FEBS Letters 588 (2014) 1821–1826
journal homepage: www.FEBSLetters.org
Structure and antimicrobial activity of platypus ‘intermediate’ defensin-like peptide Allan M. Torres a,⇑, Paramjit Bansal b, Jennifer M.S. Koh c, Guilhem Pagès d, Ming J. Wu a, Philip W. Kuchel e a
Nanoscale Organisation and Dynamics Group, School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia Queensland Tropical Health Alliance, James Cook University, Cairns, Qld 4878, Australia c Neurotoxin Research Group, School of Medical & Molecular Biosciences, University of Technology, Sydney, NSW 2007, Australia d Singapore Bioimaging Consortium, Biomedical Sciences Institutes, ASTAR, Singapore 138667, Singapore e School of Molecular Bioscience, University of Sydney, NSW 2006, Australia b
a r t i c l e
i n f o
Article history: Received 27 January 2014 Revised 12 March 2014 Accepted 23 March 2014 Available online 30 March 2014 Edited by Miguel De la Rosa Keywords: b-Defensin Defensin like peptide Intermediate-DLP NMR spectroscopy Platypus Peptide fold
a b s t r a c t The three-dimensional structure of a chemically synthesized peptide that we have called ‘intermediate’ defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and b-defensins, however the presence of a third antiparallel b-strand makes its structure more similar to the b-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency. Crown Copyright Ó 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.
1. Introduction Bioactive polypeptides such as those found in venoms, milk and other natural sources have attracted considerable attention in recent years because of their potential applications in drug discovery programs [1,2]. Such molecules can be used as tools to investigate important physiological mechanisms at the cellular and/or molecular levels. They can also be used directly as drugs, or as starting points to design new therapeutic agents. These polypeptides are often of comparatively low molecular weight and cysteine rich, and each has a specific disulfide-linked molecular framework that can be used as a scaffold to create new bioactive compounds. Defensins are good examples of such potentially useful polypeptides. These small antimicrobial proteins are found in many organisms, including mammals, birds, invertebrates and plants, Abbreviations: DLP, defensin like-peptide; Int-DLP, intermediate defensin-like peptide; Int-DLPa, intermediate defensin-like peptide analogue; OvCNP, Ornithorhyncus venom C-type natriuretic peptide; DQF-COSY, double-quantum filtered correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; RMSD, root mean square deviation ⇑ Corresponding author. Fax: +61 2 4620 3025. E-mail address: [email protected] (A.M. Torres).
and they act as antibiotics [3]. Peptides that adopt the b-defensin structural fold in particular are interesting, as they are found in a diverse range of organisms possessing disparate biological activities [4]. In humans and other mammals, b-defensins are produced in neutrophils and epithelial cells, playing an important role in the innate immune response as antimicrobial agents and as chemokines [5–9]. Polypeptides similar in fold to b-defensins have also been reported in toxins of sea anemones [10], snakes [11,12] and platypus [13,14] where they display numerous pharmacological activities, such as ion-channel inhibition, myonecrosis, and analgesia. The b-defensin-fold structure generally consists of a short helix, or turn, followed by a small twisted anti-parallel b-sheet of three strands [4]. The six cysteine residues that are paired in a 1–5, 2– 4 and 3–6 order in the primary structure are important for determining and maintaining the compact core configuration of the molecule. The low sequence similarity with other members of the same family suggests that this global fold is chemically and therefore evolutionarily robust, and that the nature of the sidechains effectively determines the functional specificity. The distinct compact fold shared by these polypeptides may therefore be useful in the design of molecules with prescribed pharmacological activity.
http://dx.doi.org/10.1016/j.febslet.2014.03.044 0014-5793/Crown Copyright Ó 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.
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The defensins from various tissues of the platypus, Ornithorhynchus anatinus, and defensin-like peptides (DLPs) from its venom present an intriguing and potentially valuable case for research study. This transpires because the platypus is a unique animal that possesses both mammalian and reptilian characteristics, thus bridging the evolutionary gap between lower and higher animal species. The male monotremes, platypus and echidna, for example, are the only known mammals to bear a venomous spur on each hind limb. Platypus envenomation in human victims causes excruciating pain, hyperalgesia, and oedema [15]. The venom contains numerous novel biologically active peptides and proteins, amongst which are natriuretic peptides called Ornithorhyncus venom C-type natriuretic peptides or OvCNPs, and a family of four polypeptides of 5 kDa called DLPs. Both the OvCNPs and DLPs are known to exist in two isomeric forms, with each pair having identical amino acid sequences but with the second amino acid residue in the D-form [16]. Various assay experiments on DLPs have so far failed to establish their role in the venom. DLPs are neither antimicrobial, myotoxic, nor do they appear to affect Na+-channel currents [14,17]. The antimicrobial defensins from the platypus are equally as intriguing as the DLPs. Six b-defensins and four a-defensins have so far been identified from platypus genome analysis [18]. The discovery of the a-defensins in the genome suggests that this type of defensin has evolved from b-defensins and that they emerged phylogenetically prior to the divergence of the three extant mammalian lineages, 210 mya. Besides this phenomenon, a new 44 amino acid residue peptide designated DEFB-VL (venom-like bdefensin) or intermediate-DLP (Int-DLP) was discovered in a gene analysis of the platypus genome [18]. Int-DLP has an amino acid sequence similar to both mammalian (including the platypus) bdefensins and the DLPs (see Fig. 1). It was suggested that this polypeptide evolved from platypus defensins to finally form the DLPs of the platypus venom, and hence we posit that it is ‘transitional’ between the two types of polypeptides. Thus, it was expected that Int-DLP would display folding characteristics that are similar to b-defensins and/or the platypus venom DLPs. Preliminary structural modeling showed that Int-DLP had a similar fold to the DLPs and may have antimicrobial activity [18]. In the present work we investigated in greater details the tertiary structure of Int-DLP in solution and its antimicrobial activity. The results obtained add to a more general understanding of the evolution of defensins and the possible role of DLPs in the platypus and potentially higher mammals. 2. Methods 2.1. Sample preparation and synthesis The 44-residue Int-DLP was synthesized on a 0.50 mmol scale using HBTU activation of Boc-amino acids with in situ neutralization chemistry, as described previously [19]. The 40-residue IntDLPa was purchased from GL Biochem Ltd. (Shanghai, China). 10
20
30
40
DLP-1
FVQHRPRDCESINGVCRHKDTVNCREIFLADCYNDGQKCCRK -----
Int-DLP
RRRRRRPPCEDVNGQCQPRGN-PC-LRLRGAC-PRGSRCCMPTVAAH C G C C RCCM
β-defensin-12 ----GPLSCGRNGGVCIPIRC-PVPMRQIGTCFGRPVKCCRSW---9
39 16
32 24
40
Fig. 1. Primary structures of DLP-1, Int-DLP, and b-defensin-12. The amino acid residues were aligned using Kalign [35] to show maximum correspondence between the structures. Shaded regions indicate identical residues. The disulfide bonding pairs are shown below the numbered sequences with dashed lines.
