Molecular insight into mechanism of antimicrobial action of the β-hairpin peptide arenicin: Specific oligomerization in detergent micelles

Abstract

Arenicins are 21-residue cationic antimicrobial peptides isolated from marine polychaeta Arenicola marina. The peptides exhibit potent broad-spectrum antimicrobial activity. In water solution arenicin-2 adopts a β-hairpin conformation, stabilized by one disulfide and nine hydrogen bonds. To determine the propensity for the peptide oligomerization in membrane mimetic systems, the recombinant arenicin-2 was overexpressed as a fused form in Escherichia coli. The arenicin-2 oligomerization and intermolecular packing in membrane mimicking environment were investigated using high-resolution NMR spectroscopy. The present studies show that arenicin-2 preserves a β-hairpin structure and forms asymmetric dimers upon incorporation into the dodecylphosphocholine micelle. Two monomers of arenicin-2 are aligned parallel to each other by the N-terminal strands of the β-hairpin (CN↑↑NC type of association). Polyacrylamide gel electrophoresis analysis indicated that in environment of anionic SDS micelles the arenicin-2 might undergo further oligomerization and form tetramers. Our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins. © 2007 Wiley Periodicals, Inc. Biopolymers 89: 455–464, 2008.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at [e-mail]

INTRODUCTION

Naturally occurring antimicrobial peptides (AMPs) were discovered in a wide range of organisms. Invertebrate animals, living without adaptive immunity in a microbe laden environment, produce AMPs as important evolutionary conserved component of their innate immune system, which play a key role in the host defense against bacterial, fungal, and viral invasion.1, 2 In marine invertebrates, a battery of AMPs were found in Spongia, Cnidaria, Annelida, Mollusca, Chelicerata, Crustacea, and Tunicata, for example, aurelin from jellyfish,³ myticin from mussels,4 tachyplesins and polyphemusins from horseshoe crabs,5, 6 penaeidins from shrimp,7 clavanins and styelins from ascidians.8–10

Earlier we discovered two novel 21-residue AMPs, termed arenicins, from coelomocytes of marine polychaeta lugworm Arenicola marina.11 Both arenicin-1 (RWCVYAYVRVRGVLVRYRRCW, 2758.3 Da) and arenicin-2 (RWCVYAYVRIRGVLVRYRRCW, 2772.3 Da) exhibit potent activity against Gram-positive and Gram-negative bacteria and fungi. Each isoform has one disulfide bond (Cys3-Cys20) forming a 18-residue ring. Our previous investigation of the spatial structure of arenicin-2 in aqueous solution by nuclear magnetic resonance (NMR) spectroscopy has shown that the peptide represents a prolonged β-hairpin formed by two β-strands (Cys3-Ile10 and Val13-Cys20) and Type I′ β-turn (Arg11-Gly12).12 The β-hairpin structure is stabilized by the disulfide bond, nine backbone-backbone hydrogen bonds and by the electrostatic π-cation interactions between aromatic and positively charged residues.12 The two-stranded β-sheet in arenicin-2 structure has significant right-handed twist in aqueous solution. This distortion effectively shields the hydrophobic side of the β-sheet from contacts with polar solvent and deprives the peptide surface of amphipathicity. Nevertheless, the peptide strongly binds to detergent micelles and anionic lipid vesicles (palmitoyl-oleoyl-phosphatidylethanolamine/palmitoyl-oleoyl-phosphatidylglycerol (POPE/POPG)) that mimic bacterial membrane. On the basis of circular dichroism (CD) investigation12 it was proposed that arenicin-2 binding to the micelleuntwists the β-hairpin and induces additional amphipathicity on the peptide surface, allowing energetically favorable contacts of the cationic and hydrophobic side chains of arenicin with polar head groups and hydrophobic interior of lipid membrane. The observed inability of arenicin-2 to bind to the neutral bilayers (palmitoyl-oleoyl-phosphatidylcholine (POPC)) might underlie the peptide selectivity toward bacterial cells.12

Arenicin has efficient and broad-spectrum antimicrobial activity.11 Yet, molecular mechanism of the peptide selective interaction with the cell membranes of the target microorganisms is obscure. Molecular details of the peptide–lipid interactions, disruption, and/or translocation across target membranes generate considerable interest. Since arenicin activity might be modulated by the peptide self-association within lipid bilayers, the present study is focused on finding evidence of the peptide oligomerization in lipid-mimicking environment, on understanding the detailed membrane-bound dimer structure of the peptide, and on gaining molecular insight into the peptide pore formation within the membrane. The results presented here is a step towards understanding the mechanism of cationic β-hairpin AMPs action.

