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: 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) 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, 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 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 (http://www.expasy.ch/cgi-bin/pi_tool) 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. References 1. Hancock, R.E. (1997) Peptide antibiotics. Lancet 349, 418–422. 2. Williams, R.J. & Heymann, D.L. (1998) Containment of anti- biotic resistance. Science 279, 1153–1154. 3. Travis, J. (1994) Reviving the antibiotic miracle? Science 264, 360–362. 4. Lehrer, R. & Ganz, T. (2002) Defensins of vertebrate animals. Curr. Opin. Immunol. 14, 96–102. 5. Lambert, J., Keppi, E., Dimarcq, J.L., Wicker, C., Reichhart, J.M.,Dunbar,B.,Lepage,P.,VanDorsselaer,A.,Hoffmann,J., Fothergill, J. et al. (1989) Insect immunity: isolation from immune blood of the dipteran Phormia terranovae of two insect antibac- terial peptides with sequence homology to rabbit lung macrophage bactericidal peptides. Proc. Natl Acad. Sci. USA 86, 262–266. 6. Ehret-Sabatier, L., Loew, D., Goyffon, M., Fehlbaum, P., Hoff- mann, J.A., van Dorsselaer, A. & Bulet, P. (1996) Characteriza- tion of novel cysteine-rich antimicrobial peptides from scorpion blood. J. Biol. Chem. 271, 29537–29544. 7. Broekaert, W.F., Terras, F.R., Cammue, B.P. & Osborn, R.W. (1995) Plant defensins: novel antimicrobial peptides as compo- nents of the host defense system. Plant Physiol. 108, 1353–1358. 8. Segura, A., Moreno, M., Molina, A. & Garcia-Olmedo, F. (1998) Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett. 435, 159–162. 9. Charlet,M.,Chernysh,S.,Philippe,H.,Hetru,C.,Hoffmann,J.A. & Bulet, P. (1996) Innate immunity. Isolation of several cysteine- rich antimicrobial peptides from the blood of a mollusc, Mytilus edulis. J. Biol. Chem. 271, 21808–21813. 10. Hubert, F., Noel, T. & Roch, P. (1996) A member of the arthropod defensin family from edible Mediterranean mussels (Mytilus galloprovincialis). Eur. J. Biochem. 240, 302–306. 11. Ganz, T. & Lehrer, R.I. (1994) Defensins. Curr. Opin. Immunol. 6, 584–589. 12. Lehrer,R.I.&Ganz,T.(1999)Antimicrobialpeptidesinmam- malian and insect host defence. Curr. Opin. Immunol. 11, 23–27. 13. Ganz,T.&Lehrer,R.I.(1999)Antibioticpeptidesfromhigher eukaryotes: biology and applications. Mol. Med. Today 5, 292–297. 14. Tang, Y.Q., Yuan, J., Osapay, G., Osapay, K., Tran, D., Miller, C.J., Ouellette, A.J. & Selsted, M.E. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286, 498–502. 15. Bonmatin, J.M., Bonnat, J.L., Gallet, X., Vovelle, F., Ptak, M., Reichhart, J.M., Hoffmann, J.A., Keppi, E., Legrain, M. & Achstetter, T. (1992) Two-dimensional 1H NMR study of recombinant insect defensin A in water: resonance assignments, secondary structure and global folding. J. Biomol. NMR. 2, 235–256. 16. Yang, Y.S., Mitta, G., Chavanieu, A., Calas, B., Sanchez, J.F., Roch, P. & Aumelas, A. (2000) Solution structure and activity of the synthetic four-disulfide bond Mediterranean mussel defensin (MGD-1). Biochemistry 39, 14436–14447. 17. Zhang, X.L., Selsted, M.E. & Pardi, A. (1992) NMR studies of defensin antimicrobial peptides. 1. Resonance assignment and secondary structure determination of rabbit NP-2 and human HNP-1. Biochemistry 31, 11348–11356. 18. Mitta,G.,Vandenbulcke,F.,Hubert,F.&Roch,P.(1999)Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge. J. Cell Sci. 112 (23), 4233–4242. 19. Cornet, B., Bonmatin, J.M., Hetru, C., Hoffmann, J.A., Ptak, M. & Vovelle, F. (1995) Refined three-dimensional solution structure of insect defensin A. Structure 3, 435–448. 20. Kagan, B.L., Selsted, M.E., Ganz, T. & Lehrer, R.I. (1990) Antimicrobial defensin peptides form voltage-dependent ion-permeable channels in planar lipid bilayer membranes. Proc. Natl Acad. Sci. USA 87, 210–214. 