Reconstitution of Holin Activity with a Synthetic Peptide
Containing the 1-32 Sequence Region of EJh, the EJ-1 Phage
Holin*
Amparo
Haroab,
Marisela
Vélezc,
Erik
Goormaghtighde,
Santiago
Lagof,
Jesús
Vázquezg,
David
Andreuh, and
María
Gassetai
From the a Insto de Química-Física
Rocasolano and g Centro de Biología Molecular Severo
Ochoa, Consejo Superior de Investigaciones Científicas, Madrid
28006, Spain, the c Facultad de Ciencias C-XVI,
University Autónoma, Madrid 28049, Spain, d Structure and
Function of Biological Membranes, University Libre Bruxelles, Bruxelles
B1050, Belgium, the f Facultad Ciencias Experimentales,
University Pablo de Olavide, Sevilla 41013, Spain, and the
h Departamento Ciencias Experimentales y de la Salud,
University Pompeu Fabra, Barcelona 08003, Spain
Received for publication, November 6, 2002, and in revised form, December 1, 2002
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ABSTRACT |
Pneumococcal EJ-1 phage holin (EJh) is a
hydrophobic polypeptide of 85 amino acid residues displaying lethal
inner membrane disruption activity. To get an insight into holin
structure and function, several peptides representing the different
topological regions predicted by sequence analysis have been
synthesized. Peptides were structurally characterized in both aqueous
buffer and membrane environments, and their potential to induce
membrane perturbation was determined. Among them, only the N-terminal
predicted transmembrane helix increased the membrane permeability. This segment, only when flanked by the positive charged residues on its
N-terminal side, which are present in the sequence of the full-length
protein, folds into a major
-helix structure with a transmembrane
preferential orientation. Fluorescein quenching experiments of
N-terminal-labeled peptide evidenced the formation of oligomers of
variable size depending on the peptideto-lipid molar ratio. The
self-assembling tendency correlated with the formation of transmembrane
pores that permit the release of encapsulated dextrans of various
sizes. When analyzed by atomic force microscopy, peptide-induced
membrane lesions are visualized as transbilayer holes. These findings
are the first evidence for a lytic domain in holins and for the nature
of membrane lesions caused by them.
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INTRODUCTION |
Holin refers to a family of bacteriophage-encoded small
hydrophobic proteins (60-145 amino acid residues) that make up the two-component lysis system used by most double-stranded DNA phages to
accomplish the release of the progeny at the last stage of the
infection (1). These proteins cause nonspecific lethal lesions (holes)
of the host cytoplasmic membrane, which allow the passage of the murein
hydrolase to the periplasm for proteoglycan degradation (1-3).
This ability makes holins the regulators of both the timing of host
lysis and the yield of the phage progeny. Despite the increasing number
of holin family members described by genetic analysis and their
promising use in biotechnology, their structure and molecular mechanism
of action are still unknown (2-6).
Most, if not all, of the current knowledge on holins is based on the
phage S protein, a type I holin characterized by a dual start motif
and three potential transmembrane segments (3, 7). Genetic studies have
provided the evidences for essential residues and the basis for
function regulation. Thus, N- and C-terminal solvent-exposed regions
have been proposed as lysis timing regulators, their positive charge
content being directly correlated with the onset retardation (8, 9). On
the other hand, the hydrophobic segments have been suggested to be
responsible for the permeation event, because they contain most of the
inactivating mutations (10, 11). In addition, biochemical
studies have determined the location of the protein at the inner
membrane, verified its predicted helical structure in detergent
micelles, and evidenced its permeating properties in model membranes
(12, 13). All these data have set the basis for a model in which a
homo- or hetero-oligomer of transmembrane helices forms a
functional pore (2, 13).
EJh,1 the holin of the
temperate pneumococcal EJ-1 phage, is an 85-amino acid polypeptide
chain ascribed to the type II holin subgroup (14). As shown for
S
holin, EJh expression is lethal for the host cell (14). In contrast to
S holin, the EJh polypeptide chain lacks a defined dual start, and
its sequence analysis only allows the prediction of two transmembrane
regions of higher hydrophobicity. To gain insight into the molecular
and structural basis of EJh membrane lesions, we have undertaken a
fragment approach to the polypeptide chain. The synthetic peptides were
conformationally characterized, both in aqueous and in membrane
environments, and their capacity to induce membrane leakage was
assayed. The body of results revealed the predicted N-terminal
transmembrane helix as the potential active region of the holin
molecule. When synthesized as a peptide, including the charged N
terminus (1-32 sequence segment), this region folded into a
transmembrane helix displaying self-assembly properties that permit
membrane permeation to dextrans of various sizes. Finally, the
study of peptide-induced bilayer lesions by atomic force microscopy has
allowed the visualization of polydisperse defects as holes. To the best
of our knowledge this is the first report on the nature of lytic domain
and the bilayer lesions carried out by holins.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Protected amino acids, resins, and other peptide
synthesis reagents were from Bachem. All lipids were purchased
from Avanti Polar Lipids. ANTS, DPX, Flu, and Flu succinimidyl ester
were purchased from Molecular Probes. D2O (99.9% isotopic
enrichment), FD-4, FD-20, and FD-70 were obtained from Sigma-Aldrich.