NMR samples were prepared by dissolving 1–2 mmol of peptide samples in 0.350 mL of H2O/D2O (9:1, v/v) in a 5-mm magnetic susceptibility matched Shigemi (Allison Park, PA, USA) NMR tube. The pH values of the samples were 3.5. 2.2. Antimicrobial activity assay Nutrient broth was prepared with 13 g of medium powder obtained from Oxoid (Australia) in 1 L of deionised H2O. The final pH of the medium was 7.0. Nutrient gel was prepared with bacteriological agar (1 g L 1), bacteriological peptone (10 g L 1), yeast extract (5 g L 1), and sodium chloride (5 g L 1). The bacteria used in the antimicrobial activity assay were Staphylococcus aureus (ATCC 12600), Escherichia coli (ATCC 11775), and Pseudomonas aeruginosa (ATCC 19582) which were obtained from the University of New South Wales [20]. Among these, S. aureus is a Gram-positive bacterium while E. coli and P. aeruginosa are Gram-negative bacteria. Liquid cultures of nutrient broth (20 mL) for each microorganism were inoculated with a single colony from the stock agar plates and grown overnight (18 h) with shaking at 150 rpm before dilution to 0.2 of OD595. Cultures were inoculated with the peptides at the final concentrations (lM) of 78, 39, 19.5, and 0, in 96-well microtitre plates with shaking at 600 rpm. Initial readings were made at the start of incubations, and the final growth was read at 24 h, using a 96-well plate reader (Multiskan EX, Thermo Electron). The screenings were carried out in duplicate. Growth inhibition values were estimated from the following expression: Average of differences in net growth between the control and treated OD595 Average of net growth of the control OD595 100%
2.3. NMR spectra and structural analysis NMR spectra were recorded on Bruker Avance III 800, 600, and 500 spectrometers with 5-mm triple resonance inverse probes, with operating temperatures of 15, 25, 30, and 35 °C. The twodimensional homonuclear proton (2D) experiments that were performed included double-quantum filtered correlation spectroscopy (DQF-COSY) [21,22]; total correlation spectroscopy (TOCSY) [23] with spin-lock periods of 60 and 90 ms; and nuclear Overhauser enhancement spectroscopy (NOESY) [24] with mixing times of 200 and 250 ms. Solvent-signal suppression was achieved by applying either presaturation or WATERGATE [25] pulse sequences. H-D exchange experiments were carried-out by adding D2O to freeze-dried NMR samples and acquiring a series of 1D spectra for at least 1 h, followed by a 5 h TOCSY spectrum. All spectra were processed using TOPSPIN software (Bruker) and were analysed using the standard protocol in the program SPARKY (T.D. Goddard and D.G. Kneller, SPARKY 3, University of California, San Francisco). 2.4. Structure calculations and analysis Distance constraints for structure calculations were obtained from cross-peak volumes in the NOESY spectra, recorded at 25 °C. Additional distant constraints from H-bonding were obtained from a hydrogen–deuterium exchange experiment after analysis of medium resolution structures. Automatic structure calculations were performed with the program CYANA [26]. The ‘best’ 20 structures with the lowest target function values were selected from the 1000 structures that were generated; these were considered to be representative of the structure of Int-DLP. The 3D structures were visualized and analyzed using the program MOLMOL [27].
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3. Results and discussion
Besides this, it was also seen that Int-DLP NMR peaks were broader than those obtained earlier for DLP-1 and DLP-2 [13,14]. To address these problems, we performed additional NMR experiments on an Int-DLP analogue that we called Int-DLPa, a shorter peptide with four arginine residues deleted from the N-terminus. It was anticipated that the deletion of these four consecutive residues would simplify and improve the quality of the NMR spectra, but not alter the overall fold of the polypeptide. NMR spectra of Int-DLPa had peaks with linewidths that were not significantly narrower than those of Int-DLP; however the deletion of the four arginine residues to make the Int-DLP analogue simplified resonance assignment by lessening the peak overlap in the pertinent regions of the spectra. Also, there were no significant changes in the chemical shifts of the remaining peaks in the IntDLPa spectra, suggesting that Int-DLPa and Int-DLP had very similar overall structural folds. By comparing the NMR spectra of Int-DLP and Int-DLPa, we able to assign most the resonances of the two polypeptides. As both sets of NMR spectra were very similar, structure calculations were performed only on the full-length Int-DLP. The summaries of the structural data from the calculated Int-DLP structures are given in Table 1.
3.1. NMR experiments
3.2. Int-DLP structures
The primary structure of Int-DLP is peculiar among those of bdefensins in that it incorporates six consecutive arginine residues at the N-terminus. This unusual property was expected to complicate the process of 1H NMR resonance (peak) assignment as it was anticipated to cause extensive peak overlap of the arginine peaks. Preliminary NMR experiments on Int-DLP indeed yielded spectra with many overlapping broad peaks (see Supplemental Fig. 1).
The 20 structures of Int-DLP of the ensemble are shown in Fig. 2A. Despite the relatively broad 1H NMR peaks, the resolution of the obtained structure was good, showing a root mean square deviation (RMSD) from the mean structure of 0.38 Å, when backbone atoms of residues 10–40 were superimposed. The N- and Ctermini of Int-DLP were also seen to be disordered in a manner similar to the DLPs [13,14,28].
Table 1 Structural statistics of the 20 Int-DLP structures. Quantity Distance restraints Intraresidue (i–j = 0) Sequential (|i–j| = 1) Medium-range (|i–j| 6 5) Long-range (|i–j| > 5) Hydrogen bonds Disulfide bonds Total Compliance with restraints CYANA average target function value Number of NOE violations > 0.30 Å Atomic rms difference with the mean (Å) Backbone atoms (10–40) Heavy atoms (10–40) Ramachandran plot statistics Most favourable region (%) Additionally allowed region (%) Generously allowed region (%) Disallowed region (%)
Value 104 138 45 130 24 9 450 0.66 0 0.38 ± 0.10 0.90 ± 0.12 58.8 38.6 2.2 0.5
Fig. 2. The calculated Int-DLP structure. (A) Ensemble of 20 Int-DLP structures aligned by superimposing the backbone atoms of residues 10–40. (B) Ribbon diagram showing secondary structures and disulfide connectivities. (C) Similar to (B) but rotated 90° about the assigned vertical axis. (D–F), Molecular surface of Int-DLP highlighted to show electrostatic potential. Surfaces with positive, negative and neutral electrostatic potentials are drawn in blue, red and white, respectively. (D) Molecular orientation similar to (B). (E) Similar to (D) but rotated 90° about the assigned vertical axis. (F) Similar to (D) but rotated 180° about the assigned vertical axis. The figures were generated using MOLMOL [27].
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DLP-1
Int-DLP
β-defensin-12
Fig. 3. Comparison of three-dimensional structural folds of DLP-1, Int-DLP and bdefensin-12. The figures were generated using MOLMOL [27].
