The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider Nicolas Mandard 1 , Philippe Bulet 2 , Anita Caille 1 , Sirlei Daffre 3 and Franc¸oise Vovelle 1 1 Centre de Biophysique Mole ´ culaire, CNRS, Orle ´ ans, France; 2 Institut de Biologie Mole ´ culaire et Cellulaire, CNRS, Strasbourg, France; 3 Departamento de Parasitologia, ICB, Universidade de Sa ˜ o Paulo, Brazil Gomesin is the first peptide isolated from s pider exhibiting antimicrobial activities. This highly cationic peptide is composed of 18 amino-acid residues including four cysteines forming two disulfide linkages. The solution structure of gomesin has been determined using proton two-dimensional NMR (2D-NMR) and restrained molecular dynamics calculations. The global fold of gomesin c onsists in a well- resolved two-stranded a ntiparallel b shee t c onnected by a noncanonical b turn. A comparison b etween the s tructures of gomesin and protegrin-1 from porcine and androctonin from scorpion outlines several common features in the distribution of hydrophobic and hydrophilic residues. The N- and C -te rmini , the b turn and one face of the b sheet are hydrophilic, but the h ydrophobicity of the other f ace depends on the peptide. The similarities suggest that the molecules interact with m embranes i n a n analogous manner. The importance of t he intramolecular d isulfide bridges in t he biological activity of gomesin is being i nvestigated. Keywords: spider; cysteine-rich; antimicrobial peptide; b sheet; NMR. In recent years, it has become widely recognized that animal defense systems rely on inducible or constitutive expression of antimicrobial peptides in response to bacterial and/or fungal infe ctions. A mong these antimicrobial molecules, open-ended cyclic cysteine-rich peptides are the most widespread. They h ave been characterized in plants, inver- tebrates and v ertebrates. S tructurally, t hey can be classified into (a) peptides adopting a b sh eet structure, namely the mammalian defensins [1]; (b) p eptides exhibiting the CSab (cysteine stabilized a helix b sheet) motif [2] such a s defen- sin A from Phormia terranovae [3], drosomycin from Drosophila melanogaster [4], heliomicin from Heliothis virescens [5], plant defensins [6]; and (c) peptides adopting a b-hairpin-like fold, such as tachyplesins from horseshoe crabs [7,8], porcine protegrins [9,10], thanatin from the bug Podisus m aculiventris [11], androctonin from the scorpion Androctonus australis [12], lactoferricin B from bovine [13] and a 20-residue antimicrobial peptide from t he plant Impatiens balsamina [14]. All the peptides adopting a b hairpin structure possess a broad antimicrobial activity spectrum. In contrast, peptides w ith a CSab motif h ave a more restricted activity spectrum; insect defensins are mainly active against Gram-positive bacteria whereas drosomycin, heliomicin and plant defensins are active exclusively against fungi. While there are numerous reports on the structural characterization and the three-dimensional structure of polypeptide toxins from spider venoms (for review see [ 15]), it is only very recently that a peptide with antimicrobial activity has been characterized from spiders [16]. This peptide, gomesin, is an 18-residue cysteine-rich antimicro- bial peptide i solated from the blood cells (hemocytes) o f the mygalomorph spider Acanthoscurria gomesiana. Gomesin has two disulfide bridges linking Cys2 to Cys15 and Cys6 to Cys11. In addition, gomesin c arries two post-translational modifications: cyclization of the N-terminal glutamine into pyroglutamic acid (pGlu or Z) and amidation of the C-terminal arginine. The molecule is highly cationic (pI ¼ 9.86 calculated by EDITSEQ from DNA STAR 4.05 software) with t he presence of five arginines, one lysine, a C-terminal amidation and no acidic amino acid. Gomesin exhibits broad activity at rather l ow concentra- tions (often below 10 l M ) against numerous microorgan- isms including bacteria, filamentous fungi and yeast. In addition, this peptide was fou nd to a ffect the v iability of t he parasite Leishmania amazonensis and to present some hemolytic activity against human erythrocytes. Sequence alignments suggest strong similarities with various anti- microbial peptides adopting a b sheet structure, such as tachyplesins, androctonin, and protegrins [16]. In this paper, we report on the elucidation of the solution structure of gomesin using two-dimensional 1 H-NMR spectroscopy and molecular modeling. Gomesin adopts a well-defined b-hairpin-like structure as it co uld b e expected from the sequence similarities with androctonin a nd prote- grins. The structure of these three peptides are compared i n order to d etermine th e s tructural f eatures r equired for their biological properties. MATERIALS AND METHODS NMR experiments Gomesin peptide was s ynthesized according to classical Fmoc chemistry as described previously [16]. Correspondence to F. Vovelle, Centre de Biophysique Mole ´ culaire, CNRS UPR 4301, Rue Charles Sadron, 45071 Orle ´ ans Cedex 2, France. Fax: + 3 3 23863 1517, Tel.: + 33 23825 5574, E-mail: vovelle@cnrs-orleans.fr Abbreviations: pGlu (Z), pyroglutamic acid; PG-1, protegrin-1. (Received 26 J uly 2001, revised 5 December 2001, accepted 2 January 2002) Eur. J. Biochem. 