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
Trang 1lipid membranes and helps to stabilize the transmembrane pore
Katarina Kristan1, Gabriella Viero2, Peter Mac˘ek1, Mauro Dalla Serra2and Gregor Anderluh1
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 non-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: mdalla@itc.it
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: gregor.anderluh@bf.uni-lj.si
(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; His6–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.
Trang 2conformational 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 His6–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
Trang 3pH 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 (cwt ¼ 0.21 lgÆmL)1± 0.03, cHisEqtII50 ¼ 0.98
lgÆmL)1± 0.04; n¼ 3–5 ± SD) (c50is 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
His6–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+⁄ PWT ¼ 9.08;
P+⁄ PS1C¼ 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+⁄ PMTSES¼ 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
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.
Trang 4(P+⁄ PMTSEAþ¼ 5.07, p ¼ 0.0003; P+⁄ PMTSETþ ¼ 4.03, p¼ 0.0002; P+⁄ Pd5¼ 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 His6–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 His6–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) (G¼ conductance), compared to
308 pS for the wild-type] The selectivity was not chan-ged (P+⁄ PHis6EqtII¼ 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 pKA of His (pKA¼ 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 m M 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 n M , under the same
experimental conditions as in (A) The representative traces of at
least two independent experiments are shown.
Trang 5drastically 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
(Urev) 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) Urevvalues 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.
Trang 6particular 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 transside 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+⁄ PMTSES)⁄ (P+⁄ PMTSEAþ)] 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 m M KCl, and 0.1 m M EDTA
(pH 8.0), on both sides, except in the lowest trace, where the buffer
was 10 m M Mes, 100 m M KCl, and 0.1 m M 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 His6–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 Urevwas monitored from the first 15 s after the addition of MTS reagents until Urevstabilized (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 Urevvalues 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.
Trang 7highest ratio of almost 10, suggesting it is located at
the constriction of the pore [24] In our case, the ratio
(P+⁄ PMTSES)⁄ (P+⁄ PMTSEAþ) 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 p M 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.
Trang 8maintained 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 His6–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 His6–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 His6–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 His6– 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 His6–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 rearea-gents 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
Trang 9Stabilization 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
His6fusion protein, which contains an N-terminal
hexa-his-tidine tag and the thrombin cleavage site (Fig 1C) His6–
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 A630¼ 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 c50, 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
Trang 10experiments with His6–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]:
GðpSÞ ¼ IðpAÞ=UðVÞ ð1Þ 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 Urev), 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]:
Pþ= ¼ ½ðatrans=acisÞ expðeUrev=kTÞ 1=
½ðatrans=acisÞ expðeUrev=kTÞ ð2Þ where atransand acis are the activities of KCl on the trans
side and the cis side, respectively [a, thermodynamic
activity on trans or cis side of membrane; Urev, reversal
potential; e, elementary charge; k, Boltzmann constant; T,
absolute temperature (at 23C 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 mass¼ 3400 Da;
Nektar Therapeutics, Huntsville, AL) were then added to
the cis or trans solution The final concentrations used were
1 or 2 mm 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)
References
1 Gouaux E (1997) Channel-forming toxins: tales of transformation Curr Opin Struct Biol 7, 566–573
2 Parker MW & Feil SC (2005) Pore-forming protein tox-ins: from structure to function Prog Biophys Mol Biol
88, 91–142
3 Tweten RK (2005) Cholesterol-dependent cytolysins, a family of versatile pore-forming toxins Infect Immun
73, 6199–6209
4 Heuck AP, Tweten RK & Johnson AE (2001) b-Barrel pore-forming toxins: intriguing dimorphic proteins Bio-chemistry 40, 9065–9073
5 Neumeyer T, Tonello F, Molin FD, Schiffler B & Benz R (2006) Anthrax edema factor, voltage-depen-dent binding to the protective antigen ion channel and comparison to LF binding J Biol Chem 281, 32335–32343
6 Anderluh G & Lakey JH (2005) Lipid interactions of a-helical protein toxins In Protein–Lipid Interactions From Membrane Domains to Cellular Networks(Tamm
LK, ed.), pp 141–162 Wiley-VCH, Weinheim
7 Lakey JH & Slatin SL (2001) Pore-forming colicins and their relatives Pore-Forming Toxins 257, 131–161
8 Anderluh G & Mac˘ek P (2002) Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actini-aria) Toxicon 40, 111–124
9 Van der Goot FG, Gonza´lez-Man˜as JM, Lakey JH & Pattus F (1991) A ’molten-globule’ membrane-insertion intermediate of the pore-forming domain of colicin A Nature 354, 408–410
10 Song LZ, Hobaugh MR, Shustak C, Cheley S, Bayley
H & Gouaux JE (1996) Structure of staphylococcal a-hemolysin, a heptameric transmembrane pore Science
274, 1859–1866
11 Viero G, Cunaccia R, Pre´vost G, Werner S, Monteil H, Keller D, Joubert O, Menestrina G & Dalla Serra M (2006) Homologous versus heterologous interactions in the bicomponent staphylococcal gamma-haemolysin pore Biochem J 394, 217–225
12 Panchal RG, Smart ML, Bowser DN, Williams DA
& Petrou S (2002) Pore-forming proteins and their application in biotechnology Curr Pharm Biotechnol
3, 99–115
13 Potrich C, Tomazzolli R, Dalla Serra M, Anderluh G, Malovrh P, Mac˘ek P, Menestrina G & Tejuca M (2005) Cytotoxic activity of a tumor protease-activated pore-forming toxin Bioconjug Chem 16, 369–376
14 Astier Y, Bayley H & Howorka S (2005) Protein com-ponents for nanodevices Curr Opin Chem Biol 9, 576– 584
15 Belmonte G, Pederzolli C, Mac˘ek P & Menestrina G (1993) Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid mem-branes J Membrane Biol 131, 11–22