Antimicrobial and conformational studies of the active and inactive analogues of the protegrin-1 peptide Sylwia Rodziewicz-Motowidło1, Beata Mickiewicz1, Katarzyna Greber2, Emilia Sikorska1, Łukasz _ Szultka2, Elzbieta Kamysz1 and Wojciech Kamysz2 ´ Faculty of Chemistry, University of Gdansk, Poland ´ Faculty of Pharmacy, Medical University of Gdansk, Poland Keywords antimicrobial peptides; IB-367; NMR; protegrin-1 analogues; three-dimensional structure Correspondence S Rodziewicz-Motowidło, Faculty of ´ Chemistry, University of Gdansk, ´ Sobieskiego 18, 80-952 Gdansk, Poland Fax: (+48 58) 523 54 72 Tel: (+48 58) 52 35 430 E-mail: sylwia@chem.univ.gda.pl (Received 11 August 2009, revised November 2009, accepted December 2009) doi:10.1111/j.1742-4658.2009.07544.x The natural antimicrobial cationic peptide protegrin-1 displays a broad spectrum of antimicrobial activity and rapidly kills pathogens by interacting with their cell membrane We investigated the structure–activity relationships of three protegrin-1 analogues: IB-367 (RGGLCYCRGRFCVCVGRNH2), BM-1 (RGLCYCRGRFCVCVG-NH2) and BM-2 (RGLCYRPRFV CVG-NH2) Our antimicrobial and antifungal activity studies of these peptides showed that BM-1 was much more active than IB-367 against Gram-positive bacteria and fungi, whereas BM-2 was inactive The BM-1 peptide showed fourfold reduced haemolysis relative to IB-367, an additional advantage of this peptide In addition, BM-1 was about 15% cheaper than IB-367 to synthesize The absence of two cysteine residues in the BM-2 sequence could be the main reason for its unstable conformation and antimicrobial inactivity The solution structures of these peptides were determined in dimethyl sulphoxide using two-dimensional NMR and restrained molecular dynamics calculations IB-367 and BM-1 formed short, antiparallel, b-hairpin structures connected by a type II¢ b-turn The shorter, inactive BM-2 analogue exhibited major and minor conformations (predominantly unordered) in the NMR spectra and was much more flexible Introduction The search for new drugs and target sites has generated interest in a group of short polypeptides, antimicrobial peptides (AMPs), compounds that can combat bacterial infections [1], and have a broad spectrum of activity against bacteria, fungi and protozoa [2,3] AMPs are positively charged molecules (there are also a few negatively charged ones [4,5]) isolated from a variety of animals and plants, where they participate in natural defence mechanisms AMPs are usually highly amphipathic molecules with hydrophobic and hydrophilic moieties segregated into distinct patches on the molecular surface Topologically, they can be grouped into linear and cysteine-bridged peptides Further subdivided according to the number of disulphide bridges in their structure, cysteine-bridged AMPs include the protegrins, first isolated in 1993 from porcine leucocytes [6] Protegrins are active against bacteria (Escherichia coli, Staphylococcus aureus [7], Pseudomonas aeruginosa, Chlamydia trachomatis, Neisseria gonorrhoeae [8]), yeasts (Candida albicans [6]) and viruses (HIV-1 [9]) The protegrin family contains the following peptides: protegrin-1, protegrin-2, protegrin-3, protegrin-4 and protegrin-5 (PG-1–PG-5) [10] They are produced from a family of antimicrobial Abbreviations AMP, antimicrobial peptide; CSI, chemical shift index; MIC, minimal inhibitory concentration; NOESY, nuclear Overhauser effect spectroscopy; PG-1, protegrin-1; ROESY, rotating frame Overhauser effect spectroscopy 1010 FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS S Rodziewicz-Motowidło et al peptide precursors known as cathelicidins [11], which are synthesized as the C-terminal portion of a cathelincontaining proregion The N-terminal cathelin domain of the precursor is highly conserved at both the amino acid and nucleotide sequence levels; this conservatism is emphasized by both inter- and intraspecific comparisons Conversely, the sequence of the C-terminal peptide carrying the antimicrobial activity is highly variable It has been shown that activated porcine neutrophils release intact pro-protegrin, which is inactive as an antimicrobial It is then processed extracellularly by elastase to form antimicrobial protegrin [12] PG-1 is an 18-amino-acid peptide with an amidated C-terminus It is thought to form an antiparallel b-sheet constrained by two disulphide bridges, Cys6– Cys15 and Cys8–Cys13 (1PG1 [13] and 1ZY6 [14] in the Protein Data Bank) [7,15] Containing six positively charged arginine residues, the native sequence of PG-1 is highly cationic The distribution of hydrophobic and hydrophilic residues at the peptide surface is a structural feature required for the cytolytic activity of PG-1 Structure–activity relationship studies of several hundred PG-1 analogues were analysed to determine the role of individual hydrophobic and hydrophilic residues in antimicrobial activity, i.e to gain an understanding of the relationship between the primary and secondary structure of protegrins and their microbial activities, and to identify a protegrin analogue for clinical development [16] The presence of the b-hairpin structure was found to be crucial to the antimicrobial activity of the protegrin The analogues – linearized or with amino acid substitutions eliminating hydrogen bonding across the b-sheet – showed a reduced biological activity, especially in the presence of physiological concentrations of NaCl [17,18] However, Tam et al [19] reported that the activity of nondisulphide-bonded analogues could be restored by the cyclization of the peptide backbone In addition, Harwig et al [17] found that, in peptides containing one disulphide bond, the ‘bullet’ analogue (cysteines one and four linked by a disulphide bond) had an activity comparable with that of PG-1, whereas the ‘kite’ analogue (cysteines two and three linked by a disulphide bond) was less active In addition, the maintenance of the amphiphilicity of the b-sheet is essential The cationic and hydrophobic clusters in PG-1 have been shown to be the structural features required for antibacterial activity [16,20] Analogues with reduced positive charge tend to be less active, which may imply that the binding of a cationic surface to a lipopolysaccharide is a key mechanistic step in the killing of bacteria [16,20] The conformations of the structural features determining the antimicrobial activity of protegrins Conformational studies of protegrin-1 analogues were calculated for 62 peptides and correlated with their experimental activity against six microbe species (E coli, N gonorrhoeae (Strain F-62), N gonorrhoeae (Strain FA-19), Listeria monocytogenes, C albicans, P aeruginosa), as well as their haemolytic and cytotoxic activities [20] Based on broad structure–activity relationship studies, only one analogue of PG-1 – IB367 – was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy [16,21,22] It displays a broad spectrum of activity, rapid microbicidal action and limited ability to induce resistance IB-367 kills a broad spectrum of bacteria and fungi, including those resistant to conventional antimicrobial drugs, by attaching to and destroying the integrity of the lipid cell membrane [23] In addition, IB-367 demonstrates enhanced bactericidal and fungicidal activity compared with that of native protegrins [16,24] It could therefore be an interesting compound for the inhibition of bacterial translocation and endotoxin release in obstructive jaundice [25] IB-367 is a 17-amino acid peptide with an amidated C-terminus (Fig 1): a peptide with such C-terminus displays greater biological activity than an analogue without one [16] Compared with other protegrin peptides of comparable activity, IB-367 has three advantages in chemical synthesis: (a) most importantly, it contains an achiral amino acid residue (glycine) at position in the b-turn – the problem of racemisation is thus avoided [16]; (b) it has only four arginine residues compared to six in PG-1 – this is significant, as arginine is expensive to purchase; (c) it contains 17 amino acids compared to 18 in PG-1 This paper elucidates the structure-activity relationship in IB-367 and two other analogues of PG-1 Fig Amino acid sequences of PG-1 and its analogues IB-367, BM-1 and BM-2 FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS 1011 Conformational studies of protegrin-1 analogues S Rodziewicz-Motowidło et al (BM-1 and BM-2) (see the sequences in Fig 1) using 2D-NMR spectroscopy and molecular dynamics The results of these investigations are compared with published structural information about PG-1 and other antimicrobial peptides in an attempt to understand which structural features are responsible for the high biological activity of AMPs Results Design of the new BM-1 and BM-2 analogues Our aim was to design new analogues of PG-1 with biological activity comparable with or better than that of PG-1, but cheaper (about 15% less) to synthesize chemically on a large scale On the basis of the structure–activity relationship studies of other PG-1 analogues (see introductory paragraphs), we designed two PG-1 analogue sequences The first analogue, BM-1, contains four cysteines occupying the nonhydrogenbonded sites of the natural b-hairpin core; Gly3, Arg4 and Arg18 were removed from the amino acid sequence Although previous studies on protegrin analogues have shown that a positive charge in the loop is essential for activity [16], we also replaced Arg10 with a glycine residue (see Fig 1) We reasoned that, by removing these residues, we would retain the cationic nature of the peptide (+4 under physiological conditions), but only in the loop (Arg7, Arg9) and in the N-terminal fragment (Arg1), not in the C-terminal fragment In PG-1, the cationic arginine residues occupy the loop, N- and C-termini In this way, we endeavoured to reduce the cost of synthesis (arginine is very expensive) The second analogue, BM-2, was designed in such a way that the relative importance of the rigidity and charge at the turn of the b-hairpin in the context of a single disulphide bridge could be assessed In BM-2, we removed Gly3 and Arg18 as in BM-1 We also removed the two cysteines forming the disulphide bond proximal to the turn Although the turn structure of single disulphide variants of PG-1 is less well defined and their activity is intermediate relative to that of PG-1 [16], we wanted to see how removal of the two cysteines (shortening the amino acid sequence) would affect their structure and activity We also wanted to promote b-hairpin formation by inducing a b-turn, and so we replaced Arg10 with Pro10 (see Fig 1) Although the proline residue is known to have high frequencies in b-turn formation [26,27], the incorporation of l-proline at position i + of the reverse turn prevents b-hairpin formation as a result of incompatibilities with the intrinsic right-handed twist 1012 of b-strands [28–30] Presumably, therefore, the BM-2 analogue would have an undefined structure and reduced antimicrobial activity An analogue similar to the BM-2 amino acid sequence has been suggested previously by Lai et al [18] They designed a peptide in which two proximal cysteines were replaced with branched residues (threonine) with a high intrinsic preference for b-sheet conformation, and with a d-proline residue instead of an arginine residue incorporated at position i + of the reverse