269, 1190–1198 (2002) Ó FEBS 2002 The sample for NMR spectroscopy was prepared by dissolving 4.5 mg of synthetic gomesin in 90%H 2 O/ 10%D 2 O to obtain a final solution at 3.3 m M . The pH was adjusted t o 3.5 with microlitre increments of HCl 1 N. For experiments in h eavy water, 90% of the volume of th e previous sample was lyophilized and then dissolved in 99.99% D 2 O. The remaining v olume (10%) was completed with H 2 O to obtain a gomesin solution at 0.3 m M . A conventional set of one-dimensional and two-dimen- sional 1 H-NMR spectra in H 2 O, including DQF-COSY [17], Clean-TOCSY [18] and NOESY [19], was acquired at a temperature of 278 K on a VARIAN INOVA NMR spectrometer equipped with a z-axis fi eld-gradient unit and operating at a proton fre quency of 600 MHz. The clean- TOCSY spe ctrum w as collected with a spin lock t ime o f 80 ms using the MLEV-17 mixing scheme [20] and NOESY spectra were recorded with mixing times of 120 ms and 3 00 ms. Water suppression was achieved either by presaturation for COSY and TOCSY experiments or using the WATERGATE pulse sequence [21] for NOESY experiments. A new series of TOCSY and NOESY spectra in D 2 O was also recorded at 278 K. In an attempt to overcome ambiguities in assignment due to spectral overlap, a second set of clean-TOCSY and NOESY spectra was performed at 285 K. Spectra were acquired over a spectral width corresponding to 9 p .p.m. and referenced to the residual H 2 O signal set as the c arrier frequency (4.964 p.p.m. at 2 78 K; 4.897 p .p.m. a t 2 85 K). All two-dimensional NMR data were processed on a Silicon Graphics Indy O2 workstation using the VNMR software package (version 6.1; Varian, Inc., Palo Alto, C A, USA). Assignments were c arried out according to classical procedures including spin-system identification and sequential assignment [22] on map s recorded at 278 K. Cross-peak intensities of the NOESY map at 278 K with the shortest mixing time 120 ms a nd recorded over 4096 data points in the F2 dimension were integrated with XEASY [23]. The unusual N -terminal residue (pyroglutamic acid) was especially built for this wo rk and its coordinates and appropriate parameters (bond length and atom charges) were included in the libraries of DYANA [24,25] and XPLOR [26] for molecular modeling. Structure calculations NOESY cross-peak in tensities were converted into upper distance limit constraints using the CALIBA program [25]. The minimum distance constraint between two p rotons was limited by their van der Waals r adi (2.0 A ˚ ). Moreover, in order t o assess possible contributions from spin diffusion effects, some NOEs only observable on the 300-ms mixing time NOESY map were taken into account with a 6-A ˚ upper limit constraint. Each of the two disulfide bridges was explicitly de fined b y t hree lower/upper distance limit restraints between the sulphur and b carbon atoms of the two cysteines i, j involved in the linkage (1.9 A ˚ < d(Sc i ,Sc j )<2.1 A ˚ ;2.5A ˚ <d(Cb i ,Sc j )<3.5 A ˚ ;2.5A ˚ < d(Sc i ,Cb j )<3.5 A ˚ ). All these constraints were brought together in a distance restraint file used as input to initial steps o f molecular modeling. Several s ets of 100 structures were generated from random-built initial models using t he annealing procedure of the variable target function program DYANA. During these rounds of calculations, restraints corresponding to the stereospecific assignment of three m ethyl p rotons proposed by GLOMSA were incorpor- ated in the data set [25]. The hydrogen bonds found at each round of calculations on a majority of structures and corresponding to atoms involved in secondary structure elements were also introduced as constraints. A final set of 50 structures was then generated in a fin al DYANA run f rom an input file taking into account the t otal set of constraints. Twenty out of these 50 DYANA structures were selected on the basis of low target function values (% 1A ˚ 2 )and subjected to e nergy minimization using Powell’s algorithm and CHARMM force field parameters [27] implemented in X - PLOR 3.1 software. The energy calculations were performed with a distance dependent dielectric function e ¼ r,a12-A ˚ cut-off distance for all nonbonded interac- tions and a force constant of 50 kcalÆmol )1 ÆA ˚ )2 for NOE restraint energy terms. All calculations were carried out on a Silicon G raphics 02 R10000 workstation and the s truc- tures were visualized with the SYBYL software (TRIPOS Inc., St Louis, MO, USA). Hydrophobic potentials were calculated with the MOLCAD option [28] implemented in SYBYL . PROCHECK [29] and PROMOTIF [30] programs were used for s tructural analysis. RESULTS AND DISCUSSION Sequence-specific assignment and secondary structure Comparison of the one-dimensional spectra of the s amples of gomesin at 0.3 m M and 3.3 m M in aqueous solution clearly shows the absence of any concentration-dependent changes in the chemical shifts or peak line widths, suggesting the monomeric state of the peptide in our experimental conditions. The two-dimensional 1 H-NMR spectra of gomesin were a ssigned via standard sequential assignment methods developed by Wu ¨ thrich [22]. The entire spin systems of individual amino-acid residues were identified through DQF-COSY and TOCSY experiments on the maps at 278 K. TOCSY and NOESY maps recorded at 285 K were used to clear up ambiguities in the a ssignment of the NH-Ha cross-peaks of Arg4 d ue to the close vicinity of its Ha chemical shift and o f the water resonance. Moreover, dipolar connectivities on the D 2 O N OESY spectra enable the best-defined Ha-Ha peaks to be obtained near the r esidual water diagonal, especially between Cys2 and C ys15, Cys6 and Cys11, Arg 4 and Thr13. The splitting of the resonance of backbone NH and Ha protons allows complete proton assignments for the fingerprint region (Fig. 1 ). 