Structural Requirements for Potent Versus Selective
Cytotoxicity for Antimicrobial Dermaseptin S4 Derivatives*,
Irina
Kustanovich
§,
Deborah E.
Shalev,
Masha
Mikhlin,
Leonid
Gaidukov, and
Amram
Mor
From the Department of Biological Chemistry and The
Wolfson Centre for Applied Structural Biology, Silberman Institute of
Life Sciences, The Hebrew University of Jerusalem, Safra Campus, Givat
Ram, 91904 Jerusalem, Israel
Received for publication, November 19, 2001, and in revised form, February 11, 2002
 |
ABSTRACT |
To better understand the structural requirements
for selective cytotoxicity of antimicrobial peptides, seven dermaseptin
S4 analogs were produced and investigated with respect to molecular organization in solution, binding properties to model phospholipid membranes, and cytotoxic properties. Native dermaseptin S4 displayed high aggregation in solution and high binding affinity. These properties correlated with high cytotoxicity. Yet, potency was progressively limited when facing cells whose plasma membrane was
surrounded by increasingly complex barriers. Increasing the positive
charge of the native peptide led to partial depolymerization that
correlated with higher binding affinity and with virtually non-discriminative high cytotoxicity against all cell types. The C-terminal hydrophobic domain was found responsible for binding to
membranes but not for their disruption. Truncations of the C terminus
combined with increased positive charge of the N-terminal domain
resulted in short peptides having similar binding affinity as the
parent compound but displaying selective activity against microbes with
reduced toxicity toward human red blood cells. Nuclear magnetic
resonance-derived three-dimensional structures of three active
derivatives enabled the delineation of a common amphipathic structure
with a clear separation of two lobes of positive and negative
electrostatic potential surfaces. Whereas the spatial positive
electrostatic potential extended considerably beyond the peptide
dimensions and was required for potency, selectivity was affected
primarily by hydrophobicity. The usefulness of this approach for the
design of potent and/or selective cytolytic peptides is discussed herein.
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INTRODUCTION |
After a half-century of virtually complete control over microbial
infections, the 1990s have brought a worldwide resurgence of infectious
diseases by evolution of antibiotic-resistant strains. So far,
resistance has developed to nearly all antimicrobial drugs (1, 2).
Levels of resistance to antibacterial agents, for instance, are
increasing at an alarming rate as antibiotics are progressively losing
their effectiveness (3, 4). Moreover, bacterial strains resistant to
all available antibacterial agents have been identified among clinical
isolates of different bacterial species, and the World Health
Organization report on essential drugs has announced antimicrobial drug
resistance as a main public health concern (5). Animal-derived
antimicrobial peptides have recently emerged as a potential class of
novel antimicrobial agents that could either complement existing
antibiotics or possibly even be used as an alternative (6, 7). So far,
more than 400 ribosomally made antimicrobial peptides have been
isolated and characterized. After extensive studies of these peptides
during the past 2 decades, it is presently becoming indisputably
evident that these ubiquitous peptides represent an essential defense component in vertebrates and invertebrates (8-11). The
three-dimensional structures of many antimicrobial peptides present an
amphipathic character, i.e. a hydrophobic face, consisting
of non-polar amino acids, topologically separated from a hydrophilic
face composed of polar and charged residues. In linear peptides, the
amphipathic character can be induced by hydrophobic media (12-14),
although conformationally constrained peptides present the
amphipathic character in both hydrophilic and hydrophobic media
(15, 16). Antimicrobial peptides vary considerably in structure, size,
amino acid sequence, and spectrum of action. Structure-activity
relationship studies have shown that the most potent peptides are
highly basic with a pronounced amphipathic character (14, 17-19).
Antimicrobial peptides are often described as membrane-active agents
although their detailed mechanism of action is not well understood.
Nevertheless, several lines of evidence exist that enable us to
stipulate a probable mechanism. Thus, isomers composed of
D-amino acids only display identical cytotoxic potency as
the L-enantiomers, implying that their mechanism of action
is not mediated by interaction with a stereospecific target such as
protein receptors, enzymes, and so on (20, 21). Rather, it seems that the peptide physicochemical properties (i.e. charge and
hydrophobicity) are the main factors affecting antimicrobial activity.
Indeed, despite a large heterogeneity in primary and secondary
structures, all antimicrobial peptides carry a net positive charge;
they can fold into well defined amphipathic structures, and they often display strong lipophilic properties and activity against a wide range
of microorganisms (6-11). In addition, microbicidal activity exhibits
strong concentration dependence. At lower concentrations, the peptides
are virtually inactive until a critical concentration is reached (22,
23) suggesting that the active form of the peptide is an oligomer. Some
of these peptides were stipulated to form ion channels or pores, as
suggested by their ability to dissipate the electric potential across
energy-transducing membranes and ion conductance across lipid bilayers
(24-30).
Despite the fact that the surrounding environment of the target cells
plasma membrane is extremely diverse, very few mechanistic studies take
into account how the mode of action of the peptides might be affected
by problems relating to the ability of the peptides to access the cell
membrane. For instance, a microbial membrane enclosed within a cell
wall would be less accessible than the virtually "naked" membrane
of protozoan cells. Correspondingly, potent antiprotozoan peptides were
shown to display weak antibacterial activity (21). Furthermore, many
antimicrobial peptides display a broad spectrum of activity (31-33),
yet many are quite inactive on normal eukaryotic cells. The basis for
this discrimination is also unclear. It appears to be related to the
lipid composition of the target membrane (i.e. fluidity,
negative charge density, and absence/presence of cholesterol), and the
possession, by the peptide-susceptible organism, of a large negative
trans-membrane electrical potential (34).
Thus, whereas the precise mechanism of action of antimicrobial peptides
is yet to be better defined, the microbicidal effect is widely believed
to result from their capacity to permeabilize the membrane of target
cells. Such a mechanism of action endows the peptide-based
antimicrobial system with attractive advantages over classical
antibiotics because it makes it extremely difficult for microbes to
develop resistance (18, 35, 36). However, a major downside of such a
mechanism is reflected in its unselective activity over a wide range of
cell types, which could be problematic, for instance, in systemic
routes of administration (37). In some ironic way therefore, a major
challenge of this field of research consists of figuring out how to
endow specificity to a system that by definition is nonspecific.