turn (see peptide 10 in [18]) The amino acid sequence of BM-2 also has a similar profile to the bullet variants studied by Harwig et al [17] Antimicrobial and haemolytic activity PG-1, IB-367, BM-1 and BM-2 were characterized with regard to their antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and fungi (see Table 1) All the test organisms were human pathogens, good selections for the initial screening of antimicrobial ⁄ antifungal activity IB-367 and BM-1 exhibited antimicrobial activity against all the bacteria, but were less active against the fungi (see Table 1) In contrast, BM-2 showed a marked decrease in activity against all the bacteria Surprisingly, BM-1 was far more active than IB-367 against Gram-positive bacteria and fungi, showing better inhibition than IB-367 of S aureus, S epidermidis and fungi The antimicrobial activity of IB-367 and BM-1 against Gram-negative bacteria was similar, however Interestingly, BM-2, inactive against bacteria, displayed a better antifungal activity than either IB-367 or BM-1 against Aspergillus niger Comparison of the PG-1 and BM-1 peptides showed that BM-1 exhibited slightly increased activity against Gram-positive bacteria and slightly decreased activity against Gram-negative bacteria and fungi than did PG-1 The comparison of the C albicans minimal inhibitory concentrations (MICs) obtained in this work with the values found by Barchiesi et al [31] showed much lower values found by Barchiesi et al [31] (2.0– 32 lgỈmL)1 compared with 256 lgỈmL)1 in our work) The differences between these MICs resulted from the fact that Barchiesi et al [31] used different experimental conditions and another strain of C albicans Barchiesi et al [31] used RPMI 1640 medium, Mops buffer (pH 7.2) and 50% reduction of the initial inoculum They also used another strain of C albicans ATCC 90029 In our work, we used Sabouraud 5% glucose medium, pH 7.4 and 99% reduction of the initial inoculum In addition, we used the reference strain of C albicans (C albicans ATCC 10231) FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS S Rodziewicz-Motowidło et al Conformational studies of protegrin-1 analogues Table Antimicrobial activity of the PG-1, IB-367, BM-1 and BM-2 peptides MBC ⁄ MFC, minimal bactericidal ⁄ fungicidal concentration; MIC, minimal inhibitory concentration MIC (lgỈmL)1) PG-1 Organism Gram-positive bacteria Bacillus subtilis ATCC 6633 Enterococcus faecalis ATCC 29212 Rhodococcus equi ATCC 6939 Staphylococcus aureus ATCC 25923 Staphylococcus epidermidis ATCC 14990 Gram-negative bacteria Escherichia coli ATCC 25922 Pseudomonas aeruginosa ATCC 27853 Fungi Aspergillus niger ATCC 16404 Candida albicans ATCC 10231 IB-367 MBC ⁄ MFC (lgỈmL)1) BM-1 BM-2 PG-1 IB-367 BM-1 BM-2 4 8 32 2 128 128 128 256 128 8 8 8 64 2 16 128 256 256 256 256 16 32 32 16 256 > 256 16 16 16 64 128 64 256 > 256 64 32 256 256 128 128 64 256 256 64 256 256 256 256 128 256 The haemolytic activity (see Table 2), using human red blood cells as targets, was measured for PG-1, IB367, BM-1 and BM-2 peptides The IC50 results showed fourfold reduced haemolysis of the BM-1 peptide (32 lgỈmL)1) relative to IB-367 (8 lgỈmL)1) BM-2 was not cytotoxic when tested against human red blood cells (> 256 lgỈmL)1) NMR results The two-dimensional NMR spectra of the peptides, obtained via the standard sequential assignment methods developed by Wuthrich [32], were assigned ă (Figs S1 and S2, see Supporting information) The proton and carbon aC chemical shifts, 3JNH–aH coupling constants and amide-proton temperature coefficients are given in Tables S1–S4 (see Supporting information) The 1H and 13C NMR chemical shifts of IB-367 and BM-1 were well dispersed, a property characteristic of b-sheet structures [32] Moreover, the good dispersion of the 1H chemical shifts permitted the identification of all the protons in the amino acid sidechains, which was of further help in obtaining the v torsion angles and in precise structural calculations One distinct set of residual proton resonances in all spectra was displayed for IB-367 and BM-1 The chemical shifts of IB-367 and BM-1 were very similar Table Haemolytic activity (lgỈmL)1) (IC50 values – concentrations that cause 50% haemolysis) of the PG-1, IB-367, BM-1 and BM-2 peptides Cells PG-1 IB-367 BM-1 BM-2 Human red blood cells 32 32 > 256 (Tables S1 and S2, see Supporting information), except for Phe10, which indicates that this phenylalanine residue in BM-1 points in the opposite direction to that in IB-367 The chemical shifts of IB-367 and BM-1 were also similar to the previously published data for PG-1 acquired at room temperature in water and in deuterated dimethyl sulphoxide [7,13] The dispersion of chemical shifts in BM-2 was not as good as in IB-367 and BM-1; there were also two sets of signals (major and minor) in the NMR spectra Analysis of rotating frame Overhauser effect spectroscopy (ROESY) and nuclear Overhauser effect spectroscopy (NOESY) revealed the trans geometry of all the peptide bonds in IB-367 and BM-1; the geometry of the Arg6–Pro7 peptide bond (major conformation) in BM-2 was cis The dHa–Ha(i,i + 1) connectivity characteristic of the cis peptide bond was seen in the NOESY spectrum, and the relevant chemical exchange cross-peaks of this bond were present in the ROESY spectrum, indicating its cis–trans isomerization (trans geometry in the minor species) The cis–trans isomerization of the Xaa–Pro peptide bonds located in the turns is a common feature of peptides [33]; it is hardly surprising to find this conformational equilibrium in BM-2 As mentioned above, at least two sets of proton resonances were present in the NMR spectrum of BM-2 Complete