunctional maleimeide-poly(ethylene
glycol)-N-hydroxysuccinimide (molecular mass ¼ 3400 Da;
Nektar Therapeutics, Huntsville, AL) were then added to
the cis or trans solution. The final concentrations used were
1or2mm for MTS reagents and 50 lm for maleimeide-
poly(ethylene glycol)-N-hydroxysuccinimide.
Acknowledgements
The Slovenian authors were supported by grants from
the Slovenian Research Agency (Ljubjana, Slovenia).
GV was supported by fellowships from the CNR Insti-
tute of Biophysics (Trento, Italy).
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