Toward better understanding the mechanism of action of
peptide-antimicrobials, particularly with respect to better defining the factors affecting potency versus selectivity, we
investigated in this study the relations between physicochemical
properties of the peptides (composition, three-dimensional
structure, and molecular organization in solution) and their
interaction with target membranes as a function of the accessibility of
the membrane. The 28-residue antimicrobial peptide dermaseptin S4 was
selected for this investigation. Dermaseptin S4 belongs to a large
family of linear polycationic peptides from frogs (12, 13, 38) displaying cytolytic activity in vitro against a large
spectrum of pathogens (21, 36, 38-44). Among natural dermaseptins, S4 is unique in displaying high cytotoxicity toward erythrocytes as well
(21, 39, 41). Previous structure-activity relationship investigations
suggested that peptide aggregation in solution, because of hydrophobic
interactions at the peptide extremities, had dramatic consequences on
cytotoxic properties including selectivity of the peptides (21). Thus,
tampering with the composition of the peptide extremities led to
disaggregation and selective activity in vitro and in animal
models for infectious disease (36, 44). Yet, the lack of
three-dimensional structural data made it impossible to draw sound
conclusions as to how the properties of the non-aggregated derivatives
contributed to selectivity (21). Here, we investigated the
three-dimensional structure of selected dermaseptin S4 derivatives. Because peptide structure might be medium-sensitive, as indicated by
circular dichroism measurements (12, 13, 31), the choice of medium for
structural studies is critical. We chose to look at the peptide
structure a fraction of time prior to the initial interaction
(i.e. the electrostatic adhesion) that we believe to
represent a most crucial moment in the mechanism of cytolytic action.
To mimic such an interface milieu, structural studies were performed in
an aqueous medium somewhat more hydrophobic than water, i.e.
20% trifluoroethanol/water (45, 46). We also investigated here the
effects of peptide alterations (length, charge, and hydrophobicity) on
cytotoxic activity as a function of accessibility to the target
membrane. The structure-dependent peptide properties were
first investigated with respect to binding to model membranes and then
correlated with their capacity to lyse erythrocytes and protozoa, for
which accessibility is maximal, or fungi, Gram-positive and
Gram-negative bacteria, cells for which accessibility is hampered due
to the presence of surrounding cell walls of increasing complexity
(47).
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EXPERIMENTAL PROCEDURES |
Peptides
The peptides were synthesized by the solid phase method as
described (21) applying the Fmoc (9-fluorenylmethyloxycarbonyl) active
ester chemistry on a fully automated, programmable model 433A Peptide
Synthesizer (Applied Biosystems).
4-Hydroxymethylphenoxymethyl-copolystyrene-1% divinylbenzene resin
(Wang resin) and 4-methylbenzhydrylamine resin (Novabiochem) were used
to obtain free carboxyl or amidated peptides, respectively. After
purification by high pressure liquid chromatography (21) the peptides
were subjected to amino acid analysis and mass spectrometry in order to
confirm their composition. Peptides were stocked as lyophilized powder
at 
20 °C. Prior to testing, fresh solutions were prepared in
water, briefly vortexed, sonicated, and centrifuged and then diluted in
the appropriate medium. Buffers were prepared using distilled water
(Milli-Q, Millipore). All other reagents were of analytical grade.
Aggregation in Solution
The aggregation properties of the peptides in aqueous solution
were investigated by static light scattering measurements on a
PerkinElmer LS-5B Luminescence Spectrometer. Peptides at an initial
concentration of 50 µM were successively diluted in 2 ml
of PBS1 (10 mM
sodium phosphate, 150 mM NaCl, pH 7.3) at room temperature, and light scattering was evaluated by measuring the reflected light at
an angle of 90°, holding both the excitation and the emission at 400 nm (5-nm slits).
To describe the dependence of scattered signal on peptide
concentration, the intensity of scattered light (mean from at least two
independent experiments) was plotted against total peptide concentrations, and a linear regression analysis was performed on the
data at the concentration range close to the monomer-micelle transition
zone. The static light scattering signal is proportional to the number
of aggregated molecules and the size of the aggregate. Therefore, the
slope is indicative of the aggregation tendency and reveals the
aggregation properties of the peptides, where a slope value above unity
indicates the presence of micellar form. For aggregating peptides, the
critical micelle concentration (CMC) was evaluated by extrapolating the
curve to the intercept with the x axis (48).
Binding Experiments
Liposome Preparation--
Small unilamellar vesicles composed of
phosphatidylcholine/phosphatidic acid (PC/PA, 1:1 molar ratio) were
prepared in PBS by the extrusion method. Briefly, dry PC and PA were
dissolved in EtOH or CHCl3, respectively, and mixed at
equimolar ratios. The solvents were evaporated under a stream of
nitrogen, and lipids were resuspended in PBS buffer at 0.5 mM total lipid concentration. The resultant suspension was
vortexed, briefly sonicated, and passed 21 times through 100 nm
polycarbonate membranes in an Avestin LiposoFast-Basic extrusion
apparatus to give a translucent solution of vesicles with a mean
diameter of 100 nm.
Surface Plasmon Resonance Analysis--
Peptide binding to
phospholipid membranes was determined using the optical biosensor
system (BIAcore 3000, Uppsala, Sweden) based on the principle of
surface plasmon resonance (49). The sensor chip L1, a carboxymethyl
dextran hydrogen derivatized with lipophilic alkyl chain anchors
(BIAcore), was used to prepare a lipid bilayer as follows. After
washing the chip with octyl glucoside (40 mM), the
liposomes (0.5 mM) were injected over the chip surface at a
flow rate of 2 µl/min at 25 °C to allow adsorption to the chip.
Irregular and loosely bound structures such as multiple lipid layers
and partially fused liposomes were washed away by a brief injection of
NaOH (50 mM) at a high flow rate (100 µl/min). Bovine
serum albumin was used to assess the degree of surface homogeneity.
Under these conditions, about 5000 resonance units of lipid were
immobilized which corresponds to a surface density equivalent to 4.6 ng/mm2 (50). Surface regeneration between consecutive
injections included the sequential injection of NaOH (50 mM) and HCl (50 mM). Association and
dissociation rate constants were calculated by nonlinear fitting of the
primary data using the BIAevaluation 3.0 software (BIAcore). The
association and dissociation rate constants were derived assuming a simple bimolecular interaction model (51). Their ratio
(ka/kd) yields the association
affinity constant Kapp.
Cytotoxic Assays
Hemolytic activity was assessed against human red blood cells.
Human blood was rinsed 3 times in PBS by centrifugation for 1 min at
2700 × g and then resuspended in PBS at 5%
hematocrit. A 50-µl suspension containing 2.5 × 108
red blood cells was added to Eppendorf test tubes containing 200 µl
of peptide solutions (serial 2-fold dilutions in PBS), PBS alone (for
base-line values), or distilled water (for 100% hemolysis). After
incubation (3 h under agitation, 37 °C) samples were centrifuged,
and the hemolytic activity was assessed as a function of hemoglobin
leakage by measuring the absorbance of 200 µl of supernatant (405 nm). Statistical data were obtained from at least two independent
experiments performed in duplicate.
Antiprotozoan activity was assessed against the promastigote form of a
Leishmania major clinical isolate (graciously provided by
Prof. Kobi Golenzer, the Parasitology Laboratory at Hadassah-Hebrew University, Jerusalem, Israel) cultured in RPMI 1640 complemented with
20% fetal calf serum, 1% penicillin, and 1% streptomycin. Inhibition
of proliferation assay was performed by adding a 100-µl suspension of
promastigotes (1 × 106 cells/ml) to 100 µl of
culture medium (non-complemented RPMI) in 96-well plates (Nunc)
containing zero or various peptide concentrations (serial 2-fold
dilutions). After the incubation period (3 h, 27 °C) the number of
viable (motile) cells was determined by counting an aliquot from each
culture on a Neubauer cell counter under a microscope (Olympus IX70).
