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Protein Science (2003), 12:438-446.
Copyright © 2003 The Protein Society

Solution structure of termicin, an antimicrobial peptide from the termite Pseudacanthotermes spiniger

Pedro Da Silva1, Laurence Jouvensal1, Mireille Lamberty2, Philippe Bulet2,3, Anita Caille1 and Françoise Vovelle1

1 Centre de Biophysique Moléculaire, UPR 4301 CNRS affiliated at Orléans University, 45071 Orléans cedex 2, France
2 Institut de Biologie Moléculaire et Cellulaire, UPR 9022 CNRS, 67084 Strasbourg, France

Reprint requests to: Françoise Vovelle, Centre de Biophysique Moléculaire, UPR 4301 CNRS affiliated at Orléans University, rue Charles Sadron, 45071 Orléans Cedex 2, France; e-mail: vovelle{at}cnrs-orleans.fr; fax: 33 (0)2 38 63 15 17.

(RECEIVED August 14, 2002; FINAL REVISION November 22, 2002; ACCEPTED December 6, 2002)

3 Present address: Atheris Laboratories, Case Postale 314, CH-1233 Bernex Geneva, Switzerland. Back

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0228303.


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
The solution structure of termicin from hemocytes of the termite Pseudacanthotermes spiniger was determined by proton two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling techniques. Termicin is a cysteine-rich antifungal peptide also exhibiting a weak antibacterial activity. The global fold of termicin consists of an {alpha}-helical segment (Phe4–Gln14) and a two-stranded (Phe19–Asp25 and Gln28–Phe33) antiparallel ß-sheet forming a "cysteine stabilized {alpha}ß motif" (CS{alpha}ß) also found in antibacterial and antifungal defensins from insects and from plants. Interestingly, termicin shares more structural similarities with the antibacterial insect defensins and with MGD-1, a mussel defensin, than with the insect antifungal defensins such as drosomycin and heliomicin. These structural comparisons suggest that global fold alone does not explain the difference between antifungals and antibacterials. The antifungal properties of termicin may be related to its marked hydrophobicity and its amphipatic structure as compared to the antibacterial defensins. [SWISS-PROT accession number: Termicin (P82321); PDB accession number: 1MM0.]

Keywords: Termite; cysteine-rich; antimicrobial peptide; insect defensin; CS{alpha}ß motif; NMR

Abbreviations: 3D, three-dimensional • 1H, proton • 2D NMR, two-dimensional nuclear magnetic resonance • RMSD, root-mean-square deviation • ARIA, ambiguous restraints for iterative assignment • AMP, antimicrobial peptide • CS{alpha}ß motif, cysteine stabilized {alpha}ß motif


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
Efficient host defense mechanisms are required to counteract the microbial challenges to which living organisms are exposed. In higher vertebrates, there are two types of immunities: innate or natural immunity, and acquired or adaptive immunity. In contrast, invertebrates and plants defend themselves against pathogens exclusively through mechanisms that are part of innate immunity. Innate immunity is triggered immediately after microbial infection, and one mechanism is the production of antimicrobial compounds, including small antimicrobial peptides (AMP). In recent years, it has become widely recognized that AMPs are powerful defensive weapons against bacteria and/or fungi, viruses, or parasites in multicellular organisms (Zasloff 2002).

The diversity of AMPs (more than 700 online at http://www.bbcm.univ.trieste.it/~tossi/antimic.html) and the continuous discovery of new structures make their classification particularly difficult. However, on the grounds of their structural features, these AMPs can be classified into three emerging families (for reviews, see Bulet et al. 1999; Bevins and Diamond 2001; Ganz 2001; Zasloff 2002). One family corresponds to linear peptides that can form amphipatic {alpha}-helices in a proper environment (for review, see Oren and Shai 1998), the second to cyclic (open-ended or fully circular) peptides containing cysteine-residues engaged in at least one cysteine bond and adopting a ß-hairpin fold (for review, see Mandard et al. 2002), and the third to peptides with a preponderance of one or two amino acids (proline, arginine, glycine, or tryptophan) (for review, see Hwang and Vogel 1998).