As shown in Fig. 2B and C, Int-DLP has a compact tertiary fold containing a helix and a b-sheet, laced together by three disulfide bonds. This overall fold was analysed using PDBefold structure search algorithm [29] and was found to be very similar with that of b-defensins and the DLPs, as illustrated in Fig. 3. The disulfide connectivities of 1–5, 2–4 and 3–6 (revealed by NMR spectroscopy) are identical with that of the DLPs. The presence and locations of the short sections of secondary structures in Int-DLP and the DLPs were also very similar. The structures of DLP-1, -2 and -4 incorporate anti-parallel b-sheets from residues 15–18 and 37–40, and a helix-like structure from residues 10–12 [13,14,28]. In comparison, the Int-DLP structure contained a definite anti-parallel b-sheet that encompassed residues 15–17 and 35–38 and it also contained an a-helix from residues 8–11. 3.3. Presence of third antiparallel b-strand as in b-defensin Unlike the DLPs from the platypus, which possess only two b-strands, many anti-microbial b-defensins from mammalian sources, such as hBD-1, hBD-2 and b-defensin 12, incorporate three b-strands [4,9,30,31]. Int-DLP displayed a third b-strand similar to the b-defensins encompassing residues 24–27 with a b-bulge at residues 26–27 (see Fig. 3). The chemical shift deviations from random coil values, as shown in Supplemental Fig. 2, also support the presence of this b-strand but suggest that that this strand could be longer, perhaps spanning residues 21–26.
20 15 10 5 0
E. coli (ATCC 11775)
It is clear that the third b-strand in Int-DLP makes its overall structure more similar to b-defensins than to DLPs. Although the cysteine spacing in Int-DLP is more similar to other b-defensins in the platypus, the amino acid sequence of Int-DLP is more similar to DLPs than to b-defensins [18]. The N-terminus that incorporates four arginine residues was found to be disordered (and could be flexible) due to the dearth of medium and long-range NOESY cross-peaks corresponding to this part of the molecule. These Nterminal residues did not appear to have a significant role in the overall fold of Int-DLP as the chemical shifts of the 1H atoms of Arg 5 and Arg 6 in Int-DLP and Int-DLPa were very similar. 3.4. Antimicrobial activity The antimicrobial activity of Int-DLP and Int-DLPa were tested against three bacterial species viz., S. aureus, E. coli, and P. aeruginosa. Fig. 4 shows the antimicrobial activity of the Int-DLP polypeptide with various concentrations, expressed as a percentage of growth inhibition in the three different bacterial cultures. Although growth inhibition was observed for all three species, there were significant differences in the potency of the peptides. Both peptides showed consistent activity against S. aureus and the least activity against E. coli. For E. coli, the very slight activity (at less than 5% growth inhibition at 39 lM polypeptide) observed for both polypeptides suggests insignificant or no activity against this microorganism. For S. aureus, a consistent activity of 10% growth inhibition occurred with the two peptides at concentrations of 20–78 lM. Int-DLPa showed uniform activity of 8–9% at concentrations of 20–78 lM, while Int-DLP showed slightly higher activity of 12% at 39 and 78 lM; but its activity decreased by 30% when its concentration was decreased to 20 lM. With P. aeruginosa, there were significant differences in the antimicrobial activities of the two polypeptides. With 78 lM IntDLP the growth inhibition was 18%, which was 3 times higher than the 7% obtained with Int-DLPa. Unlike that seen with S. aureus, growth inhibitions in P. aeruginosa for the two polypeptides decreased almost linearly with concentration. As clearly shown by our results, Int-DLP and Int-DLPa had antimicrobial properties, unlike the DLPs which had none [13,14]. Int-DLP was seen to be more potent than Int-DLPa against
20 15 10 5 0
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM) 20 15 10 5 0
S. aureus (ATCC 12600)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM) 20 15 10 5 0
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM)
20 15 10 5 0
P. aeruginosa (ATCC 19582)
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM)
E. coli (ATCC 11775)
S. aureus (ATCC 12600)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM)
20 15 10 5 0
P. aeruginosa (ATCC 19582)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM)
Fig. 4. Antimicrobial activities of Int-DLP and Int-DLPa. The antimicrobial activities of both of the peptides against E. coli (ATCC 11775), S. aureus (ATCC 12600), and P. aeruginosa (ATCC 19582) are expressed as a percentage of growth inhibition. Data represent the average of two replicates with very small standard deviations shown by the horizontal bars.
A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826
P. aeruginosa. It is surmised that the deletion of the four arginine residues in Int-DLPa caused the observed decrease in activity and that these arginine residues are important for Int-DLP antimicrobial potency. This is consistent with the known activity of stretches of arginine and lysine residues that mediate cell entry of membrane-crossing peptides like penetratin [32]. The antimicrobial activity of Int-DLP is not surprising as it can be readily explained by the obtained 1H NMR-based structure. As shown in the surface diagrams in Fig. 2D–F, there is a clear separation between various types of amino acid in the Int-DLP molecule. The membrane-active cationic arginine residues (blue surface) are largely situated on one side of the molecule while clusters of hydrophobic residues (white surface) are situated on the other side. The amphipathic nature of Int-DLP is similar to that of defensins, such as b-defensin-12. It is believed that this structural feature is responsible for defensins antimicrobial activities as it facilitates interaction with targets such as bacterial membranes [5]. An interesting feature of the Int-DLP structure is the cluster of cationic residues. In human b-defensins, hBD-1, hBD-2, and hBD-3, these membrane active basic residues are located at the C-terminus [3] while in Int-DLP they are at the N-terminus. Note that in antimicrobial studies of hBD-3 analogues [33] a linear peptide fragment corresponding to the C-terminus gave enhanced activity against E. coli, a Gram-negative bacterium, while the linear fragment corresponding to the N-terminus was active against S. aureus, a Gram-positive bacterium. Since the position of the basic residues in Int-DLP is reversed, in contrast to that of human b-defensins, it might be expected that the N-terminus of Int-DLP is important for ‘attacking’ Gram-negative bacteria. While no significant activity against E. coli was detected for Int-DLP, it was evident that the four arginine residues at its Nterminus were important for activity against P. aeruginosa, a Gram-negative bacterium. In addition to the previously mentioned structural feature, we suggest that the proline residues in Int-DLP play a significant part in the overall fold and hence the antibacterial activity. As shown in Fig. 1, b-defensin-12 and Int-DLP have four and five proline residues, respectively, but DLP-1 has only one. The presence of two consecutive proline residues near the N-terminus of Int-DLP probably gives a specific orientation to the six cationic arginine charged groups so that they interact with the cell membrane leading to enhanced antimicrobial activity. In the same manner, the presence of other proline residues at the same, or nearly same, position in the sequence of Int-DLP and b-defensin-12 makes their structures more similar and hence a similar antimicrobial activity or spectrum. 