The influence of ion composition, pH, and peptide
concentration on the conformation and activity of the 37-residue human
antibacterial peptide LL-37 has been studied. At micromolar
concentration in water, LL-37 exhibits a circular dichroism spectrum
consistent with a disordered structure. The addition of 15 mM HCO3
,
SO42
, or
CF3CO2
causes the peptide
to adopt a helical structure, with approximately equal efficiency,
while 160 mM Cl
is less efficient. A
cooperative transition from disordered to helical structure is observed
as the peptide concentration is increased, consistent with formation of
an oligomer. The extent of
-helicity correlates with the
antibacterial activity of LL-37 against both Gram-positive and
Gram-negative bacteria. Two homologous peptides, FF-33 and SK-29,
containing 4 and 8 residue deletions at the N terminus, respectively,
require higher concentrations of anions for helix formation and are
less active than LL-37 against Escherichia coli D21. Below
pH 5, the helical content of LL-37 gradually decreases, and at pH 2 it
is entirely disordered. In contrast, the helical structure is retained
at pH over 13. The minimal inhibitory concentration of LL-37 against
E. coli is 5 µM, and at 13-25
µM the peptide is cytotoxic against several eukaryotic cells. In solutions containing the ion compositions of plasma, intracellular fluid, or interstitial fluid, LL-37 is helical, and hence
it could pose a danger to human cells upon release. However, in the
presence of human serum, the antibacterial and the cytotoxic activities
of LL-37 are inhibited.
 |
INTRODUCTION |
During the past decade, the widespread appearance of naturally
occurring antibacterial peptides has been firmly established. Their
abundance, tissue distribution, and in vitro activity
suggest an essential role in biological defense systems (1). In
mammals, antibacterial peptides such as defensins have been located in circulating leukocytes, where they are a part of the intracellular bactericidal machinery (2). Other peptides, such as those belonging to
the cathelicidin family, are released upon stimulation and exert their
activity extracellularly (3). Recently, several broad spectrum
bactericidal peptides have been found to be expressed or induced at
surface epithelia, probably providing an effective barrier for invading
bacteria (4-7).
Sequence comparison of gene-encoded antibacterial peptides from
vertebrate reveals a pronounced heterogeneity. In general, they can be
divided into four major groups according to composition and secondary
structure. One group, which includes the defensins, is folded into an
antiparallel
-sheet structure, containing three disulfide bridges
(2). A second group, which includes cecropins and magainins, exhibits
an
-helical structure (8, 9). A third group comprises peptides that
form loop structures with one or more disulfide bridges, such as
bactenecin (10). The fourth group comprises peptides with a high
content of specific amino acid, such as the proline-arginine-rich
peptide PR-39 (11) and the tryptophan-rich peptide indolicidin (12).
Despite these very diverse structural motifs, many of these peptides
are membrane-active, containing an amphiphilic secondary structure (13,
14). They kill bacteria mainly by lysis, and some are also cytotoxic to eukaryotic cells. However, the nonlytic PR-39 adopts a nonamphiphilic polyproline-helix at low temperature (15) and kills bacteria by
interrupting both DNA and protein synthesis (16).
The human cathelicidin peptide LL-37 was originally predicted from a
cDNA clone, and the putative active peptide was synthesized as
FA-LL-37 (14). Later, the mature active peptide LL-37 (two residues
shorter at the N-terminal end than the predicted peptide) was isolated
from degranulated granulocytes (17). LL-37 exhibits moderate
antibacterial activity in Luria-Bertani
(LB)1 medium, but upon the
addition of medium E (a salt medium used for culturing
Escherichia coli (18)) a pronounced increase of the
antibacterial activity was noticed. This enhancement of the activity
was shown to correlate with induction of an
-helical structure
(14).