Statistical data were obtained from at least two independent
experiments performed in duplicate.
Antifungal activity was assessed against a clinical isolate of
Cryptococcus neoformans (strain B3501, graciously provided by Prof. Itzhac Polacheck, the Mycology Laboratory at Hadassah-Hebrew University, Jerusalem, Israel) cultured in RPMI supplemented with 150 mM MOPS. Inocula of 106 cells/ml were used. The
cell populations were estimated by absorbance measurements at
620 nm referred to a calibration curve. 100-µl suspensions of the
yeast cells were added to 100 µl of culture medium containing zero or
various peptide concentrations (serial 2-fold dilutions) in 96-well
plates (Nunc). Inhibition of proliferation was determined by absorbance
measurements (620 nm) after incubation overnight at 30 °C.
Statistical data were obtained from at least two independent
experiments performed in duplicate.
Antibacterial activity was assessed against clinical isolates
(graciously provided by Dr. Yehuda Carmeli, Division of Infectious Diseases, Sourasky Medical Center, Tel Aviv, Israel) of
Staphylococcus aureus (strain B38302) and Escherichia
coli (strain U16318) representatives for Gram-positive and
Gram-negative bacteria, respectively. Growth inhibition was assessed
essentially as described for the antifungal assay except that the
culture medium used was 2xty (16 g/liter tryptone, 10 g/liter
yeast extract, 5 g/liter NaCl, pH 7.4), and incubations were performed
at 37 °C.
CD Measurements
CD spectra in millidegrees were measured with an Aviv model 62A
DS spectrometer (Aviv Associates, Lakewood, NJ) using a 0.020-cm rectangular QS Hellma cuvette at 25 °C (controlled by thermoelectric Peltier elements with an accuracy of 0.1 °C). The CD spectrum was
scanned for peptide samples (280 µM, determined by UV
using standard curves of known concentrations for each peptide) that were dissolved in 20% (v/v) trifluoroethanol/water. CD data represent average values from three separate recordings.
NMR Measurements
For the NMR studies, a 1 mM solution of each S4
derivative (Table I) was prepared by dissolving lyophilized powder in
an aqueous solution containing 20% (v/v)
trifluoroethanol-d3 (Aldrich) at the apparent
pH = 2.8-3.5.
NMR experiments were carried out on a Bruker Avance 600 MHz DMX
spectrometer operating at the proton frequency of 600.13 MHz using a
5-mm selective probe equipped with a self-shielded xyz gradient coil. The transmitter frequency was set on the HDO signal. The
residual water resonance was suppressed using a Watergate sequence (52)
for TOSCY experiments and by low power cw irradiation during the
relaxation delay for the double quantum filtered COSY and the NOESY
experiments, and the mixing time of the latter. Two-dimensional
homonuclear spectra were acquired in the phase-sensitive mode with
2K complex data points in t2 and 400 or
512 t1 increments depending on the experiment.
The spectral width was 13 ppm, and the relaxation delays were set to
1.2 and 2 s in the TOCSY and NOESY experiments, respectively.
TOCSY spectra were recorded using the MLEV-17 pulse scheme for the spin
lock at several isotropic mixing periods ranging over 50-110 ms with
48-88 scans per t1 increment depending on the
peptide used (53). The NOESY experiments were collected with mixing
times varying from 100 to 550 ms in order to gain maximal NOE buildup
with a minimal contribution from spin diffusion. The build-up curves
were monitored for several well resolved backbone amide protons and the
tryptophan aromatic protons when present (Table I). Between 112 and 224 transients were acquired for each t1 for
structural analysis. The water signal was used as a reference for all
samples, and its chemical shift was corrected at low temperatures
relative to external trimethylsilyl propionate (sodium salt; Cambridge
Isotope Laboratories) as the zero point.
The NH-C
H scalar couplings were obtained by fitting the Lorentzian
line shapes (Aurelia program) to the in-phase doublets in TOCSY spectra
collected with 8K points in the F2
dimension and zero-filled to 16K points. For several overlapping
signals it was impossible to derive
3JNH
couplings with an adequate
fit (Fig. 4).
Spectra were processed and analyzed with the XWINNMR and Aurelia
software packages (Bruker Analytische Messtechnik GmbH) on Silicon
Graphics Indy R4000 and Indigo2 R10000 workstations. Zero filling in
the t1 dimension and data apodization with a
shifted squared sine bell window function in both dimensions was
applied prior to Fourier transformation. The base line was further
corrected in the F2 dimension with a quadratic
polynomial function.
NOE Measurements and Experimental Distance Restraints
NOESY spectra used for the determining structural organization
of the S4 dermaseptin short and substituted derivatives (Table I) in
20% TFE/water solution were recorded at 285 K. A more elevated temperature of 310 K was required to obtain an adequate spectrum for
the K4K20-S4 analog because at lower
temperatures severe overlap of the resonance lines was observed,
presumably due to the aggregation of the peptide. The mixing time was
set to 250 ms for all peptides, although different values were
carefully inspected with respect to the possible contribution of spin
diffusion for all the peptides used in this study (see above).
Furthermore, NOESY spectra of the short derivatives collected at longer
mixing times did not reveal any additional significant NOE
connectivities. Integrated peak volumes were converted into distance
restraints using an r
6 dependence and were
calibrated relative to the fixed distance of 2.66 Å between the two
adjacent protons of the tryptophan indole ring. Pseudoatom corrections
were used for degenerated protons of the methyl groups (54, 55). For
the structural calculations, a total of 181 distance restraints were
used for K4K20-S4 peptide, among which 66 were
sequential and 47 were between i,i + 2, i,i + 3 and i,i + 4 pairs
of interacting protons (for details see Fig. 4). Among the 131 restraints obtained for the K4-S4(1-16)a analog, 52 were
sequential and 40 medium range restraints stemming from the NOE
connectivities between residues further than two residues apart. The
shortest analog, K4-S4(1-13)a, gave a total 146 restraints
comprising 46 intra-residual, 59 sequential, and 41 medium ranged restraints.
Structure Calculation
The structures were calculated by the hybrid distance
geometry-dynamical simulated annealing method using XPLOR version 3.856 (56). The NOE energy was introduced as a square well potential with a
force constant of 50 kcal/mol·Å2 that was kept constant
throughout the protocol. Each round of simulated annealing refinement
consisted of 1500 3-fs steps at 1000 K and 3000 1-fs steps during
cooling to 300 K. Finally the structures were minimized using conjugate
gradient energy minimization for 4000 iterations. These initial low
resolution structures were used to assign stereospecifically ambiguous
peaks. The InsightII program (Molecular Modeling System version 97.0, Molecular Simulations, Inc.) was used for visualization and analysis of
the NMR-derived structures. Their quality was assessed using Procheck
(57).
