Tài liệu Báo cáo khoa học: Key role of the loop connecting the two beta strands of mussel defensin in its antimicrobial activity docx

Key role of the loop connecting the two beta strands of mussel
defensin in its antimicrobial activity
Bernard Romestand
1
, Franck Molina
2
,Ve
´
ronique Richard
1
, Philippe Roch
1
and Claude Granier
2
1
DRIM, Universite
´
Montpellier 2, France;
2
Centre de Biotechnologie et Pharmacologie pour la Sante
´
, CNRS UMR Montpellier,
France
To elucidate the structural features of the mussel defensin
MGD1 required for antimicrobial activity, we synthesized a
series of peptides corresponding to the main known secon-
dary structures of the molecule and evaluated their activity
towards Gram-positive and Gram-negative bacteria, and
filamentous fungi. We found that the nonapeptide corres-
ponding to residues 25–33 of MGD1 (CGGWHRLRC)
exhibited bacteriostatic activity once it was cyclized by a

non-naturally occurring disulfide bridge. Longer peptides
corresponding to the amino acid sequences of the a-helical
part or to the b-strands of MGD1 had no detectable activity.
The bacteriostatic activity of the sequence 25–33 was strictly
dependent on the bridging of Cys25 and Cys33 and was
proportional to the theoretical isoelectric point of the pep-
tide, as deduced from the variation of activity in a set
of peptide analogues of the 25–33 sequence with different
numbers of cationic charges. By using confocal fluorescence
microscopy, we found that the cyclic peptides bound to
Gram-positive bacteria without apparent lysis. However, by
using a fluorescent dye, we observed that dead bacteria
had been permeated by the cyclic peptide 25–33. Sequence
comparisons in the family of arthopod defensins indicate
that MGD1 belongs to a subfamily of the insect defensins,
characterized by the constant occurrence of both positively
charged and hydrophobic amino acids in the loop. Model-
ling studies showed that in the MGD1 structure, positively
charged and hydrophobic residues are organized in two
layered clusters, which might have a functional significance
in the docking of MGD1 to the bacterial membrane.
Keywords: defensin; antimicrobial peptide; solid-phase syn-
thesis; active loop; cyclic peptide.
Antimicrobial peptides are essential actors of innate immu-
nity that have been conserved throughout evolution. Many
such molecules have been purified over the past decade,
from vertebrates, invertebrates, plants and bacteria. Some
of these compounds have been investigated with a view to
possible therapeutic use [1], as an alarming increase of
resistance of microorganisms to classical antibiotics has

been reported [2,3]. Defensins are antimicrobial peptides
isolated from mammals [4], arthropods [5,6], plants [7,8]
and more recently from molluscs [9,10]. They are cationic
molecules belonging to the cysteine-rich family of anti-
microbial peptides. Mammalian defensins comprise human
neutrophil peptides (HPN-1–4), human defensins (HD-5
and 6), two human b defensins (HBD-1 and 2) [11–13] and a
cyclic rhesus theta defensin (RTD-1) [14]. Although all
defensins display antibacterial activity, mammalian and
other vertebrate defensins are quite different from the
arthropod/mollusc defensins in terms of both sequence and
structure [15–17].
MGD1 is a defensin of 39 residues, which has been
isolated from plasma and haemocytes of the edible Medi-
terranean mussel, Mytilus galloprovincialis [10,18]. MGD1
shares the so-called cysteine-stabilized alpha-beta motif
(Csab) with arthropod defensins [19], but it is characterized
by the presence of an additional disulfide bond. The three-
dimensional solution structure of MGD1 has been estab-
lished using
1
H-NMR and mainly consists of a helical part
(residues 7–16) and two antiparallel b-strands (residues
20–25 and 33–39) [16]. The a-helix and the b1-strand are
connected by a distorted type II turn (loop 2), whereas the
loop connecting both strands of the b-sheet (residues 25–33)
includes a type III¢ turn (loop 3) and points out of the core
of the protein.
There is a consensus view that defensins act by disrupting
the cytoplasm membrane [20–24], although the exact mode of