In recent decades, a new family of cysteine rich peptides, the defensins, has been identified, and has emerged as the most widespread. Defensins are compact (3 to 5 kD) protease-resistant molecules containing three or four disulfide bridges. They have been isolated from invertebrates (Dimarcq et al. 1998), plants (Garcia-Olmedo et al. 1998), and vertebrates (Lehrer and Ganz 2002). Mammalian defensins ({alpha} and ß defensins) adopt a ß-sheet structure including a three-stranded antiparallel ß-sheet with an amphiphilic character, while the architecture of the group of invertebrate and plant defensins includes an {alpha}-helix and a ß-sheet linked by two disulfide bridges. Together, the structural elements of the latter group make up the so-called CS{alpha}ß motif, for cysteine-stabilized {alpha}-helix/ß-sheet motif (Kobayashi et al. 1991; Cornet et al. 1995). This motif is found in antibacterial and antifungal insect defensins (Hanzawa et al. 1990; Cornet et al. 1995; Landon et al. 1997, 2000; Lamberty et al. 2001a), and in the mussel defensin MGD-1 (Yang et al. 2000). Compared to the antibacterial defensins with a {alpha}ßß scaffold, the particularity of drosomycin (Landon et al. 1997, 2000) and heliomicin (Lamberty et al. 2001a), the two strictly antifungal defensins from insects, is the presence of a third strand in the ß-sheet, resulting in a ß{alpha}ßß scaffold. In addition, it has been pointed out that all antifungal defensins from insect and plants exhibit a common amphipathic character at the surface of the molecules (Lamberty et al. 2001a).

Recently, a novel cysteine-rich peptide, named termicin, has been isolated from the fungus-growing termite Pseudacanthotermes spiniger, a heterometabolous insect of the order of isoptera (Lamberty et al. 2001b). Termicin is not induced by bacterial challenge as observed in all insect species but is constitutively expressed. Although termicin does not show sequence similarities with drosomycin or heliomicin, its activity spectrum is closer to that of the antifungal defensins than to that of the antibacterial defensins. In fact, the peptide exhibits a potent antifungal activity but only weakly affects Gram-positive bacteria. This 36-residue peptide shows no obvious similarities with other AMPs except for the consensus motif of cysteine residues forming the disulfide scaffold characteristic of the CS{alpha}ß motif.

The past decades have witnessed a dramatic growth in knowledge of natural AMPs, and the goal now is to obtain as much information as possible regarding the structures of the molecules and their biologic properties. In an attempt to design strain specific AMPs, the first step is to understand the structural elements required for an antifungal activity or an antibacterial activity. In this study, we have determined the structure of termicin in aqueous solution using two-dimensional NMR spectroscopy and molecular modeling. The global fold involves an {alpha}-helix and a double-stranded ß-sheet. This solution structure was compared to drosomycin and heliomicin (the two known antifungal defensins from insects), to the antibacterial Phormia defensin A, and to MGD-1, the mussel antibacterial defensin. In contrast to its biologic properties (mainly antifungal), the global fold of termicin is reminiscent of the antibacterial defensins.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Electronic supplemental material
 References
 
NMR
Following the production of recombinant termicin in the yeast Saccharomyces cerevisiae (see the general procedure reported by Lamberty et al. 2001b), termicin was purified to homogeneity by reverse-phase HPLC and dissolved in 90% H2O:10% D2O for NMR experiments. Well-resolved spectra were obtained for the termicin sample in the conditions reported in the Materials and Methods section.