Electrostatic free energies were derived from finite difference
solutions of the Poisson-Boltzman equation using the DelPhi program
from within the InsightII software package (58). The AMBER forcefield
(59) was employed using Van der Waals radii, and a full Coulombic
calculation was performed using a boundary extending 10 Å beyond the
longest axis of the peptide. The internal peptide dielectric constant
was 5.0; the water dielectric constant was 80, and its radius was 1.4 Å. When a dielectric constant of 60 was used to reflect the addition
of trifluoroethanol, no significant change in the calculated
electrostatic potential distribution resulted. An energy convergence
criterion of 1 × 106 kcal/mol was applied.
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RESULTS |
Toward a better understanding of the structural requirements for
selective cytotoxicity, we generated a series of dermaseptin S4
derivatives (the peptide sequences are listed in Table
I). Peptide purity was in the range of 95 to >99% as determined by analytical high pressure liquid
chromatography, and their identity was confirmed by mass spectrometry
and amino acid analysis (data not shown). The peptides were
investigated with respect to organization in solution, binding
properties to model phosphomembranes, and cytotoxic properties, and the
three-dimensional solution structure of selected peptides was
determined.
Aggregation in Solution--
Peptide aggregation was investigated
using their static light scattering properties in PBS as a function of
the range of concentrations used for bioassays (up to 50 µM). Fig. 1 shows the
concentration-dependent light scattering profile of the
native dermaseptin and its derivatives. The results indicate that
dermaseptin S4 is in the highest aggregation state (slope = 12, a
slope of 1 is expected for monomers) with a CMC value of 0.2 µM as estimated from the intersection with the peptide
concentration axis (inset of Fig. 1). This is in close agreement with the value found (0.1 µM) using a
fluorescence method (39). The truncated derivatives, S4(9-28) and
S4(5-16), did not display aggregative properties. This is indicated by
the linear relationship between their light scattering and
concentrations (slopes = 0.8-0.9).
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Fig. 1.
Aggregation properties of dermaseptin S4 and
derivatives in PBS. Aggregation state is revealed from the
concentration dependence light scattering of the peptides. Data
represent mean from at least two independent experiments. For
aggregating peptides, critical micelle concentration was estimated by
extrapolating the curve to the intercept with the x axis as
depicted in the inset. Aggregated S4 and monomeric
K4-S4(1-16)a (asterisks and
rectangles, respectively) are shown as typical
examples.
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K4K20-S4 was clearly less aggregated than
native dermaseptin S4 (slope = 7.4 with the CMC estimated at
0.3-0.4 µM) suggesting that the introduction of a
positive charge in each of the hydrophobic domains was responsible for
limiting inter-monomer interactions. Similarly, replacing the 12 C-terminal residues of K4K20-S4 with a
carboxamide resulted in additional loss of aggregation. Further truncating the C terminus of the peptide by 3 (or 6) residues resulted
in a similar outcome; both K4-S4(1-13)a and
K4-S4(1-10)a displayed the light scattering profile
expected for monomers.
Binding Experiments--
To investigate the lipophilic properties
of the peptides, their association affinity constant
(Kapp) was determined using the BIAcore system
whereby small unilamellar vesicles composed of PC/PA were adsorbed onto
a sensor chip (L1) to form a supported lipid bilayer, which chemically
and physically resembles the surface of a cell membrane. Fig.
2 portrays a typical binding
(association/dissociation) profile for various concentrations of
dermaseptin S4 from which the Kapp was derived.
Nonlinear analysis of the sensorgram data was performed assuming a
simple bimolecular association model (60) as used previously to
describe the binding affinity of the antimicrobial peptide cecropin
(61). Although this represents a certain simplification, a satisfactory
fit of the binding curves to this model was obtained. Because the level
of immobilized membrane varied slightly between the injection cycles,
the maximal response (Rmax) was set to a local
parameter, i.e. unique for each sensorgram. The resulting
data are summarized in Table I. Note that the mathematical model used
for calculating the rate constants assumes that the reactants are
monomers, whereas aggregation reduces the apparent monomeric
concentration by an unknown extent. Therefore, the
Kapp values obtained for aggregated peptides can
only be considered indicative. For the sake of comparison,
phosphatidylcholine and bovine serum albumin were assayed under the
same experimental conditions and found to have affinity constants of
4.2 × 1010 and <1 × 103
M1, respectively.
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Fig. 2.
Overlay of sensorgrams of the binding to
PC/PA bilayer for various concentrations of dermaseptin S4. The
membrane bilayer, composed of PC/PA (1:1), was immobilized onto an L1
chip. The plot depicts the interaction kinetics for increasing peptide
concentrations in PBS at 25 °C. Each sensorgram represents an
average curve of two injections.
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Native dermaseptin displayed a high binding affinity (3.0 × 106 M1). Deletion of the eight
N-terminal residues resulted in increased binding affinity (8.2 × 106 M1). Deletion of both
hydrophobic extremities resulted in a binding level below detection,
i.e. a loss of binding affinity by more than 3 orders of
magnitude (<1 × 103 M1).
The substituted derivative K4K20-S4 displayed
the highest binding affinity (5.2 × 107
M1). Compared with
K4K20-S4, the shorter derivative,
K4-S4(1-16)a, displayed a 10-fold reduced binding affinity
(2.9 × 106 M1). It is
noteworthy that truncating the C terminus of the peptide by an
additional 3 residues did not affect the binding significantly, but
further truncation, to 10 residues, led to loss of binding affinity by
2 orders of magnitude.
Cytotoxicity--
To assess the membranolytic potential of the
dermaseptin S4 derivatives, their ability to induce hemoglobin leakage
was investigated in PBS. In parallel, the ability of the peptides to
affect the viability of protozoan cells was investigated in a minimal
culture medium to minimize irrelevant peptide interactions with medium components. The resulting data are summarized in Table I. Incubation of
erythrocytes in the presence of dermaseptin S4 resulted in massive
hemoglobin release corresponding to 50% hemolysis at a peptide
concentration of 1.5 µM. An identical peptide
concentration was needed to kill 50% of leishmanial cells. However,
deletion of the N-terminal hydrophobic domain resulted in a 15-fold
reduction of hemolytic activity and a 33-fold reduction of
leishmanicidal activity. Truncating both hydrophobic extremities
resulted in a general loss of activity. Substitution of positions 4 and
20 by lysines resulted in a 3-fold increase of hemolytic activity, but
leishmanicidal activity remained unaffected. Compared with K4K20-S4, K4-S4(1-16)a displayed a
30-fold reduction in hemolytic activity but only a 4-fold reduction of
leishmanicidal activity. Further C-terminal truncations resulted in
further loss of activity with similar proportions. Thus, compared with
K4K20-S4, K4-S4(1-13)a displayed a
reduced hemolytic activity by more than 100-fold but only a 6-fold
reduction of leishmanicidal activity. K4-S4(1-10)a was
inactive in both assays up to the highest concentration tested (50 µM).