RESULTS

Arenicin-2 Expression and Purification

E. coli BL-21 (DE3) cells transformed with the recombinant plasmid pET-His8-TrxL-Ar2, containing arenicin-2 sequence fused with thioredoxin A as a carrier protein, and induced by IPTG demonstrated satisfactory level of expression of the fusion protein, constituting up to 20–25% of the total cell protein (Figure 1). The strain allowed to produce about 5 mg of the recombinant arenicin-2 per liter of the cell culture. Decreasing the induction temperature down to 25–30°C resulted in several-fold multiplication of the expression level of the arenicin-containing fusion protein. Changing of other process variables such as inductor concentration, induction time, glucose concentration did not show much efficacy.

Figure 1. Expression and IMAC purification of arenicin-2 containing fusion protein (SDS-PAGE). 1 – Total cell proteins 5 h after IPTG induction, 2 – Ni-NTA flow-through, 3 – proteins eluted from Ni-NTA column.

Purification of the solubilized inclusion bodies using immobilized metal affinity chromatography (IMAC) provided two-fold enrichment of the sample with the fusion protein (up to 40–50% of the total protein). Efficiency of the fusion protein cleavage with CNBr constituted 60–70%. Fine purification of the cleavage products was performed using reverse phase high performance liquid chromatography (RP-HPLC) (Figure 2). The parameters of the recombinant arenicin-2 elution (retention time 33–34 min, acetonitrile concentration 21.5–22.0%) matched those of the native and synthetic analogues under the same conditions. MS analysis of the major HPLC peak demonstrated the presence of a peptide with M.m. of 2772.0 Da (Figure 3A) corresponding to the mature arenicin-2. The minor satellite HPLC peak contained a peptide with M.m.of 2788.1 Da (Figure 3B) which might represent an oxidized (most probably, at tryptophan residue) arenicin-2. Subsequent experiments showed that arenicin molecule spontaneously formed its native conformation. This property of the peptide allowed us to produce its fully active recombinant analogue without introducing the refolding step into the downstream process.

Figure 2. RP-HPLC purification of the recombinant arenicin-2. 1 – The supposed oxidized form of the peptide, 2 – the recombinant arenicin-2 in the cyclic form.

Figure 3. MALDI-TOF MS analysis of RP-HPLC fractions. (A) Fraction containing the recombinant arenicin-2 in the cyclic form (m/z 2772.0). (B) Fraction containing the supposed oxidized form of arenicin-2 (m/z 2788.1). Besides the main peaks the double-charged molecular ions are also seen.

Characteristics of Recombinant Arenicin-2

The recombinant peptide homogeneity and its identity to natural arenicin-2 were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), MALDI-TOF mass-spectrometry, automated microsequencing, and antimicrobial assay. It was shown that the recombinant arenicin-2 was identical to the natural peptide in respect of its molecular mass, amino acid sequence, and antimicrobial activity.