4. Concluding remarks Overall, we determined the structural fold and antimicrobial activity of another polypeptide from the platypus genome: IntDLP displays the robust structural fold of the b-defensin family of polypeptides, incorporating a helix and a triple stranded b-sheet. It also exhibited antibacterial activity although gene and amino acid sequence analyses show that Int-DLP is more similar to DLPs, which are devoid of antimicrobial activity, than to b-defensins. It is possible that Int-DLP constitutes an evolutionary link between the b-defensins and the DLPs. Further phylogenetic study similar to that performed on fungal defensin-like peptides [34] will help determine if this ‘intermediate’ molecule has indeed evolved from b-defensins and is the precursor of the DLPs. It would also be interesting to characterise the other unique defensins that have been identified in the platypus genome [18] and to compare their structures and antimicrobial activities with those of other mammalian defensins, and with Int-DLP. Such future studies may yield
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effective antimicrobials that can be used to treat newly emergent drug-resistant bacteria that have been inadvertently selected by overuse of conventional antibiotics. Acknowledgements The work was supported by a Discovery Project Grant from the Australian Research Council to PWK. We thank Dr Ann Kwan for implementing some of the 2D NMR experiments. We also thank Professor Kathy Belov and Dr Camilla Whittington for valuable discussions on platypus genomics. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. 03.044. References [1] Harvey, A.L. (2002) Toxins ‘R’ Us: more pharmacological tools from nature’s superstore. Trends Pharmacol. Sci. 23, 201–203. [2] Harvey, A.L., Bradley, K.N., Cochran, S.A., Rowan, E.G., Pratt, J.A., Quillfeldt, J.A. and Jerusalinsky, D.A. (1998) What can toxins tell us for drug discovery? Toxicon 36, 1635–1640. [3] Pazgier, M., Hoover, D.M., Yang, D., Lu, W. and Lubkowski, J. (2006) Human bdefensins. Cell. Mol. Life Sci. 63, 1294–1313. [4] Torres, A.M. and Kuchel, P.W. (2004) The b-defensin-fold family of polypeptides. Toxicon 44, 581–588. [5] Zimmermann, G.R., Legault, P., Selsted, M.E. and Pardi, A. (1995) Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the betadefensins is identical to that of the classical defensins. Biochemistry (Mosc.) 34, 13663–13671. [6] Hoover, D.M., Rajashankar, K.R., Blumenthal, R., Puri, A., Oppenheim, J.J., Chertov, O. and Lubkowski, J. (2000) The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J. Biol. Chem. 275, 32911– 32918. [7] Hoover, D.M., Chertov, O. and Lubkowski, J. (2001) The structure of human beta-defensin-1: new insights into structural properties of beta-defensins. J. Biol. Chem. 276, 39021–39026. [8] Schibli, D.J., Hunter, H.N., Aseyev, V., Starner, T.D., Wiencek, J.M., McCray Jr., P.B., Tack, B.F. and Vogel, H.J. (2002) The solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus. J. Biol. Chem. 277, 8279–8289. [9] Bensch, K.W., Raida, M., Magert, H.J., Schulz-Knappe, P. and Forssmann, W.G. (1995) HBD-1: a novel beta-defensin from human plasma. FEBS Lett. 368, 331– 335. [10] Wilcox, G.R., Fogh, R.H. and Norton, R.S. (1993) Refined structure in solution of the sea anemone neurotoxin ShI. J. Biol. Chem. 268, 24707–24719. [11] Siqueira, A.M., Martins, N.F., De Lima, M.E., Diniz, C.R., Cartier, A., Brown, D. and Maigret, B. (2002) A proposed 3D structure for crotamine based on homology building, molecular simulations and circular dichroism. J. Mol. Graph. Model. 20, 389–398. [12] Nicastro, G., Franzoni, L., de Chiara, C., Mancin, A.C., Giglio, J.R. and Spisni, A. (2003) Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur. J. Biochem. 270, 1969–1979. [13] Torres, A.M., Wang, X., Fletcher, J.I., Alewood, D., Alewood, P.F., Smith, R., Simpson, R.J., Nicholson, G.M., Sutherland, S.K., Gallagher, C.H., King, G.F. and Kuchel, P.W. (1999) Solution structure of a defensin-like peptide from platypus venom. Biochem. J. 341, 785–794. [14] Torres, A.M., de Plater, G.M., Doverskog, M., Birinyi-Strachan, L.C., Nicholson, G.M., Gallagher, C.H. and Kuchel, P.W. (2000) Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold. Biochem. J. 348, 649–656. [15] Fenner, P.J., Williamson, J.A. and Myers, D. (1992) Platypus envenomation – a painful learning experience. Med. J. Aust. 157, 829–832. [16] Torres, A.M., Tsampazi, M., Tsampazi, C., Kennet, E.C., Belov, K., Geraghty, D.P., Bansal, P.S., Alewood, P.F. and Kuchel, P.W. (2006) Mammalian L-to-D-aminoacid-residue isomerase from platypus venom. FEBS Lett. 580, 1587–1591. [17] Torres, A.M., Bansal, P., Alewood, P.F., Bursill, J.A., Kuchel, P.W. and Vandenberg, J.I. (2003) Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin. FEBS Lett. 539, 138–142. [18] Whittington, C.M., Papenfuss, A.T., Bansal, P., Torres, A.M., Wong, E.S.W., Deakin, J.E., Graves, T., Alsop, A., Schatzkamer, K., Kremitzki, C., Ponting, C.P., Temple-Smith, P., Warren, W.C., Kuchel, P.W. and Belov, K. (2008) Defensins and the convergent evolution of platypus and reptile venom genes. Genome Res. 18, 986–994. [19] Schnolzer, M., Alewood, P., Jones, A., Alewood, D. and Kent, S.B. (1992) In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40, 180–193.
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[28] Torres, A.M., Tsampazi, C., Geraghty, D.P., Bansal, P.S., Alewood, P.F. and Kuchel, P.W. (2005) D-amino-acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties. Biochem. J. 391, 215–220. [29] Krissinel, E. and Henrick, K. (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268. [30] Harder, J., Bartels, J., Christophers, E. and Schroder, J.M. (1997) A peptide antibiotic from human skin. Nature 387, 861. [31] Selsted, M.E., Tang, Y.Q., Morris, W.L., McGuire, P.A., Novotny, M.J., Smith, W., Henschen, A.H. and Cullor, J.S. (1993) Purification, primary structures, and antibacterial activities of b-defensins, a new family of antimicrobial peptides from bovine neutrophils. J. Biol. Chem. 268, 6641–6648. [32] Derossi, D., Joliot, A.H., Chassaing, G. and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J. Biol. Chem. 269, 10444–10450. [33] Hoover, D.M., Wu, Z., Tucker, K., Lu, W. and Lubkowski, J. (2003) Antimicrobial characterization of human b-defensin 3 derivatives. Antimicrob. Agents Chemother. 47, 2804–2809. [34] Zhu, S. (2008) Discovery of six families of fungal defensin-like peptides provides insights into origin and evolution of the CSab defensins. Mol. Immunol. 45, 828–838. [35] Lassmann, T. and Sonnhammer, E.L. (2005) Kalign – an accurate and fast multiple sequence alignment algorithm. BMC Bioinformatics 6, 298.