Susceptibility of cell wall-containing microorganisms (fungi and
bacteria) to dermaseptin S4 and its derivatives was assessed by
measuring the minimal inhibitory concentration (MIC) of the peptides
against three clinical isolates including yeast cells C. neoformans, Gram-positive bacteria S. aureus, and
Gram-negative bacteria E. coli. The resulting data are
summarized in Table I. Dermaseptin S4 displayed a MIC at the peptide
concentration of 4.5 µM against C. neoformans,
and the peptide had a 5-fold reduced potency against Gram-positive
bacteria and was virtually inactive against Gram-negative bacteria.
Truncation of one or both hydrophobic domains resulted in a general
loss of activity. Unlike dermaseptin S4, the derivative
K4K20-S4 displayed a rather similar potency against the three types of targets (MICs of 2.25, 2, and 1.5 µM, respectively). The short derivatives,
K4-S4(1-16)a and K4-S4(1-13)a, also displayed
a nearly homogeneous potency in all three assays with a progressive
loss of potency compared with K4K20-S4.
Finally, K4-S4(1-10)a was weakly active against C. neoformans and E. coli but was virtually inactive
against S. aureus up to the highest concentration tested (50 µM).
CD Measurements--
Preliminary indications of the peptide
structures in solution were obtained with CD measurements in water and
in 20% TFE/water. All peptides displayed typical spectra of unordered
structure in water (data not shown). In the more hydrophobic medium
however, the CD profile of some peptides indicated a clear shift toward an ordered structure, possibly of typical -helix as characterized by
double minima at 208 and 220 nm. The CD spectra are summarized in Fig.
3. The structure of native dermaseptin
contained -helical components, whereas the CD profiles of S4(9-28)
and S4(5-16) progressively shifted toward random structures. The CD
profile of K4K20-S4 suggested that charge
increase led to increased helical content. Interestingly, the data also
suggested that the 16-mer and, surprisingly, even the 13-mer derivative
maintained a helical structure. The concentration dependence was
investigated for K4-S4(1-13)a in the range of 100-400 µM. No dependence was observed. We cannot exclude the
possibility that this is because the lowest concentration tested was
already above the concentration at which aggregation is complete. We
were unable to obtain unambiguous quantitative data at lower
concentrations due to reduced signal strength. The shortest derivative
K4-S4(1-10)a displayed random structure.
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Fig. 3.
Far-UV circular dichroism spectra of
dermaseptin S4 and its derivatives in 20% trifluoroethanol/water.
Left panel compares the CD spectra of the 28-residue
native dermaseptin S4 with two truncated derivatives: S4(9-28) and
S4(5-16). Right panel compares the CD spectra of
K4K20-S4 with three truncated derivatives:
K4-S4(1-16)a, K4-S4(1-13)a, and
K4-S4(1-10)a.
|
|
NMR Measurements and Resonance Assignment--
Further insight
into the molecular organization of the dermaseptin analogs was obtained
by conducting NMR structural studies. The proton spectrum of the native
S4 peptide in water showed broad unresolved signals indicative of
severe aggregation. Furthermore, although various dermaseptin analogs
dissolved well in water as shown by the presence of their sharp
spectral lines, their two-dimensional NOESY spectra did not contain any
NOEs characteristic of a defined stable conformation, in agreement with
the CD findings (see above). Titrating the aqueous samples with TFE
showed that increasing the alcohol content from 0 to 20% v/v induced a
marked difference in the appearance of the spectra of
K4K20-S4, K4-S4(1-16)a and K4-S4(1-13)a substituted analogs (group 1) and S4(9-28),
S4(5-16) and K4-S4(1-10)a peptides (group 2). The NOESY
spectra of the first group showed numerous strong NOE connectivities
suggesting the presence of a stable preferred structure. Further
increasing the alcohol content to 100% did not lead to additional
changes in their NOESY spectra. On the other hand, the corresponding
spectra of the second group were unaffected by addition of TFE
indicating that this membrane-mimicking environment does not perturb
peptides with no propensity toward helix formation and leaves them in
flexible, random coil conformations that are in fast exchange on the
time scale of the NMR. The behavior of the S4 peptide was exceptional. Even at high alcohol concentrations, the peptide remained aggregated and therefore was unsuitable for structural studies. Based on these
observations, combined with the supporting CD results (see above), an
aqueous solution containing 20% v/v trifluoroethanol was chosen for
the structural NMR determinations.
The resonance assignments of all dermaseptin analogs were carried out
based on the TOCSY and NOESY spectra measured consecutively and under
the same experimental conditions, according to the sequential assignment methodology developed by Wüthrich (62). Although no
line broadening was detected from the increased viscosity upon addition
of TFE (63, 64), strong signal overlap was observed due to the presence
of multiple copies of several amino acids, in particular Ala, Lys, and
Leu. The weak dispersion of their NH and H chemical shifts
constituted a major difficulty in peak assignment and necessitated
acquiring TOCSY spectra at several temperatures. The line width of the
signals remained narrow throughout the measured temperature range
(280-315 K), allowing us to assign the vast majority of the signals
with the exception of a small number of degenerated 
H protons of Lys
and Leu (see Table II for the chemical
shifts of the first group; chemical shifts of the second group are
reported in Supplemental Table I). Moreover, no broadening indicative
of aggregation and/or slow exchange was observed for any of the
peptides.
Secondary Structure Determination for
K4K20-S4, K4-S4(1-16)a, and
K4-S4(1-13)a--
The NOESY spectra of the S4(9-28),
S4(5-16), and K4-S4(1-10)a derivatives did not contain
any substantial structural information over the entire temperature
range in a 20% TFE/water solution. Cooling these samples to 277 K
resulted in the appearance of several weak sequential HN-H
connectivities (data not shown). Their chemical shifts (Supplemental
Table I) were consistent with the reported random coil values, and
3JNH coupling constants ranged
between 5.5 and 8.0 Hz indicating an equilibrium average of the
different conformers.
Fig. 4, A-C,
summarize the NOE cross-peaks detected for the first group of
substituted analogs. Analyzing these patterns revealed a remarkable
similarity between the spectral features of the N terminus of the
K4K20-S4 peptide and those of the
K4-S4(1-16)a and K4-S4(1-13)a analogs. Almost
continuous stretches of
dNN(i,i+1) connectivities
were observed between Leu2 and Ala15 (K12 for
the 13-mer analog), and nearly complete sets of
dN(i,i+1) and
dN(i,i+3) cross-peaks
were spread throughout each entire peptide. These spectral patterns are
typical of an -helical structure. This finding was further
substantiated by the observation of a series of strong NOE cross-peaks
of the dN(i,i+4) type which are
specifically found in -helices. There were a few resonances that
could not be assigned unambiguously (Fig. 4), presumably because of the
small dispersion of their chemical shifts due to the -helix
structure. In particular, an aliphatic region of the NOESY spectra
showed severe overlap of the resonances, thus preventing unambiguous
assignment of the 
(i,i + 3) pairs which also occur in
-helices. In addition, several
dN(i,i+2) and
N(i,i+2) connectivities,
characteristic of a 310-helix, were also identified. In
comparison, the NOESY spectrum of the C-terminal part of the
K4K20-S4 peptide showed only a few, mainly
sequential, NOE connectivities that could not be attributed to any
defined structural organization. The
3JNH coupling constants of
residues Lys16-Ala28 varied from 5 to 11 Hz
suggesting a conformational dispersion of random coil structures in
this region.