We have found the
-helical conformation of LL-37 to be anion-, pH-,
and concentration-dependent. The extent of
-helical content correlates well with the observed antibacterial activity. The
minimal inhibitory concentration (MIC) of LL-37 against E. coli D21 was determined to 5 µM, and at 3-5 times
this concentration, the peptide also exhibits cytotoxic activity toward
eukaryotic cells. This means that LL-37, when released, could cause
host cell damage. A mechanism for protection from such potentially harmful effects appears to be in place in the circulation, since we
find that the cytotoxic activity of LL-37 is inhibited by human serum.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Porcine and human sera were purchased from Sigma
and from the Department of Transfusion Medicine and Clinical
Immunology, Karolinska Hospital (Stockholm, Sweden), respectively. The
composition of medium E (18) is 0.8 mM MgSO4,
9.6 mM citric acid, 57.4 mM K2HPO4, and 16.7 mM
NaNH4HPO4. Compositions of the physiological salt solutions used were as follows: for plasma, 113.0 mM
NaCl, 24.0 mM NaHCO3, 0.6 mM
MgCl2, 1.3 mM CaCl2, 3.9 mM KCl; for interstitial fluid, 117.0 mM NaCl,
27.0 mM NaHCO3, 0.6 mM
MgCl2, 1.1 mM CaCl2, 4.0 mM KCl; for intracellular fluid, 70.0 mM
K2HPO4, 12.0 mM NaHCO3, 17.0 mM MgCl2, 2.0 mM
CaCl2 (19). All physiological salt solutions were adjusted
to pH 7.3 with 2 M HCl.
Peptide Synthesis--
The amino acid sequences of LL-37 and the
N-terminally truncated forms FF-33 and SK-29 are shown in Fig.
1. Peptide synthesis was performed with
an Applied Biosystems model 430A peptide synthesizer using standard
solid-phase procedures (for review, see Ref. 20). Starting from
t-butoxycarbonyl-Ser(benzyl)-OCH-phenylacetamidomethyl resin
(0.67 mmol/g), t-butoxycarbonyl amino acid derivatives were used with reactive side chains protected as follows: serine and threonine, benzyl; lysine, 2-chlorobenzyloxycarbonyl; glutamate and
aspartate, benzyl ester; and arginine, 4-toluenesulfonyl. Double
couplings were performed for arginine, glutamine, and asparagine residues. The synthesis was halted after 29 cycles, and
of
the resin was removed, yielding the 29-residue peptide, SK-29. For the
remaining part of the resin, the synthesis continued for another four
cycles, and again
of the resin was removed, yielding the
33-residue peptide, FF-33. The synthesis was then completed after
another four cycles, giving the 37-residue peptide LL-37. The peptides
were cleaved from the resins with liquid
hydrogenfluoride/anisole/methylsulfide (10:1:1, v/v/v) for 60 min at
0 °C. The cleavage products were washed with diethylether to remove
scavengers and protecting groups, extracted in 30% acetic acid, and
lyophilized. The peptides were further purified by HPLC (Waters) on a
reversed phase Vydac C18 column (The Separation Group),
using a linear gradient of acetonitrile (15-60% in 40 min) in 0.1%
trifluoroacetic acid. The molecular masses were determined with a
matrix-assisted laser desorption/ionization instrument (Lasermat 2000, Finnigan MAT) and were in all cases in agreement with the calculated
masses.

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Fig. 1.
Helical net diagram and amino acid sequence
of LL-37. In the lower helical net diagram, basic
residues are in triangles, acidic residues are in
squares, and neutral residues are circled. Complimentary and repulsive ion pairs, from residues in positions i, i + 3/i + 4 are indicated by
thick solid and dashed lines, respectively. The
upper net diagram depicts the helix in a different orientation to show the continuous nonpolar surface. The unpolar part
of the amphipathic helix encompassing positions 11-31 is black, and the nonpolar residues in the N-terminal part,
which are located outside the perfect amphipathic helix, are in
gray. The amino acid sequences of the two N-terminally
truncated versions FF-33 and SK-29 are underlined.
|
|
CD Spectroscopy--
CD spectra were recorded on either a Jasco
J-720 (Jasco Inc.) or an Aviv 62DS (Aviv Associates Inc.)
spectropolarimeter. All spectra were recorded at 0.5 nm/point
resolution, and data were reported as differences in molar
absorptivities (
=
L
R) of the backbone
amide bond (M
1 cm
1). Peptide
concentration was 40 µM unless otherwise noted. Helical content was estimated by obtaining the average of the following values:
percentage of helix = (
208
1.21)/
11.22 (21)
and percentage of helix = (
222
0.91)/
11.82
(22). For determination of the concentration dependence of the peptide
CD, concentrations between 10
7 and 10
3
M were measured in cuvettes with path lengths between 0.1 and 50 mm.