Sephadex G-75 and Sephacryl S-300 were purchased from Amersham
Biosciences. All reagents for buffer preparation and other experimental
procedures were of the highest commercial quality.
Peptide Synthesis and Purification--
EJh peptides were
synthesized using standard solid-phase methods with either
Fmoc (N-(9-fluorenyl)methoxycarbonyl) (15) or
tert-butyloxycarbonyl (16) chemistry. Flu labeling on the N
terminus of the resin-bound peptides was performed as previously described (17). Peptides were purified by reverse-phase
high-performance liquid chromatography (>97% pure, except about 89%
for EJh-M2), and their molecular masses were confirmed by mass
spectrometry (18). Water-soluble peptides were extensively dialyzed
against 25 mM Hepes-HCl, pH 7.0, buffer containing 0.1 M NaCl and 0.5 mM EDTA using 500-kDa cutoff
dialysis tubing and kept frozen. For CD experiments NaCl was
replaced by KF. Hydrophobic peptides were first washed with 10 mM HCl, then dissolved in TFE and aliquoted. When required,
TFE was evaporated under N2 stream, and the solid peptide
was redissolved in Me2SO. Peptide concentrations were determined by quantitative amino acid analysis performed at the Protein
Chemistry Facility of the Centro de Investigaciones
Biológicas.
Liposome Preparation and Complex Formations--
Vesicles were
prepared by hydration of lipid dry films in 25 mM
Hepes-HCl, pH 7.0, containing 0.1 M NaCl and 0.5 mM EDTA, followed by five freeze-thaw cycles and extrusion
through polycarbonate membranes of 0.1-µm pore diameter (18). The
lipid concentration was determined by phosphorus assays (19).
Lipid-peptide complexes were formed by external addition of the
peptides to the liposome suspensions. For hydrophobic peptides,
peptides were added from a TFE stock solution (final TFE < 0.2%,
v/v) except for permeability assays and AFM analysis in which
Me2SO stock solutions were used to avoid membrane damage.
Circular Dichroism--
CD spectra were recorded in a Jasco 810 spectropolarimeter at 25 °C (20). The measurements were performed
using samples of 0.07-0.2 mM peptide concentration and
0.1-cm path length cells. Spectra are reported as averages of seven
scans recorded at a scan rate of 0.33 nm/s. After base line correction
and noise reduction the observed ellipticities were converted to mean
residue ellipticity in units of
degree·cm2·(decimoles of amino acid
residue)
1 using the molecular weight per residue
calculated from the sequence with ExPASy Tools. The secondary structure
composition was determined from the best fit using the
least-square fitting methods provided by Jasco and by DICROPROT
software. The best fit was considered in terms of goodness of
experimental spectrum reproduction with the resulting evolvent
and of converge between different fitting methods.
Infrared Spectroscopy--
ATR-FTIR spectra were recorded in a
Bruker IFS-55 FTIR spectrometer equipped with a nitrogen-cooled mercury
cadmium telluride detector and continuously purged with dried air. The
internal reflection element was a germanium ATR plate (50 × 20 × 2 mm) tilted 45° relative to the incident beam. For each
spectrum, 256 scans at 2 cm
1 nominal resolution were
averaged. Samples were prepared by slow evaporation of the analyte
solution (peptides, lipids, peptide-lipid complexes) under nitrogen
stream on one side of the germanium plate and then sealed in a
universal sample holder. Isotopic exchange (hydrogen/deuterium) was
allowed for 1 h under a stream of nitrogen saturated with heavy
water, before spectrum acquisition. Solvent and water vapor absorptions
were compensated for by subtracting the respective spectra. Secondary
structure analyses of the peptides in the absence and presence of
phospholipids were performed as described previously (21). The
determination of the molecular orientation was estimated from the
linear dichroic spectrum using a KRS-5 polarizer mount assembly (22,
23). The dichroic spectrum is the difference between the spectra
recorded with parallel and perpendicular polarizations. The
perpendicular spectrum was multiplied by a factor for zeroing the
carbonyl-stretching band (1750-1700 cm
1). A larger
absorbance for the parallel polarization (positive amide I' band) of an
-helix indicates a dipole oriented preferentially near the normal of
the ATR plate (23). Conversely, a larger absorbance for the
perpendicular polarization (positive amide I' band) of a
-sheet
might indicates its preferential orientation parallel to the ATR plate
(24).
Fluorescence Spectroscopy--
All fluorescence emission
measurements were performed on an SLM-8100 spectrofluorimeter at
25 °C, unless otherwise stated. Conventionally, quartz cells
(0.5 × 0.5 or 1 × 1 cm) and Glan-Thompson polarizers in
magic angle configuration were used. In quenching experiments,
Flu-labeled peptide was mixed in TFE with increasing amounts of
unlabeled peptide and then added to POPG:POPE (70:30) suspensions at 1:100 and 1:1000 final P:L (0.05-0.1 µM
Flu-labeled peptide final concentration). After an overnight incubation
at 25 °C, the emission spectra (480-nm excitation wavelength) were recorded before and after the addition of 0.1% SDS. Detergent lysis
was used to correct for the slight deviations in concentration. Spectra
were corrected for base line and instrument and optical factors.