journal homepage: www.FEBSLetters.org
Structure and antimicrobial activity of platypus ‘intermediate’ defensin-like peptide Allan M. Torres a,⇑, Paramjit Bansal b, Jennifer M.S. Koh c, Guilhem Pagès d, Ming J. Wu a, Philip W. Kuchel e a
Nanoscale Organisation and Dynamics Group, School of Science and Health, University of Western Sydney, Penrith, NSW 2751, Australia Queensland Tropical Health Alliance, James Cook University, Cairns, Qld 4878, Australia c Neurotoxin Research Group, School of Medical & Molecular Biosciences, University of Technology, Sydney, NSW 2007, Australia d Singapore Bioimaging Consortium, Biomedical Sciences Institutes, ASTAR, Singapore 138667, Singapore e School of Molecular Bioscience, University of Sydney, NSW 2006, Australia b
a r t i c l e
i n f o
Article history: Received 27 January 2014 Revised 12 March 2014 Accepted 23 March 2014 Available online 30 March 2014 Edited by Miguel De la Rosa Keywords: b-Defensin Defensin like peptide Intermediate-DLP NMR spectroscopy Platypus Peptide fold
a b s t r a c t The three-dimensional structure of a chemically synthesized peptide that we have called ‘intermediate’ defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear magnetic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overall structural fold of Int-DLP was similar to that of the DLPs and b-defensins, however the presence of a third antiparallel b-strand makes its structure more similar to the b-defensins than the DLPs. Int-DLP displayed potent antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. The four arginine residues at the N-terminus of Int-DLP did not affect the overall fold, but were important for its antimicrobial potency. Crown Copyright Ó 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.
1. Introduction Bioactive polypeptides such as those found in venoms, milk and other natural sources have attracted considerable attention in recent years because of their potential applications in drug discovery programs [1,2]. Such molecules can be used as tools to investigate important physiological mechanisms at the cellular and/or molecular levels. They can also be used directly as drugs, or as starting points to design new therapeutic agents. These polypeptides are often of comparatively low molecular weight and cysteine rich, and each has a specific disulfide-linked molecular framework that can be used as a scaffold to create new bioactive compounds. Defensins are good examples of such potentially useful polypeptides. These small antimicrobial proteins are found in many organisms, including mammals, birds, invertebrates and plants, Abbreviations: DLP, defensin like-peptide; Int-DLP, intermediate defensin-like peptide; Int-DLPa, intermediate defensin-like peptide analogue; OvCNP, Ornithorhyncus venom C-type natriuretic peptide; DQF-COSY, double-quantum filtered correlation spectroscopy; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; RMSD, root mean square deviation ⇑ Corresponding author. Fax: +61 2 4620 3025. E-mail address: [email protected] (A.M. Torres).
and they act as antibiotics [3]. Peptides that adopt the b-defensin structural fold in particular are interesting, as they are found in a diverse range of organisms possessing disparate biological activities [4]. In humans and other mammals, b-defensins are produced in neutrophils and epithelial cells, playing an important role in the innate immune response as antimicrobial agents and as chemokines [5–9]. Polypeptides similar in fold to b-defensins have also been reported in toxins of sea anemones [10], snakes [11,12] and platypus [13,14] where they display numerous pharmacological activities, such as ion-channel inhibition, myonecrosis, and analgesia. The b-defensin-fold structure generally consists of a short helix, or turn, followed by a small twisted anti-parallel b-sheet of three strands [4]. The six cysteine residues that are paired in a 1–5, 2– 4 and 3–6 order in the primary structure are important for determining and maintaining the compact core configuration of the molecule. The low sequence similarity with other members of the same family suggests that this global fold is chemically and therefore evolutionarily robust, and that the nature of the sidechains effectively determines the functional specificity. The distinct compact fold shared by these polypeptides may therefore be useful in the design of molecules with prescribed pharmacological activity.
http://dx.doi.org/10.1016/j.febslet.2014.03.044 0014-5793/Crown Copyright Ó 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.
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The defensins from various tissues of the platypus, Ornithorhynchus anatinus, and defensin-like peptides (DLPs) from its venom present an intriguing and potentially valuable case for research study. This transpires because the platypus is a unique animal that possesses both mammalian and reptilian characteristics, thus bridging the evolutionary gap between lower and higher animal species. The male monotremes, platypus and echidna, for example, are the only known mammals to bear a venomous spur on each hind limb. Platypus envenomation in human victims causes excruciating pain, hyperalgesia, and oedema [15]. The venom contains numerous novel biologically active peptides and proteins, amongst which are natriuretic peptides called Ornithorhyncus venom C-type natriuretic peptides or OvCNPs, and a family of four polypeptides of 5 kDa called DLPs. Both the OvCNPs and DLPs are known to exist in two isomeric forms, with each pair having identical amino acid sequences but with the second amino acid residue in the D-form [16]. Various assay experiments on DLPs have so far failed to establish their role in the venom. DLPs are neither antimicrobial, myotoxic, nor do they appear to affect Na+-channel currents [14,17]. The antimicrobial defensins from the platypus are equally as intriguing as the DLPs. Six b-defensins and four a-defensins have so far been identified from platypus genome analysis [18]. The discovery of the a-defensins in the genome suggests that this type of defensin has evolved from b-defensins and that they emerged phylogenetically prior to the divergence of the three extant mammalian lineages, 210 mya. Besides this phenomenon, a new 44 amino acid residue peptide designated DEFB-VL (venom-like bdefensin) or intermediate-DLP (Int-DLP) was discovered in a gene analysis of the platypus genome [18]. Int-DLP has an amino acid sequence similar to both mammalian (including the platypus) bdefensins and the DLPs (see Fig. 1). It was suggested that this polypeptide evolved from platypus defensins to finally form the DLPs of the platypus venom, and hence we posit that it is ‘transitional’ between the two types of polypeptides. Thus, it was expected that Int-DLP would display folding characteristics that are similar to b-defensins and/or the platypus venom DLPs. Preliminary structural modeling showed that Int-DLP had a similar fold to the DLPs and may have antimicrobial activity [18]. In the present work we investigated in greater details the tertiary structure of Int-DLP in solution and its antimicrobial activity. The results obtained add to a more general understanding of the evolution of defensins and the possible role of DLPs in the platypus and potentially higher mammals. 2. Methods 2.1. Sample preparation and synthesis The 44-residue Int-DLP was synthesized on a 0.50 mmol scale using HBTU activation of Boc-amino acids with in situ neutralization chemistry, as described previously [19]. The 40-residue IntDLPa was purchased from GL Biochem Ltd. (Shanghai, China). 10
20
30
40
DLP-1
FVQHRPRDCESINGVCRHKDTVNCREIFLADCYNDGQKCCRK -----
Int-DLP
RRRRRRPPCEDVNGQCQPRGN-PC-LRLRGAC-PRGSRCCMPTVAAH C G C C RCCM
β-defensin-12 ----GPLSCGRNGGVCIPIRC-PVPMRQIGTCFGRPVKCCRSW---9
39 16
32 24
40
Fig. 1. Primary structures of DLP-1, Int-DLP, and b-defensin-12. The amino acid residues were aligned using Kalign [35] to show maximum correspondence between the structures. Shaded regions indicate identical residues. The disulfide bonding pairs are shown below the numbered sequences with dashed lines.