Oligomerization of Arenicin-2 in SDS

SDS-PAGE of the recombinant arenicin-2 showed some unexpected patterns when conducted with insufficient concentrations of the reducing agent (2-mercaptoethanol) in a sample buffer or without proper heating of the mixture before PAGE. Along with the distinct ∼3 kDa band, in some experiments an additional smeared band of 10–15 kDa with an approximate “maximum” of 12 kDa was observed (Figure 4A). In the absence of 2-mercaptoethanol the “12 kDa form” dominated (Figure 4B), but sometimes a faint band around 6 kDa was visible (Figure 4C). Notably, the last one was observed when an aliquot of the sample, previously used for NMR studies and containing dodecylphosphocholine (DPC), was applied to SDS-PAGE. It was possible to partially transform the “12 kDa form” to monomer “3 kDa form” without the reducing agent in the sample, but at extremely harsh conditions (boiling in >1% SDS and 9M urea for 20–30 min). Addition of an excess of 2-mercaptoethanol allowed to obtain a single 3 kDa band (Figure 4D). The AU-PAGE in nonreducing conditions demonstrated two main bands (Figure 4E). The SDS-PAGE examination allowed us to hypothesize that the ∼6 and ∼12 kDa bands were formed correspondingly by the peptide dimer and tetramer, stabilized by noncovalent intermolecular interactions. MALDI-TOF mass-spectrometry of the peptide sample prepared at the temperature and pH values mimicking the SDS-PAGE conditions, revealed the molecular mass corresponding to the monomer of arenicin, thus confirming the noncovalent nature of interactions that stabilize arenicin-2 oligomers. In sum, it seems that arenicin molecule has a definite oligomerization tendency.

Figure 4. Electrophoretic analysis of the purified arenicin-2. (A) SDS-PAGE of the partially reduced peptide; 1 – M.m. standard, 2 – recombinant arenicin, 3 – synthetic arenicin. (B) SDS-PAGE of nonreduced samples; 1 – M.m. standard, 2 – recombinant arenicin, 3 – synthetic arenicin. (C) SDS-PAGE of DPC-containing sample; 1 – M.m. standard, 2 – recombinant arenicin. (D) SDS-PAGE of the fully reduced peptide; 1 – M.m. standard, 2 – recombinant arenicin. (E) Acid-urea PAGE of the nonreduced sample; 1 – cytochrome c, 2 – recombinant arenicin.

Formation of the Arenicin-2/DPC Micelle Complex

The spatial structure of arenicin-2 in aqueous solution was determined in our previous NMR investigation.12 The peptide was described as a highly twisted β-hairpin without pronounced amphipathicity on its surface. The significant changes in arenicin-2 secondary structure were observed by CD spectroscopy upon incorporation of the peptide into the detergents micelles (DPC or SDS).12 The present observation of the oligomeric states of arenicin-2 by SDS-PAGE indicates that these changes are coupled with oligomerization of the peptide in membrane mimicking environment. To investigate the detailed structure of the arenicin-2 oligomers by NMR spectroscopy, we decided to choose DPC as a mild and more convenient detergent.13 Besides, taking into account the similarity of the CD spectra of arenicin-2 in SDS and DPC micelles,12 it was assumed that oligomerization of the peptide also takes place in DPC micelles.

Formation of the complex between arenicin-2 and DPC micelle was studied using diffusion measurements and 1D NMR spectroscopy at 30°C. 1D NMR spectra of the peptide were dramatically affected under variation of detergent/peptide molar ratio (D:P). Behavior of the signals belonging to H^ε1 protons of Trp2 and Trp21 is illustrated in Figure 5A. Upon increase of D:P, amplitude of the signals belonging to the free peptide was gradually decreased and additional broad signals corresponding to arenicin-2/DPC complexes appeared. The line-widths, positions, and multiplicities of these signals depended upon the detergent concentration in the sample, thus indicating the presence of exchange between several arenicin-2 conformations or formation of arenicin-2 oligomers within the DPC micelles. Finally, at D:P of 80:1 the arenicin-2/DPC complex spectrum contained four signals for two H^ε1 protons of Trp2 and Trp21, and further increase of DPC concentration to 100:1 did not change appearance of the spectrum (Figure 5A). Probably, a stable oligomer of arenicin-2 was formed in DPC solution at the detergent concentration of 80:1. The basic building block of this oligomer could be a dimer with slightly different environments for all four Trp residues (Trp2A, Trp21A, Trp2B, and Trp21B).

Figure 5. Titration of 1.0 mM arenicin-2 with DPC, H₂O (10% D₂O), pH 3.3, 30°C. (A) Fragment of 1D ¹H NMR spectrum with signals from indole H^ε1 protons of Trp2 and Trp21 at different DPC/arenicin-2 molar ratio. (B) Translation diffusion coefficient D_T of arenicin-2 molecule is shown versus DPC/arenicin-2 molar ratio.