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Fig. 4.
Summary of the NOE connectivities observed
for K4K20-S4 (A),
K4-S4(1-16)a (B), and
K4-S4(1-13)a (C) in 20% TFE/water.
Coupling constants are indicated with filled circles for
3JNH smaller than 5 Hz,
open circles for 5 Hz < 3JNH < 7.5 Hz, and filled
squares for 3JNH larger than
8 Hz. Residues Ala18 (in A) and
Lys16 (B) could not be fitted due to
overlap.
|
|
The -helix spanning the N-terminal residues Leu2 to
Ala15 of K4K20-S4 and the entire
16- and 13-mer peptides was confirmed by the small values observed for
the 3JNH coupling constants; most
residues had coupling constants below 5 Hz (filled circles
in Fig. 4). A few exceptions (open circles) were found
within these helical regions, in particular the C terminus of the
K4-S4(1-16)a analog, indicating some flexibility at the
ends and/or equilibrium between a helical structure and a more random organization.
It is well established that the up-field deviations of the chemical
shifts of the H protons from their random coil values are
characteristic of residues adopting an -helical structure (65, 66).
The H chemical shifts (Table II) of the three analogs were compared
with (i) the random coil values in TFE/water solution reported by
Merutka et al. (67) and to (ii) the comparative values
reviewed by Richards and co-workers (66). Although the latter values
were observed for small peptides dissolved in water, it has been shown
that random coil H values are insensitive to the addition of
trifluoroethanol up to 30% (68). In Fig.
5 the up-field shifts are summarized for
the two short analogs and the N terminus of the longer peptide. The
corresponding values of the C-terminal residues of the
K4K20-S4 peptide did not show any significant
structure-related deviations (data not shown). By setting a threshold
value of 0.2 ppm from the random coil H values as indicative of an
-helical structure, it can be seen that the -helix is confined to
the N terminus of the K4K20-S4 peptide,
i.e. from Ala1 to Lys16, and
extends over almost the entire K4-S4(1-16)a peptide
(except for the values of Leu2, as observed for all three
peptides). The magnitudes of the chemical shift deviations found for
the majority of the K4-S4(1-16)a residues indicates that
even this short peptide adopts a well defined -helical structure.
The deviations of the three C-terminal residues were found to be below
the threshold value suggesting conformational flexibility in this
region.
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Fig. 5.
The deviation of the 1H chemical
shifts from the random coil values for the N terminus of
K4K20-S4 and the two short analogs.
|
|
NMR-calculated Structures of K4K20-S4,
K4-S4(1-16)a, and K4-S4(1-13)a--
The NMR
data collected for the S4(9-28), S4(5-16), and
K4-S4(1-10)a peptides (as described above) clearly
indicated the lack of any molecular conformation in the 20% TFE/water
solution. Molecular modeling of the three-dimensional structures of the
K4K20-S4, K4-S4(1-16)a, and
K4-S4(1-13)a peptides was carried out. The results of the
final simulated annealing stage in the XPLOR program for the calculated
structures yielded 68, 87, and 60 low energy structures for three
peptides, respectively, which had no NOE restraint violations above 0.5 Å, and bond lengths were correct to within 0.05 Å and angles within
5°, and dihedral violations were within 5° of ideal values. These
calculated structures were subjected to validation by the Procheck
statistical analysis (57). For each peptide, 30 best low-energy
structures with residues located only in the "most favored" and
"allowed" regions of the Ramachandran plot were chosen for
structural analysis. The superpositions of the 15 low-energy calculated
structures on their average structure for each peptide are presented in
Fig. 6. For the
K4K20-S4 analog (Fig. 6A), the
alignment was optimized for the N-terminal residues Ala1 to
Lys16, whereas for the other two short analogs (Fig. 6,
B and C) all residues were included. As
anticipated from the very limited number of NOE connectivities detected
for the C terminus of the K4K20-S4 peptide
(Fig. 4), the NMR-derived structures showed no convergence in this
region of the molecule, confirming the lack of a well defined secondary
structure and the inherent flexibility in this region of the molecule.
However, the N-terminal segment of the peptide structures lines up into
a well defined -helical structure. The r.m.s.d. values calculated
between residues 1 and 16 of the 30 low-energy structures and their
corresponding average structure are 2.3 Å for the backbone atoms. The
structures of the two K4-S4(1-16)a and
K4-S4(1-13)a peptides contain a long -helix spanning
the entire length of each peptide with the exception of the terminal residues which typically are more flexible. The r.m.s.d. values, calculated for the entire length of each peptide as above, were 1.9 and
1.3 Å for the backbone atoms, respectively. The fact that the
truncated peptides K4-S4(1-16)a and
K4-S4(1-13)a show a defined and stable conformation is
unusual considering their short lengths, but the most compelling
evidence for the importance of the helical structure conformation of
the N termini of the peptides for antimicrobial action is the fact that
the first 16 residues of the K4K20-S4 show a
well defined helical structure, whereas the C-terminal half of the
peptide shows flexibility and no clear structure. Numerous hydrogen
bonds between the backbone NH-O atoms were identified in the
calculated structures of all three helices. The most abundant donor-acceptor pairs (appearing in over 80% of the calculated structures which fulfilled all the NOE and Ramachandran validation criteria as described above) were found between residues
Thr5-Leu2, Leu6-Trp3,
Leu7-Lys4, Lys8-
Lys4, Lys9-Thr5,
Val10-Leu7, and
Lys16-Ala14 located at the N terminus of the
K4K20-S4 peptide;
Leu6-Trp3, Lys8-
Lys4, Lys9-Thr5,
Leu11-Leu7,
Leu11-Lys8,
Lys12-Lys8,
Ala13-Lys9, and
Ala14-Val10 for the K4-S4(1-16)a;
and Thr5-Leu2,
Leu6-Trp3, Leu7-Lys4,
Lys8-Lys4, Lys9-Thr5,
Lys12- Lys8, and
Ala13-Val10, for the K4-S4(1-13)a
analog.
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Fig. 6.
Superposition of 15 low energy NMR-derived
structures. K4K20-S4 (A) was
aligned over residues Ala1-Lys16 at the N
terminus. Peptides K4-S4(1-16)a (B) and
K4-S4(1-13)a (C) were superimposition in their
entirety.