1 H c hemical s hifts o f g omesin are reported i n Table 1 and the complete pathway Ha(i) ) NH(i +1)is shown i n Fig. 2. The NOE c onnectivity diagram exhibits d NN (i,i +2)andd aN (i,i+ 2) N OEs between the c entral residues (Tyr7–Arg10), suggesting the presence of a turn in this region (Fig. 3A). Strong d aN (i,i +1)NOEsinseg- ments (Cys2–Cys6 and Cys11–Cys15) a re indicative of two extended strands of b sheet. This hypothesis is c onfirmed by the presence of long-distance Ha(i)-Ha(j) co nnectivities detected on D 2 O maps even if d euterium exchange studies revealed that all amide protons were quickly exchanging with the solvent. Figure 3B shows the number of NOEs between t wo residue s i and j with r espect to the d ifference |i ) j|. The enhancement o f the nu mber of NOEs observed Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1191 for 5 <|i ) j|<13 is m ainly due to connectivities between the protons of residues Cys6 a nd Cys11 (|i ) j| ¼ 5), L eu5 and Val12 (| i ) j| ¼ 7), Arg4 a nd Thr13 (|i ) j| ¼ 9), Arg3 and Tyr14 (|i ) j| ¼ 11), Cys2 and Cys15 (|i ) j| ¼ 13). Finally, no NOE cross-peak, indicative of an oligomeric association in solution, could b e d etected, which is consis- tent with the high abundance of positively charged r esidues (five arginines and one lysine) in the primary structure of the peptide. Structure evaluation The three-dimensional structure of gomesin was determined using the standard simulated annealing p rotocol of DYANA AND energy minimization with X - PLOR , as described in Materials and methods. The final restraint file c omprised a set o f 289 distanc e restraints including 82 intraresidual, 102 sequential, 32 medium-range (2 < |i ) j| < 5) and 73 long range (|i ) j| ‡ 5) restraints (with an average of 16 restraints per resid ue). Long-range limits con cern mainly residues located in t he segments corresponding to the t wo strands o f the b sheet (pGlu1–Tyr7; Arg10–Arg16) (data not shown). As shown i n Table 2, the 20 selected structures are in very good agreement with all experimental data and the standard covalent geometry. There are no violations larger than 0.3 A ˚ and t he root-mean-square deviations (rmsd) with respect to the standard geometry are low. Both negative van der Waals and electrostatic energy te rms are indicative of favorable non-bonded interaction s. Moreover, the Rama- chandran plot exhibits nearly 91% of the (/,w) angles of all structures in the most favored regions and additional allowed regions according to the PROCHECK software nomenclature. The structure files have been deposited at the Protein Data Bank (http://www.rcsb.org/pdb) with the accession number 1KFP. Structure description The overall fold of gomesin is formed by a hairpin-like structure with a two-residue extension at the C-terminal end. This hairpin-like structure c onsists of two antiparallel b strands (pGlu1–Tyr7 facing Arg10–Arg16) forming a twisted s heet and connected by a four-residue turn (Tyr7– Arg10). A s shown in the structural statistics (Table 2) and Fig. 1. Fingerprint region of a TOCSY spec- trum of gomesin in 9 0%H 2 O/10%D 2 Oat 5°C, pH 3.5. The spin s ystems of the am ide protons are designated by the amino acid one- letter code, upper case letters. The spin system of side chain nitrogen-bond protons is indi- cated with the amino acid one-letter, lower case letters. 1192 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 by superimposition of the 20 structures (Fig. 4 ), the structures are extremely well defined. The pairwise rmsd on the N , Ca,C¢backbone atoms of residues 1–16 i s only 0.34 A ˚ and drops to 0.17 A ˚ when calculated in the b sheet region. Several main structural elements contribute to a strong stabilization of the sheet. Six regular backbone- backbone hydrogen bonds characteristic of the b sheet structure, NH(Arg3)–O(Tyr14), O(Arg3)–NH(Tyr14), Table 1. 1 H chemical shifts (p.p.m.) for gomesin in aqueous solution at 278K, pH 3.5. Residue Chemical shifts NH Ha Hb Others pGlu1 8.15 4.44 2.40, 2.05 Hc 2.57, 2.57 Cys2 8.88 5.48 3.02, 2.63 Arg3 9.04 4.64 1.79, 1.69 Hc 1.54, 1.54; Hd 3.18, 3.18; NHe 7.20 Arg4 8.80 5.00 1.73, 1.58 Hc 1.42, 1.42; Hd 3.03, 3.03; NHe 7.18 Leu5 9.13 4.74 1.60, 1.60 Hc 1.51; Hd 0.81, 0.81 Cys6 9.03 5.44 2.98, 2.70 Tyr7 8.76 4.59 2.94, 2.94 Hd 7.15; He 6.78 Lys8 9.17 3.58 1.69, 1.69 Hc 0.91, 0.75; Hd 1.51, 1.51; He 2.88, 2.88; NHe 7.56 Gln9 8.53 3.94 2.21, 2.21 Hc 2.25, 2.25 Arg10 7.92 4.63 1.97, 1.85 Hc 1.61, 1.50; Hd 3.21, 3.21; NHe 7.24 Cys11 8.98 5.60 2.99, 2.48 Val12 8.92 4.35 2.00 Hc 0.86, 0.71 Thr13 8.65 4.83 3.91 Hc 1.07 Tyr14 9.17 4.80 2.94, 2.85 Hd 7.05; He 6.73 Cys15 8.97 5.16 2.86, 2.86 Arg16 8.10 4.22 1.83, 1.75 Hc 1.65, 1.65; Hd 3.18, 3.18; NHe 7.21 Gly17 8.69 3.94, 3.94 Arg18 8.41 4.27 1.84, 1.70 Hc 1.59, 1.59; Hd 3.15, 3.15; NHe 7.21 8.08.59.0 3.5 4.0 4.5 5.0 5.5 11 6 15 2 14 5 3 12 7 13 8 17 9 18 1 16 10 4 Fig. 2. A mide- a region of a 120-ms mixing time NOESY spectrum of g omesin. For the sake of clarity, only th e intraresidue a-amide cross-peaks are labeled. Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1193 NH(Leu5)–O(Val12), O(Leu5)–NH(Val12) are found between the d isulfide bridges as well as O(pGlu1)– NH(Arg16) and NH(Tyr7)–O(Arg10) located at each extremity of the b sheet. Two i nterstrand disulfide bridges adopt a well-defined right-handed conformation with v SS , v 1 , v 2 torsion angles close to the expected values for favorable energy conformers (Table 2). Moreover, whatever the model co nsidered, the average distance between the Ca atoms of the cysteine residues is small (3.75 ± 0.10 A ˚ ). This often o ccurs when d isulfide bridges link antiparallel b-strands [31]. The backbone of the loop (Tyr7-Lys8- Gln9-Arg10) also exhibits a well-defined conformation. When t he structures are best fi tted on the four backbone residues of the turn, the local pairwise rmsd of this turn is 0.22 A ˚ . The (i,i + 3) hydrogen bond between the CO group of Tyr7 and the NH group of Arg10 closing classical b turns is found only o n 10 out of the 20 structures. Whatever the nomenclature used ([32] or [33]), this turn appears to be particularly difficult to classify as Lys8 exhibits positive / and w angles as observed i n a le ft-handed helix and t he /, w average values ()150°,–60°)ofGln9areveryunusual. Owing to a lack of NOE data, the conformation of the t wo C-terminal residues Gly17 and Arg18, which are not included in the b sheet, i s poorly defined. Most side chains of s trand residues adopt a well-defined conformation due to the p resence of numerous i nterstrand NOEs. In particular, significantly low circular variances [33] for v 1 and v 2 angles are observed for the four cysteines, for Tyr7, Val12, Thr13 and Tyr14 residues (CV < 0.1). Low v 1 and v 2 circular variances are also observed for long chain or bulky resid ues such as Arg4, Leu5, and Arg10 but, in these cases, the e xtremity of their side chain i s rather fl oppy. I n contrast, the side chains of Arg16 and Arg18 at the Table 2. S tructural statistics o f t he 2 0 models of go mesin. R amachandran plots were calculated with PROCHECK and t he energy te rms were calculated using the CHARMM force field. Restraint violations, mean number per structure (min, max) Distance restraints > 0.3 A ˚ 0.7 (0, 2) Distance restraints > 0.2 A ˚ 1.6 (1, 4) Deviation from standard geometry, mean number per structure (min, max) Bond lengths > 0.05 A ˚ 0.3 (0, 1) Bond angles > 10° 0.2 (0, 2) Ramachandran Maps (%) Most favourable regions 77.0 Additional regions 13.7 Cysteine side chain torsion angles (average values in degrees) i ) j v i 1 v i 2 v SS v j 2 v j 1 Cys2-Cys15 )60.3 ± 3.1 )84.4 ± 4.5 103.6 ± 3.0 )84.7 ± 3.5 )70.7 ± 3.5 Cys6-Cys11 )65.7 ± 2.5 )96.0 ± 3.2 96.0 ± 1.4 )70.6 ± 2.7 )67.7 ± 3.4 Final energies (kcalÆmol )1 ) E total )163 ± 11 E electrostatic )251 ± 11 E vdw )50.0 ± 2.5 E NOE 13.2 ± 2.0 Average rmsd (N-Ca-C¢) Pairwise (A ˚ ) Mean structure (A ˚ ) Whole 0.79 ± 0.32 0.51 ± 0.19 Hairpin 0.34 ± 0.08 0.24 ± 0.07 b sheet 0.17 ± 0.07 0.14 ± 0.05 Turn 0.22 ± 0.10 0.15 ± 0.07 A d NN (i,i+1) d αN (i,i+1) d βN (i,i+1) d NN (i,i+2) d αN (i,i+2) d αN (i,i+4) 5 Z CRRL CYKQ 10 RC VT Y 15 CRGR 0 4 8 12 16 0 20 40 60 80 100 120 Range |i-j| Number of NOEs B Fig. 3. N OE connectivities and number. (A) Summary of the s eq uential NH(i) ) NH( i +1), Ha(i))NH(i +1), Hb(i))NH(i +1), and medium range NH(i) ) NH(i +2), Ha(i))NH(i +2), Ha(i) ) NH(i + 4) connectivities i d entified for gomesin. Th e heigh t of the bars reflects the strength of the N OE correlation as strong, medium and weak. ( B) Number of NOEs vs. difference |i ) j|. 1194 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 C-terminus, but also of Gln9 in the turn, display l arge conformational variability. Hydrophobic potentials The distribution of hydrophobic potentials at the Connolly surface of gomesin are presented on Fig. 5 . The lack of definition of the extremity of several side chains does not significantly modify the distribution of hydrophobic potential on the surface whatever the model chosen. Gomesin b sheet is amphipathic, its structure clearly displays (a) an hydrophobic face formed by a large aggregate of hydrophobic residues (Leu5, Tyr7, Val12, and T yr14) w hich are located on the concave surface of t he peptide; and (b) a second face showing a globally interme- diate potential through the presence of the two apolar disulfide bridges, the polar (Thr13) a nd the c harged (Arg4) side chains. T wo hydrophilic regions are located at the two spatial extremities of the molecule, at the C-te rminus with Arg16 and Arg18, and in the turn with the presence of Lys8, G ln9 and Arg10. Comparison to b-hairpin-like antimicrobial peptides with two disulfide bridges Gomesin shares several physico-chemical properties with most antimicrobial peptides adopting a b-hairpin-like structure with two disulfide bridges [2]. All of them have a molecular mass of % 2 kDa, including a