The proton resonances of termicin were assigned using sequential assignment methods proposed by Wüthrich (1986). Most spin systems of the individual amino acid residues were identified through DQF-COSY and TOCSY experiments on the maps recorded at 300 K. The DQF-COSY spectrum was particularly useful for assigning the protons of aliphatic and aromatic side chains; TOCSY and NOESY maps recorded at 286 K were used to resolve ambiguities in the assignment of several peaks. The proton resonances were quasi-totally assigned, except for {zeta}CH of phenylalanine residues (at positions 4, 15, 23, and 33), {delta}1NH of histidine 15, {eta}NH2 of arginine residues (at positions 20, 21, 26, and 35), and {zeta}NH2 of lysine 30.

Secondary structure
Deviations of the {alpha} proton chemical shifts from their statistical values in a random coil conformation were used to identify probable secondary structure elements of termicin (Wishart and Sykes 1994). The chemical shift deviations are reported in Figure 1Go. Positive deviations for the Ala22–Asp25 and Gln28–Val34 segments suggest the presence of a ß-sheet structure, while negative deviations for residues Phe4–Gln14 are indicative of a helical structure.



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  		Figure 1. H{alpha} chemical shift deviations with respect to a random coil structure for termicin: Negative and positive deviations are indicative of helical and ß-stranded structures, respectively. (Upper) Schematic representation of the secondary structures, cylinder for the {alpha}-helix, and arrows for the ß-sheet strands. (Lower) Empty circles indicate slowly exchanging amide protons.

 
These results are confirmed by the NOE connectivity diagram (Fig. 2Go) exhibiting d{alpha}N(i,i+4) peaks in the segment Phe4–Gln14, typical of an {alpha}-helix. Strong d{alpha}N(i,i+1) NOEs in the segments Phe19–Arg25 and Gln28–Phe33 suggest the presence of two extended strands of ß-sheet. Besides, sequential dNN(i,i+1) peaks are not observed in this region, which is typical of an extended conformation. This hypothesis is further validated by the presence of many long-range NOEs typical of a double stranded ß-sheet: long-range H{alpha}(i)-H{alpha}(j) connectivities between Phe19 and Phe33, Ala22 and Cys31, and Cys24 and Cys29; NH(i)–NH(j) peaks between Phe23 and Lys30, Asp25 and Gln28, on the one hand, and H{alpha}(i)–NH(j) connectivities between Ala22 and Val32, Cys31 and Phe23, and Cys25 and Lys30, on the other hand. Finally, dNN(i,i+2) and d{alpha}N(i,i+2) NOEs between the central residues (Asp25 to Gln28) indicate the presence of a turn in this region.



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  		Figure 2. NOE connectivity diagram: Sequential NH(i)-NH(i+1), H{alpha}(i)-NH(i+1), Hß(i)-NH(i+1), and medium range NH(i)-NH(i+2), H{alpha}(i)-NH(i+3), H{alpha}(i)-NH(i+4) NOE data of termicin.

 
When the freeze-dried sample of termicin was solubilized in deuterated water, the signals of 12 amide protons, namely Cys7, Trp8, Ala9, Thr10, Cys11, Gln12, Ala13, Phe23, Asp25, Ser27, Lys30, and Val32, were still observed after 24 h (Fig. 1Go). These 12 residues belong to the secondary structure elements defined above, strongly suggesting that their amide protons are involved in significant hydrogen bonds.

Structure evaluation
After eight ARIA iterations, the three-dimensional structure of termicin was refined by molecular dynamics in an explicit solvent environment. The final data file comprised a set of 796 distance restraints, among which 793 are nonambiguous, including 371 intraresidue, 184 sequential, 102 medium-range (2 < |i-j| < 5), 136 long-range (|i-j| >= 5) restraints (with an average of 22 restraints per residue), 26 dihedral angle restraints, and 12 restraints from main-chain hydrogen bonds. Long-range limits concern mainly residues located in the segments corresponding to the two strands of the ß-sheet (Phe19–Asp25; Gln28–Phe33). Finally, a family of 20 accepted 3D structures is in very good agreement with all the experimental data and the standard covalent geometry (Table 1Go): no experimental violation greater than 0.3 Å was observed, and the root-mean-square deviations (RMSD) with respect to the standard geometry are low. Both negative van der Waals and electrostatic energy terms are indicative of favorable nonbonded interactions. Moreover, the Ramachandran plot exhibits nearly 96% of the ({phi},{xi}) angles of the 20 converged structures in the most favored regions and additional allowed regions according to the PROCHECK software nomenclature. The residues in the disallowed region (3%) correspond to the ({phi},{xi}) of Arg26 involved in the {gamma}-turn.