Parallel experiments with POPC vesicles excluded effects of changes in
the ionization state of fluorescein on the emission intensities.
Membrane Permeability Assays--
Disruption of the membrane
permeability barrier was measured using the ANTS/DPX leakage assay
(25). Briefly, LUVs were prepared in 10 mM Hepes-HCl, pH
7.0, containing 12.5 mM ANTS, 45 mM DPX, 20 mM NaCl, and 1 mM EDTA and then separated from
non-encapsulated material on Sephadex G-75 using 10 mM
Hepes-HCl, 0.1 M NaCl, 1 mM EDTA, pH 7.0, as
elution buffer. The fluorescence intensity increase of vesicle solution
(60-70 µM phospholipid concentration) upon peptide
addition was measured through an OG-550 Schott cutoff filter (>530 nm)
upon excitation at 386 nm. The 0 and 100% levels of leakage were taken
as the intensities of the corresponding vesicle suspension before and
after addition of Triton X-100 (0.5% final concentration),
respectively. Pore size determination was performed as described (26).
Briefly, POPG:POPE (70:30) lipid films were hydrated in 10 mM Hepes-HCl, pH 7.0, 20 mM NaCl, and 1 mM EDTA containing either 3 mg/ml FD-20 and 2 mg/ml FD-4 or 4 mg/ml FD-70 and 0.5 mg/ml Flu, then frozen and thawed 20 times and
extruded through 0.2-µm pore diameter membranes. Untrapped dextrans
were removed by gel filtration using Sephacryl S-300 HR packed into a
17 × 0.7-cm column and equilibrated in 10 mM Hepes-HCl, pH 7.0, containing 70 mM NaCl and 1 mM EDTA (elution buffer). Fluorescein derivative-containing
LUVs were then incubated for 2 h at 37 °C in the absence or
presence of 0.5% Triton X-100 and EJh-L1M1 at 1:1500 and 1:750 P:L
molar ratio. The treated LUVs were loaded onto a Sephacryl S-300 HR
37 × 1.2-cm column run at 19.8 ml/h in elution buffer. Fractions
of 0.5 ml were collected, and their fluorescence emission intensity at
520 nm (480-nm excitation wavelength) was measured. The area under each
peak was determined from the analysis of the elution profiles using a
three-peak log·normal distribution with Origin 5.0 software.
Atomic Force Microscopy of Supported Bilayer
Preparations--
POPG and POPG:POPE (70:30) liposomes, prepared by
extrusion through polycarbonate membranes of 0.2-µm pore diameter,
were incubated in the absence and presence of EJh-L1M1 at a 1:300 P:L molar ratio. The supported membranes were prepared by depositing 50 µl of 1 mg/ml solution of liposomes in 20 mM Hepes-HCl,
pH 7.0, containing 0.1 M NaCl and 20 mM
CaCl2 on top of freshly cleaved circular pieces of mica
glued onto a Teflon surface (27). The liposomes were allowed to fuse on
the mica for 30-60 min at room temperature and then extensively washed
with buffer. AFM images were taken with an Atomic Force microscope
(Nanotec Electronica, Madrid, Spain) operated in the contact or jump
mode (28, 29). Silicon nitride tips with a force constant of 0.12 newtons/m (DI instruments) were used. The samples were maintained under
buffer solution while imaging. The number of peptide molecules
constituting a hole was estimated from the linear packing of spheres of
an
-helix-like radius (0.5 nm) in the perimeter of the
lesion. The number of lipid molecules was estimated considering a lipid
head group area of 65 Å2 (30).
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RESULTS |
EJh Sequence as Template for Peptide Design--
Hydropathy
profiles of EJh polypeptide chain predicted the presence of three
solvent-exposed regions and two hydrophobic segments compatible with
membrane-spanning
-helices (14). This structural trend would sustain
the folding of the polypeptide chain as a transbilayer helix-turn-helix
that could hypothetically act as a scaffold unit for the formation of
an oligomeric assembly displaying membrane-permeating properties (Fig.
1). In the context of this model, at
least two types of active oligomers differing in the nature of the
transmembrane segment forming the aqueous cavity can be considered. To
elucidate the secondary structure and the molecular basis of membrane
permeation, the EJh chain was chemically synthesized as
different individual fragments (Table
I and Fig. 1).

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Fig. 1.
EJh sequence, predicted folding, and
hypothetical models for its biological action. A, EJh
sequence as template for peptide synthesis following the hydrophobic
analysis previously performed (14). Regions predicted as putative
transmembrane regions are depicted as thick rectangles.
B, folding model for EJh molecule assuming a two
transmembrane -helix scaffold for oligomerization (2). Top
views of the possible oligomeric structures. Model a
involves a heterogeneous pore wall made of both transbilayer segments.
Model b involves a homogeneous pore wall (a
priori either gray or white segments would
be consistent with the model).