rather high percentage of basic r esidues (over 30%). In addition, their three-dimensional structures a re stabilized by t he presence of two internal disulfide bridges in a parallel arrangement: Cys 1 –Cys 4 and Cys 2 –Cys 3 . Interestingly, they all have a broad s pectrum o f activity affecting the growth of various microorganisms as w ell as parasites. Sequence alignments reveal high similarities between gomesin and peptides belonging to the families of tachyplesins and polyphemusins from horseshoe crabs [34,35], to androctonin from scorpion [36], a nd to PG-1 from porcine leukocytes [37]. We have compared the three-dimensional structure of gomesin to androctonin a nd to protegrin (PG-1) w hich coordinates a re available in the Protein Data Bank. The three-dimensional structure o f a ndroctonin h as been determined recently in aqueous solution ([12], PDB code 1CZ6). The structure o f PG-1 has been studied in aqueous solution [9,10], in (CD 3 ) 2 SO [9] a s we ll as in the presence of micelles of dodecylphosphocholine [38]. Like gomesin, PG-1 contains 18 amino acids whereas androctonin i s s ignificantly l onger with 25 residues. Although the spacing o f the cysteine residues differs in gomesin, androctonin and PG-1, t he three molecules adopt a s imilar rigid pleated b sheet structure. The two pairs of cysteine re sidues a re separated by t hree re sidues in gomesin instead of only one in protegrin [37]. Androctonin presents an unequal number of residues on each s trand b etween the two bridges, fi ve in the N-terminal strand and t hree in the C-terminal strand. This leads to a higher t wist of the b sheet of androctonin compared to the two other peptides. Despite such differences, the rmsd of the coordinates of the b strands of the three peptides when superimposed on the backbone atoms N, Ca,C¢are very low, 0.85 A ˚ between gomesin and androctonin and 0.87 A ˚ between gomesin and prote- grin (1.25 A ˚ between protegrin and androctonin). On t he basis of this best-fit superposition, we were able to perform a structural alignment of the three molecules (Fig. 6) which differs slightly from the sequence alignment presented by Silva Jr. et al. [16]. The three structures are stabilized by two tight disulfide linkages and a regular pattern of backbone- backbone hydroge n bonds typical of antiparallel b strands (pGlu–Tyr7 and Arg10–Arg11 in gomesin vs. Leu5–Arg9 Fig. 4. R epresentations of the polypeptide bac kbone of go mesin and o f the central hydrophobic c luster. (A) stereoview of a superposition of the backbones of the 20 fin al structures. T he structures are b est fitted o n the N -Ca-C¢ atoms of the well-defined b shee t. (B) schematic representation of the overall fold with the b strands represented a s arrows. Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1195 and P he12–Val16 in PG-1; Arg5–Arg11 and Gly15–Thr21 in androctonin). As with gomesin, the b turn of PG-1 is locally well d efined and adopts an unclassified conforma- tion. Nevertheless, the conformations of the two turns a re different. In the c ase o f PG-1, it seems subjected to a r igid- group Ôhinge movementÕ relative to the b sheet [9,10] and can adopt different orientations with respect to the rigid remaining part of the molecule. In androctonin, the two strands of the b shee t are not c onnected by a b turn, but instead, the ch ain re versal is ensured by a fi ve membered - turn locally well defined. The structures of gomesin and androctonin are particularly w ell defined in the b sheet region. PG-1 shows a higher flexibility in water as pointed out by much larger rmsd (with respect to the average structure), 1.38 A ˚ and 0.8 A ˚ for the hairpin region in references [9,10], respectively, compared to 0.14 A ˚ for gomesin. Nevertheless, addition of (CD3) 2 SO reduces the flexibility of the PG-1 molecule [9]. Comparison of hydrophilic/hydrophobic properties on the molecule surfaces shows that gomesin and PG-1 structures share two highly hydrophilic and positively charged poles located in the N- and C-terminal regions and i n the turn (PG-1: Arg9, Arg10, A rg11; gomesin: L ys8, Gln9, A rg10) (Fig. 5). The turn of androctonin i nvolving three arginines is h ighly hydrophilic and positively c harged as well as its N-terminus (Arg1, Ser2). In contrast, t he doublet Pro24–Tyr25 gives a hydrophobic character to the C-terminus. A large difference concerns the d istribution of hydrophobic/hydrophilic potentials on the surface of the b sheet between the tails and the turn. The gomesin b sheet is divided into two nonequivalent faces: hydrophobic s ide chains are clustered on the concave face (Leu5, Tyr7, Val12 and Tyr14), whereas two polar side chains (Arg4, Thr13) flanked by the apo lar disulfide bridges are located o n the other face of gomesin. The central portion of PG-1 is particularly hydrophobic as it contains only a polar residues Leu5, Cys6, Tyr7, Cys8, Phe12, Cys13, Val14, Cys15 and Val16 alternatively distributed on each side of the b sheet [9,10]. In a ndroctonin, the highly t wisted character of t he b sheet does n ot suggest a clear dichotomy in the d istribu- tion of polar and apolar residues. The presence of three charged residues (Arg5, Lys8, and Lys19) distributed on each side of the s heet reduces