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  		Table 1. Structural statistics for the 20 models of termicin
 
Structure description and hydrophobic potentials
The overall fold of termicin is formed by an {alpha}-helix (Phe4–Gln14) and two antiparallel ß-strands (Phe19–Asp25; Gln28–Phe33) forming a twisted ß-sheet. The long turn T1, which connects the {alpha}-helix to the first strand of the ß-sheet, corresponds to a noncanonical turn, whereas the sequence (Asp25–Ser27), linking the two strands of the ß-sheet, forms a {gamma}-turn. The {alpha}-helix and the second strand of the ß-sheet (Gln28–Phe33) are cross linked by Cys7–Cys29 and Cys11–Cys31 disulfide bridges localized in the core of the termicin structure. This arrangement constitutes the typical CS{alpha}ß motif. In addition to this motif, the 3D structure is further stabilized by a third disulfide bridge linking Cys2 to Cys24 and connecting the N-terminal segment to the C-terminal part of the first strand of the ß-sheet. As evidenced by the structural statistics (Table 1Go) and by a superposition of the backbone coordinates for the 20 converged structures (Fig. 3Go), the structure of termicin is particularly well defined. The pairwise RMS deviations on the N, C{alpha}, C' backbone atoms of residues 1 to 36 are 0.59 Å (on all heavy atoms: 1.18 Å) and drop to 0.26 Å (on all heavy atoms: 0.93 Å) when calculated on the secondary structure elements. The presence of hydrogen bonds typical of the secondary structures are observed on all 20 models and confirmed by the 1H-2H exchange experiments. There are seven regular backbone–backbone hydrogen bonds characteristic of the {alpha}-helix structure: O(Asn3)–HN(Cys7), O(Phe4)–HN(Trp8), O(Gln5)–HN(Ala9), O(Ser6)–HN(Thr10), O(Cys7)–HN(Cys11), O(Trp8)–HN(Gln12), O(Ala9)–HN(Ala13), and four regular backbone–backbone hydrogen bonds characteristic of the ß-sheet: O(Arg21)–NH(Val32), HN(Phe23)–O(Lys30), O(Phe23)–HN(Lys30), HN(Asp25)–O(Gln28). Finally, the hydrogen bond HN(Ser27)–O(Asp25) suggested by proton exchange experiments, which contributes to the stabilization of the {gamma}-turn, is present on the 20 structures.



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  		Figure 3. The NMR structure of termicin. (A) Orthographic view of a best-fit superposition of the backbones of all 20 termicin structures. (B) Ribbon representation of the lowest energy model of the termicin solution structures. These figures were generated by the programs SYBYL and MOLSCRIPT, respectively.

 
The distribution of the hydrophobic potentials at the accessible surface of termicin presented in Figure 4AGo is characteristic of an amphipathic structure. Termicin structure clearly displays a hydrophobic face formed by a large aggregate of hydrophobic residues (Ile17, Tyr18, Val32, Phe33, and Val34) and a hydrophilic one, including three charged residues (Asp25, Arg26, and Lys30).