analysis, however, would require a separate conformational analysis for each set of resonances; too few proton resonances were obtained for the minor species to perform reliable three-dimensional structural calculations The chemical shift index (CSI), defined as the deviation of the random-coil chemical shift from the experimental value, is a very sensitive indicator of the secondary structure of peptides and proteins [34] In FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS 1013 Conformational studies of protegrin-1 analogues S Rodziewicz-Motowidło et al Fig CSIs relative to sodium 3-(trimethylsilyl)-(2,2,3,3-2H4)-propionate, summary of intra- and inter-residual NOEs among the backbone NH, aH and bH, vicinal coupling constants 3JHN–Ha measured in deuterated dimethyl sulphoxide at 22 °C, and temperature coefficients of amide protons measured in deuterated dimethyl sulphoxide at 22, 25, 27, 30, 32, 35 and 37 °C for IB-367 (A), BM-1 (B), BM-2 major (C) and BM-2 minor (D) CSIs were equal to zero or were not calculated for amino acid residues with open squares Bar height indicates the strength of the NOE correlation as strong, medium or weak Filled squares show 3JNH–Ha coupling constants > 9.0 Hz, and filled circles the temperature coefficients of amide protons higher than –3.0 ppbỈdeg)1 the case of IB-367, most of the amino acids had CSIs of –1 (Leu4–Arg8 and Arg10–Arg17), a value characteristic of b-sheet structure formation (see Fig 2) The CSI pattern of IB-367 resembled that of PG-1, where the Leu5–Arg10 and Cys13–Gly17 regions exhibited typical b-strand chemical shifts, whereas the Arg9– Arg11 region exhibited deviations from the b-strand chemical shifts [7,13] The CSIs of the aC atoms in BM-1 and BM-2 displayed no regularity, indicating a predominantly random structure Figure summarizes the NOE pattern, vicinal couplings 3JNH–aH and temperature coefficients of the amide protons in the investigated peptides In IB-367 and BM-1, the presence of strong daN(i,i + 1) and weak (or absence of) dMN(i,i + 1) and dbN(i,i + 1) NOE connectivities in the Gly3–Arg8 and Cys14– Gly16 regions of IB-367 and in the Gly2–Cys6 and Val12–Val14 regions of BM-1 indicates a significant population of conformers with dihedral angles in the b-strand region of (/,w) space [35–37] In both peptides, the weak or absent dNN(i,i + 1) NOE effects indicate an unordered structure in the N-terminal fragments and bend structures in the Arg10–Val13 region of IB-367 and the Cys6–Cys11 region of BM-1 The daa(5–14; 7–12) of IB-367 and daa(4–13; 6–11) of BM-1 NOEs strongly suggest a disulphide pattern in both peptides; this results from subsequent calculations Several long-range NOEs for IB-367 and BM-1 (Fig 2) were found, which involved residues from N- and C-termini, in agreement with a two-stranded antiparallel b-structure Moreover, the high values of the vicinal coupling constants 3JNH–aH (> 9.0 Hz) in whole peptide sequences and the temperature coefficients of many amide protons higher than – 3.0 ppbỈK)1 (Fig S2A, B, Tables S1 and S2, see 1014 Supporting information) strongly confirmed the presence of a b-sheet structure in IB-367 and BM-1 Inspection of the NOE pattern of BM-2 (major conformation, Fig 2C) showed a lack of diagnostic dMN(i,i + 1) NOEs and provided evidence for the unstructured conformation of this peptide in the middle part of the peptide (Tyr5–Val10) Some strong daN(i,i + 1) NOEs in the Gly2–Cys4 and Val10–Gy13 regions suggested a b-structure In addition, the lack of hydrogen bonds in BM-2 indicated a flexible structure Finally, no NOE cross-peaks, indicative of oligomeric association in solution, could be detected, consistent with the high abundance of positively charged residues (four arginines in IB-367 and three arginines in BM-1 and BM-2) in the primary structure of the peptides Structural analysis Conformational analysis was performed for the three PG-1 analogues, in an attempt to correlate their structure and activity in comparison with native PG-1, which has been shown previously to form a highly stable, rigid b-hairpin [7,13,18] IB-367 also adopts a well-defined b-hairpin structure, as expected from the sequence similarities with PG-1 (Fig 3A, B) The 300 structures of IB-367 were well defined, with an rmsd value of the ˚ ˚ Ca atoms of all residues of 2.57 A, falling to 1.30 A in the Cys5–Cys14 region (Table S5, see Supporting information) The solution structure of IB-367 consisted of two antiparallel b-strands in the Tyr6–Arg8 and Phe11–Val13 regions linked by two residues – Gly9 and Arg10 The Arg8–Phe11 fragment formed a well-defined type II¢ b-turn, stabilized by a hydrogen bond between FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS S Rodziewicz-Motowidło et al Conformational studies of protegrin-1 analogues A B C D E F Fig Superimposed conformations of the aC atoms of residues Cys5–Cys13 of IB-367 (130 conformations) (A), Cys4–Cys13 of BM1 (93 conformations) (C) and Cys4–Cys11 of BM-2 (95 conformations) (E) The most populated families of conformations are shown Averaged structures of IB-367 (B), BM-1 (D) and BM-2 (F) peptides The backbone is shown in ribbon representation, the side-chains in stick representation Arginine residues are shown in blue, disulphide bonds in yellow the CO group of Arg8 and the NH group of Phe11, found in all the calculated structures The b-sheet was strongly stabilized by five regular backbone–backbone hydrogen bonds – NH(Tyr6)–O(Val13), NH(Arg8)– O(Phe11), NH(Arg10)–O(Arg8), NH(Phe11)–O(Arg8) and NH(Val13)–O(Tyr6) – found between the antiparal- lel strands in most of the calculated structures All of these structures adopted a very similar b-hairpin structure in the middle