considerably the hydropho- bicity of the surface of androctonin when compared to the two other peptides (Fig. 5 ). Mode of action The mode of action of the t hree peptides is not yet clearly understood. It has been established t hat androctonin a nd PG-1 interact with the bacterial membrane. Concerning androctonin, biochemical e xperiments have sh own t hat the peptide induces permeabilization of the c ytoplasmic m em- brane and interacts with negatively charged membranes in a monomeric form [39], suggesting a mode of action similar to a detergent effect. O n the basis o f N MR structures, s everal models of binding of PG-1 to the cellular membrane h ave been proposed, some possibly with an o ligomerization of Fig. 5. D istribution of hydrophobic potentials. Middle and right: orthographic view of the hydrophobic potentials at the connolly surfaces (radius 1.4 A ˚ ) of gomesin (top), protegrin (middle) and androctonin (bo ttom). Left: schematic representations of the p eptide backbones indicating the orientation in the left o rth ographic view pictures. Hyd rophobicity i ncreases from blue to brown while green is a colour halfway for intermediate potentials. Fig. 6. Structural alignment a nd schematic representation of gomesin, protegrin- 1 a nd an dr octo nin. (A) Structural alignment of the sequences. The alignment is obtained from the best-fitted three-dimensional superposition of the backbone atoms. The letters in italics and bold correspond to residues used for the best-fitted three-dimensional superposition. The zone ingrey indicates the b sheet strand limits for the three molecules. (B) Schematic representations of the three molecules. 1196 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the peptides [10]. These models are supported by recent NMR studies of the peptide in the presence of dodecyl- phosphocholine mice lles [38], s uggesting the formation of a dimeric structure and possibly of h igher o rder associations. Oriented C D studies on PG-1 are indicative of two possib le orientations of the peptide with respect to the membrane, depending on the peptide concentration, on the membrane components and on the hydration conditions [40]. This two- state model corresponds to (a) a functionally inactive binding state, when protegrin in low concentration tends to adsorb in the headgroup region of the membrane, leading to a decrease of the thickness of the lipid bilayer, and then to (b) an active state, when the peptide penetrates the hydrocarbon core of the bilayer leading to the disruption of the m embrane integrity, p robably through the formation of pores [40]. The similarities between the three-dimensional structure of g omesin and those o f PG-1 a nd androctonin suggest that gomesin e xerts antibacterial activity by inter- acting with the cytoplasmic bacterial membrane. Differences in t he distribution of hydrophilic and hydro- phobic residues at the surface of the three peptides may indicate different modes of action on the membrane. This may also a ccount for differences in the hemolytic activity of the peptide. I ndeed, it has been suggested that, when compared, peptides with a high content of hydrophobic residues are more hemolytic [41]. In this respect, the different levels of hemolytic activity in androctonin, gome- sin and protegrin could be linked to the difference of hydrophobicity of their central part. The prerequisite for antibacterial activity is still contro- versial. Over the last few years, a growing opinion argues that only the maintenance of the hydrophobic-hydro philic balance in those highly cationic peptides is the key point for activity. This viewpoint has to be t aken with cau tion; in some cases, as with tachyplesins, the presence of disulfide bridges leading to the formation of a w ell-folded amphi- pathic b sheet structure does not seem essential for activity [42]. For other peptides, such as protegrins, disulfide bridges would be necessary to ensure an antiparallel b sheet conformation leading to an active peptide [43]. In addition, protegrin analogues with particular amino-acid substitu- tions that eliminate hydrogen bonding across the b sheet have shown reduced activities [44]. To obtain a better understanding of the importance of disulfide bridges and the hydrophobic-hydrophilic balance on the antimicrobial activity of gomesin, synthetic analogues of this peptide t hat lack on e o r both cysteine d isulfides have been designed and are now being testing against s everal strains of microorgan- isms and euckaryotic cells. The first results obtained suggest that both disulfide bridges are important for the mainten- ance of the full biological a ctivity. Gomesin a nalogs with only one bridge or linear g omesin remain active b ut with a specificity towards particular microorganisms (S. Daffre, Departmento d e P arasitologia, ICB, Universidade d e Sa ˜ o, Paulo, Brazil, personal communication). The hydrophobic/ hydrophilic balance on the antimicrobial activity of gomesin is also investigated. In conclusi on, we h ave determined the three-dimensional structure of gomesin which adopts a well-defined b sheet structure like other open-ended c yclic peptides. Gomesin is active at low concentration (below 10 l M ) against a large number of bacterial and