NMR samples were prepared by dissolving 1–2 mmol of peptide samples in 0.350 mL of H2O/D2O (9:1, v/v) in a 5-mm magnetic susceptibility matched Shigemi (Allison Park, PA, USA) NMR tube. The pH values of the samples were 3.5. 2.2. Antimicrobial activity assay Nutrient broth was prepared with 13 g of medium powder obtained from Oxoid (Australia) in 1 L of deionised H2O. The final pH of the medium was 7.0. Nutrient gel was prepared with bacteriological agar (1 g L 1), bacteriological peptone (10 g L 1), yeast extract (5 g L 1), and sodium chloride (5 g L 1). The bacteria used in the antimicrobial activity assay were Staphylococcus aureus (ATCC 12600), Escherichia coli (ATCC 11775), and Pseudomonas aeruginosa (ATCC 19582) which were obtained from the University of New South Wales [20]. Among these, S. aureus is a Gram-positive bacterium while E. coli and P. aeruginosa are Gram-negative bacteria. Liquid cultures of nutrient broth (20 mL) for each microorganism were inoculated with a single colony from the stock agar plates and grown overnight (18 h) with shaking at 150 rpm before dilution to 0.2 of OD595. Cultures were inoculated with the peptides at the final concentrations (lM) of 78, 39, 19.5, and 0, in 96-well microtitre plates with shaking at 600 rpm. Initial readings were made at the start of incubations, and the final growth was read at 24 h, using a 96-well plate reader (Multiskan EX, Thermo Electron). The screenings were carried out in duplicate. Growth inhibition values were estimated from the following expression: Average of differences in net growth between the control and treated OD595 Average of net growth of the control OD595 100%
2.3. NMR spectra and structural analysis NMR spectra were recorded on Bruker Avance III 800, 600, and 500 spectrometers with 5-mm triple resonance inverse probes, with operating temperatures of 15, 25, 30, and 35 °C. The twodimensional homonuclear proton (2D) experiments that were performed included double-quantum filtered correlation spectroscopy (DQF-COSY) [21,22]; total correlation spectroscopy (TOCSY) [23] with spin-lock periods of 60 and 90 ms; and nuclear Overhauser enhancement spectroscopy (NOESY) [24] with mixing times of 200 and 250 ms. Solvent-signal suppression was achieved by applying either presaturation or WATERGATE [25] pulse sequences. H-D exchange experiments were carried-out by adding D2O to freeze-dried NMR samples and acquiring a series of 1D spectra for at least 1 h, followed by a 5 h TOCSY spectrum. All spectra were processed using TOPSPIN software (Bruker) and were analysed using the standard protocol in the program SPARKY (T.D. Goddard and D.G. Kneller, SPARKY 3, University of California, San Francisco). 2.4. Structure calculations and analysis Distance constraints for structure calculations were obtained from cross-peak volumes in the NOESY spectra, recorded at 25 °C. Additional distant constraints from H-bonding were obtained from a hydrogen–deuterium exchange experiment after analysis of medium resolution structures. Automatic structure calculations were performed with the program CYANA [26]. The ‘best’ 20 structures with the lowest target function values were selected from the 1000 structures that were generated; these were considered to be representative of the structure of Int-DLP. The 3D structures were visualized and analyzed using the program MOLMOL [27].
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3. Results and discussion
Besides this, it was also seen that Int-DLP NMR peaks were broader than those obtained earlier for DLP-1 and DLP-2 [13,14]. To address these problems, we performed additional NMR experiments on an Int-DLP analogue that we called Int-DLPa, a shorter peptide with four arginine residues deleted from the N-terminus. It was anticipated that the deletion of these four consecutive residues would simplify and improve the quality of the NMR spectra, but not alter the overall fold of the polypeptide. NMR spectra of Int-DLPa had peaks with linewidths that were not significantly narrower than those of Int-DLP; however the deletion of the four arginine residues to make the Int-DLP analogue simplified resonance assignment by lessening the peak overlap in the pertinent regions of the spectra. Also, there were no significant changes in the chemical shifts of the remaining peaks in the IntDLPa spectra, suggesting that Int-DLPa and Int-DLP had very similar overall structural folds. By comparing the NMR spectra of Int-DLP and Int-DLPa, we able to assign most the resonances of the two polypeptides. As both sets of NMR spectra were very similar, structure calculations were performed only on the full-length Int-DLP. The summaries of the structural data from the calculated Int-DLP structures are given in Table 1.
3.1. NMR experiments
3.2. Int-DLP structures
The primary structure of Int-DLP is peculiar among those of bdefensins in that it incorporates six consecutive arginine residues at the N-terminus. This unusual property was expected to complicate the process of 1H NMR resonance (peak) assignment as it was anticipated to cause extensive peak overlap of the arginine peaks. Preliminary NMR experiments on Int-DLP indeed yielded spectra with many overlapping broad peaks (see Supplemental Fig. 1).
The 20 structures of Int-DLP of the ensemble are shown in Fig. 2A. Despite the relatively broad 1H NMR peaks, the resolution of the obtained structure was good, showing a root mean square deviation (RMSD) from the mean structure of 0.38 Å, when backbone atoms of residues 10–40 were superimposed. The N- and Ctermini of Int-DLP were also seen to be disordered in a manner similar to the DLPs [13,14,28].
Table 1 Structural statistics of the 20 Int-DLP structures. Quantity Distance restraints Intraresidue (i–j = 0) Sequential (|i–j| = 1) Medium-range (|i–j| 6 5) Long-range (|i–j| > 5) Hydrogen bonds Disulfide bonds Total Compliance with restraints CYANA average target function value Number of NOE violations > 0.30 Å Atomic rms difference with the mean (Å) Backbone atoms (10–40) Heavy atoms (10–40) Ramachandran plot statistics Most favourable region (%) Additionally allowed region (%) Generously allowed region (%) Disallowed region (%)
Value 104 138 45 130 24 9 450 0.66 0 0.38 ± 0.10 0.90 ± 0.12 58.8 38.6 2.2 0.5
Fig. 2. The calculated Int-DLP structure. (A) Ensemble of 20 Int-DLP structures aligned by superimposing the backbone atoms of residues 10–40. (B) Ribbon diagram showing secondary structures and disulfide connectivities. (C) Similar to (B) but rotated 90° about the assigned vertical axis. (D–F), Molecular surface of Int-DLP highlighted to show electrostatic potential. Surfaces with positive, negative and neutral electrostatic potentials are drawn in blue, red and white, respectively. (D) Molecular orientation similar to (B). (E) Similar to (D) but rotated 90° about the assigned vertical axis. (F) Similar to (D) but rotated 180° about the assigned vertical axis. The figures were generated using MOLMOL [27].