action is not clearly established. To gain further insight into
the structural requirements for antimicrobial activity, we
designed a number of peptide fragments based on the
knowledge of thestructure of MGD1 [16]. Syntheticpeptides,
including amino acid substitutions, were tested for bacterio-
static activity and revealed the crucial role of loop 3 and the
effect of positive charges. Loop 3-derived peptides were
found to bind to Gram-positive bacteria resulting in
permeation of the membrane and bacterial killing.
Materials and methods
Synthesis of soluble peptides
All soluble peptides were synthesized on an Abimed AMS
422 synthesizer by Fmoc chemistry [25,26]. Peptides were
deprotected and released from the Rink amide resin
Correspondence to P. Roch, Laboratoire DRIM, CC080, Universite
´
Montpellier 2, Place E. Bataillon, 34095 Montpellier, France.
Fax: + 33 4 67 14 46 73, Tel.: + 33 4 67 14 47 12,
E-mail:
Abbreviations: MGD, Mytilus galloprovincialis defensin;
MIC, minimal inhibitory concentration.
(Received 14 February 2003, revised 28 April 2003,
accepted 08 May 2003)
Eur. J. Biochem. 270, 2805–2813 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03657.x
(Novabiochem) by trifluoroacetic acid treatment in the
presence of appropriate scavengers. Peptides were lyophi-
lized and then purified on a preparative C18 reverse-phase
HPLC column (Waters) by elution with a mixture of water/
acetonitrile [both containing 0.1% (v/v) trifluoroacetic acid].
Homogeneity of purified peptides was analysed on analyti-

cal C18 HPLC column and peptide integrity was checked by
MALDI-TOF mass spectrometry. The N-terminal residue
of every peptide was blocked by acetylation, and the
C-terminal residue was amidated. Disulfide bond formation
in cysteine-containing peptides was performed by dilution of
the peptide in 20% dimethylsulfoxide, 0.05
M
ammonium
acetate, pH 7.5, for 24 h at room temperature under
agitation [27]. Peptide Q, which comprises two disulfide
bonds, was obtained by sequential formation of the Cys25-
Cys33 disulfide bond, as described above, then removal of
the acetamidomethyl group (0.1
M
iodine in water acidified
to pH 4 with dilute acetic acid) that was introduced during
synthesis at Cys21 and Cys38. When disulfide bond
formation was not desired, the Cys residues in the original
MGD1 sequence were replaced by Ser. Peptides were
labelled with biotin by elongation during the solid-phase
synthesis with the spacer motif Ser-Gly-Ser followed by
N-terminal biotinylation. Isoelectric points were computed
from the amino acid sequences using the internet tool,
/>Sequence and structural analysis
Sequences of the arthropod defensin family were extracted
from the Pfam database [28]. The N- and C-termini of the
sequences corresponding to the defensin structural domain
that were sometimes missing in the Pfam alignments were
added manually. Sequence analysis was performed using
CLUSTAL X

software for multiple alignment and
SEAVIEW
software for manual adjustment. Three dimensional struc-
ture analysis and drawing were done with
SWISS
-
PDB VIEWER
v3.7 using the 1jfn file from the Protein Data Bank.
Antibacterial assays
Antibacterial activity towards four Gram-positive bacteria
(Micrococcus lysodeikticus ATCC 4698, Staphylococcus
aureus ATCC 25293, Staphylococcus epidermidis ATTC
12228 and Bacillus megaterium ATCC 17749) and four
Gram-negative bacteria (Vibrio alginolyticus ATCC 17749,
Vibrio metchnikowkii NTCC 8483, Escherichia coli 363
ATCC 11775 and Salmonella newport (isolated from the
e
´
tang de Thau by P. Monfort, Universite
´
Montpellier 2,
France) was monitored by a liquid growth inhibition assay
[18]. Briefly, 10 lL of native or synthetic peptides was
incubated in 96-well microtiter plates with 100 lLof
bacteria suspension, at a starting D
600
¼ 0.001, in poor
broth nutriment medium [1% bactotryptone, 0.5% (w/v)
NaCl, pH 7.5]. Bacterial growth was assayed by measure-
ment at 600 nm after 24 h incubation at 30 °C. The