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  		Figure 4. Lipophilic map of termicin and best-fit superposition of the global folds and lipophilic potentials of various antimicrobial peptides. (A) Left: Lipophilic potentials calculated with the MOLCAD option of SYBYL at the accessible surface of termicin; hydrophobic and hydrophilic areas are displayed in brown and in blue, respectively. Green surfaces represent an intermediate hydrophobicity. Right: Tube representation of termicin global fold showing the hydrophobic (orange) and hydrophilic (blue) side chains; disulfide bridges are displayed in yellow. (B) Best-fit superposition of the global folds and lipophilic potentials of (a) termicin (yellow), (b) drosomycin (red), (c) heliomicin (green), (d) Phormia defensin A (magenta), (e) MGD-1 (blue). The best fit was obtained for the backbone heavy atoms corresponding to the common secondary structure elements.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Electronic supplemental material
 References
 
As expected from the disulfide bridges array, the global fold of termicin includes the CS{alpha}ß motif observed in the defensins isolated from cell-free hemolymph of insects (Cornet et al. 1995), from mollusks (Yang et al. 2000) and from plants (Bruix et al. 1993). This motif, initially reported in scorpion toxins (Bontems et al. 1991), has also been found in a sweet-tasting protein from fruit (Caldwell et al. 1998). This simple motif constitutes a structural platform on which different kinds of biologic activities can be grafted, depending on the nature and on the distribution of the residues in the structure. One of our challenges is to determine the optimal arrangement of residues accounting for a specific activity, antibacterial versus antifungal. Most of the AMPs of known structure adopting the CS{alpha}ß motif reveal some selectivity towards micro-organisms. For example, sapecin and Phormia defensin A (Matsuyama and Natori 1988; Lambert et al. 1989) were found to be active against Gram-positive bacteria at micromolar concentration, while high concentrations are required to affect the growth of a limited number of Gram-negative bacteria and fungi. The mussel defensin MGD-1 is active against Gram-positive bacteria, and several Gram-negative strains at a concentration close to the micromolar but is not active on the fungus N. crassa (P. Bulet, unpubl.). In contrast, termicin possesses a marked activity against filamentous fungi and yeast strains (MICs <10 µM) and, at a higher concentration (MICs >50 µM), also affects the growth of some Gram-positive bacteria but is not effective on Gram-negative bacteria (Lamberty et al. 2001b). As opposed to these molecules with an extended activity spectrum, drosomycin and heliomicin are purely antifungal, sharing analogous activity spectra. Previous studies revealed that heliomicin and drosomycin present a very similar scaffold (Lamberty et al. 2001a). A comparison of the structural features of these two peptides and of the antifungal plant defensin Rs-AFP1 evidences similarities in the distribution of hydrophobic potentials that may account for their common antifungal activity.

When the properties of termicin are compared to those of other AMPs, it is evident that the termite molecule is on the borderline between antifungal defensins and antibacterial defensins. The first noticeable difference between termicin structure ({alpha}ßß topology) and antifungal defensins structures (ß{alpha}ßß topology) is the absence of the first strand of the ß-sheet together with the loop linking this strand to the {alpha}-helix. The {alpha}-helix of termicin starts readily at Phe4, so that termicin constitutes a minimal representation of proteins with the CS{alpha}ß motif. This makes the global fold ({alpha}ßß) of termicin closer to that of the defensins from primitive arthropods (Bulet et al. 1992; Cociancich et al. 1993; Charlet et al. 1996; Ehret-Sabatier et al. 1996; Hubert et al. 1996) and of the mollusk defensin MGD-1 (Yang et al. 2000), which are preferentially active against Gram positive bacteria. A second feature makes termicin closer to these defensins: like MGD-1 the peptide is constitutively expressed in the saliva and in blood cells, and is released into the termite hemolymph exclusively after an immune-challenge (Mitta et al. 1999; Lamberty et al. 2001b). This contrasts with AMPs from evolutionary more recent insects, which are induced upon a septic injury in the fat body, and released into the hemolymph (for review, see Bulet et al. 1999).