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DLP-1
Int-DLP
β-defensin-12
Fig. 3. Comparison of three-dimensional structural folds of DLP-1, Int-DLP and bdefensin-12. The figures were generated using MOLMOL [27].
As shown in Fig. 2B and C, Int-DLP has a compact tertiary fold containing a helix and a b-sheet, laced together by three disulfide bonds. This overall fold was analysed using PDBefold structure search algorithm [29] and was found to be very similar with that of b-defensins and the DLPs, as illustrated in Fig. 3. The disulfide connectivities of 1–5, 2–4 and 3–6 (revealed by NMR spectroscopy) are identical with that of the DLPs. The presence and locations of the short sections of secondary structures in Int-DLP and the DLPs were also very similar. The structures of DLP-1, -2 and -4 incorporate anti-parallel b-sheets from residues 15–18 and 37–40, and a helix-like structure from residues 10–12 [13,14,28]. In comparison, the Int-DLP structure contained a definite anti-parallel b-sheet that encompassed residues 15–17 and 35–38 and it also contained an a-helix from residues 8–11. 3.3. Presence of third antiparallel b-strand as in b-defensin Unlike the DLPs from the platypus, which possess only two b-strands, many anti-microbial b-defensins from mammalian sources, such as hBD-1, hBD-2 and b-defensin 12, incorporate three b-strands [4,9,30,31]. Int-DLP displayed a third b-strand similar to the b-defensins encompassing residues 24–27 with a b-bulge at residues 26–27 (see Fig. 3). The chemical shift deviations from random coil values, as shown in Supplemental Fig. 2, also support the presence of this b-strand but suggest that that this strand could be longer, perhaps spanning residues 21–26.
20 15 10 5 0
E. coli (ATCC 11775)
It is clear that the third b-strand in Int-DLP makes its overall structure more similar to b-defensins than to DLPs. Although the cysteine spacing in Int-DLP is more similar to other b-defensins in the platypus, the amino acid sequence of Int-DLP is more similar to DLPs than to b-defensins [18]. The N-terminus that incorporates four arginine residues was found to be disordered (and could be flexible) due to the dearth of medium and long-range NOESY cross-peaks corresponding to this part of the molecule. These Nterminal residues did not appear to have a significant role in the overall fold of Int-DLP as the chemical shifts of the 1H atoms of Arg 5 and Arg 6 in Int-DLP and Int-DLPa were very similar. 3.4. Antimicrobial activity The antimicrobial activity of Int-DLP and Int-DLPa were tested against three bacterial species viz., S. aureus, E. coli, and P. aeruginosa. Fig. 4 shows the antimicrobial activity of the Int-DLP polypeptide with various concentrations, expressed as a percentage of growth inhibition in the three different bacterial cultures. Although growth inhibition was observed for all three species, there were significant differences in the potency of the peptides. Both peptides showed consistent activity against S. aureus and the least activity against E. coli. For E. coli, the very slight activity (at less than 5% growth inhibition at 39 lM polypeptide) observed for both polypeptides suggests insignificant or no activity against this microorganism. For S. aureus, a consistent activity of 10% growth inhibition occurred with the two peptides at concentrations of 20–78 lM. Int-DLPa showed uniform activity of 8–9% at concentrations of 20–78 lM, while Int-DLP showed slightly higher activity of 12% at 39 and 78 lM; but its activity decreased by 30% when its concentration was decreased to 20 lM. With P. aeruginosa, there were significant differences in the antimicrobial activities of the two polypeptides. With 78 lM IntDLP the growth inhibition was 18%, which was 3 times higher than the 7% obtained with Int-DLPa. Unlike that seen with S. aureus, growth inhibitions in P. aeruginosa for the two polypeptides decreased almost linearly with concentration. As clearly shown by our results, Int-DLP and Int-DLPa had antimicrobial properties, unlike the DLPs which had none [13,14]. Int-DLP was seen to be more potent than Int-DLPa against
20 15 10 5 0
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM) 20 15 10 5 0
S. aureus (ATCC 12600)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM) 20 15 10 5 0
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM)
20 15 10 5 0
P. aeruginosa (ATCC 19582)
Int-DLPa Int-DLPa Int-DLPa Int-DLPa (78 µM) (39 µM) (19.5 µM) (0 µM)
E. coli (ATCC 11775)
S. aureus (ATCC 12600)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM)
20 15 10 5 0
P. aeruginosa (ATCC 19582)
Int-DLP Int-DLP Int-DLP Int-DLP (78 µM) (39 µM) (19.5 µM) (0 µM)
Fig. 4. Antimicrobial activities of Int-DLP and Int-DLPa. The antimicrobial activities of both of the peptides against E. coli (ATCC 11775), S. aureus (ATCC 12600), and P. aeruginosa (ATCC 19582) are expressed as a percentage of growth inhibition. Data represent the average of two replicates with very small standard deviations shown by the horizontal bars.