minimal inhibitory concentration (MIC) was evaluated by
testing serial doubling dilutions and defined as the lowest
peptide concentration that prevented any growth [29]. The
bactericidal capacity of peptides was assessed using the
Live/Dead Bac Light Bacterial viability kit (Molecular
Probes). The fluorescence given by live (FITC SYTO9Ò,
green) or dead bacteria (propidium iodide, red) was
observed using a fluorescent microscope (Leica) equipped
with Omega filters XF22 and XF 32.
Antifungal assay
Susceptibility of Fusarium oxysporum (a gift from A. Vey,
INRA Saint Christol-le
`
s-Ale
`
s, France) and Candida sp.
(a gift from O. Thaler, Universite
´
Montpellier 2, France)
was tested by a liquid growth inhibition assay as described
by Fehlbaum [30]. Briefly, 80 lL of fungal spores (final
concentration 10
4
sporesÆmL
)1
) suspended in potato dex-
trose broth (Difco) containing 0.1 mg tetracycline was
addedto20lL of peptide dilutions in microtiter plates.
Peptides were replaced by 20 lL of sterile water in
controls. Growth inhibition was observed under the

microscope after 24 h incubation at 30 °C and quantified
by D
600
measurement after 48 h. The MIC was defined as
described above.
Confocal laser-scanning observations
M. lysodeikticus (10
5
CFU in midlogarithmic phase) were
immobilized on a glass slide by a 10 min centrifugation at
2500 g at room temperature, and incubated for 3 h at 37 °C
with biotin-labelled peptides. Slides were then washed with
phosphate-buffered saline (NaCl/P
i
), pH 7.5, and incubated
for 5 min in NaCl/P
i
containing 0.2% (v/v) Triton X-100.
After three washes in NaCl/P
i
, the slides were incubated for
30 min at room temperature with 10 lgÆmL
)1
streptavidin-
FITC (Pierce-Interchim) and observed with a laser-scanning
confocal microscope (Bio-Rad 1024, CRIC Centre d’Ima-
gerie Re
´
gionale de Montpellier) equipped with a 488 nm
filter.

Cytotoxicity tests on the human lymphoma K562 cell line
Peptide concentrations corresponding to 10 times the MIC
for M. lysodeikticus were incubated with K562 cells. Toxi-
city was evaluated after 48 h of incubation by measuring the
optical density of the culture at 570/690 nm using the
In Vitro Toxicology Assay Kit (Sigma), based on conver-
sion of the yellow tetrazolium salt MTT into purple
formazan crystals by metabolically active cells.
Results
Anti-Gram-positive bacteria activity is conveyed
by the cyclized loop 3
Figure 1A shows the three-dimensional structure of
MGD1 [16] and Fig. 1B the designed set of peptides.
Peptides with two cysteines were oxidized so as to be
cyclised. Dilutions of the purified peptides were further
tested for growth inhibition of the Gram-positive bacteria
M. lysodeikticus (Fig. 1B). Peptides corresponding to the
a-helical part of MGD1 (peptide T) or to the a-helical
part prolonged by the N-terminal turn (GFGSP) and by
the short sequence (IPGR) connecting the a-helix to the
first strand of the b-hairpin (peptide S) did not exhibit
measurable activity, although the latter peptide repre-
sents almost 50% of the MGD1 amino acid sequence.
2806 B. Romestand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
However, the 9-mer peptide B, CGGWHRLRC, corres-
ponding to the sequence of the MGD1 loop 3 occurring
between the two b-strands, had an MIC of 28 l
M
(i.e.
about 2.5% of the activity of the synthetic MGD1,

peptide A). Peptide B was active only when a disulfide
bond was formed between the two cysteine residues
(Cys25 and Cys33 of MGD1; these two cysteines are not
linked together in natural MGD1) as shown by the fact
that peptide E did not show any measurable activity. A
complementary assay involving peptide B incubated in
the presence of 10 m
M
dithiothreitol, known to open the
disulfide bridges by reducing the cysteines, confirmed the
absolute necessity of cyclization for activity (data not
shown). The inhibitory activity of peptide B cannot be
simply attributed to the basic or looped characteristics, as
peptide V, a mimetic of loop 2, located between the C-end
of the a-helix and the beginning of the first b-strand, was
inactive. Two peptides consisting of peptide B plus
extensions from the b-strands domains (peptide I,
B extended by the b2-sheet sequence and peptide K, B
prolonged by b1andb2-sheet sequences) displayed
slightly greater bacteriostatic activity than peptide B
(Fig. 1B). Peptide H had almost the same activity as B,
despite the added b1-sheet sequence. Peptide Q was the
most active molecule of the series, possibly due to the
presence of two disulfide bonds obtained by stepwise
formation (one to form the loop 25–33, another linking
the N and C termini), which might rigidify the peptide
structure. Meanwhile, peptide M, which corresponds to
peptide B prolonged by the unlinked sequences of the two
b-strands, displayed a MIC similar to that of peptide H. It
is important to note that the sequences of the two