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|
Spatial Distribution of the Electrostatic Potential for
K4-S4(1-16)a and K4-S4(1-16)a--
The
striking correlation between the presence of the helix in the N
terminus of K4K20-S4 and the stable helices of
the smaller K4-S4(1-16)a and K4-S4(1-13)a is
exemplified by the superposition of their representative low-energy
structures (Fig. 7A). Residues Lys2-Ala15 and
Lys2-Ala13 within the well defined region of
the average structure of K4K20-S4 peptide were
aligned with the corresponding segments of the entire ensembles of
K4-S4(1-16)a and K4-S4(1-13)a peptides. The
r.m.s.d. of the backbone atoms of the ensemble of truncated peptides
from the average non-truncated analog were 1.6 and 2.1 Å,
respectively. The helical conformations govern a unique amphipathic
organization of the hydrophobic (red) and hydrophilic
(blue) amino acids shown in Fig. 7A.
Because the biological and structural data both indicate that the
active portion of the peptide is the N-terminal region, we present data
for the two truncated peptides that span this region without the
complication of the flexible C-terminal region. The hydrophobic and
hydrophilic regions within the peptides are clearly delineated, as seen
from the hydrophobic surface in Fig. 7B, where the
K4-S4(1-16)a peptide (left) and
K4-S4(1-13)a peptide (right) are shown from the
side (top of panel) and along the long axis of
the helix (bottom of panel). The spatial
electrostatic potential distribution was calculated for the peptides,
and the surfaces corresponding to +1 kT/e (blue) and
1 kT/e (red) potentials are shown in Fig. 7C.
The most striking observation is the clear separation of the
electrostatic potentials into a positive and negative region. This
feature is seen in the N-terminal region of the 28-mer peptide (data
not shown) and is amplified in the truncated analogs, presumably due to
the contribution of an additional positive charge from the amidated C
terminus (Table I). These continuous regions of the monovalent positive
and negative lobes lie along the long axes of the peptides. The
positive potential extends far beyond the Van der Waals radii of the
peptides suggesting that it is likely to take part in the initial long
range recognition of negatively charged pathogenic membranes.
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Fig. 7.
Comparison of amphipathicity,
hydrophobicity, and electrostatic distribution of dermaseptin S4
derivatives. A, truncated derivatives,
K4-S4(1-16)a and K4-S4(1-13)a, superimposed
on the N terminus of K4K20-S4. The
blue and red colors represent hydrophilic and
hydrophobic amino acids. B, van der Waals surfaces of
K4-S4(1-16)a and K4-S4(1-13)a colored
according to their Kyte-Doolittle hydrophobicity profile (72).
C, spatial electrostatic potential distribution of
K4-S4(1-16)a and K4-S4(1-13)a; red
and blue lined surfaces indicate the negative 1 kT/e and
positive 1 kT/e surfaces, respectively.
|
|
| |
DISCUSSION |
Toward better defining the factors affecting selective
cytotoxicity, we investigated in the present study the relation between peptide molecular organization in solution upon encountering its target
and peptide cytolytic effect, taking into account the ability of the
peptide to access the target membrane. We first tried to understand how
physicochemical parameters affected binding to an idealized charged
membrane model. Then peptide properties were correlated with their
capacity to affect cells whose plasma membrane is readily accessible
(erythrocytes and protozoa), as well as cells for which accessibility
is hampered due to the presence of surrounding cell walls (fungi and
bacteria). Extensive NMR data were taken for all dermaseptin peptides
under investigation. The ensembles of the structured peptides and
peptide regions composed a large number of structures and showed good
convergence with low r.m.s.d. values clearly indicating the presence of
high resolution, well defined, preferred helical structures at the N
terminus of K4K20-S4, and for the entire
K4-S4(1-16)a and K4-S4(1-13)a peptides. The
Procheck statistical evaluations completely corroborated these findings. The data provided clear information regarding the
conformational state of the S4 analogs constituting a solid basis for
analyzing the chemical-physical properties of these peptides and
allowing us to address certain aspects of the mechanism of action.
NMR-derived structures enabled the delineation of structural
characteristics that we believe form the basis for the activity of
dermaseptin S4 and, perhaps, of many other antimicrobial peptides to an
extent. From a purely structural point of view, the data revealed the
fact that the amino acid sequence common to the three peptides has a
strong inclination to fold in the environment represented by 20%
TFE/water. Folding enabled the topological segregation of charged
residues from uncharged ones, thus forming a bi-lobed amphipathic
structure that, besides its inherent hydrophobic/hydrophilic characteristics, engendered a positive electrostatic potential that
extended well beyond the physical dimensions of the peptides. The fact
that this potential distribution is common to all the active peptides
indicates a role in interactions of the cytolytic peptide with target membranes.
Native dermaseptin S4 displayed highly lipophilic properties as
suggested by its high Kapp to the model membrane
and as indicated by its tendency to aggregate in solution (NMR and
light scattering data). Furthermore, dermaseptin S4 displayed high
membranolytic activity against red blood cells and
Leishmania, with very rapid kinetics (lysis of both types of
these cells can be observed under microscope within seconds, data not
shown). These properties may be rationalized with respect to the steps
involved in the theoretical mechanism of action where monomers are
stipulated to adhere to the membrane until reaching a threshold
concentration. Our data suggest that aggregation is responsible for the
rapid and highly efficient activity because naked membranes are
contacted by a pre-formed active entity. Therefore, the relative
inactivity of such an entity against E. coli may be directly
related to the inability of the peptide to cross the external membrane
as supported by the progressive loss of activity with increasingly
impermeable cell walls (C. neoformans > S. aureus > E. coli). The fact that aggregated
peptides become fully active against E. coli if tested in
the presence of EDTA further supports this
possibility.2
Aggregation in solution and its consequences could affect the activity
of many other antimicrobial peptides in a similar fashion. For
instance, the bee venom peptide, melittin (69), or the human peptide,
LL-37 (70), were reported to form aggregates in aqueous solutions. Both
peptides are highly hemolytic, whereas their non-aggregated derivatives
display significantly reduced hemolytic activity (70). Surprisingly,
deleting the eight N-terminal residues of S4 resulted in increased
binding affinity. Although this could reflect inaccurate determination
of the affinity constant of dermaseptin S4 (because of its aggregated
state), it could also mean that monomeric S4(9-28) is genuinely
endowed with increased binding properties. This possibility is
supported by the fact that S4(9-28) displayed a lower dissociation constant (data not shown). Regardless, although S4(9-28) was endowed with high binding affinity, the peptide was inefficient in affecting cell viability, and both NMR and CD data showed its low tendency toward
folding in solution. This indicates that binding does not require particular folding and is required for but does not necessarily lead to membrane disruption. To induce membrane disruption the peptide
needed the properties encompassed by the missing N-terminal sequence,
including the ability to fold properly. It is therefore not surprising
that truncating both hydrophobic domains resulted in dramatic loss of
structure, binding, and all activities assessed even though the
original net charge of +4 was preserved (Table I).