Antibacterial Activity--
An inhibition zone assay in thin
(1-mm) agarose plates seeded with E. coli D21 or
Bacillus megaterium Bm11 was used. 1% (w/v) agarose in LB
broth was supplemented with different single salt solutions or medium
E. Antibacterial activity in "salt-free" medium was determined by
omitting NaCl from the LB medium. Bacteria were grown overnight on agar
plates containing streptomycin (100 µg/ml) and were then inoculated
in LB medium. Bacteria were added to the agarose mixture just before
plating to a concentration of 6 × 104 cells/ml. Small
wells (3-mm diameter, 3-µl volume) were punched out of the plates,
and a dilution serie of a given peptide (starting with 5 µg/µl in
water) was applied. For serum inhibition studies, LL-37 was dissolved
at a concentration of 1 µg/µl in water, human serum, or porcine
serum, with 3 µl applied of each sample. After overnight incubation
at 30 °C, the diameters of bacteria-free zones were measured.
For the MIC value, E. coli D21 was grown to late log phase.
Approximately 2000 bacteria were incubated in LB supplemented with
medium E and different peptide concentrations (ranging from 20 to 0.31 µM in 1:1 dilution steps) for 3 h. The lowest
concentration inhibiting bacterial growth was taken as the MIC
value.
Analysis of Cellular Viability by Flow Cytometric
Analysis--
The cytotoxic effect of LL-37 was determined by the
ability of intracellular eukaryotic esterases to hydrolyze fluorescein diacetate (FDA) to free fluorescein, followed by flow cytometric analysis (23). LL-37 was incubated with either trypomastigote forms of
Trypanosoma cruzi (Tulahuén strain (24)), obtained from the culture supernatant of L-929 infected cells, human peripheral blood leukocytes (PBL), or the T-cell line MOLT. Cells were resuspended in RPMI 1640 medium containing 5% fetal calf serum, penicillin, and
streptomycin, in the absence or presence of indicated dilutions of
LL-37, at 37 °C and 5% CO2. Serum inhibition of
cytotoxicity was analyzed by using serial dilutions of human serum
(40-0%), 50 µM LL-37, and PBL. After 12-16 h, cells
were centrifuged, and the pellet was washed once in phosphate-buffered
saline. Cells were resuspended to 2 × 106/ml and
incubated with 1 mg/ml FDA in phosphate-buffered saline, (a 100-fold
dilution from a stock FDA in acetone). After incubating 15 min at
37 °C, cells were washed once in phosphate-buffered saline and fixed
in 2% paraformaldehyde for 10 min at room temperature, and
105 events were then analyzed in a FACScan flow-cytometer
(Beckton & Dickinson). The percentage of nonviable cells was determined as the number of events showing fluorescein staining after LL-37 incubation as compared with control cells incubated with RPMI 1640 medium alone.
 |
RESULTS |
Concentration-, Anion-, and pH-dependent Transition
from Disordered to Helical Structure of LL-37--
In 20 mM SO42
at a concentration
of 10
3 M, the CD spectrum of LL-37 is
dominated by double minima (222 and 208 nm) and a single maximum (195 nm) characteristic of an
-helical secondary structure (25) (Fig.
2A). At this concentration,
the
-helical content is estimated to be 50%. This helical
conformation is lost in a cooperative fashion upon dilution of the
peptide in 20 mM SO42
buffer. The existence of an isodichroic point (204 nm) is consistent with a two-state helix-coil equilibrium (26). At the lowest concentration measured (10
7 M), the CD
spectrum of LL-37 has lost essentially all
-helical character and
now contains a single pronounced minimum at about 200 nm. The position
of the minimum undergoes a blue shift with decreased peptide
concentration (Fig. 2A). This is expected as the small
amount of remaining helical structure is lost and further supports the
notion of a largely disordered peptide at concentrations below
10
6 M. Taken together, this highly
cooperative concentration-dependent helix-coil equilibrium
is highly reminiscent of the monomer-oligomer transitions common to
peptide sequences capable of forming amphipathic
-helices
(e.g. melittin; Ref. 27). Preliminary analysis of the
concentration-dependent CD and sedimentation equilibrium
experiments indicates that a significant portion of the helical form of
LL-37 is a tetramer (data not shown). We cannot at present rule out the
presence of higher molecular weight oligomers.