A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826
P. aeruginosa. It is surmised that the deletion of the four arginine residues in Int-DLPa caused the observed decrease in activity and that these arginine residues are important for Int-DLP antimicrobial potency. This is consistent with the known activity of stretches of arginine and lysine residues that mediate cell entry of membrane-crossing peptides like penetratin [32]. The antimicrobial activity of Int-DLP is not surprising as it can be readily explained by the obtained 1H NMR-based structure. As shown in the surface diagrams in Fig. 2D–F, there is a clear separation between various types of amino acid in the Int-DLP molecule. The membrane-active cationic arginine residues (blue surface) are largely situated on one side of the molecule while clusters of hydrophobic residues (white surface) are situated on the other side. The amphipathic nature of Int-DLP is similar to that of defensins, such as b-defensin-12. It is believed that this structural feature is responsible for defensins antimicrobial activities as it facilitates interaction with targets such as bacterial membranes [5]. An interesting feature of the Int-DLP structure is the cluster of cationic residues. In human b-defensins, hBD-1, hBD-2, and hBD-3, these membrane active basic residues are located at the C-terminus [3] while in Int-DLP they are at the N-terminus. Note that in antimicrobial studies of hBD-3 analogues [33] a linear peptide fragment corresponding to the C-terminus gave enhanced activity against E. coli, a Gram-negative bacterium, while the linear fragment corresponding to the N-terminus was active against S. aureus, a Gram-positive bacterium. Since the position of the basic residues in Int-DLP is reversed, in contrast to that of human b-defensins, it might be expected that the N-terminus of Int-DLP is important for ‘attacking’ Gram-negative bacteria. While no significant activity against E. coli was detected for Int-DLP, it was evident that the four arginine residues at its Nterminus were important for activity against P. aeruginosa, a Gram-negative bacterium. In addition to the previously mentioned structural feature, we suggest that the proline residues in Int-DLP play a significant part in the overall fold and hence the antibacterial activity. As shown in Fig. 1, b-defensin-12 and Int-DLP have four and five proline residues, respectively, but DLP-1 has only one. The presence of two consecutive proline residues near the N-terminus of Int-DLP probably gives a specific orientation to the six cationic arginine charged groups so that they interact with the cell membrane leading to enhanced antimicrobial activity. In the same manner, the presence of other proline residues at the same, or nearly same, position in the sequence of Int-DLP and b-defensin-12 makes their structures more similar and hence a similar antimicrobial activity or spectrum. 4. Concluding remarks Overall, we determined the structural fold and antimicrobial activity of another polypeptide from the platypus genome: IntDLP displays the robust structural fold of the b-defensin family of polypeptides, incorporating a helix and a triple stranded b-sheet. It also exhibited antibacterial activity although gene and amino acid sequence analyses show that Int-DLP is more similar to DLPs, which are devoid of antimicrobial activity, than to b-defensins. It is possible that Int-DLP constitutes an evolutionary link between the b-defensins and the DLPs. Further phylogenetic study similar to that performed on fungal defensin-like peptides [34] will help determine if this ‘intermediate’ molecule has indeed evolved from b-defensins and is the precursor of the DLPs. It would also be interesting to characterise the other unique defensins that have been identified in the platypus genome [18] and to compare their structures and antimicrobial activities with those of other mammalian defensins, and with Int-DLP. Such future studies may yield
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effective antimicrobials that can be used to treat newly emergent drug-resistant bacteria that have been inadvertently selected by overuse of conventional antibiotics. Acknowledgements The work was supported by a Discovery Project Grant from the Australian Research Council to PWK. We thank Dr Ann Kwan for implementing some of the 2D NMR experiments. We also thank Professor Kathy Belov and Dr Camilla Whittington for valuable discussions on platypus genomics. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2014. 03.044. References [1] Harvey, A.L. (2002) Toxins ‘R’ Us: more pharmacological tools from nature’s superstore. Trends Pharmacol. Sci. 23, 201–203. [2] Harvey, A.L., Bradley, K.N., Cochran, S.A., Rowan, E.G., Pratt, J.A., Quillfeldt, J.A. and Jerusalinsky, D.A. (1998) What can toxins tell us for drug discovery? Toxicon 36, 1635–1640. [3] Pazgier, M., Hoover, D.M., Yang, D., Lu, W. and Lubkowski, J. (2006) Human bdefensins. Cell. Mol. Life Sci. 63, 1294–1313. [4] Torres, A.M. and Kuchel, P.W. (2004) The b-defensin-fold family of polypeptides. Toxicon 44, 581–588. [5] Zimmermann, G.R., Legault, P., Selsted, M.E. and Pardi, A. (1995) Solution structure of bovine neutrophil beta-defensin-12: the peptide fold of the betadefensins is identical to that of the classical defensins. Biochemistry (Mosc.) 34, 13663–13671. [6] Hoover, D.M., Rajashankar, K.R., Blumenthal, R., Puri, A., Oppenheim, J.J., Chertov, O. and Lubkowski, J. (2000) The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J. Biol. Chem. 275, 32911– 32918. [7] Hoover, D.M., Chertov, O. and Lubkowski, J. (2001) The structure of human beta-defensin-1: new insights into structural properties of beta-defensins. J. Biol. Chem. 276, 39021–39026. [8] Schibli, D.J., Hunter, H.N., Aseyev, V., Starner, T.D., Wiencek, J.M., McCray Jr., P.B., Tack, B.F. and Vogel, H.J. (2002) The solution structures of the human beta-defensins lead to a better understanding of the potent bactericidal activity of HBD3 against Staphylococcus aureus. J. Biol. Chem. 277, 8279–8289. [9] Bensch, K.W., Raida, M., Magert, H.J., Schulz-Knappe, P. and Forssmann, W.G. (1995) HBD-1: a novel beta-defensin from human plasma. FEBS Lett. 368, 331– 335. [10] Wilcox, G.R., Fogh, R.H. and Norton, R.S. (1993) Refined structure in solution of the sea anemone neurotoxin ShI. J. Biol. Chem. 268, 24707–24719. [11] Siqueira, A.M., Martins, N.F., De Lima, M.E., Diniz, C.R., Cartier, A., Brown, D. and Maigret, B. (2002) A proposed 3D structure for crotamine based on homology building, molecular simulations and circular dichroism. J. Mol. Graph. Model. 20, 389–398. [12] Nicastro, G., Franzoni, L., de Chiara, C., Mancin, A.C., Giglio, J.R. and Spisni, A. (2003) Solution structure of crotamine, a Na+ channel affecting toxin from Crotalus durissus terrificus venom. Eur. J. Biochem. 270, 1969–1979. [13] Torres, A.M., Wang, X., Fletcher, J.I., Alewood, D., Alewood, P.F., Smith, R., Simpson, R.J., Nicholson, G.M., Sutherland, S.K., Gallagher, C.H., King, G.F. and Kuchel, P.W. (1999) Solution structure of a defensin-like peptide from platypus venom. Biochem. J. 341, 785–794. [14] Torres, A.M., de Plater, G.M., Doverskog, M., Birinyi-Strachan, L.C., Nicholson, G.M., Gallagher, C.H. and Kuchel, P.W. (2000) Defensin-like peptide-2 from platypus venom: member of a class of peptides with a distinct structural fold. Biochem. J. 348, 649–656. [15] Fenner, P.J., Williamson, J.A. and Myers, D. (1992) Platypus envenomation – a painful learning experience. Med. J. Aust. 157, 829–832. [16] Torres, A.M., Tsampazi, M., Tsampazi, C., Kennet, E.C., Belov, K., Geraghty, D.P., Bansal, P.S., Alewood, P.F. and Kuchel, P.W. (2006) Mammalian L-to-D-aminoacid-residue isomerase from platypus venom. FEBS Lett. 580, 1587–1591. [17] Torres, A.M., Bansal, P., Alewood, P.F., Bursill, J.A., Kuchel, P.W. and Vandenberg, J.I. (2003) Solution structure of CnErg1 (Ergtoxin), a HERG specific scorpion toxin. FEBS Lett. 539, 138–142. [18] Whittington, C.M., Papenfuss, A.T., Bansal, P., Torres, A.M., Wong, E.S.W., Deakin, J.E., Graves, T., Alsop, A., Schatzkamer, K., Kremitzki, C., Ponting, C.P., Temple-Smith, P., Warren, W.C., Kuchel, P.W. and Belov, K. (2008) Defensins and the convergent evolution of platypus and reptile venom genes. Genome Res. 18, 986–994. [19] Schnolzer, M., Alewood, P., Jones, A., Alewood, D. and Kent, S.B. (1992) In situ neutralization in Boc-chemistry solid phase peptide synthesis. Rapid, high yield assembly of difficult sequences. Int. J. Pept. Protein Res. 40, 180–193.
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