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Table I
Major secondary structure of EJh-based peptides
The secondary structure composition was determined by analysis of CD
and ATR-FTIR spectra as described under "Experimental
Procedures." The structure indicated is that which the peptide
populates to at least 40%. In CD experiments, aqueous
buffer refers to both 20 mM Hepes-HCl, pH 7.0, containing 0.1 M KF, 0.5 mM EDTA, and 20 mM phosphate, pH 7.0. For ATR-FTIR, spectra were collected
in peptide films obtained from 10 mM Tris-HCl, pH 7.0, initial solutions, except in the case of EJh-M1 and EJh-M2 where the
transfer was carried out from 40% TFE solutions. Membranes refer to
the presence of POPG:POPE (70:30) at a 1:10 P:L molar
ratio.
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Secondary Structure of EJh Peptides--
The secondary structure
of EJh-based peptides was probed by both far-UV CD and ATR-FTIR
spectroscopies in aqueous and membrane environments (Figs.
2 and 3 and
Table I). In aqueous buffers, EJh-L1 and EJh-L3 displayed CD spectral
features of mainly non-ordered conformations. Under similar conditions,
EJh-L2 spectral shape revealed the coexistence of both turns and
non-ordered conformations. In the three cases, spectral analysis by
least-square fitting supported the previous structural trend, even
though the contribution from non-amide chiral components arising from
Trp and Tyr chains was not considered (31, 32). When inspected by
ATR-FTIR, amide I' band analysis also confirmed the conformational
trend for EJh-L2 and EJh-L3 but clearly revealed the presence of
-sheet structure (peak at 1628 cm
1) in EJh-L1 (Fig.
3A). EJh-M1 and EJh-M2, both representing the predicted
transmembrane regions, displayed limited solubility in aqueous buffer.
Dilution of the peptides from TFE stock solutions into aqueous buffers
revealed the formation of extended structures, which were soluble in
the case of EJh-M1 but insoluble in the case of EJh-M2 (Fig. 2). When
analyzed by ATR-FTIR as films dried from 40% TFE solutions, the
amide I' band of EJh-M1 suggested a helix-random mixture, whereas that
of EJh-M2 revealed signatures of turn (1675 cm
1) and
-sheets (1628 cm
1) (Fig. 3A).

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Fig. 2.
Far-UV CD spectra of EJh peptides in aqueous
and membrane environments. A, CD spectra in aqueous
buffer of: a, EJh-L1; b, EJh-M1; c,
EJh-L2; and d, EJh-L3. EJh-M2 precipitated, and its spectrum
is not shown. B, CD spectra of: a, EJh-L1 in the
presence of POPG:POPE (70:30), EJh-M1 in the presence of POPG:POPE
(70:30) (b) and SDS micelles (c), and EJh-M2 in
the presence of SDS micelles (d). The CD spectra of EJh-L2
and Ejh-L3 in the presence of POPG:POPE membranes were similar if not
identical to traces c and d of A,
respectively. Peptide concentrations were about 0.05-0.1
mM. POPG:POPE (70:30) vesicles were used in a 10:1 L:P
molar ratio. Aqueous buffer refers to 20 mM Hepes-HCl, pH
7.0, containing 0.1 M KF and 0.5 mM
EDTA, although similar results were obtained in 20 mM
phosphate, pH 7.0. SDS micelles refers to 17 mM SDS
in 20 mM phosphate, pH 7.0.
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Fig. 3.
ATR-FTIR spectra of EJh peptides in both
aqueous solutions and membranes. A, ATR-FTIR spectra of
lipid free (from bottom to top) EJh-L1, EJh-M1,
EJh-L2, EJh-M2, and EJh-L3. EJh-L1, EJh-L2, and EJh-L3 were dried on
the Ge crystal from 10 mM Tris-HCl solutions with
the pH adjusted to 7.0. EJh-M1 and EJh-M2 were dried from solutions
prepared in 40% TFE. B, ATR-FTIR spectra of (from
bottom to top) EJh-L1, EJh-M1, EJh-L2, EJh-M2,
and EJh-L3 in the presence of POPG:POPE (70:30) membranes at a 10:1
lipid-to-peptide molar ratio. Samples were prepared by mixing the
required amounts of lipid vesicles and peptides. EJh-Li peptides were
added from aqueous stock solutions, whereas EJh-Mi and EJh-L1M1
peptides were prepared in 40% TFE. C, linear dichroic
ATR-FTIR spectra of EJh-L1, EJh-M1, and EJh-M2 complexed with POPG
(solid) and POPG:POPE (70:30) (dotted) membranes
at a 10:1 lipid-to-peptide molar ratio.
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In the presence of POPG:POPE (70:30) vesicles (1:10 P:L to avoid light
scattering interference), the CD spectra of both EJh-L2 and EJh-L3
remained unchanged (data not shown). The ATR-FTIR spectra agree with
the corresponding CD spectra, and underline the preference of EJh-L2
for adopting a turn structure as evidenced from the band at 1675 cm
1 (Fig. 3B). In contrast to the previous
cases, addition of POPG-containing vesicles to EJh-L1 caused the
appearance of a negative band in the CD spectrum and an increase in
resolution of the amide I' band compatible with
-sheet structures
(Fig. 3B). Furthermore, the linear dichroism ATR-FTIR
spectrum of lipid-bound EJh-L1 revealed a negative band characteristic
of extended structures lying parallel to the membrane surface (Fig.