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Fig. 2.
Concentration- and salt-dependent
conformation of LL-37. A, CD spectra of LL-37 at the
indicated concentrations in 20 mM
Na2SO4, pH ~6. B, CD spectra of
LL-37 (40 µM at 20-25 °C, pH ~6) in 15 mM solutions of the indicated salts. The spectrum in NaCl
is very similar to that of LL-37 in water (cf. Fig.
5A).
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|
At a concentration of 40 µM, the CD spectrum of LL-37 in
water exhibits a minimum around 200 nm, which is indicative of a highly
disordered conformation (see Fig. 5A). Upon the addition of
15 mM Na2SO4, NaHCO3,
or NaCF3CO2, a conformational change occurs, as
evidenced by spectra containing minima at 208 and 222 nm and a maximum
around 195 nm (Fig. 2B). Based on these spectra, the
helicities in the presence of these salts are similar (Fig. 2B) and are estimated to be about 40%. The addition of up
to 40% (v/v) trifluoroethanol to LL-37 in water results in
approximately 30% helical content (data not shown). 15 mM
NaCl affects the structure of LL-37 to a very limited extent (Fig.
2B), and replacement of Na+ with
Mg2+ does not have any effect on the structural transitions
observed, which shows that the structural changes are predominantly
caused by the SO42
,
HCO3
, and
CF3CO2
anions.
Given the clear cut effects of the anion composition and concentration
on the secondary structure of LL-37, we next examined the peptide
conformation in solutions with ion compositions similar to those of
various physiological environments. Fig.
3 shows that LL-37 adopts an
-helical
conformation virtually identical to that in medium E in solutions
containing ion compositions that mimic those of human plasma,
interstitial fluid, and intracellular fluid.

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Fig. 3.
Secondary structure of LL-37 in physiological
ion compositions. CD spectra of LL-37 (40 µM) in
solutions with similar ion compositions as plasma, intracellular fluid
(ICF), and interstitial fluid (ISF) are shown.
See "Experimental Procedures" for the exact compositions and pH
values of these solutions.
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|
The helicity of LL-37 in salt solutions as inferred from CD
measurements is reduced at low pH. Below pH 5, a transition to a
disordered conformation is evident, and at pH 2-3, the CD spectrum is
practically identical to that in water (Fig.
4). The original
-helical structure is
regained upon raising the pH (not shown). In contrast, the helical
content is retained at pH values over 13 (Fig. 4), and the addition of
50 mM NaOH to LL-37 dissolved in water causes a transition
to a helical conformation (not shown).

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Fig. 4.
pH effects on the secondary structure of
LL-37. CD spectra of LL-37 in an ion composition corresponding to
that of interstitial fluid (cf. Fig. 3) at the indicated pH
values are shown.
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|
The Helical Content Correlates with Antibacterial
Activity--
Fig. 5A shows
the CD spectra of LL-37 (40 µM) in water and after the
addition of various salts. The addition of medium E or 84 mM SO42
results in the
most pronounced helix formation. 160 mM Cl
induces formation of helical structure, but the helical content is only
about half of that observed with medium E or 84 mM
SO42
(Fig. 5A). The
antibacterial activity of LL-37 in the presence of these ions was
determined using an inhibition zone assay. The activity toward the
Gram-positive bacterium B. megaterium Bm11 closely
correlates with the helical content (Fig. 5B). The
activities in medium E, 5 and 84 mM
Na2SO4, and 12 mM
NaHCO3 are similar and significantly higher than that in
160 mM NaCl, which in turn is higher than the activity
observed in "salt-free" medium (Fig. 5B). The same trend
is observed for the activity toward the Gram-negative bacterium
E. coli D21 (Fig. 5C), except that the activity
in 12 mM NaHCO3 is reduced, probably due to the
different compositions of the membranes for Gram-positive and
Gram-negative bacteria. Finally, the MIC value for LL-37 against
E. coli D21 in LB supplemented with medium E was found
to be approximately 5 µM.