The translational diffusion coefficient of arenicin-2 (D_T) also steeply went down upon rising the detergent content in the sample (Figure 5B) indicating formation of arenicin-2/DPC complexes and the possible peptide oligomerization. The initial fast decay was ended at D:P of 40:1, and subsequent addition of DPC did not significantly influence the D_T value. To estimate the stoichiometry of the arenicin-2/DPC complex at the D:P of 80:1, the hydrodynamic Stokes radius (R_H) was calculated from the measured D_T (0.82 ± 0.03 × 10⁻¹⁰ m²/s). The obtained R_H value (33.8 ± 1.2 Å) corresponds to the mixed arenicin-2/DPC micelle with molecular weight 67 ± 7 kDa, that in turn fits well to the arenicin-2 dimer (∼6 kDa) in complex with 160 DPC molecules (∼62 kDa). Thus, the diffusion data are also in agreement with formation of the arenicin-2 dimer in DPC micelle solution with detergent concentration above certain threshold (80:1).

Complexity of the peptide spectra and a small value of the D_T at intermediate detergent concentrations (from 30:1 to 60:1) can be explained by formation of the arenicin-2 complexes with large aggregation numbers (trimers, tetramers, etc.). In this case, increase in DPC concentration leads to dissociation of these aggregates and formation of the peptide dimers. Interestingly, D_T of the peptide, and consequently, the mass of the arenicin-2/DPC complex were changed slightly upon the process, indicating the delicate balance between the peptide molecules releasing from the complex and newly incorporating detergent molecules.

Structure of the Arenicin-2 Dimer in DPC Solution

The peptide/detergent complex with the mass above 60 kDa is a challenge for structural investigation by homonuclear high-resolution NMR spectroscopy. The main problem is a relatively fast transverse relaxation of magnetization, which induces significant line broadening and makes ineffective the magnetization transfers through scalar J-couplings in correlation spectroscopy and total correlation spectroscopy (TOCSY) experiments. Possible solution of this problem is to conduct all structural studies at elevated temperatures. Additionally, the broadening of the HN proton signals, induced by exchange with solvent, could be significantly decreased at pH in the range 3.0–4.0. The additional difficulties in investigation of spatial structure of the arenicin-2/DPC complex arise from possible asymmetric dimerization of the peptide. The number of spin systems observed in 2D NMR spectra is doubled as compared with spectra of the peptide in aqueous solution. The amino acid composition of arenicin-2 also leads to strong overlap in NMR spectra and significantly hinders their analysis. Twelve out of 42 spin systems, observed in the arenicin-2 dimer, belong to Arg residues (∼29%), 10 belong to aromatic residues (Tyr/Trp, ∼24%) and 12 belong to hydrophobic residues (Val/Leu/Ile, ∼29%). Taking into account the above mentioned problems, the present investigation was focused on the qualitative description of the arenicin-2 dimer structure. Partial assignment of the ¹H resonances of the arenicin-2 was obtained by standard techniques14 using 2D nuclear Overhauser effect spectroscopy (NOESY) and TOCSY spectra (Figure 6). The obtained assignment involves approximately all backbone resonances (HN and H^α) and partially covers side-chain resonances (H^β, H^γ, and H^δ) for two monomers of the peptide (A and B). The obtained NOE connectivity pattern (Figure 7) indicates that both arenicin-2 monomers preserve the β-hairpin structure upon incorporation into the DPC micelle. The observed intermolecular NOE contacts (Figure 7) and H^α chemical shift data (Figure 8) confirmed the formation of the peptide dimer. Two monomers (A and B) of arenicin-2 associate by the N-terminal strands in a parallel asymmetric fashion (CN↑↑NC type of association). Noncovalent association between two fragments of the monomers A and B is possibly stabilized by eight intermolecular hydrogen bonds (Figure 7). Interestingly, the dimerization interface involves mainly aromatic and hydrophobic residues and is restricted by two positively charged residues Arg1 and Arg9.