3C). In the presence of membranes EJh-M1 exhibited spectral
features typical of helical structures (a major narrow amide I' band
centered at 1655 cm
1) but lacking a preferential
orientation with respect to the lipid bilayer as judged by the flat
linear dichroism ATR-FTIR spectrum. On the contrary, the spectral
signatures of EJh-M2 in the presence of both membranes and SDS micelles
supported a complex mixture of turn and
-sheet structures. Larger
versions of EJh-M2, such as DIGNK-EJh-M2 and EJh-M2-NDPT, designed to
increase net peptide charge, did not change the conformational features
(data not shown).
Taken together these results indicated that the EJh polypeptide chain
can be described, from the N terminus to the C terminus, by: (i) an
11-residue segment that distributes between a non-ordered aqueous phase
and a membrane-bound
-sheet; (ii) a 22-residue segment folded into a
helix structure; (iii) a segment that folds into a membrane-stabilized
turn structure; (iv) a second hydrophobic region that collapses into a
structure; and (v) a non-ordered C-terminal tail. This structural
trend is in good agreement with the prediction analysis, except for the
-sheet-forming tendencies of both EJh-L1 and EJh-M2. In the case of
EJh-L1 the formation of
-sheet structures upon acidic lipid binding
can be explained by the i, i+2 amphipathicity of its sequence. On the
other hand, EJh-M2 behavior can be explained in terms of the absence of
regulatory long range interactions as previously described for other
helical segments when handled as isolated peptides (33-35).
Membrane Permeabilization Activity of EJh-based Peptides--
Once
EJh fragments were structurally characterized we addressed their
capacity to produce membrane lesions on ANTS-DPX-loaded vesicles (25).
Co-encapsulation of ANTS probe with DPX extinguishes its fluorescence,
which, if released to the medium, increases due to dequenching upon DPX
dilution. Among all peptides tested, only EJh-L1 and EJh-M1 were
capable of inducing an increase in ANTS fluorescence compatible with a
membrane lesion event (Fig. 4).

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Fig. 4.
Effect of EJh peptides on lipid vesicle
permeability assessed by ANTS-DPX dequenching leakage assay.
A, kinetics of ANTS fluorescence dequenching by
co-encapsulated DPX in POPG:POPE (70:30) vesicles. Depicted traces
correspond to: (a) EJh-L1 at 1:10 P:L; EJh-M1 at
(b) 1:1000, (c) 1:100 and (d) 1:50
P:L; EJh-L1M1 at (e) 1:1000 and (f) 1:100 P:L;
(g) peptide-free lipid vesicles. Traces obtained upon
addition of EJh-L2, EJh-M2, and EJh-L3 are superimposed on trace
g. B, percentage of leakage from POPG:POPE (70:30)
vesicles induced by EJh-L1 (open squares), EJh-M1
(solid circles), and EJh-L1M1 (open circles) as a
function of P:L molar ratio. C, variation of vesicle leakage
with the membrane POPG content: EJh-L1 at 1:10 P:L (open
squares), EJh-M1 at 1:100 P:L (solid circles), and
EJh-L1M1 at 1:100 P:L (open circles). Displayed data
correspond to POPG:POPE mixtures, but those made of POPG:POPC yielded
identical results.
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Using either POPG:POPE (70:30) or POPG:POPC (70:30), the ANTS
fluorescence relief induced by EJh-L1 was fast and saturated at about
1:10 P:L molar ratio. On the contrary, the vesicle leakage produced by
EJh-L2 followed biphasic kinetics compatible with the existence of two
steps, a fast membrane insertion and a slow in-membrane oligomerization
event (Fig. 4A). In this case, end-point titrations at 60 min revealed that saturation of probe release after 1 h of
incubation was achieved at a P:L ratio of 1:1000 (Fig. 4B).
This latter figure must be taken cautiously, because increasing the
peptide concentration in solution can favor the formation of peptide
aggregates non-competent for leakage. In fact, the lack of leakage
completion at increasing peptide concentrations might support side
reactions involving peptide aggregation in aqueous media that would
decrease the yield of the membrane interaction process.
Fig. 4C shows the lipid composition dependence of EJh-L1-
and EJh-M1-induced vesicle leakage. EJh-L1-perturbing activity highly depended on the acidic lipid content of the target membrane, with the
maximum leakage being observed for pure POPG vesicles. For this lipid,
leakage was accompanied by a dramatic increase in solution turbidity
(data not shown), suggesting that the permeability perturbation occurs
as a consequence of a major liposome membrane rearrangement. Decreasing
POPG content in the target vesicle decreased the leakage extent to a
negligible value that made EJh-L1 activity incompatible with lytic
activity at the cellular level unless a segregation of acidic lipids is
assumed. Contrary to EJh-L1, EJh-M1-induced leakage was independent on
the lipid composition of the target vesicle. This feature made EJh-M1
the candidate for housing the hole formation capacity of the entire
molecule. It should be emphasized that EJh-L2, EJh-M2, and
EJh-L3 did not cause any change in a variety of modifications of the
assay, including lipid composition, pH, ionic strength, and temperature.