Interestingly, the CS{alpha}ß motif in termicin is very close to that observed for the peptides depicted in Figure 4BGo. The RMS deviations between the backbone atoms of the CS{alpha}ß secondary structure elements of termicin, drosomycin, heliomicin, MGD-1, and Phormia defensin A are rather low, ranging between 0.66 and 1.22 Å (Table 2Go). A detailed analysis of the motifs reveals dissimilarities in the lengths of the secondary structure elements. Compared to the other molecules, termicin has a long {alpha}-helix linked to the first strand of the ß-sheet by a long-turn T1 (four residues versus three) (Fig. 3Go). The turn T2, linking the two strands of the ß-sheet, is very short (two residues only), while the two strands, involving six residues each, are the longest of the series. The special length of turn T1 is due to the presence of cumbersome residues (His15, Ile17, Tyr18, Phe19, Arg20, and Arg21), whereas in most defensin sequences, the Arg–Arg dipeptide is replaced by a doublet of glycines favoring the packing of the strand of the ß-sheet on the helix (Fig. 3Go). Previous studies showed that antifungal defensins display a common distribution of hydrophobic residues at their surfaces. Examination of the lipophilic potential at the accessible surface of termicin evidences that, like drosomycin and heliomicin (Fig. 4BGo), termicin displays an amphiphilic character. However, in the 3D structure, the positions of the hydrophilic and hydrophobic zones are opposite to those found for heliomicin and drosomycin. As a matter of fact, hydrophobic and aromatic residues are mainly concentrated in the T1 turn and at the C-terminus, while in heliomicin and drosomycin they are found at the pole constituted by the turn T2 and by the long loop L1 linking the first strand of the ß-sheet to the {alpha}-helix (Lamberty et al. 2001a) (Fig. 4BGo). Also, relying on sequence comparisons between heliomicin and antibacterial defensins, a mutant of heliomicin (Hel-LL) has been designed in which two basic residues (Lys23 and Arg 34) were mutated into leucines, conferring a neutral character to the molecule. Hel-LL shows, compared to heliomicin, a reduction of its antifungal activity in favor of its antibacterial activity, and does not exhibit an obvious amphipathy (Lamberty et al. 2001a). The distribution of hydrophobic residues is more patchy and rather similar to that observed for Phormia defensin A. Interestingly, defensin A and MGD-1, showing (as far as we know for MGD-1) no antifungal activity, do not display such a distribution of lypophilic potentials, and are on the whole more hydrophilic than termicin. This result confirms the importance of the amphipathic character in defining the antifungal properties of a peptide. However, it is rather disturbing to notice that the distribution of hydrophobic and hydrophilic residues on the CS{alpha}ß motif does not really matter.


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  		Table 2. RMS deviations between the backbone atoms of the CS{alpha}ß secondary structure elements for termicin, drosomycin, heliomicin, Phormia defensin A, and MGD-1
 
Drosomycin shares with several morphogenic plant defensins like Rs-AFP2 a potential interaction site with an unknown fungal receptor, including a positively charged lysine residue embedded in a hydrophobic cluster (Landon et al. 2000). Even though the hydrophobic cluster is conserved, this site is not found in the heliomicin structure (Lamberty et al. 2001a), the lysine being replaced by an asparagine. Termicin, like both antifungal molecules, behaves as a morphogenic defensin, reducing the hyphal elongation but increasing the hyphal branching (Banzet et al. 2002). On the termicin structure, a possible interacting site is suspected, which involves the side chains of Arg20 and Arg21 clustered in the hydrophobic core formed by Phe4, Trp8, Phe19, Phe23, Val32, and Val34, but the location of this site on the 3D structure is also opposite to that found for drosomycin. Nevertheless, such a hypothesis has to be validated by mutational experiments. So far, nothing is clearly demonstrated about the mode of action of these antifungal peptides. Even if the amphipathy of the molecule is in favor of a detergent-like mode of action, the specificity of the peptides for various fungal strains and the variety of effects on fungal hyphaes support the existence of a second step in the mode of action presumably, involving a more specific target at the surface of the fungal membranes (Thevissen et al. 1996). The arrangement of hydrophobic residues at the surface of the peptide is probably essential for a correct positioning of the peptide, allowing favorable interactions with its receptor.