b-strands apparently did not convey activity by themselves
as peptide X was inactive and peptide K active
(MIC ¼ 12 l
M
). Peptide X corresponds to peptide K in
which the sequence of the b-strands was maintained but
the amino acids from loop 3 had been replaced by
multiple Ser and Gly residues. Therefore, in the sequence
of the whole b-hairpin structure of MGD1, only the loop
part seems to convey activity.
Activity is directed mainly against Gram-positive bacteria
and fungi
The activity spectra of peptides B, K, M and Q were
compared with that of synthetic MGD1 (peptide A).
Although less active than the entire molecule, peptides
derived from loop 3 were active on all the Gram-positive
bacteria tested (Table 1). Gram-negative bacteria were not
inhibited by any peptide, with the exception of E. coli 363,
which was sensitive to peptides K and M (MIC ¼ 62 l
M
),
and Q (MIC ¼ 22 l
M
). The fungus F. oxysporum was
inhibited by all four peptides, especially by peptides Q and
M(MIC¼ 13–15 l
M
) and peptide K (MIC ¼ 17 l
M
).

Curiously, the Candida sp. was found to be sensitive to
peptide M (one disulfide bond 25–33) but not to peptide K
(one disulfide bond 21–38) or to peptide Q (two disulfide
bonds). In addition, peptide M was sevenfold more active
on the Candida sp. than peptide B. On the contrary, no
lethality on the human lymphomyeloid K563 cell line was
detected, even with peptide concentrations equal to 10 times
the MIC for M. lysodeikticus (data not shown), strongly
suggesting that toxicity of peptides was specific for pro-
karyotic cells and fungi.
Fig. 1. Molecular dissection of MGD1 peptide. (A) Representation of the three dimensional structure of MDG1 (as determined by Yang et al.[16])
and its main secondary features. (B) Main synthetic peptides used in the study and their minimal inhibitory concentration (MIC in l
M
)forthe
growth of M. lysodeikticus cells.
Ó FEBS 2003 Role of the hairpin loop in mussel defensin activity (Eur. J. Biochem. 270) 2807
Activity is correlated with the isoelectric point
of peptides
The influence of the overall positive charge of the active
peptide on the growth inhibition of M. lysodeikticus was
investigated. Several peptides derived from the loop 3-based
peptide B were designed to include various proportions of
positively charged residues. As a quantitative index of the
cationic character, the theoretical isoelectric point of each
peptide was computed (Fig. 2). In peptide F, Arg30 and
Arg32 were replaced by two isosteric but nonionisable
citrulline residues, thus lowering the pI from 9.02 to 8.06. As
a result, the bacteriostatic activity was almost completely
lost. In contrast, peptides with one, two or four of their
naturally occurring residues from peptide B substituted by

Lys (peptides C, D and J, respectively) had higher pI values
and displayed greater inhibitory activity than that of
peptide B. A strict quantitative relationship between the
theoretical isoelectric points and the logarithm of the
corresponding experimental bacteriostatic activities (corre-
lation coefficient of 0.999) was observed (Fig. 2). Finally,
the increase in bacteriostatic activity observed with loop
3-based peptides as a function of their increasing pI (cationic
charges) was also observed for the activity of larger peptides
containing the substituted peptide B (Table 2, L–K, N–M
and P–Q), indicating that the properties of the loop drive the
properties of larger peptides enclosing the loop.
Binding capacity of loop 3-derived peptides
on
M. lysodeikticus
The aforementioned results showed that synthetic peptides
corresponding to fragments of the MGD1 defensin
reproduced the behaviour of the entire molecule with
regard to its specificity. Thus, an active synthetic peptide
can be used instead of the natural molecule to study the
interaction of defensin with the bacterial membrane. To
monitor the mode of action on bacteria, biotin-labelled
peptides B and D were incubated with M. lysodeikticus
and binding of the peptides to bacteria was examined
using FITC-streptavidin. At concentrations of 1–60 l
M
,
both biotinylated peptide D (Fig. 3A) and biotinylated
peptide B (not illustrated) decorated the cell surface but
apparently did not penetrate the bacteria. Even using