region of the molecule with unordered N- and C-termini In addition, the N- and C-terminal fragments pointed in opposite directions as a result of the electrostatic repulsions of Arg1 and Arg17 (see Fig 3) The side-chains in all the structures were well defined, particularly in the middle region of the peptide (Fig 3A), owing to the presence of numerous interstrand NOEs In contrast, the side-chains at the N- and C-termini displayed large conformational variability Two interstrand disulphide bridges adopted a well-defined, right-handed conformation IB-367 formed a characteristic amphipathic structure, displaying a hydrophobic face formed by the bulked, hydrophobic residues (Leu4, Tyr6, Phe11, Val13, Val15) located on the concave surface of the peptide Two apolar disulphide bridges and charged Arg17 side-chains formed the second face of the peptide Two hydrophilic regions were located at the two spatial tips of the molecule, at both termini with Arg1 and Arg17, and in the turn in the presence of Arg8 and Arg10 (see Figs and 4) Our conformational studies showed that BM-1 formed a twisted b-sheet structure, similar to that of native PG-1 and IB-367 (Fig 3C, D) The structures of BM-1 were well defined in the backbone, with rmsd ˚ values of the Ca atoms of all residues of 2.60 A and ˚ in the Cys4–Cys13 region (Table S6, see 1.82 A Supporting information) The BM-1 structures consisted of two antiparallel b-strands in the Leu3–Cys6 and Cys11–Cys13 regions, linked by Arg7–Phe10 residues The turn region was formed by a type II¢ b-turn, as in IB-367 b-Hairpin stabilization was guaranteed by three regular backbone–backbone hydrogen bonds – NH(Leu3)–O(Cys13), NH(Cys13)–O(Leu3) and NH(Cys13)–O(Cys4) – between the disulphide bridges in most calculated structures Although the backbone of BM-1 was well defined, the side-chains were much less clearly defined than in IB-367 (cf Fig 3A, C) Two interstrand disulphide bridges adopted a right-handed or extended conformation In most of the calculated structures, the disulphide bonds were located at the same peptide face, but, in some, the disulphide bonds were on the opposite side of the peptide face Thus, it was difficult to state unequivocally which residues were located on any given face of the peptide BM-1 also formed a characteristic amphipathic structure, which displayed a hydrophobic face formed by the hydrophobic residues and the disulphide bridges, and a second polar face formed by charged Arg1, Arg7 and Arg9 side-chains (see Figs and 4) In general, BM-1 was more flexible than PG-1 and IB-367, but, as FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS 1015 Conformational studies of protegrin-1 analogues S Rodziewicz-Motowidło et al in the last two peptides, two hydrophilic regions were located at the two spatial tips of the molecule, at the N-terminus with Arg1 and in the turn in the presence of Arg7 and Arg9 (see Figs and 4) The shorter analogue, BM-2, was the most flexible of all the peptides studied in this work BM-2 formed major and minor conformations – this was easily read from the NMR spectra The calculated structures of the major BM-2 conformation were predominantly unordered, especially in the turn region of the peptide with the cis peptide bond between Arg6 and Pro7 (Fig 3E, F) There were no hydrogen bonds in the calculated structures and no regularities in the secondary structure The rmsd values of all the aC atoms in the 300 calcu˚ ˚ lated structures were 2.71 A and 1.54 A in the Cys4– Cys11 region (Table S7, see Supporting information) The conformational ensemble of peptides, determined by molecular dynamics simulation restraints from NMR experiments, was clustered into families Ten families were found for IB-367 and BM-2, and six ˚ for BM-1, at an rmsd cut-off of 5.0 A, one of which was dominant (130 molecules) for IB-367, one (93 molecules) for BM-1 and two (95 and 65 molecules) for BM-2 The conformations in all the families for IB-367 and BM-1 had one feature in common: the central part of the structure was better defined than the C- and N-terminal parts The conformational differences between the structural families of these peptides applied mainly to the varied structures of the N- and C-termini All the features of the structures calculated for all three peptides were in very good agreement with the experimental NMR data Discussion The presence of a cationic, amphiphilic b-sheet is key to maintaining the biological activity and stability of PG-1-like peptides (IB-367 and BM-1) The highly flexible analogue without a b-sheet structure (BM-2) has no antimicrobial activity Previous studies on protegrin variants have also shown that the antimicrobial activity is highly dependent on b-hairpin stabilization by disulphide bonds or backbone cyclization [16– 19,38,39] IB-367 and BM-1 share characteristic physicochemical properties with most antimicrobial peptides, adopting a b-hairpin-like structure with two disulphide bridges [39,40] Sequence alignments revealed great similarities between IB-367 or BM-1 and PG-1 from porcine leucocytes [6], gomesin from mygalomorph spider haemocytes [41] and androctonin from scorpions [42] All have a molecular mass of approximately kDa, including a rather high percentage (> 20%) of basic residues Their three-dimensional structures are stabi1016 lized by two internal disulphide bridges Most have a broad spectrum of antimicrobial activity against various microorganisms Comparison of the PG-1 structure from the Protein Data Bank (1PG1 [13] in water and 1ZY6 [14] in dodecylphosphocholine micelles) with the structures of IB-367 and BM-1 obtained here reveals several characteristic differences (see Fig 4) The b-sheet structures