Key role of the loop connecting the two beta strands of musseldefensin in its antimicrobial activity Bernard Romestand1, Franck Molina2, Ve´ronique Richard1, Philippe Roch1and Claude Gra
Trang 1Key role of the loop connecting the two beta strands of mussel
defensin in its antimicrobial activity
Bernard Romestand1, Franck Molina2, Ve´ronique Richard1, Philippe Roch1and Claude Granier2
1
DRIM, Universite´ Montpellier 2, France;2Centre 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 using1H-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 the structure of MGD1 [16] Synthetic peptides, 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: proch@univ-montp2.fr
Abbreviations: MGD, Mytilus galloprovincialis defensin;
MIC, minimal inhibitory concentration.
(Received 14 February 2003, revised 28 April 2003,
accepted 08 May 2003)
Trang 2(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, 0.05M 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 (0.1Miodine 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,
http://www.expasy.ch/cgi-bin/pi_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 withSWISS-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 lL of
bacteria suspension, at a starting D600¼ 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 30C 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 SYTO9,
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 104sporesÆmL)1) suspended in potato dex-trose broth (Difco) containing 0.1 mg tetracycline was added to 20 lL 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 30C and quantified
by D600measurement after 48 h The MIC was defined as described above
Confocal laser-scanning observations
M lysodeikticus(105CFU 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 37C with biotin-labelled peptides Slides were then washed with phosphate-buffered saline (NaCl/Pi), pH 7.5, and incubated for 5 min in NaCl/Picontaining 0.2% (v/v) Triton X-100 After three washes in NaCl/Pi, 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 VitroToxicology 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
Trang 3However, 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 lM (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 mMdithiothreitol, 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 b1 and b2-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 (MIC¼ 12 lM) 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 (MIC¼ 62 lM), and Q (MIC¼ 22 lM) The fungus F oxysporum was inhibited by all four peptides, especially by peptides Q and
M (MIC¼ 13–15 lM) and peptide K (MIC¼ 17 lM) 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 ) for the growth of M lysodeikticus cells.
Trang 4Activity 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
onM 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 lM, 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 concentration up to 60 lM (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
Gram-positive bacteria
Gram-negative bacteria
Vibrio alginolyticus >75 >75 >75 >75 >75 Vibrio metschnikowii >75 >75 >75 >75 >75
Fungi
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 (http://www.expasy.ch/cgi-bin/pi_tool) and plotted against the log of the measured MIC.
Trang 5status of M lysodeikticus bacteria treated with 30 lM
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 theCLUSTAL 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.
K [Ser25,Ser33,Ser35] MGD1 (21–39) CGGYSGGWHRLRSTSYRCG 12
L [Ser25,Ser33,Ser35][Lys29,Lys31]MGD1(21–39) CGGYSGGWKRKRSTSYRCG 9
N [Ser21,Ser35][Lys29,Lys31]MGD1(21–39) SGGYCGGWKRKRCTSYRSG 12
P [Lys29,Lys31][Ser35]MGD1(21–39) CGGYCGGWKRKRCTSYRCG 8
Trang 6Fig 3 Effect of loop 3-derived peptides on M lysodeikticus cells (A) M lysodeikticus cells were treated with 1, 10, 30 and 60 lgÆmL)1of 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 SYTO9 (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.
Trang 7important 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 charged residues as compared with the b-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.
Trang 8From 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 residues in the protruding domain consisting
of the type VI b-turn and the first part of b-strand 3 (i.e the
b-hairpin loop and part of the adjoining b-sheet) and
residues in the loop connecting b-strand and a-helix and
contiguous residues on the a-helix and the last part of
b-strand 3 [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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