EJh-L1M1 as a Miniholin Model: Oligomer Assembly and Pore
Sizing--
In light of the previous results, EJh-M1 fulfilled the
secondary structure and functional criteria to be considered the holin domain of the EJh molecule. However, the absence of a preferential orientation with respect to the membrane is puzzling. We considered therefore a longer synthetic peptide, EJh-L1M1, consisting of the
natural tandem of EJh-L1 and EJh-M1 (Table I and Fig. 1). In the
presence of POPG-containing membranes, EJh-L1M1 folded into a major
helical structure as judged by the narrow amide I' band at 1657 cm
1 (Fig. 5). Analysis of
the structure composition by amide I' band resolution enhancement
methods resulted in a 46%
-helix, 22%
-sheet, 17% turn, and
15% random structure estimated for different lipid composition
(POPG:POPE (70:30) and POPG:POPC (70:30)) and P:L molar ratios (1:10
and 1:100). This similarity allowed us to minimize the contribution
from different peptide populations over the secondary structure of a
single polypeptide chain. Furthermore, the linear dichroism ATR-FTIR
spectrum showed a positive band in the amide I' region centered at 1657 cm
1 (Fig. 5), revealing that the helical structure in
EJh-L1M1 is oriented mainly with its axis parallel to the bilayer
normal. With regard to membrane-damaging activity, the ANTS-DPX leakage assay showed that EJh-L1M1 retains the membrane-permeating properties of EJh-M1 but with a faster kinetics (Fig. 4).

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Fig. 5.
ATR-FTIR analysis of EJh-L1M1 secondary
structure and membrane orientation. EJh-L1M1 was incorporated into
POPG:POPE (70:30) vesicles at 1:10 P:L, dried on the Ge plate
and subjected to hydrogen/deuterium exchange for 1 h (upper
spectrum). The linear dichroism ATR-FTIR spectrum was calculated
as described under "Experimental Procedures."
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To test the assembly properties of EJh-L1M1, we used fluorescence
quenching experiments (Fig. 6). The
experiments were performed using peptide solutions containing a fixed
concentration of Flu-labeled and increasing amounts of unlabeled
EJh-L1M1 and POPG:POPE (70:30) vesicles. Fluorescence emission at 520 nm diminished with the increase of Flu-EJh-L1M1 molar fraction
indicating the existence of an in-membrane oligomerization process.
These oligomers were not resistant to detergents, because the addition
of SDS or
-octyl glucoside (0.1-1%, w/v final concentration)
reverted the fluorescence decreases to a value that was independent of
Flu-EJh-L1M1 (data not shown). Furthermore, for a given molar fraction
of fluorescent peptide, the extent of quenching was dependent on the
total P:L, suggesting that oligomeric species exhibit polydispersity in
size.

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Fig. 6.
EJh-L1M1 oligomerization in membranes
followed by the self-quenching of fluorescein-labeled peptide.
Mixtures of Flu-labeled and unlabeled EJh-L1M1 were incubated with
POPG:POPE (70:30) vesicles at 1:100 and 1:1000 P:L, and the
fluorescence intensity at 520 nm was taken from the emission spectra.
Flu-EJh-L1M1 concentration was kept in the 0.05-0.1 µM
range. Quenching was calculated as
F520/F ratio,
where F520 and F
are the fluorescence intensities of Flu-labeled peptide at a given
molar fraction and at infinite dilution, respectively.
F was obtained from the fit of the
experimental data. Displayed data are the average of two independent
measurements.
|
|
To get an idea of the pore size we followed a procedure developed by
Ladhokin et al. (26) that allows simultaneous measurement of
the leakage of fluorescein derivatives of different sizes. Fig.
7A shows that incubation of
EJh-L1M1 at a P:L of 1:1500 with vesicles (200-nm average diameter)
containing co-encapsulated FD-4 and FD-20 resulted in the spilling to
the medium of both dextrans to an equal extent (35 ± 7%). Under
similar conditions, the leakage of co-encapsulated FD-70 and Flu
differed notably. The spilling of Flu was almost complete (85 ± 9%), whereas the leakage of FD-70 amounted to only 17 ± 6%
(Fig. 7B). Increasing the amount of incorporated EJh-L1M1
enhanced the extent of leakage of the encapsulated compounds,
suggesting the existence of pores of various sizes. The compromise
between assay sensitivity and peptide incorporation under conditions
preserving vesicle integrity precluded the study of higher P/L molar
ratio. With the available data, and assuming dextrans as prolate
ellipsoids of 20-Å short and 40- to 440-Å long axes and a fluorescein
hydrodynamic radius of about 6 Å, the permeabilization data suggested
a maximum diameter of 25-30 Å (26, 37). Membrane lesions of such size
would allow the spill to the medium of Flu and the escape of the
dextrans through a slippery mechanism.

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Fig. 7.
Sizing the membrane lesion induced by
EJh-L1M1 by gel filtration chromatography. A, typical
elution profile of POPG:POPE (7:3) vesicles containing co-encapsulated
FD-20 and FD-4, followed by fluorescein emission
(F520) as a function of elution volume.