Figure 6. Fragments of the 2D TOCSY (A, τ_m = 40 ms) and NOESY (B, τ_m = 100 ms) spectra of arenicin-2 (DPC/arenicin-2 ratio 100:1, pH 3.3, 50°C). The assignments for Val8-Arg11 tetrapeptide from monomer A and Tyr7-Ile10 tetrapeptide from monomer B are shown in red and blue, respectively. The intermolecular NOE cross-peaks are shown by oblique black crosses and connected by dotted lines.

Figure 7. Overview of NOE data defining the structure of the arenicin-2 dimer in DPC micelles solution. The strong, weak, and possibly overlapped NOE contacts observed in the 50 ms NOESY spectrum are shown by solid, open, and dashed arrows, respectively. The hydrogen bonds that stabilize the dimer structure are shown by wavy lines. Two peptide copies that form dimer are denoted as monomers A and B.

Figure 8. Changes of arenicin-2 chemical shifts of H^α protons (ΔδH^α) upon incorporation into DPC micelle and dimerization. The black and gray bars for each residue denote changes in H^α chemical shift for monomers A and B, respectively.

Chemical shifts of H^α protons from the C-terminal strands, that directed sideways from dimerization interface (odd residues: Val13, Val15, Tyr17, Arg19, and Trp21), do not significantly change their values upon detergent induced peptide oligomerization (Figure 8). This observation proves the absence of an additional peptide association by the C-terminal strands and confirms assumption that arenicin-2 forms dimers in the DPC micelles at D:P above 80:1, but does not form aggregates of higher order.

The present structural data were measured at rather extreme experimental conditions (pH 3.3, 50°C). To validate their applicability to more physiological conditions, a set of the 1D NMR spectra for the arenicin-2/DPC sample (1:100) was acquired. Results indicate that neither variation of the sample pH in the range 2.0–8.0, nor variation of the sample temperature in the range 30–50°C influence dimeric structure of the peptide. The characteristic pattern of four H^ε1 Trp signals was observed at all conditions tested (data not shown).

DISCUSSION

Arenicin-2 Expression and Purification

Various heterologous expression systems for AMPs production were developed during the last two decades. The majority of them utilize E. coli as an expression host. The intrinsic cytotoxicity and small sizes of AMPs require almost obligatory use of fusion partners to enhance productivity of prokaryotic expression systems. It cannot be said in advance which carrier protein will be suited well for this task in the case of each specific AMP. Incomplete list of the carrier proteins tested for this purpose includes: L-ribulokinase fragment,15 glutathione-S-transferase,16Pseudomonas aeruginosa outer membrane protein,16Staphylococcus aureus protein A, and the dimer of its IgG-binding domain,17 subtilisin inhibitor,18 β-galactosidase,18 maltose-binding protein,18 prochymosin,19E.coli replication protein RepA and its fragment,20 cellulose-binding domains,20 human defensin HNP-1 preprodomain,20 modified magainin intervening sequence (MIS),21 GABA-transaminase,22 truncated E.coli amidophosphoribosyltransferase,23Pseudomonas testosteroni ketosteroid isomerase,24 fluorescent proteins GFP and obelin,25, 26 light meromyosin fragment,27 bacteriophage PaP3 protein 30,28 baculoviral polyhedrin,29E. coli thioredoxin A,30E. coli TrpE protein,31 and ubiquitin.32 An intein-based expression and purification system was also described.33 More detailed review on heterologous systems for recombinant AMPs production was recently published.34