21. Cociancich, S., Ghazi, A., Hetru, C., Hoffmann, J.A. & Letellier, L. (1993) Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus. J. Biol. Chem. 268, 19239–19245. 22. Lehrer, R.I., Barton, A. & Ganz, T. (1988) Concurrent assessment of inner and outer membrane permeabilization and bacteriolysis in E. coli by multiple-wavelength spectrophotometry. J. Immunol. Methods 108, 153–158. 23. Shimoda, M., Ohki, K., Shimamoto, Y. & Kohashi, O. (1995) Morphology of defensin-treated Staphylococcus aureus. Infect. Immun. 63, 2886–2891. 24. Yeaman, M.R., Bayer, A.S., Koo, S.P., Foss, W. & Sullam, P.M. (1998) Platelet microbicidal proteins and neutrophil defensin dis- rupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action. J. Clin. Invest. 101, 178–187. 25.Gausepohl,H.,Boulin,C.,Kraft,M.&Frank,R.W.(1992) Automated multiple peptide synthesis. Pep. Res. 5, 315–320. 26. Laune, D., Molina, F., Ferrieres, G., Villard, S., Bes, C., Rieunier, F., Chardes, T. & Granier, C. (2002) Application of the Spot method to the identification of peptides and amino acids from the 2812 B. Romestand et al.(Eur. J. Biochem. 270) Ó FEBS 2003 antibody paratope that contribute to antigen binding. J. Immunol. Methods 267, 53–70. 27. Tam, J.P., Wu, C., Liu, W. & Zhang, J.W. (1991) Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applica- tions. J. Am. Chem. Soc. 113, 6657–6662. 28. Bateman, A., Birney, E., Cerruti, L., Durbin, R., Etwiller, L., Eddy, S.R., Griffiths-Jones, S., Howe, K.L., Marshall, M. & Sonnhammer, E.L. (2002) The Pfam protein families database. Nucleic Acids Res. 30, 276–280. 29. Hancock, R.E., Falla, T. & Brown, M. (1995) Cationic bacteri- cidal peptides. Adv. Microb. Physiol. 37, 135–175. 30. Fehlbaum, P., Bulet, P., Michaut, L., Lagueux, M., Broekaert, W.F.,Hetru,C.&Hoffmann,J.A.(1994)Insectimmunity.Septic injury of Drosophila induces the synthesis of a potent antifungal peptide with sequence homology to plant antifungal peptides. J. Biol. Chem. 269, 33159–33163. 31. Fant, F., Vranken, W., Broekaert, W. & Borremans, F. (1998) Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR. J. Mol. Biol. 279, 257–270. 32. Vogel, H.J., Schibli, D.J., Jing, W., Lohmeier-Vogel, E.M., Epand, R.F. & Epand, R.M. (2002) Towards a structure-function analysis of bovine lactoferricin and related tryptophan- and argi- nine-containing peptides. Biochem. Cell Biol. 80, 49–63. 33. White, S.H., Wimley, W.C. & Selsted, M.E. (1995) Structure, function, and membrane integration of defensins. Curr. Opin. Struct. Biol. 5, 521–527. 34. Tam, J.P., Lu, Y.A. & Yang, J.L. (2002) Correlations of cationic charges with salt sensitivity and microbial specificity of systine- stabilized beta-strand antimicrobial peptides. J. Biol. Chem. 277, 50450–50456. 35. De Samblanx, G.W., Goderis, I.J., Thevissen, K., Raemaekers, R.,Fant,F.,Borremans,F.,Acland,D.P.,Osborn,R.W., Patel, S. & Broekaert, W.F. (1997) Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity. J. Biol. Chem. 272, 1171–1179. 36. Thennarasu, S. & Nagaraj, R. (1999) Synthetic peptides corre- sponding to the beta-hairpin loop of rabbit defensin NP-2 show antimicrobial activity. Biochem. Biophys. Res. Commun. 254, 281–283. 37. Schaper, W.M., Posthuma, G.A., Plasman, H.H., Sitsma, L., Fant, F., Borremans, F.A.M., Thevissen, K., Broekaert, W., Meloen,R.H.&vanAmerongen,A.(2001)Synthetic peptides derived from the beta 2-beta 3 loop of Raphanus sativus antifungal protein that mimic the active site. Peptide Res. 57, 409–418. Ó FEBS 2003 Role of the hairpin loop in mussel defensin activity (Eur. J. Biochem. 270) 2813 . 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. 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