K4K20-S4 also exhibited high membranolytic
activity against red blood cells and Leishmania. This
correlated nicely with the increased binding affinity of the peptide
compared with dermaseptin S4. Although the NMR structure of S4 was not
resolved due to its high aggregation state, it is possible that the
increase in potency is also related to a quantitative increase in the
electrostatic potential which in turn could be responsible for enhanced
binding due to the enlarged charged surface displayed by
K4K20-S4. This possibility will hopefully be
elucidated in future studies. In contrast,
K4K20-S4 was about 2-fold less aggregated in
PBS and displayed nearly equipotent cytotoxicity against all types of cells tested. The following interpretation can account for our observations. If we consider the peptide solution under
monomer/oligomer equilibrium, the data can be interpreted as follows:
twice as many molecules of K4K20-S4 are present
as monomers in the solution compared with S4. If there are enough
monomers in the solution (note that estimated CMC is approaching the
average MIC), then the cell wall containing pathogens could become
permeable to monomeric K4K20-S4,
i.e. the peptide will be able to reach the plasma membrane and exert its membranolytic potential that is increased due to its
higher charge.
To obtain a quantitative estimation of monomer units (n)
within the aggregate, an attempt to determine n for S4 and
K4K20-S4 was made using SDS-PAGE as described
previously (71). However, both peptides gave smears of molecular weight
that increased with increasing peptide concentration. The range of
molecular mass was estimated between 6 and 21 kDa for the
highest concentration tested (350 µM). Under these
conditions n may be estimated as being between 2 and 6.
The high binding affinity displayed by S4(9-28) suggested that binding
affinity is mostly influenced by the C-terminal sequence. Yet,
truncation of the C-terminal sequence did not abolish binding. This
reflects the contribution of the N-terminal sequence to binding, particularly because its net charge content, was increased (from +4 to
+6 in K4-S4(1-16)a), which seems to have greatly
compensated for the truncated C terminus. Moreover, the reduced binding
affinity displayed by K4-S4(1-16)a compared with
K4K20-S4 correlated nicely with the loss of
hemolytic and anti-leishmanial activities but contrasted the fact that
K4-S4(1-16)a hardly lost any potency against yeast and
bacteria. This may be rationalized in light of the aggregation and
structural results. Because the peptide is no longer aggregated in
solution, its activity can be expected to follow the experimental
"rule" that endows potent membranolytic properties to highly
charged peptides that have the ability to fold into an amphipathic
structure. Our structural data suggest that when
K4-S4(1-16)a enters into an interaction with the target membrane, the peptide presents a structure similar to the amphipathic structure of K4K20-S4, although more compact
and with modified hydrophobic properties (due to the truncated C
terminus). Accordingly, monomeric K4-S4(1-16)a exerts more
potency on membranes of microorganisms than on red blood cell membranes
for the "classical" (and yet poorly understood) reasons related for
instance to the presence of a transmembrane potential, negative charge
density, and membrane fluidity.
Similar observations can be made regarding K4-S4(1-13)a.
Compared with K4-S4(1-16)a, the shorter derivative was
only slightly less active despite having lost both charge and
hydrophobicity. Its preserved activity seems to result from maintaining
the same three-dimensional structure and a similar electrostatic
potential. As for K4-S4(1-10)a, having further lost both
charge and hydrophobicity in addition to having lost the ability to
fold into an amphipathic structure, the peptide became incapable of
interacting properly with membranes and efficiently affecting target
cell integrity.
The biological and structural data are well correlated. The N-terminal
regions of all active, structured S4 analogs have similar three-dimensional features that govern the spatial distribution of the
electrostatic potential. Our findings indicate that this property is
necessary for cytotoxicity and without it the activity is severely
curtailed; S4(5-16) has all the four lysine residues of active S4 and
the same net positive charge as active K4-S4(1-13)a, but
because it is unstructured it is inactive. K4-S4(1-10)a,
lacking one positively charged residue, loses its ability to maintain the bi-lobal potential distribution leading to a severe reduction in
activity. The extent of this positive electrostatic lobe correlates the
degree of cytolytic activity as seen in the increase of activity between K4-S4(1-13)a and K4-S4(1-16)a.
Another essential factor is the ability of the peptide to bind to the
target membrane. Our studies show that the C terminus enhances binding
properties. The S4(9-28)-truncated analog represents the highly
hydrophobic C-terminal region of S4 and shows the strongest binding
affinity but is unstructured and has no activity. The combined effect
of these two characteristics, the extended positive electrostatic potential and the binding affinity of the hydrophobic C terminus, is
essential for efficient cytolytic activity.
K4K20-S4 is the most potent of the studied
peptides as it incorporates both these features. In conclusion, the
data presented support the idea that cytotoxic activity of
dermaseptins, and possibly of other antimicrobial peptides, arises from
the generation of a bi-lobed electrostatic potential field. Activity is
expected to persist as long as this amphipathic organization is
maintained. Whereas potency is a direct function of this electrostatic
potential field, selectivity (the spectrum of action) is affected
primarily by the hydrophobic properties of the peptides. The usefulness
of our approach for the design of selective cytolytic peptides is
demonstrated by our having transformed the native dermaseptin S4 from a
weak antibacterial and highly hemolytic peptide to a range of peptides
that exert enhanced antibacterial activity but reduced hemolysis. Such
peptides could be useful in various applications in the antimicrobial field.
| |
ACKNOWLEDGEMENTS |
The expert assistance of Dr. Eugene
Gussakovsky (Bar Ilan University) and Josephina Silfen
(Hebrew University) in CD and peptide synthesis, respectively, is
gratefully acknowledged.
| |
FOOTNOTES |
*
This work was supported in part by the DA'AT consortium, a
Magnet project administered by the Office of the Chief Scientist at the
Ministry of Industry and Trade, and in part by the Israel Science
Foundation Grant 523/98.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.
The on-line version of this article (available at
http://www.jbc.org) contains Table I.
§
To whom correspondence should be addressed: Dept. of Biological
Chemistry, Institute of Life Sciences, The Hebrew University of
Jerusalem, Givat Ram 91904, Jerusalem, Israel. Tel.: 972-2-65-85-575; Fax: 972-2-65-85-573; E-mail: irinak@macbeth.ls.huji.ac.il.
Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M111071200
2
I. Kustanovich, D. E. Shalev, M. Mikhlin, L. Gaidukov, and A. Mor, unpublished observations.
| |
ABBREVIATIONS |
The abbreviations used are:
PBS, phosphate-buffered saline;
PC, egg yolk
L--phosphatidylcholine;
PA, L--phosphatidic acid;
TFE, trifluoroethanol;
TOCSY, total correlation spectroscopy;
NOE, nuclear Overhauser enhancement;
NOESY, two-dimensional NOE spectroscopy;
MOPS, 4-morpholinepropanesulfonic acid;
r.m.s.d., root mean square deviation;
CMC, critical micelle concentration;
MIC, minimal inhibitory
concentration.
| |
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TOP
ABSTRACT
INTRODUCTION