Concerning the weak antimicrobial activity against Gram-positive strains, it has been shown (Yang et al. 2000) that MGD-1 and Phormia defensin A share rather similar electrostatic properties, with a number of Arg and Lys residues distributed at the surfaces of the molecules. MGD-1 (five arginines and one lysine), is more basic than Phormia defensin A (three arginines and one lysine), and displays a wider activity spectrum because it shows efficacy against Streptococcus aureus at a low concentration (0.6 µM) and also affects the growth of some Gram-negative strains. Despite a distribution of basic residues (four arginines and one lysine) close to that found for Phormia defensin A and MGD-1, termicin is only weakly active against the Gram-positive strain Micrococus luteus. There is no doubt that the activity spectra of defensins depend in an acute manner on the balance between hydrophobic and hydrophilic and/or charged residues. The number of known antibacterial defensins is not yet sufficient to draw precise structure–activity relationships. However, from this study it appears that the features that make a molecule active against fungi (an amphipathic structure) are not fully compatible with the features conferring an antibacterial activity to the molecule (hydrophilic character with hydrophobic patches). This may explain why insect defensins and defensins from other arthropods are so specific. Among arthropod defensins of known 3D structures, termicin is a particular molecule. Its activity spectrum is close to that of heliomicin and drosomycin but its structure, including only the CS{alpha}ß motif with a minimal N-terminal extension formed by three residues, seems less distant from that of antibacterial defensins.

In the future, the particular amino acids required to have a specific activity against a precise strain of fungus or bacterium can be explored by experiments using point mutagenesis or DNA shuffling strategies (Raillard et al. 2001; Joern et al. 2002). In vitro recombination of homologous synthetic genes coding AMPs may represent a particularly interesting strategy to investigate the potential use of the invertebrate AMPs as therapeutic agents. This may be particularly relevant when the number of individuals belonging to the same family of AMPs is high, which is the case for the defensin family.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Electronic supplemental material
 References
 
Nuclear magnetic resonance experiments
Sample preparation
Recombinant termicin from P. spiniger was produced by a strain of the yeast Saccharomyces cerevisiae and purified to homogeneity according to procedures described previously (Lamberty et al. 2001b). The sample used for NMR spectroscopy was prepared by dissolving 5 mg of recombinant termicin powder in 600 µL 90% H2O:10% D2O, resulting in a final concentration of 2 mM. The pH of the peptide solution was adjusted to 3.9 with minute increments of HCl 1 N to observe the amide protons. For D2O experiments, the sample was lyophilized and redissolved in 99.9% D2O.

NMR spectroscopy
All 1H-NMR spectra were recorded on a VARIAN INOVA NMR spectrometer equipped with a z-axis field-gradient unit and operating at a proton frequency of 600 MHz. All NMR data sets were processed on a Silicon Graphics Indy O2 workstation using the VNMR software package (version 6.1; Varian, Inc., Palo Alto, CA). A 1H DQF-COSY spectrum (Piantini et al. 1982), a clean-TOCSY spectrum with a spin lock of 80 msec produced by the MLEV-17 sequence (Bax and Davis 1985; Griesinger et al. 1988), and NOESY spectra collected at mixing times of 120 and 300 msec (Jeener et al. 1979), were acquired with a spectral width of 7.5 kHz in each dimension at a temperature of 300 K. For spectra recorded in H2O, the water resonance suppression was achieved either by low-power irradiation for DQF-COSY experiments, or using the WATERGATE method (Piotto et al. 1992) for TOCSY and NOESY experiments. 1H DQF-COSY, TOCSY, and NOESY spectra were also recorded in D2O at 300 K. Moreover, in an attempt to assign ambiguous peaks due to spectral overlap, 1H-NMR spectra, including clean-TOCSY and NOESY spectra, were obtained in H2O at 286 K as well. All spectra were referred to the residual H2O signal set as the carrier frequency, 4.888 ppm at 286 K and 4.754 ppm at 300 K. 1H DQF-COSY, TOCSY, and NOESY spectra were assigned according to classical procedures including spin-system identification, sequential assignment, and secondary structure identification. To identify slowly exchanging amide protons in termicin, 1H-2H exchanging experiments were performed by dissolving the freeze-dried H2O sample in D2O and recording 1D and 2D spectra at 286 K. The amide protons that were not exchanged 24 h after dissolution were identified as slowly exchanging protons and interpreted either as hydrogen-bond donors or as protons not accessible to the solvent.