After a series of experiments with several strain-plasmid combinations we settled on E.coli BL-21 (DE3) transformed with the plasmid pET-His8-TrxL-Ar2 for a laboratory-scale arenicin-2 production. Interestingly, even such a highly soluble expression partner as thioredoxin accumulated in inclusion bodies, being fused with the arenicin sequence. Common methods of increasing recombinant proteins solubility (lowering cultivation temperature, addition of glucose to the growth medium, decreasing IPTG concentration or substitution it with lactose) did not change this tendency. Taking into consideration the small size and simple organization of the arenicin molecule, its refolding was supposed to be not a difficult task. Anyway, the spontaneous disulfide bridge forming, no matter either it occurred simultaneously with expression or during the postprocessing stages, resulted in the properly folded peptide possessing the expected antimicrobial activity. This allowed us to skip the refolding step and avoid using an expression host with oxidizing cytoplasmic environment. In all the recombinant constructions the arenicin sequence was placed downstream of the carrier protein in order to obtain a complete analogue of the natural peptide after the CNBr cleavage. It was supposed that the presence of homoserine residue at the C-terminus of arenicin would impair its antimicrobial activity by disturbing the mechanism of the peptide insertion into lipid bilayer. Introduction of a short anionic MIS between the carrier protein and arenicin-2 did not give any advantage in respect of the protein yield. It should be noted that the cell culture of BL-21(DE3)/pET-His8-TrxL-Ar2 was grown in a simple laboratory thermal shaker without using forced aeration or automatic pH adjustment, so the yield obtained with this strain could be possibly improved in the future.

PAGE examination of the native (oxidized, cyclic) arenicin in the presence of SDS (0.1–1.0%) in a weakly alkaline medium at nonreducing conditions revealed the ∼12 kDa band. We suppose that the observed “12 kDa” band represents arenicin tetramer, stabilized by noncovalent interactions between the peptide monomers. SDS-PAGE mobility of the native arenicin is highly sensitive to conditions of the sample preparation. The “12 kDa form” was transformed to monomer “3 kDa form” in the presence of 2-mercaptoethanol. Under more harsh conditions (boiling in >1% SDS and 9M urea for 20–30 min) the “12 kDa form” was partially transformed to the monomer “3 kDa form” in the absence of the reducing agent. Thus the complex can be disintegrated as well under nonreducing conditions. Along with the results of MALDI-TOF analysis (see Results), this confirms the assumption about noncovalent nature of arenicin tetramers. Disappearance of the tetramer band in SDS-PAGE under reducing conditions can be explained with disruption of the peptide β-hairpin structure, essential for its selective oligomerization. The ∼6 and ∼12 kDa bands might represent the arenicin dimer and tetramer, respectively. It seems that the peptide tends to form aggregates, probably of a variable composition. As shown in our previous NMR investigation of arenicin in aqueous solution, the reduction of intramolecular disulfide bond disrupts the β-structural organization of the peptide and transforms it to virtually unfolded state.12 Most probably the same process takes place in SDS solution containing the reducing agent, and the resulting unfolded arenicin monomers do not retain the ability to form oligomers. It is possible that association and dissociation of the aggregate is taking place during electrophoresis process, so the peptide bands are sometimes smeared.

Probable Structure of the Arenicin-2 Tetramer, and Implications for the Mode of Action

Most AMPs are considered to kill pathogenic microbes by destroying their cell membranes. Three main models, such as barrel-stave, carpet-like, and toroidal-pore, hypothesize the mode of AMPs action.35–37 Despite specific aggregation and oligomerization of AMPs are central to these models, there is little information about structure of these aggregates and mechanisms of the AMP-membrane interactions up to now.

Our previous NMR investigation showed that in water solution arenicin-2 displayed a prolonged β-hairpin, stabilized by the system of nine hydrogen bonds.12 A significant right-handed twist in the β-sheet is deprived the peptide surface of amphipathicity. CD spectroscopic analysis indicates that arenicin-2 binds to the SDS and DPC micelles, and conformation of the peptide was significantly changed upon binding. Arenicin strongly binds to anionic lipid (POPE/POPG) vesicles in contrast with zwitterionic (POPC) ones.12 These results suggest that arenicins are membrane active peptides. To better understand arenicin-induced membrane disruption, the present study used NMR spectroscopy to investigate specific oligomerization of the peptide in detergent micelles.