concentrationupto60l
M
(i.e. 7.5-fold the MIC of
peptide D), and incubation times up to 14 h, peptides B
and D remained associated with the outer parts of cells
and no lysis was observed. Furthermore, the live or dead
Table 1. Antimicrobial activity of synthetic MGD1 (peptide A) and several fragments on various Gram-positive and Gram-negative bacteria, and fungi.
The numbers correspond to MIC values in l
M
; NT, not tested.
Species
Peptides
ABKMQ
Gram-positive bacteria
Micrococcus lysodeikticus 0.6 28 12 16 8
Staphylococcus aureus 0.6 49 22 17 28
Staphylococcus epidermidis 3.1 43 43 41 33
Bacillus megaterium 0.8 51 29 34 NT
Gram-negative bacteria
Vibrio alginolyticus >75 >75 >75 >75 >75
Vibrio metschnikowii >75 >75 >75 >75 >75
Escherichia coli 363 >75 >75 62 62 22
Salmonella newport >75 >75 >75 >75 >75
Fungi
Fusarium oxysporum 5 30 17 13 14.8
Candida species NT 59 >75 8 >75
Fig. 2. Relationships between the bacteriostatic activity of a series of
charge variants of the b-hairpin loop 3 (peptide B) with their isoelectric
points. The bacteriostatic activity is expressed as MIC in l
M

on Gram-
positive bacteria M. lysodeikticus. The theoretical isoelectric point was
computed ( and plotted against
the log of the measured MIC.
2808 B. Romestand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
status of M. lysodeikticus bacteria treated with 30 l
M
peptides E, B and D was assessed using a double
fluorescence labelling (Fig. 3B). In the absence of any
peptide or in the presence of peptide E (noncyclized
loop 3), an important number of live bacteria was
observed and practically no dead cells. However, both
peptides B and D inhibited the growth of bacteria, leading
to a low number of observable green fluorescence. In
addition, the few detectable bacteria were dead.
Common features of sequences of loop 3 in arthropod
and mussel defensins
Figure 4 shows the
CLUSTAL
format alignment of arthro-
pod defensins. Two subfamilies were identified by this
analysis, one including MGD1 (structural PDB code
1FJN) as a prototype and one including the insect
defensin A (PDB code 1ICA). In the MGD1 subfamily,
some striking features of the loop 3 sequences (comprised
between conserved cysteines Cys25 and Cys33 in MGD1)
are evident: (a) this part of the molecule contains at least
one (often two) positively charged residue (Lys or Arg;
boxed characters in Fig. 4); (b) it contains one or several
hydrophobic amino acids (Phe, Trp, Leu; greyed charac-

ters in Fig. 4); and (c) it is flanked by two highly
conserved sequences GGY and TCYR. This part of the
defensin molecule therefore constantly encloses basic and
hydrophobic residues, which are considered to be import-
ant for membrane binding and disruption. Note that
basicity and hydrophobicity are not exclusive characteri-
stics of this part of the molecule, but only loop 3-derived
peptides have demonstrated activity. In the insect defensin
subfamily, the loop is much shorter, it does not always
include basic residues and aromatic residues were never
found; just as in the MGD1 subfamily, the loop sequence
was also flanked by highly conserved amino acid stretches.
One can also notice that loop 1 (the four residues that
precede the a-helix) is highly conserved in the MGD1
subfamily (Fig. 4) and is close in space to the tip of loop 3
(Fig. 1). In Fig. 5, the solvent-accessible surface of
residues from loop 1 and loop 3 from MDG1 is color-
coded. The surface contribution of positively charged
residues (blue) forms a long and quite continuous patch,
whereas the surface contribution of hydrophobic residues
(yellow) forms a second, distinct patch. Remarkably in the
MGD1 model, the two types of accessible surfaces
(positively charged and hydrophobic) seem to be layered
one on top of the other.
Discussion
The MGD1 protein was isolated from the edible mussel
Mytilus galloprovincialis [10] and shown to belong to the
arthropod defensin family. It has bactericidal activity on
Gram-positive bacteria. Although killing of bacteria
occurred through cytoplasmic membrane permeation