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Fig. 5.
Salt-dependent conformation and
activity of LL-37. A, CD spectra of LL-37 (40 µM at 20-25 °C, pH ~6) in water, 160 mM
NaCl, 84 mM MgSO4, or medium E. B,
the antibacterial activity against the Gram-positive bacterium B. megaterium Bm11 of LL-37 in LB medium containing the indicated
salt solutions, or omitting the NaCl in the case of H2O.
C, same as B except against the Gram-negative bacterium
E. coli D21.
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Further support for a direct relation between helical content and
antibacterial activity comes from analyses of N-terminally truncated
versions of LL-37. The helical content of LL-37, FF-33, or SK-29 each
increases with increasing SO42
concentration (Fig. 6A). All
peptides show a isodichroic point at 203-204 nm upon salt titration
(not shown), consistent with a two-state helix-coil transition. The
shorter peptides require significantly higher concentrations of
SO42
for induction of maximal helical
content, approximately 300 mM for FF-33 and SK-29 compared
with about 30 mM for LL-37. This is reflected in their
respective activities against E. coli D21 in 5 mM Na2SO4, where LL-37 is more
active than FF-33 or SK-29 (Fig. 6B). In medium E, LL-37 and
FF-33 are equally active and significantly more active than the shorter
SK-29 peptide (Fig. 6C).

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Fig. 6.
Folding and activity of N-terminally
truncated forms of LL-37. A, CD at 222 nm as a function of
SO42 concentration for LL-37, FF-33,
and SK-29 at a peptide concentration of 40 µM,
20-25 °C, pH ~6. B, the antibacterial activity of the peptides LL-37, FF-33, and SK-29 in LB medium supplemented with 5 mM Na2SO4 against E. coli D21. C, same as B except the LB was supplemented
with medium E.
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Correlation of pH with the antibacterial activity could not be
determined, since the bacterial test strains do not grow well at
extremes of pH. However, values were obtained for E. coli
D21 at pH 4.3, where the inhibition zones were, depending on the
peptide concentration, 40-85% of the zones obtained at pH 7.
Cytotoxic Effects of LL-37--
The cytotoxic effect of LL-37 was
studied by FDA incorporation using three different types of eukaryotic
cells: trypomastigotes of the protozoan parasite T. cruzi, a
T lymphocyte cell line (MOLT), and PBL. For these cell types, toxicity
is clearly observed at 13-25 µM of LL-37, and gradually
increases at higher concentrations (Fig.
7).

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Fig. 7.
Cytotoxic effect of LL-37 against eukaryotic
cells. The toxic effect for different peptide concentrations was
measured by FDA staining. The cell types are T. cruzi, the T
lymphocyte cell line MOLT, and PBL.
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The Antibacterial and Cytotoxic Activities of LL-37 Are Inhibited
in Serum--
Our data show that the active helical conformation of
LL-37 is cytotoxic to eukaryotic cells in the ion compositions and at pH values that are encountered under physiological conditions. Thus,
LL-37, if released extracellularly, could be harmful to human cells
in vivo. However, such potential effects are apparently attenuated by one or several factors present in human serum, since the
cytotoxic effect is reduced when human serum is included in the assay.
In the absence of serum, 50 µM LL-37 results in 81% nonviable cells, while in the presence of 40% serum, 38% nonviable cells are found. In addition, the antibacterial activity of LL-37 is
lost completely when the peptide is first dissolved in human serum and
significantly reduced in porcine serum (Fig.