The obtained structure of the arenicin-2 dimer in DPC micelles permits to speculate about structure of the peptide tetramers in SDS solution. Assuming that the basic building block is identical in both environments, we can envisage the tetramer as an associate of the two dimers. The only interface available for further association is the C-terminal strands. Taking into account the segregation of hydrophobic (Val13-Val15) and cationic (Arg16-Arg-19) residues in the C-terminal part of the peptide we can conclude that the most probable tetramer structure is a parallel aggregate of dimers (CN↑↑NC↑↑CN↑↑NC type of association) (Figure 9A). The requirement of the anionic detergent (SDS) for the tetramerization points to the involvement of the detergent head groups in the aggregate formation. Possibly, these negatively charged groups are intercalated between positively charged fragments of the C-terminal strands (Figure 9A). The proposed mechanism of the arenicin-2 dimer aggregation points to the possibility that in anionic lipid environment arenicin can exist not only in a tetrameric form but also in a form of higher order aggregates (hexamers, octamers, etc.).

Figure 9. (A) The proposed structure of arenicin-2 tetramer in SDS micelles. The residues of the peptide are color coded according to their properties. The aromatic, hydrophobic, positively charged, and other residues are shown by green, pink, blue, and gray circles, respectively. The residues with side-chains directed up from the picture plane (toward reader) are marked by black labels; the residues with side chains directed down from the picture plane are marked by white labels. The negatively charged SDS head groups are shown by red ovals. (B) The proposed model of arenicin-2 membrane action (see text for details). The arenicin-2 dimers are shown by different shades of blue just to improve visual appearance of the picture. The negatively charged and neutral lipids head groups are shown by red and yellow spheres, respectively.

The proposed structure of the arenicin-2 tetramer is closely resembles the tetramerization motif of protegrin-1 revealed by solid-state NMR spectroscopy in anionic lipid membranes.38 This β-hairpin peptide forms the tight dimers by parallel association of the C-terminal strands. These dimers, in turn, pack in higher-order aggregates by loose parallel association of the N-terminal strands with anionic lipid head groups, also possibly intercalated between them.38

Using all the available structural data on arenicin-2 (structure of the monomer in aqueous solution,12 structure of the dimer and proposed model of the tetramer in anisotropic membrane mimetics) we can easily adapt the modern model describing mechanism of AMP membrane action (carpet/toroidal- pore model)35–37 to the arenicin case (Figure 9B). Initially, the peptide in the twisted β-hairpin conformation without pronounced amphipathicity approaches the anionic membrane surface (Figure 9B). The peripheral binding to the bilayer surface induces dimerization of the arenicin-2 monomers, which in turn leads to formation of amphipathic structure with the hydrophobic β-structural core and positively charged residues situated on its edges. The subsequent increase in concentration of the bound peptide induces the tetramerization of the arenicin with the aid of anionic lipid head groups (Figure 9B, carpet-like step). The accumulation of the peptide molecules in one of the membrane leaflets invokes the significant curvature strain and causes transformation of the arenicin aggregates into transmembrane (TM) arrangement (the length of the peptide ∼30 Å) and formation of the ion-conducting pores in complex with anionic lipids head groups (Figure 9B, toroidal-pore step).

In the adapted toroidal-pore model, the TM state of the peptide dimers is stabilized by electrostatic interactions between negatively charged lipid head groups and positively charged residues at the termini and tip of the β-hairpin (Arg1, Arg9, and Arg11). The hydrophobic interactions between β-structural core of the dimers and fatty lipid tails are also contribute to stability of the TM pore. At the same time, the positive charges on the long edges of the dimers (Arg17, Arg18, and Arg19) possibly interact with anionic lipid head-groups that are wrapped between the membrane leaflets in toroidal fashion. In this case these head-groups line the interior of the pore.

To confirm the proposed model and determine the structure of the active arenicin oligomer the further biophysical and structural investigations are clearly needed. The most promising approach for structure determination of the higher order arenicin oligomer implies the NMR investigation of the peptide in solution of anionic detergent micelles. Unfortunately, the present study of the arenicin dimer in complex with detergent is in the upper edge of the applicability of natural abundance NMR spectroscopy, and for the further structural studies the uniformly isotope labeled peptide analogue is needed. The availability of effective expression system for the recombinant arenicin-2 makes this task feasible. In summary, our results afford further molecular insight into possible mechanism of antimicrobial action of arenicins and contribute to better understanding the peptide behavior in biological membrane.