Displayed traces correspond to: untreated vesicles (solid
line), vesicles treated with EJh-L1M1 at 1:1500 P:L (open
circles), and vesicles lysed with 0.5% Triton X-100.
B, percentage of leakage of Flu, FD-4, FD-20, and FD-70 from
POPG:POPE (70:30) containing co-encapsulated FD-4/FD-20 and
Flu/FD-70 mixtures upon incorporation of EJh-L1M1 at the indicated P:L.
Column runs were performed in sequential duplicates using the following
order: lipids, lipids plus peptide at 1:1500, lipids plus peptide at
1:750, and lipids plus Triton. Percentage of leakage was calculated
from the area under each peak and is referenced to the area of the
peaks after detergent lysis.
|
|
To assess the size distribution of membrane lesion we obtained their
image with AFM (Fig. 8). Lipid vesicles
of various compositions (POPG, POPG:POPC (70:30)) in the absence and
presence of EJh-L1M1 (externally added from an Me2SO stock
solution) were fused onto freshly prepared mica surfaces using
Ca2+ as fusogen (27). Pure lipid bilayers,
regardless of composition, were observed as smooth surfaces with
statistically negligible defects (Fig. 8A). These defects
allowed an estimate of 5-6 nm for the thickness of the bilayer plus
the water layer between the support and the contact leaflet, in
agreement with previously reported data (27, 38). In contrast, when the
lipid-peptide assemblies were fused onto the mica, the formed bilayers
displayed numerous holes of different sizes, shapes, and depths. Fig.
8B illustrates the typical aspect of the surface of a fused
POPG:POPC (70:30) bilayer containing EJh-L1M1 at a 1:300 P:L molar
ratio. This selected field was fully reproducible with other lipid
compositions except for the heterogeneity of the observable
lesions. The holes ranged in diameter from 3 to 44 nm. Their
depth was also variable, the largest being as deep as the bilayer and
the smallest ones shallower. Given that the diameter of some of the
smaller holes was of the same order of magnitude as the expected radius
of the tip (10 nm), we could rule out that in those cases the depth
measured was limited by the access of the tip into the hole. Therefore, EJh-L1M1 generated membrane lesions of polydisperse size.

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Fig. 8.
AFM images of EJH-L1M1 incorporated into
POPG-containing bilayers. A, POPG bilayers fused onto
mica; B, POPG bilayers containing EJh-L1M1 at a 1:300 P:L
and fused onto mica. The horizontal bar represents 150 nm.
The depth profiles, depicted at the right-hand side of each
panel, correspond to the dark lines shown on the
images. The total gray scale represents 10 nm.
|
|
A quantitative analysis of the perimeter of the observed holes
indicated that the amount of peptide expected to be present in the
membrane at the P:L molar ratio used would be enough to line only a
third of the perimeter of the total amount of holes observed. This
unexpected observation suggested that the presence of EJh-L1M1
transbilayer pores on the liposomes affects the bilayer upon fusion on
the mica allowing the enlargement of the defects detected as smaller
and shallower holes through the inclusion of peptide-free lipid edges.
 |
DISCUSSION |
Genetic studies have provided evidence for the existence of a
growing family of phage-encoded small hydrophobic proteins named holins, which are tailored for causing nonspecific membrane lesions (3,
7, 39). These lesions have always been thought as membrane holes but
their nature was unknown. The simple constitution of holins as either
two or three potential transmembrane helical segments, and the
exchangeability of those segments in chimeric molecules, prompted the
elaboration of a model in which a protein oligomer is fully responsible
for the formation of a membrane pore (7). This in-membrane
oligomerization process is particularly important, because it has been
correlated with the requirement of a critical mass action pool of
protein in the membrane that it is achieved along the lysis timing
period (39, 40).
EJh, the holin of the temperate pneumococcal EJ-1 phage is one of the
best conserved members of the type-II holin group (7, 14). In contrast
to
S, the EJh sequence does not allow the existence of two
translation initiation sites (two-start motif) and the consequent
activity regulation by the polypeptide chain length (7, 14). Moreover,
EJh sequence contains only two hydrophobic regions, whereas
S
displays three (7, 14). This apparent simplicity makes EJh an adequate
candidate to approach holins in molecular terms.
Theoretical analysis of the EJh sequence predicts the existence of two
hydrophobic segments with a length compatible with transmembrane
helices separated by a putative turn that permits a minimal
helix-turn-helix folding with both chain ends projected into the
cytoplasmic side of an imaginary inner membrane (14). All these
topological regions, when synthesized as individual peptides and
studied by CD and ATR-FTIR spectroscopies, in both aqueous and membrane
environments, essentially reproduced the predicted tendencies except in
three aspects. First, EJh-L1 (the 11-amino acid N-terminal region)
displays an environment-sensitive secondary structure characterized by
the stabilization of an extended conformation in the presence of acidic
lipids that can be explained on the basis of its sequence
amphipathicity. Second, the second predicted transmembrane helix,
EJh-M2, collapses into extended aggregates even in the presence of SDS
micelles. Extending the chain length with four or five N- and/or
C-terminal flanking residues from the protein sequence, and changing
the acyl chain of the target membrane has no impact on the observed
behavior, thus excluding hydrophobic mismatching effects and solubility
impairments. This discrepancy between the theoretical and experimental
secondary structure propensity of the isolated EJh-M2 might arise from
the dependence of folding on long range interactions, as suggested for
other membrane-spanning sequences (33-35, 41). Third, the transbilayer
disposition of the first transmembrane region is achieved only in a
longer form containing charged amino acids. Therefore, we can conclude
that, with the limitations imposed by the absence of long range
interaction and the sequential amphiphilicity, the EJh molecule might
fold essentially as predicted.