of our peptides are shorter than that of PG-1 The positive charge in PG-1 in water turns almost the whole of one side of its structure into a cationic surface, whereas, in PG-1 in dodecylphosphocholine micelles, IB-367 and BM-1, the positive charge is distributed separately The b-sheet structures of IB-367 and BM-1, as in all the PG-1 peptide family, are characteristically amphipathic, with one surface hydrophilic and one hydrophobic Such a structure is essential to both Gram-positive and Gram-negative antimicrobial activity [16] PG-1, IB-367 and BM-1 form four-residue b-turns, but, in PG-1, there is an atypical b-turn, possibly caused by the presence of Arg10 in position i + of the turn, whereas, in IB-367 and BM-1, a type II¢ b-turn is formed In PG-4, IB-367 and BM-1 analogues, the Arg10 residue is replaced by a glycine and, in PG-5, by a proline residue Glycine and proline residues are better suited than arginine to induce a canonical b-turn conformation [43] In addition, there is only one cationic Arg1 residue on the N-terminus in BM-1, rather than two arginines (Arg1 on the N-terminus and Arg18 in PG-1 or Arg17 in IB-367 on the C-terminus), one at the N- and the other at the C-terminus; this is sufficient to ensure the cationic nature of the peptide at its terminus The structure of BM-1 is very compact, like the PG-1 structure, whereas that of IB-367 is more expanded These structural features could be responsible for the better antimicrobial activity of BM-1 than IB-367 against Gram-positive bacteria Natural b-hairpin-like antimicrobial peptides other than PG-1 (gomesin [41], tachyplesin I [44], polyphemusin I [45] and androctonin [46]), with two disulphide bridges, are structurally similar to IB-367 and BM-1 Like PG-1, gomesin and polyphemusin contain 18 amino acids, but tachyplesin contains 17 residues and androctonin is significantly longer with 25 residues Although the spacing of the cysteine residues differs in these peptides from those studied here, all the molecules adopt a similar rigid plated b-sheet structure Androctonin has an unequal number of residues on each strand between the two bridges – five in the N-terminal strand and three in the C-terminal strand This causes a greater twist in the b-sheet of androctonin compared with the other peptides Despite such differences, FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS S Rodziewicz-Motowidło et al Conformational studies of protegrin-1 analogues Fig Structures of PG-1 (Protein Data Bank code 1PG1 [13]), PG-1 (Protein Data Bank code 1ZY6 [14]), IB-367, BM-1 and BM-2 The backbone is shown in ribbon representation, the side-chains of arginine (blue) and cysteine (yellow) residues in stick representation The electrostatic potential was calculated on the van der Waals’ surface Positive potential is shown in blue, neutral in grey the rmsd value of the coordinates of the peptide b-strands (IB-367, BM-1, PG-1, gomesin, tachyplesin I, polyphemusin I and androctonin), when superimposed ˚ on the backbone atoms, is approximately A Comparison of the hydrophilic ⁄ hydrophobic properties on the molecule surfaces shows that the structures of IB-367, BM-1, PG-1, gomesin, tachyplesin I and polyphemusin I share two highly hydrophilic and positively charged poles in the N- and C-terminal regions and in the turn Androctonin also has a highly hydrophilic and positively charged turn and N-terminus, but, in contrast, the C-terminus containing Pro24–Tyr25 is hydrophobic [46] There is a large difference in the distribution of hydrophobic ⁄ hydrophilic potentials on the b-sheet surface between the tails and the turn The central portion of IB-367, BM-1 and PG-1 is particularly hydrophobic, as it contains only apolar residues distributed on either side of the b-sheet The b-sheet in gomesin is divided into two nonequivalent faces: the hydrophobic side-chains are clustered on the concave face, whereas the two polar side-chains flanked by the apolar disulphide bridges are located on the other face [41] In tachyplesin I and polyphemusin I, the cationic arginine and lysine FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS 1017 Conformational studies of protegrin-1 analogues S Rodziewicz-Motowidło et al residues are also located in the central part of the b-hairpin structures In androctonin, the highly twisted character of the b-sheet does not suggest a clear dichotomy in the distribution of polar and apolar residues [46] Differences in the distribution of hydrophilic and hydrophobic residues at the surface of the peptides may indicate different modes of action on the membrane This may also account for differences in the haemolytic activity of the peptides A better understanding of the mode of action of these peptides is crucial for the development of a new generation of antibiotics It is known that, in the absence of both disulphide bonds, or even one disulphide bond, the b-sheet structure is less stable, and the antimicrobial activity is much reduced [16–19,47,48] This was also found in BM-2, which has no b-hairpin structure and is inactive against bacteria The single disulphide bond and the proline residue in position i + of the reverse b-turn prevent b-hairpin formation and are responsible for the great flexibility of BM-2 in solution Lai et al [18] obtained similar results with their analogue 12 Interestingly, BM-2 is more active than IB-367 and BM-1 against A niger; the considerable plasticity of the BM-2 structure may permit better activity against this fungus The current study shows that, with its broad spectrum of antimicrobial activity, especially against Grampositive bacteria, the BM-1 analogue could be a good molecule for clinical development