MATERIALS AND METHODS

Arenicin-2 Expression and Purification

The recombinant arenicin-2 was expressed in E. coli and purified as described earlier.12 Briefly, the expression cassette was composed of a T7 promoter, a ribosome binding site and a sequence encoding the recombinant protein that included octahistidine tag, TrxL carrier protein (E. coli thioredoxin A with M37L mutation), a methionin residue and the mature arenicin-2. This sequence was ligated with BglII/XhoI fragment of pET-20b(+) vector, containing pBR322 origin of replication, β-lactamase gene and T7 terminator, resulting in pET-His8-TrxL-Ar2 expression plasmid. The E. coli strain DH-10B (Life Technologies) was used for plasmid preparation and E. coli BL-21 (DE3) (Novagen) was used as an expression host. The cells were induced by IPTG at 25–30°C and incubated at this temperature for 4–6 h.

Following the cells harvesting, sonication in a neutral buffer containing 1% Triton X-100 and preparative centrifugation of the cell lysate the insoluble fraction was subjected to consecutive washing steps, solubilization and Ni-NTA affinity purification. The affinity purification was performed in 100 mM phosphate buffer (pH 7.8) containing 6M guanidine hydrochloride, using 0.5M imidazole as an eluent. Arenicin was obtained by CNBr cleavage of the fusion protein in 80% trifluoroacetic acid (TFA) under standard conditions. Lyophilized products of cleavage reaction were dissolved in 10% acetonitrile containing 0.1% TFA and loaded onto a RP-HPLC semipreparative column C4 (Waters). The chromatography was performed in a linear gradient of acetonitrile in water containing 0.1% TFA: 5–35% for 60 min, 35–80% for 15 min, and 80–5% for 15 min.

Purified recombinant arenicin-2 was analyzed by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) using Reflex III mass-spectrometer (Bruker Daltonics) and automatic Edman degradation using Procise cLC491 protein sequencing system (PE Applied BioSystems). Quantity and purity of the fusion protein and the peptide of interest on different stages of isolation process were monitored using tris-tricine SDS-PAGE39 in SE 250 vertical gel electrophoresis unit (Amersham Biosciences). The DPC-containing sample used in NMR studies was mixed with three volumes of a buffer containing 1% SDS and 8M urea and incubated at 37°C for 10 min before applying to SDS-PAGE.

Continuous acid-urea PAGE of arenicin-2 was conducted in 16.5% T, 6% C slabs containing 5M urea and 5% acetic acid, with 5% acetic acid used as an electrode buffer. Gels were subjected to pre-electrophoresis at 150 V for 1 h. Samples were dissolved in a mixture of 5% acetic acid, 9M urea and 0.01% methyl green and then applied to electrophoresis for about 1 h at the same voltage. The gels were fixed and stained in 0.025% Coomassie G-250 solution containing 50% methanol and 10% acetic acid, and then washed in 5% acetic acid.

NMR Spectroscopy and Spectral Assignment

All the NMR experiments were performed on a Bruker AVANCE-700 spectrometer equipped with TCI cryoprobe. A relaxation delay of 1.4 s was used. The WATERGATE technique was used to suppress strong solvent resonance. The titration of arenicin-2/H₂O (10% D₂O) sample (1.0 mM) with perdeuterated DPC (98% deuterium, CIL) was performed at 30°C, pH 3.3. At each detergent concentration (from 5 to 100 mM) 1D NMR spectrum was acquired, and diffusion rate of arenicin-2/DPC complex was measured using a stimulated echo experiment with bipolar gradients.40 Structure of the arenicin-2 dimer was investigated using the same peptide sample at detergent to peptide molar ratio (D:P) of 100:1 (pH 3.3, 50°C). For this end, the 2D TOCSY (τ_m = 40 and 60 ms) and NOESY (τ_m = 100 and 200 ms) spectra were recorded in the States-TPPI manner.41 Proton resonance assignments for arenicin-2 were obtained by the standard procedure14 using the XEASY program.42¹H chemical shifts were measured relative to the residual protons of H₂O, the chemical shift of the signal being arbitrary chosen as 4.55 ppm at 50°C.