fungal strains. The presence of two disulfide bridges, C-terminal amidation as well as N-terminus cyclization tends to protect gomesin from proteolytic degradatio n. These properties, associated t o a rapid killing o f various bacterial and fungal strains a nd to a relatively low hemolytic activity [16], are enc ouraging for potential applications of gomesin as a therapeutic agent. In addition, gomesin constitutes a novel probe for further studies of the interaction between b sheet peptides and membranes, since most biochemical and biophysical s tudies have been done on a helical structures. A better under- standing of the action mode of these peptides is c rucial for the development of a new generation of antibiotics. ACKNOWLEDGEMENTS The authors thank Dr C. Landon for her helpful comments. This work was supported by C NRS , the University L ouis Pasteur o f Strasbourg. We are indebted t o Dr J P. Briand for gomesin synthesis (UPR 9 021 CNRS, IBMC S trasbourg France). REFERENCES 1. Ganz, T. (1999) Defensins and h ost defense. Science 286, 421–421. 2. Dimarcq, J L., Bulet, P., Hetr u, C. & Hoffmann, J.A. (1998) Cysteine-rich antimicrobial peptides in invertebrates. Biopolymers 47, 465 –477. 3. Cornet, B., Bonmatin, J.M., Hetru, C., Hoffmann, J.A., Ptak, M. & Vovelle, F. (1995) Refined three-dimensional solution structure of insect defensin A. Structur e 3, 435 –448. 4. Landon, C., So dano, P., Hetru, C., Hoffmann, J. & P tak, M. (1997) Solution structure o f drosomycin, th e first in ducible anti- fungal protein from insects. Protein Sci. 6, 1878–1884. 5. Lamberty, M ., Caille, A., Landon, C., Tass in-Moindrot, S., Hetru, C., Bulet, P. & Vovelle, F. (2001) Solution structures of the antifungal heliomicin and a selected variant with both anti- bacterial and antifun gal activities. Biochemistry 40, 11995–12003. 6. Fant, F., Vranken, W.F. & Borremans, F.A.M. (1999) The three- dimensional solution structure of Aesculus hippocastanum anti- microbial protein 1 d etermin ed b y 1 H nuclear magnetic r esonance. Proteins 37, 388–403. 7. Kawano, K., Yoneya, T., Miyata, T., Yoshikawa, K., Tokunaga, F., Terada, Y. & Iwanaga, S. (1990) Antimicrobial peptide, tachyplesin I, isolated from hemocytes of horseshoe crab (Tachyplesus t ridentatus). J. Biol. Chem. 265, 1 5365–15367. 8. Tamamura, H., Kuroda, M ., Ma suda, M., Ot aka, A ., Funa koshi , S., Nakashima, H., Yam amoto, N., Waki, M., Matsumoto, A., Lancelin, J M., K ohda, D., Tate, S ., Inaga ki, F . & Fujii, N . ( 1993) A c omparative study of the solution structures of tachy plesin I and a novel anti-HIV synthetic peptide, T 22 ([Tyr 5,12 ,Lys 7 ]-polyph- emusin II), determined by nu clear magnetic resonance. Biochim. Biophys. Ac ta 1163, 209–216. 9. Aumelas, A., Mangoni, M., Roumestand, C., Chiche, L., Desp- aux, E., G rassy, G ., Ca las, B. & Chavanieu, A. (1996) S ynthesis and solution s tructure o f the antimicrobial peptide prot egrin-1. Eur. J. Biochem. 237, 575–583. 10. Fahrner, R.L., Dieckmann, T., Harwig, S., Lehrer, R.I., Eisen- berg, D. & Fe igon, J. (1 996) Solution s tructure of p rotegrin-1, a broad-spectrum antimicrobial peptide f rom p orcine l eucocytes. Chem. Biol. 3, 543–550. 11. Mandard, N., Sodano, P., Labbe ´ , H., Bonmatin, J., Bulet, P., Hetru, C., Ptak, M. & V ovelle, F . (1998) Solution structure o f thanatin, a potent bactericidal and fungicidal insect peptide, determined from proton two-dimensional nuclear magnetic resonance data. Eur. J. Biochem. 256, 404– 410. 12. Mandard, N., Sy, D., Maufrais, C., Bonmatin, J M., Bulet, P., Hetru, C. & V ovelle, F. (1999) Androctonin, a novel antimicrobial peptide f rom scorpion And roctonus australis: s olutio n structure Ó FEBS 2002 Solution structure of gomesin (Eur. J. Biochem. 269) 1197 and molecular dynamics simulations in the presence of a lipid monolayer. J. Biomol. Struct. Dyn. 17, 3 67–380. 13. Hwang, P.M., Zhou, N., Shan, X., Arrowsmith, C.H. & Vogel, H.J. ( 1998) Three-dimensional s olution structure of lactoferricin B, an antimicrobial pep tide derived from bovine l actoferrin. Bio- chemistry 37, 428 8–4298. 14. Patel, S.U., Osborn, R., Rees, S. & T hornton, J.M. (1998) Structural studies o f Impatiens balsamina antimicrobial protein (Ib-AMP1). Biochemistry 37, 983–990. 15. Grishin, E. (1999) Polypeptide neurotoxins from spider venoms. Eur. J. Biochem. 264 , 276–280. 16. Silva, P.I.J., Daffre, S. & B ulet, P. (2000) Isolation and char- acterization of gomesin, an 18-residue cysteine-rich defense pep- tide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family. J. Biol. Chem. 275 , 33464–33470. 17. Piantini, U., Sorensen, O.W. & Ernst, R.R. (1982) Multiple quantum fi lters for elucidating NMR c oupling networks. J. Am. Chem. S oc. 104, 6 800–6801. 18. Griesinger, C., Otting, G., Wu ¨ thrich, K. & Ernst, R.R. (1988) Clean TOCSY for 1 Hspinsystemidentificationinmacro- molecules. J. Am. Chem. Soc. 110, 7870–7872. 19. Kumar, A., Ernst, R.R. & Wu ¨ thrich, K. (1980) A tw o-dimen- sional nuclear overhauser enhancement (2D NOE) experiment for the elucidation o f complete p roton -proton cro ss-relaxation net- works in biological macromolecules. Biochem. Biophys. Res. Commun. 95 ,1–6. 20. Bax, A. & D avis, D.G. (1985) MLEV-17-Based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355–360. 