In the quest for finding the polypeptide region responsible for the
membrane-damaging activity we tested the previous peptides in a
conventional leakage assay. Among them, only those peptides containing
the N-terminal hydrophobic region, namely EJh-M1 and EJh-L1M1,
permeabilized the membranes to solutes with Stokes radii of about 6 Å (36, 37) in a lipid composition-independent fashion. This assay allows
the assignment of the membrane lesion activity to the N-terminal
hydrophobic region but does not rule out the cooperation of EJh-M2 in
the entire EJh molecule. In terms of the alternative models for the
active oligomer displayed in Fig. 1B, the present data
support a holin oligomer structure in which the pore lining is formed
by EJh-M1 units (top view b). Interestingly, EJh-M1 does
contain a proportion of polar amino acids large enough to be considered
amphipathic, but its N terminus contains a cluster of aromatic residues
that have been referred to as functional ion sinks (42, 43). Adoption
of this model requires also the verification of the capacity of EJh-M1
to oligomerize. In this sense, the relief of the self-quenching of
fluorescein-labeled EJh-L1M1 upon co-incorporation into membranes with
non-fluorescent peptide verifies this hypothesis. In this context, it
is worth emphasizing the low stability displayed by the oligomers,
because both
-octyl glucoside and SDS treatments reversed their association.
To investigate the nature of the membrane lesions produced by holins,
the conventional leakage used in activity screening is probably an
inadequate model. On a conceptual basis, holins are believed to
generate membrane discontinuities large enough to allow the passage of
a peptidoglycan hydrolase, which for EJ-1 phage is a 36-kDa amidase
exhibiting a monomer-to-dimer equilibrium (44, 45). Both pore sizing
using encapsulation relief strategies and the topographic analysis by
atomic force microscopy of lipid-peptide assemblies evidenced membrane
lesions large enough to allow the passage of the amidase partner across
the bilayer. Interestingly, the large lesions that concur with
biological activity seem to result from the enlargement of smaller
holes upon the dynamic bilayer deformations required for the attachment
to the mica. Probably the treatment used in these in vitro
experiments would not be operative enough in a cellular environment,
but the differences in composition between prokaryote cytoplasm and
periplasm ensures a variety of ways to accomplish the needed bilayer
stress. The observed slit enlargement implies the generation of
peptide-naked lipid edges as shown in the nascent pores made by
streptolysin O (46) and, consequently, their contemplation in the
original model of holin action. In this sense, transbilayer holin
oligomers could be considered as seeds for membrane deformation events.
 |
ACKNOWLEDGEMENTS |
We thank E. Díaz, J. L. García, and P. García for advice and helpful
discussions. We also thank Douglas Laurents for the careful reading
of the manuscript and for grammar corrections.
 |
FOOTNOTES |
*
This work was supported in part by Grants PB96/0850 and
BIO2000-1664 of the Ministerio de Ciencia y Tecnologia (to
M. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
e
Research Director of the Fonds National de Recherche
Scientific (Belgium).
b
Present address: Biotools BM Laboratories, Valle de
Tabalinas 52 N-43, Madrid 28021, Spain.
i
To whom correspondence should be addressed: Instituto
Química-Física Rocasolano, Consejo Superior de
Investigaciones Científicas, Serrano 119, Madrid 28006, Spain.
Tel.: 34-915-619-400; Fax: 34-915-642-431; E-mail:
mgasset@iqfr.csic.es.
Published, JBC Papers in Press, December 2, 2002, DOI 10.1074/jbc.M211334200
 |
ABBREVIATIONS |
The abbreviations used are:
EJh, EJ-1 phage
holin;
EJh-Li and EJh-Mi, synthetic peptides representing the i
solvent-exposed and i membrane-spanning predicted regions,
respectively;
ANTS, aminonaphtalene-3,6,8-trisulfonic acid;
DPX, p-xylenebis(pyridium bromide);
Flu, 5-(and
6)-carboxyfluorescein;
FD-70, FD-20, and FD-4, fluorescein
isothiocyanate dextrans of 70, 20, and 4 kDa, respectively;
POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine;
POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine;
POPG, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol;
P:L, peptide-to-lipid molar ratio;
LUV, large unilamellar vesicle;
TFE, 2,2,2-trifluoroethanol;
CD, circular dichroism;
ATR-FTIR, attenuated
total reflection Fourier transform infrared;
AFM, atomic force
microscopy.
 |
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Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.