Moreover, the BM-1 peptide shows fourfold reduced haemolysis relative to IB-367, an additional advantage of this peptide This peptide is easy to synthesize; it is 15% cheaper to produce than IB-367 Materials and methods Peptide synthesis The peptides were solid-phase synthesized on Polystyrene AM-RAM resin (0.76 mmolặg)1, Rapp Polymere, Tubingen, ă Germany) using 9-uorenylmethoxycarbonyl chemistry [49], all the relevant reagents being obtained from Sigma-Aldrich ´ (Poznan, Poland) The procedure was as follows: (a) and 20 deprotection steps using 20% piperidine in dimethylformamide in the presence of 1% Triton X-100; (b) the coupling reactions were carried out with the protected amino acid diluted in dimethylformamide in the presence of 1% Triton X using diisopropylcarbodiimide as coupling reagent in 1-hydroxybenzotriazole for h The completeness of each coupling reaction was monitored by the chloranil test [50] If positive, the coupling reaction was repeated using O-(benzotriazol-1-yl)-N,N,N¢,N¢-tetramethyluronium tetra- 1018 fluoroborate and 1-hydroxybenzotriazole in diisopropylethylamine and mixed for h The protected peptidyl resin was treated with a mixture of 90% trifluoroacetic acid, 2.5% ethanedithiol, 2.5% phenol, 2.5% water and 2.5% triisopropylsilane for h After cleavage, the solid support was removed by filtration, and the filtrate was concentrated under reduced pressure The cleaved peptide was precipitated with diethyl ether The linear product was oxidized with 0.1 m I2 in CH3OH The peptide was purified by HPLC on a Knauer K501 two-pump system with a Kromasil C8 column [10 · 250 mm; particle diameter, lm; pore size, ˚ 100 A; flow rate, mLỈmin)1; gradient, 10–60% A ⁄ 120 (A, 0.1% trifluoroacetic acid in acetonitrile; B, 0.1% aqueous trifluoroacetic acid), absorbance at 226 nm] The resulting fractions with a purity of better than 96–98% were tested by HPLC (Hypersil C18 column, 4.6 · 250 mm) The peptides were analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry Organism and antimicrobial assay The reference strains were supplied by the Polish Collection of Microorganisms (Polish Academy of Sciences, Institute of Immunology and Experimental Therapy, Wrocaw, Poland) MICs were determined using a broth microdilution method with Mueller–Hinton broth (Becton Dickinson, Le Pont de Claix, France) at initial inocula of · 105 colonyforming units (cfu)ỈmL)1 for bacteria, and Sabouraud 5% dextrose broth pH 7.4 (Sigma-Aldrich) at initial inocula of · 103 cfmL)1 for fungi, according to the procedures of the Clinical and Laboratory Standards Institute (formerly National Committee on Clinical Laboratory Standards) Polystyrene 96-well plates (Sigma-Aldrich) were incubated in air at 37 °C for 18 h (bacteria) and at 25 °C for 72 h (fungi) MIC was taken as the lowest drug concentration at which observable growth was inhibited The minimum bactericidal concentration was taken as the lowest concentration of each drug resulting in > 99.9% reduction of the initial inoculum Experiments were performed in triplicate on three different days Haemolytic activity Freshly collected human blood was washed with NaCl ⁄ Pi (pH 7.4) until the supernatant became colourless A suspension was made of 0.5 mL packed cells in 10 mL of NaCl ⁄ Pi Peptides were dissolved in NaCl ⁄ Pi and serially diluted on a 96-well polystyrene microtiter plate The final concentration of peptides ranged from 256 to 0.5 lgỈmL)1 Twenty microlitres of cell suspension were added As a positive control (100% lysis), a 10% solution of Triton X was used After incubation for h at 37 °C, haemolysis was observed and compared with the positive control FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS S Rodziewicz-Motowidło et al (Triton X) A positive result (IC50) was defined when 50% of haemolysed red blood cells were taken NMR experiments The NMR samples were prepared by dissolving mg peptide in 0.5 mL deuterated dimethyl sulphoxide (peptide concentration,  mm) The same solvent was used in conformational studies of native PG-1 [7] to enable a further three-dimensional structural comparison of the studied peptides with PG-1 Deuterated dimethyl sulphoxide was also used in conformational studies of other bioactive peptides A sample contained a small amount of trifluoroacetic acid in order to downfield shift the vestigial water signal and to retard amide-proton exchange Chemical shifts are given relative to sodium 3-(trimethylsilyl)-(2,2,3,3-2H4)propionate, the internal chemical shift standard H-NMR spectra (Varian Unity+ 500 NMR spectrometer, Varian Inc., Palo Alto, CA, USA) were obtained at a proton frequency of 500 MHz and the following temperatures: 22, 25, 27, 30, 32, 35 and 37 °C Two-dimensional spectra, including double-quantum filtered correlation spectroscopy, total correlation spectroscopy (60 ms), NOESY (100 and 200 ms), ROESY (200 ms) and 1H–13C heteronuclear single quantum coherence spectroscopy, were obtained at 22 °C NMR data were processed with vnmr [51] and analysed with xeasy software [52] Both ROESY spectra (200 ms) were compared with the NOESY spectra (200 ms) and no additional peaks demonstrating the appearance of a spindiffusion effect were found The NOESY spectra showed better quality and, for this reason, were used for further calculations Assignments were carried out according to standard procedures, including spin-system identification and sequential assignment [32] In the case of the one-dimensional NMR spectra, 16 000 data points were collected and a spectral width of kHz was used The two-dimensional homonuclear experiments were measured using a proton spectral width of 4.5 kHz, collecting 2000 data points JNH-aH vicinal