8). In contrast, the porcine
antibacterial peptide PR-39, which is nonlytic (16), is equally active
if the peptide is first dissolved in water, human serum, or porcine
serum (Fig. 8). We interpret the bactericidal activity of the peptide
dissolved in water (Fig. 8) as being the result of the conformational
inducing effects of medium E in the agarose plate. Control experiments
indicate that the observed inhibition is not likely to be caused by
proteolytic degradation of LL-37. After incubation in human plasma for
20 h at 25 °C, the peptide elutes at the identical position as
untreated LL-37 upon reversed phase HPLC, and the mass value
determined by matrix-assisted laser desorption/ionization time of
flight mass spectrometry is identical to that of intact LL-37.

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Fig. 8.
Inhibition of antibacterial activity of LL-37
in serum. An inhibition zone assay on E. coli D21 in
the presence of medium E with the human peptide LL-37 and the porcine
nonlytic peptide PR-39. The peptides were dissolved at a concentration of 1 µg/µl in water, human serum, or porcine serum as indicated. 3 µl were loaded in each well. The diameter of the inhibition zone for
LL-37 in water is 10.5 mm.
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 |
DISCUSSION |
Salt-, pH-, and Concentration-dependent Folding of
LL-37--
The cooperative concentration-dependent
-helix formation of LL-37 is consistent with a system in which
largely unfolded monomeric peptide (the dominant form at
<10
5 M) is in equilibrium with an
-helical oligomer form. This is indeed the expected behavior of an
amphiphilic
-helix such as LL-37 (Figs. 1 and 2). The driving force
of oligomerization and concomitant
-helix formation is then expected
to be due largely to the hydrophobic effect, i.e. efficient
removal of the apolar face from contact with the solution to form the
interior of the oligomer, leaving the polar face exposed. In this
context, the effect of some ions
(SO42
,
HCO3
,
CF3CO2
and to a
significantly lesser extent Cl
) in promoting helix
formation in LL-37 can be rationalized from their ability to "salt
out" nonpolar groups, commonly referred to as the Hofmeister effect
(see Refs. 28 and 29). The salting out of apolar groups is thought to
result from the effect of ions on the surface tension of water. By
increasing the surface tension of water the exposure of hydrophobic
surface becomes more unfavorable. In proteins, the salting out effect
is often a manifestation of reduced solubility at high salt
concentrations (>1 M), hence the name. However, the
salting out of an amphiphilic
-helix like LL-37 should result in a
shift of the equilibrium toward the oligomer form at lower salt
concentrations, which we observe as increased helical content of the
peptide.
Ionic interactions along the polar face of the amphiphilic helix also
appear to play a role in the conformation of LL-37. Salt bridges in
helices, formed by location of residues with acidic and basic side
chains, respectively, in positions i and i + 3 or i + 4, which are close in a helical conformation,
specifically stabilize the helical conformation (30). LL-37 contains
nine such potential ion pairs (Fig. 1). In addition, LL-37 contains seven locations where residues with basic side chains are located both
in positions i and i + 3 or i + 4,
but no instances where two negative charges are in such positions (Fig.
1). It is likely that the anion-induced folding of LL-37 into a helical
structure at near neutral pH (Figs. 2B and 5A) is
at least in part caused by reducing repulsive forces between positively
charged residues located in the seven (i,
i + 3/i + 4) positions. The unfolding of
-helical LL-37 at low pH (Fig. 4) could be brought about by protonation of acidic side chains with concomitant losses of
stabilizing complimentary (i,
i + 3/i + 4) side chain ion pairs, since
destabilizing interactions between positive residues are expected to be
largely unaffected at low pH. This in turn suggests that, in the
present case, i, i + 3/i + 4 ion
pairs are more helix-stabilizing than the corresponding hydrogen bonds
between protonated Asp or Glu side chains and positively charged side
chains. In contrast, Marqusee and Baldwin (31) found that, in
16-17-residue model peptides, Glu0-Lys+
hydrogen bonds are roughly as effective as the
Glu
-Lys+ salt bridge in stabilizing the
helix. However, an important caveat is that in their model system all
peptides were shown to be monomeric, whereas LL-37 is clearly an
oligomer when helical. Finally, at pH > 13, where the side chains
of Lys and Arg are uncharged, complimentary side chain ion pairs are
again lost, but so are repulsive forces between basic side chains. This
does not cause a net destabilization of the LL-37 helical fold (Fig.