[20,21], the mode of action of defensins requires an initial
binding step on the outer membrane. The way this contact
takes place and the molecular features of the protein
involved are yet to be deciphered. The lack of information
about the mode of action and the availability of a refined
three-dimensional model [16] prompted us to prepare a
series of synthetic fragments designed on the basis of the
secondary structure elements of MGD1.
A remarkable result is that only peptides including
residues 25–33 of MGD1 displayed activity against Gram-
positive bacteria and fungi, after this short peptide had
been cyclized by disulfide bridging. Three series of
arguments suggest that the b-hairpin loop of MGD1,
i.e. residues 25–33 (CGGWHRLRC), plays a major role
in the binding of MGD1 to M. lysodeikticus. First, among
the synthetic fragments that we designed from the
available three-dimensional structure, only the cyclic
peptide CGGWHRLRC showed bacteriostatic activity
whereas larger fragments, corresponding either to the
a-helix sequence, or to the a-helix sequence extended by
the loop between the a-helix and the first b-strand (loop
2), or to the sequence of the whole b-hairpin with residues
from the loop substituted by serine and glycines, had no
detectable activity. This cyclic peptide CGGWHRLRC
was observed in confocal microscopy to bind to M. lyso-
deikticus, inhibiting the bacterial growth without lysing the
bacteria. Therefore, it is speculated that the binding of
MGD1 to the bacterial membrane is mediated by the
loop 3 region of the defensin, thus participating in the
early events of bactericidal activity. Our construction of

the model of MGD1, showing that positively charged and
hydrophobic residues are clustered in two discrete
domains, led to the hypothesis that the positive cluster
could initially dock the molecule onto the phospholipids
and that the surrounding hydrophobic cluster could
initiate the slipping of MGD1 into the hydrophobic part
of membrane lipids. Second, the activity of the synthetic
peptide CGGWHRLRC was detectable only when it was
cyclized by pairing two Cys residues (not linked in the
natural defensin, but close in space [16]), indicating that
the loop structure present in MGD1 (a type III¢ turn) is
Table 2. Antimicrobial activity (MIC for M . lysodeikticus) of peptides containing either the natural sequence of loop 3 of MGD1 or sequences with
increased number of positively charged residues. In the amino acid sequence, C indicates a half-cystine residue (Cys engaged in a disulfide bridge) and
underlined residues indicate amino acid changes from the native MGD sequence.
Peptide Identity Amino acid sequence MIC (l
M
)
K [Ser25,Ser33,Ser35] MGD1 (21–39) CGGY
SGGWHRLRSTSYRCG 12
L [Ser25,Ser33,Ser35][Lys29,Lys31]MGD1(21–39) CGGY
SGGWKRKRSTSYRCG 9
M [Ser21,Ser35] MGD1 (21–39) SGGYCGGWHRLRCT
SYRSG 16
N [Ser21,Ser35][Lys29,Lys31]MGD1(21–39) SGGYCGGW
KRKRCTSYRSG 12
Q [Ser35]MGD1(21–39) CGGYCGGWHRLRCTSYRCG 10
P [Lys29,Lys31][Ser35]MGD1(21–39) CGGYCGGW
KRKRCTSYRCG 8
Ó FEBS 2003 Role of the hairpin loop in mussel defensin activity (Eur. J. Biochem. 270) 2809
Fig. 3. Effect of loop 3-derived peptides on M. lysodeikticus cells. (A) M. lysodeikticus cells were treated with 1, 10, 30 and 60 lgÆmL

)1
of
biotinylated peptide D at 37 °C for 24 h and the binding of peptides to bacteria visualized with FITC–streptavidin. Confocal microscopic images
show the localization of the biotinylated peptide D on the cell surface. Similar results were obtained for peptide B. Control experiments were
performed in the presence of FITC–streptavidin and absence of peptide, and in the presence of FITC–streptavidin and 30 l
M
of an irrelevant
biotinylated peptide (Biot-YKKWINTFSGVPTYA). (B) Viability of M. lysodeikticus in the presence of 30 l
M
peptide E, B or D after overnight
incubation at 37 °C. The live or dead status of bacteria was assessed by labeling with FITC SYTO9Ò (green fluorescence, living bacteria) and
propidium iodide (red fluorescence, dead bacteria). Bacterial growth in the absence of any peptide was used as a control. Note the absence of killing
in the presence of peptide E and the important number of green living bacteria. In contrast, both peptides B and D inhibited bacterial growth and
the few observed bacteria were dead.
2810 B. Romestand et al.(Eur. J. Biochem. 270) Ó FEBS 2003
important for the mode of action. In addition, this loop
has been found to be highly solvent exposed in MGD1
[16], defensin A [19] and the Raphanus sativus defensin
[31]. It is worth noting that many other cysteine-rich
antibacterial peptides, not belonging to the defensin
family, exhibit a cysteine-bridged loop that also contain
basic and hydrophobic residues: e.g. CRIVVIRVC (bac-
tenecin), CYRGIGC (tachyplesin), CRRRFC (buthinin),
CTMIPIPRC (tigerinin), etc. Also remarkable is the
observation that lactoferricin B (a tryptophan/arginine
rich antibiotic peptide), when enzymatically cleaved from
lactoferrin adopts a twisted b-sheet structure, the loop
part of which includes one tryptophan and two arginine
residues [32], thus resembling loop 3 derived peptides.
Third, the relationship between the isoelectric point of the