21. Piotto, M., Saudek, V. & S klenar, V. (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661–665. 22. Wu ¨ thrich, K. (1986) NMR of p roteins and nucleic acids. Wiley Interscience, New Y or k. 23. Bartels, C., Xia, T.E., B illeter, M., Gu ¨ ntert, P. & Wu ¨ thrich, K. (1995) The program XEASY for computer-supported NMR spectral analysis of biological macromolecules. J. Biomol. NMR 5, 1 –10. 24. Gu ¨ ntert, P., Mumenthaler, C. & Wu ¨ thrich, K. ( 1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283– 298. 25. Gu ¨ ntert, P., Mumenthaler, C. & Herrmann, T. (1998) DYANA - User’s ma nual. Version 1. ET H, Honggerberg, Zu ¨ rich. 26. Bru ¨ nger, A.T. (1992) The X-PLOR Software Manual. Version 3.1 Yale University, New Haven, CT, USA. 27. Brooks, B., Brucoli, R., Olafson, B.O., States, D ., Swaminathan, S. & Karplus, M. (1983) CH ARMM: a program for macromolecular energy minimization and dynamics calculations. J. Comp. Che m. 4, 1 87–217. 28. Viswanadhan, V.N., Ghose, A.K., R evankar, G.R. & Robins, R.K. (1989) Atomic physicochemical parameters for three dimensional structure directed quantitative structure–activity relationships. 4. Additional parameters for h ydrophobic and dispersive interactions and their application f or an automated superposition of certain naturally occuring nucleoside a ntibiotics. J. Che m. Inf. C omput. Sci. 29, 163–172. 29. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. (1993) PROCHECK: a program to check the stereochemical quality of p roteins structures. J. Ap pl. Crystallogr. 26, 283–291. 30. Hutchinson, E.G. & T hornton, J.M. (1996) PROMOTIF – a program to identify and analyse structural motifs i n proteins. Protein Sci. 5, 212–220. 31. Srinivasan, N., Sowdhamini, R ., Ramakrishnan, C. & Balaram, P. (1990) Conformations of disulfide bridges in proteins. Int. J. Peptide Prot. Res. 35 , 147–155. 32. Efimov, A.V. (1993) Standard structures in proteins. Prog. Biophys. M olec. Biol. 60 , 201–239. 33. MacArthur, M.W. & Thornton, J.M. (1993) Conformational analysis of protein structures d erived from NMR data. Prote ins 17, 232–251. 34. Nakamura, T., Furunaka, H., M iyata, T., Tokunaga, F., M uta, T., I wanaga, S., Niwa, M., Takao, T. & Shimonishi, Y. (1988) Tachyplesin, a c lass of antimicrobial peptide from th e hemocytes of the horseshoe crab Tachypleus tridentatus.Isolationand chemical structure. J. Biol. Chem. 263, 16709–16713. 35. Miyata, T ., Tokunaga, F., Yoneya, T., Yoshikawa, K ., Iwanaga, S., Niwa, M., Takao, T. & Shimonishi, Y. (1989) Antimicrobial peptides, i solated from horseshoe cra b hemocytes, ta chyplesin II, and polyphemusins I and II: chemical structures a nd biological activity. J. Biochem. 106, 6 63–668. 36. Ehret-Sabatier, L., L oew, D., Goyffon, M., Fehlbaum, P., Hoff- mann, J.A., van Dorsselaer, A. & Bulet, P. (1996) Characteriza- tion of no vel cysteine-rich antimicrobial peptides fro m scorpion blood. J. Biol. Chem. 271, 29537–29544. 37. Kokryakov, V.N., Harwig, S.S.L., Panyutich, E.A., S hevchenko, A.A., Aleshina, G.M., Shamova, O.V., Korneva, H.A. & Lehrer, R.I. (1 993) Protegrins: leuk ocyte antimicrobial p eptides that combine featu res of corticostatic defen sins and tachyplesins. FEBS Lett. 327, 231–236. 38. Roumestand, C., Louis, V., Aumelas, A., Grassy, G., Calas, B. & Chavanieu, A. (1998) Olig omerization of protegrin-1 in the pre- sence of DPC micelles. A proton high-resolution NMR study. FEBS Lett. 421, 263–267. 39. Hetru,C.,Letellier,L.,Oren,Z.,Hoffmann,J.A.&Shai,Y.(2000) Androctonin, a hydrophilic disulphide-bridged non-haemolytic antimicrobial peptide: a plausible mode of action. Bi ochem. J. 345 , 653–664. 40. Heller, W.T., Waring, A.J., Lehrer, R.I. & Huang, H.W. (1998) Multiples states of beta-sheet peptide protegrin i n lipid bilayers. Biochemistry 37 , 17331–17338. 41. Saberwal, G . & Nagaraj, R. ( 1994) Cell-lytic and antibacterial peptides that act by perturbing t he barrier function of membranes: facets of thei r c onformation al featu res, st ructure–function c o rre- lations and membr ane-perturbing facilities. Biochim. Biophys. Acta 1197, 109–131. 42. Rao, A.G . (1 99 9) Conformation an d antimicrobial activity of linear derivatives of t achyplesin lacking disulfide bonds. Arch. Biochem. Biop hys. 361, 1 27–134. 43. Harwig, S.S.L., Waring, A., Yang, H .J., Cho, Y., Tan, T . & Lehrer, R.I. (1996) Intramolecular disulfide bonds enhance the antimicrobial and lytic activities of protegrins at physio- logical sodium chloride concentrations. Eur. J. Biochem. 240, 352–357. 44. Chen, J ., Falla, T .J., Liu, H., H urst, M.A., Fujii, C.A., Mosca, D.A., E mbree, J.R., L oury, D.J., Radel, P.A., Cheng Chang, C., Gu,L.&Fiddes,J.C.(2000)Developmentofprotegrinsforthe treatment and prevention of oral mucositis: structure–activity relationships of s ynthetic protregrin analogues. Biopolymers 55, 88–98. 1198 N. Mandard et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . better understanding of the importance of disulfide bridges and the hydrophobic-hydrophilic balance on the antimicrobial activity of gomesin, synthetic analogues of. of the b sheet of androctonin compared to the two other peptides. Despite such differences, the rmsd of the coordinates of the b strands of the three peptides