4).
Conformational Requirement for Antibacterial Activity--
The
helical, oligomeric conformation of LL-37 is apparently a requirement
for activity, since the highest antibacterial activity correlates to
maximal helical content, while intermediate and low activities
correspond to less helical content and disordered secondary structure
(Fig. 5). This indicates that optimal antibacterial activity of LL-37
requires an oligomeric
-helical structure prior to interacting with
the bacterial membrane. Hence, for LL-37 the bacterial membrane alone
cannot be a major determinant for folding into an active conformation,
as has been claimed for the cecropins (32).
In cystic fibrosis, the innate immunity of the lung is compromised,
leading to repeated bacterial infections. This defect has been claimed
to depend on inactivation of human
-defensin-1, which is inactive at
high NaCl concentrations (33). Our data suggest that changes in the
microenvironment, such as e.g. those encountered in cystic
fibrosis lung, could influence the conformation of antibacterial
peptides and thus modulate their activities.
To determine if peptide length influences the conformation and/or
biological activity, the two N-terminally truncated variants FF-33 and
SK-29 were synthesized. In 5 mM
Na2SO4, LL-37 has considerable helicity, while
FF-33 is significantly less helical (Fig. 6A). Accordingly,
LL-37 is more active against E. coli D21 than FF-33 at this
salt concentration (Fig. 6B). The antibacterial activity of
FF-33 in medium E, however, is similar to that of LL-37 (Fig. 6C), showing that the four N-terminal amino acids as such
are not essential for the antibacterial activity. Recently, a mouse cathelicidin, murine cathelin-like protein, was identified (34), where
the C-terminal 37 residues have 43% sequence identity with LL-37,
indicating that murine cathelin-like protein is the mouse homologue of
human LL-37. A 29-residue putative antibacterial peptide derived from
the C-terminal part of murine cathelin-like protein exhibited a
disordered structure both in water and in medium E and had no
antibacterial activity (34). In contrast, the corresponding human
peptide SK-29 does adopt an
-helical secondary structure and is
antibacterial in salt solutions. However, SK-29 requires higher
concentrations of SO42
for induction
of maximal helical content compared with LL-37 (Fig. 6A). In
line with this result, the antibacterial activity of SK-29 against
E. coli D21 is lower than that of LL-37 both in 5 mM Na2SO4 and medium E (Fig. 6,
B and C).
Cytotoxic Activity of LL-37 and Serum Inhibition--
Many
antibacterial peptides (e.g. defensins (2), indolicidin
(35), BMAP-27 (36), and LL-37) also exhibit cytotoxic effects against
eukaryotic cells, but usually at higher concentrations compared with
the bactericidal activity. The differences in effective bactericidal
and cytotoxic concentrations could lie in the different membrane
compositions of eukaryotic and prokaryotic cells. However, the primary
structures of BMAP-27 and BMAP-28 influence their membrane
specificities, since the hydrophobic C-terminal tail of these peptides
is needed for cytotoxic, but not antibacterial, effect (36). LL-37
lacks a hydrophobic tail, suggesting a different mechanism for its
cytotoxicity. Cytotoxic effects may well be physiologically relevant at
sites of inflammation, where antibacterial/cytotoxic peptides are
induced in epithelial cells and/or recruited from granulocytes (5, 7).
This could result in a high local concentration of peptide, leading to
cytotoxicity. Examples of protective mechanisms against cytotoxic
activities in the circulation do exist, where for example
2-macroglobulin works as a scavenger for the defensins (37). We have
demonstrated that the antibacterial (Fig. 8) and cytotoxic activities
of LL-37 are inhibited by human serum. Since we have no indication of
degradation of LL-37 in human serum, it is possible that this
inhibition reflects binding of LL-37 to a serum protein.
In conclusion, LL-37 requires an
-helical, oligomeric conformation
for optimal antibacterial activity, and this conformation is dependent
on LL-37 concentration and several factors in the microenvironment.
Potentially harmful cytotoxic effects exerted by LL-37 against host
cells are apparently attenuated by one or several factors in human
serum.