coupling constants were determined by two-dimensional double-quantum filtered correlation spectroscopy experiments Distance constraints and coupling constants were used in the habas program [53] of the dyana package [53] to generate /, w and v1 dihedral angle constraints and stereospecific assignments Dihedral angle constraints were calculated from the Karplus equation with A = 6.4, B = )1.4 and C = 1.9 [54] NOE intensities were determined from the NOESY (200 ms) spectra of the PG-1 analogues NOE volumes were integrated and calibrated with xeasy software [52] After internal calibration, the cross-peaks from the NOESY experiments were converted into upper distance limits with the caliba program of the dyana package [53] Based on the experimental chemical shifts of aC nuclei, CSIs were calculated relative to the sodium 3-(trimethylsilyl)-(2,2,3,3-2H4)-propionate reference [55] For cysteines, Conformational studies of protegrin-1 analogues the reference random-coil chemical shift was not reported in [55]; hence, CSIs of cysteine amino acid residues were not calculated The temperature dependence of the amide proton chemical shifts was measured in order to determine whether any of the amide protons were involved in hydrogen-bonding interactions The temperature coefficients (Dd ⁄ DT) of the amide proton chemical shifts were measured from onedimensional NMR spectra for the following temperatures: 22, 25, 27, 30, 32, 35 and 37 °C About 80% of all hydrogen-bonded amides in proteins occur in the range )5 to ppbỈdeg)1, and their average value is )3.2 ± 2.0 ppbỈdeg)1 [56] In our studies, we used the criterion of hydrogen bond formation by amide protons as a value higher than )3.0 ppbỈdeg)1, and all values more negative than )3.0 ppbỈdeg)1 indicated a lack of hydrogen bond formation Structures of the peptides studied The structures of the peptides studied were determined with xplor software, Version 3.1 [57], each structure being produced using distance and torsion angle restraints For the xplor three-dimensional structure calculations, the NOESY experiments provided 558 distance restraints for IB-367, 427 for BM-1 and 213 for the major conformation of BM-2 The habas program provided 36, 24 and 21 torsion angles for IB-367, BM-1 and BM-2 (major conformation), respectively The structures were calculated with the standard xplor program modules, as well as the charmm force field [58] in vacuo, starting from a random structure For all three molecules, 300 cycles of simulated annealing were carried out, each with 27 000 iterations of 80 ps with fs steps The molecule was maintained at 1000 K for 50 ps and annealed at 100 K for 29 ps In the last 200 iterations (1 ps), energy was minimized with Powell’s algorithm [59] During simulated annealing refinement, the molecule was slowly cooled from 1000 to 100 K over 30 ps Finally, 300 energy-minimized conformations were obtained The set of final conformations was clustered using the graphic molmol program [60]; this was also used to draw, analyse and display the electrostatic potential on the van der Waals’ surface All conformations for each peptide were ˚ divided into families at an rmsd cut-off of 5.0 A Acknowledgements The authors wish to thank the State Committee for Scientific Research for grants DS 8440-4-0172-10 and DS 8452-4-0135-9 This research was conducted using the resources of the Linux cluster at the Informatics Centre of the Metropolitan Academic Network (IC ´ MAN) in Gdansk, Poland Beata Mickiewicz expresses FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS 1019 Conformational studies of protegrin-1 analogues S Rodziewicz-Motowidło et al her gratitude to EFS, project No ZPORR ⁄ 2.22 ⁄ II ⁄ 2.6 ⁄ APR ⁄ U ⁄ ⁄ 05 References Toke O (2005) Antimicrobial peptides: new candidates in the fight against bacterial infections Biopolymers 80, 717–735 Hancock REW & Chapple DS (1999) Peptide antibiotics Antimicrob Agents Chemother 43, 1317–1323 Cho Y, Turner JS, Dinh NN & Lehrer RI (1998) Activity of protegrins against yeast-phase Candida albicans Infect Immun 66, 2486–2493 Brodgen KA, Ackermann M, McCray PB & Tack BF (2003) Antimicrobial peptides in animals and their role in host defences Int J Antimicrob Agents 22, 465–478 Lai R, Liu H, Lee WH & Zhang Y (2002) A novel proline rich bombesin-related peptide (PR-bombesin) from toad Bombina maxima Peptides 23, 437–442 Kokryakov VN, Harwig SS, Panyutich EA, Shevchenko AA, Aleshina GM, Shamova OV, Korneva HA & Lehrer RI (1993) 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dimethyl sulphoxide at 22 °C Table S4 Proton and aC chemical shifts and 3JNH–Ha coupling constants of BM-2 (minor conformation) measured in deuterated dimethyl sulphoxide at 22 °C Table S5 Structural statistics for the bundle of all calculated IB-367 structures Table S6 Structural statistics for the bundle of all calculated BM-1 structures Table S7 Structural statistics for the bundle of all calculated BM-2 (major conformation) structures This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 1010–1022 ª 2010 The Authors Journal compilation ª 2010 FEBS ... activity of the peptides A better understanding of the mode of action of these peptides is crucial for the development of a new generation of antibiotics It is known that, in the absence of both... at the two spatial tips of the molecule, at the N-terminus with Arg1 and in the turn in the presence of Arg7 and Arg9 (see Figs and 4) The shorter analogue, BM-2, was the most flexible of all the. .. mechanistic step in the killing of bacteria [16,20] The conformations of the structural features determining the antimicrobial activity of protegrins Conformational studies of protegrin-1 analogues were