loop 3 sequence and its bacteriostatic activity is in
agreement with the general observation that the basicity
of defensins is an important parameter for activity, in
particular because it is thought that binding to negatively
charged membrane phospholipids is favoured by electro-
static interactions [33]. We report here that this parameter
is indeed important, but we found that the electrical
charge of the b-hairpin loop, as compared with that of
other domains of the molecule, might be a key feature for
the activity of defensins. It must be noted that the a-helix
(residues 7–16) contains the same number of positively
chargedresiduesascomparedwiththeb-hairpin loop 3
peptide and even has two His residues which could also
be ionized under certain pH conditions; meanwhile,
peptides including the a-helix sequence did not display
any antimicrobial activity. This clearly indicates that the
positive charge density is not sufficient for binding to
Gram-positive bacteria, but rather that these charges must
be presented in an appropriate structural context [34].
Fig. 4. Alignment of arthropod defensin sequences. The alignment was
performed using the
CLUSTAL X
(1.8) algorithm on the PFAM arth-
ropod defensin family. The boxed parts indicate the sequences com-
prised between the half-cystines forming the b-strand loop of defensins.
Characters corresponding to basic amino acids are boxed and to
hydrophobic amino acids are greyed.
Fig. 5. Solvent accessible surface of loop 1 and loop 3 residues from
MDG1 (1FJN [16]). The surface contribution of positively charged
residues is coloured in blue and the contribution of hydrophobic

residues in yellow. (A) View from the top of the molecule. Accessible
and positively electrically charged residues form a linear patch. (B) Side
view showing layered hydrophobic and surface accessible residues.
Ó FEBS 2003 Role of the hairpin loop in mussel defensin activity (Eur. J. Biochem. 270) 2811
From our observations, it is not possible to infer whether
the key role of the b-hairpin loop of MGD1 (loop 3) in
antimicrobial activity of MGD1 is of general value in the
mechanism of action of defensins. However, our results
could be compared with results obtained by other groups
who also point to the role of the connecting loop of the
b-hairpin of defensins. A combination of mutational
analysis [35] and structural analysis of the plant defensin
Rs-AFP1 [31] identified two subsites on this molecule
comprising Ôresidues in the protruding domain consisting
of the type VI b-turn and the first part of b-strand 3Õ (i.e. the
b-hairpin loop and part of the adjoining b-sheet) and
Ôresidues in the loop connecting b-strand and a-helix and
contiguous residues on the a-helix and the last part of
b-strand 3Õ [35]. Our observations were similar, although
obtained by a completely different approach. Note that we
did not succeed in demonstrating activity with synthetic
peptides corresponding to the second subsite of the
Rs-AFP1; this might be due to methodological differences.
Finally, two reports using synthetic peptides derived from
the amino acid sequence of rabbit [36] and plant defensins
[37] led to the conclusion that the whole b-hairpin could be
an important structural feature of the mode of action,
although the precise role of the loop was not elucidated.
In conclusion, our results indicated that residues 23–35 of
mussel defensin MGD1 play a key role in the binding to

Gram-positive bacteria, inhibiting bacterial growth and
permeabilizing bacteria. Moreover, and in agreement with
other reports using similar or different approaches, our
results argue in favour of the critical importance of the loop
sequence bridging the two common b-strands of defensins in
their biological activity. Our hypothesis is that this protru-
ding part of the molecule might initiate the molecular
process by which defensins bind to and penetrate microbial
membranes.
Acknowledgements
We are greatly indebted to Dr S. L. Salhi for editing the manuscript. We
thank C. Nguyen for his skilful help in peptide synthesis.
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Ó FEBS 2003 Role of the hairpin loop in mussel defensin activity (Eur. J. Biochem. 270) 2813