Structure calculations
The cross peak intensities on the NOESY spectrum recorded with 120 msec mixing time were integrated and partially assigned within the XEASY software (Bartels et al. 1995). These NOEs, along with a table of chemical shifts, were used as input to ARIA 1.1 (Linge et al. 2001) implemented in the software CNS 1.1 (Brünger et al. 1998). In addition, 26 {phi} dihedral angle restraints, deduced from 3JNH-H{alpha} coupling constants determined with the INFIT program (Szyperski et al. 1991), were added to the input data base. These {phi} angles were restrained to -60 ± 30° for 3JNH-H{alpha} < 5 Hz; -60 ± 40° for 5 <= 3JNH—H{alpha} <=6 Hz; -120 ± 30° for 8 <= 3JNH—H{alpha} <= 9 Hz; and -120 ± 20° for 3JNH-H{alpha} > 9 Hz. Because the disulfide pairing of termicin has been determined by mass spectroscopy (Lamberty et al. 2001b), and further confirmed unequivocally on the basis of interesidual NOE connectivities for the three disulfide bridges, the following distance restraints between two cysteine residues involved in the linkage were added for C2–C24, C7–C29, and C11–C31 disulfide pairs: 2.0 Å < d(S{gamma}i,S{gamma}j) < 2.1 Å; 3.0 Å < d(Cßi,S{gamma}j) < 3.1 Å; 3.0 Å <= d(S{gamma}i,Cßj) < 3.1 Å. Moreover, hydrogen bond restraints were imposed between the NH and C=O groups for pairs of residues involved in secondary structure elements when the amide proton was not completely exchanged with D2O after 24 h. For each hydrogen bond, the HN—O upper limit restraint was set to 2.4 Å and the lower limit to 1.6 Å. A large number of NOEs were calibrated and assigned automatically during the structure calculation by ARIA (Linge et al. 2001), a method combining an iterative NOE interpretation scheme with a dynamical assignment of ambiguous NOE cross peaks. This is completed by treating ambiguous NOEs as the sum of contributions from all possible assignments. This procedure of assignment/refinement is repeated iteratively eight times.

In this study, after the eight cycles of structure calculations, ARIA generated a set of 796 nonredundant data. Nine restraints that were systematically violated in the calculated structures of lowest energy were removed. These excluded peaks, randomly distributed along the sequence, were carefully checked and their presence in the 120-msec NOESY spectrum was examined manually. These signals of weak intensities may be due to noise. In the final iteration, 100 structures were calculated using this set of data, from which the best fifty were further refined in a shell of water to remove the artefacts due to the simplified representation of the force field in the simulated annealing steps (Nilges et al. 1997). Finally, a set of 20 structures with the minimum number of residual violations on distance restraints were considered as representative of the solution structure of termicin. These 20 structures were displayed with the SYBYL software (TRIPOS Inc.) and used for further analysis with PROCHECK (Laskowski et al. 1996) and PROMOTIF (Hutchinson and Thornton 1996) programs. Hydrophobic potentials were calculated using the MOLCAD option (Viswanadhan et al. 1989) implemented in SYBYL.


   Electronic supplemental material
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
References
 
1H chemical shifts (ppm) table of termicin.


   Acknowledgments
 
This work was financially supported by the Centre National de la Recherche Scientifique, the University Louis Pasteur, and EntoMed SA.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.


   References
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Electronic supplemental material
 References
 
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