Structure and antimicrobial activity of platypus ‘intermediate’ defensin-like peptide

FEBS Letters 588 (2014) 1821–1826

journal homepage: www.FEBSLetters .org

Structure and antimicrobial activity of platypus ‘intermediate’defensin-like peptide

http://dx.doi.org/10.1016/j.febslet.2014.03.0440014-5793/Crown Copyright � 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical Society. All rights reserved.

Abbreviations: DLP, defensin like-peptide; Int-DLP, intermediate defensin-likepeptide; Int-DLPa, intermediate defensin-like peptide analogue; OvCNP, Ornitho-rhyncus venom C-type natriuretic peptide; DQF-COSY, double-quantum filteredcorrelation 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).

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, Australiab Queensland Tropical Health Alliance, James Cook University, Cairns, Qld 4878, Australiac Neurotoxin Research Group, School of Medical & Molecular Biosciences, University of Technology, Sydney, NSW 2007, Australiad Singapore Bioimaging Consortium, Biomedical Sciences Institutes, A�STAR, Singapore 138667, Singaporee School of Molecular Bioscience, University of Sydney, NSW 2006, Australia

a r t i c l e i n f o

Article history:Received 27 January 2014Revised 12 March 2014Accepted 23 March 2014Available online 30 March 2014

Edited by Miguel De la Rosa

Keywords:b-DefensinDefensin like peptideIntermediate-DLPNMR spectroscopyPlatypusPeptide fold

a b s t r a c t

The three-dimensional structure of a chemically synthesized peptide that we have called ‘interme-diate’ defensin-like peptide (Int-DLP), from the platypus genome, was determined by nuclear mag-netic resonance (NMR) spectroscopy; and its antimicrobial activity was investigated. The overallstructural fold of Int-DLP was similar to that of the DLPs and b-defensins, however the presenceof 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 Pseudomonasaeruginosa. 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 � 2014 Published by Elsevier B.V. on behalf of Federation of European Biochemical

Society. All rights reserved.

1. Introduction and they act as antibiotics [3]. Peptides that adopt the b-defensin

Bioactive polypeptides such as those found in venoms, milkand other natural sources have attracted considerable attentionin recent years because of their potential applications in drugdiscovery programs [1,2]. Such molecules can be used as toolsto investigate important physiological mechanisms at the cellularand/or molecular levels. They can also be used directly as drugs,or as starting points to design new therapeutic agents. Thesepolypeptides are often of comparatively low molecular weightand cysteine rich, and each has a specific disulfide-linked molec-ular framework that can be used as a scaffold to create new bio-active compounds.

Defensins are good examples of such potentially useful poly-peptides. These small antimicrobial proteins are found in manyorganisms, including mammals, birds, invertebrates and plants,

structural fold in particular are interesting, as they are found in adiverse range of organisms possessing disparate biological activi-ties [4]. In humans and other mammals, b-defensins are producedin neutrophils and epithelial cells, playing an important role in theinnate immune response as antimicrobial agents and as chemo-kines [5–9]. Polypeptides similar in fold to b-defensins have alsobeen reported in toxins of sea anemones [10], snakes [11,12] andplatypus [13,14] where they display numerous pharmacologicalactivities, such as ion-channel inhibition, myonecrosis, andanalgesia.

The b-defensin-fold structure generally consists of a short helix,or turn, followed by a small twisted anti-parallel b-sheet of threestrands [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 deter-mining and maintaining the compact core configuration of themolecule. The low sequence similarity with other members ofthe same family suggests that this global fold is chemically andtherefore evolutionarily robust, and that the nature of the side-chains effectively determines the functional specificity. The dis-tinct compact fold shared by these polypeptides may thereforebe useful in the design of molecules with prescribed pharmacolog-ical activity.

1822 A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826

The defensins from various tissues of the platypus, Ornithorhyn-chus anatinus, and defensin-like peptides (DLPs) from its venompresent an intriguing and potentially valuable case for researchstudy. This transpires because the platypus is a unique animal thatpossesses both mammalian and reptilian characteristics, thusbridging the evolutionary gap between lower and higher animalspecies. The male monotremes, platypus and echidna, for example,are the only known mammals to bear a venomous spur on eachhind limb. Platypus envenomation in human victims causes excru-ciating pain, hyperalgesia, and oedema [15]. The venom containsnumerous novel biologically active peptides and proteins, amongstwhich are natriuretic peptides called Ornithorhyncus venom C-typenatriuretic peptides or OvCNPs, and a family of four polypeptidesof �5 kDa called DLPs. Both the OvCNPs and DLPs are known to ex-ist in two isomeric forms, with each pair having identical aminoacid sequences but with the second amino acid residue in theD-form [16].

Various assay experiments on DLPs have so far failed to estab-lish their role in the venom. DLPs are neither antimicrobial, myo-toxic, nor do they appear to affect Na+-channel currents [14,17].The antimicrobial defensins from the platypus are equally asintriguing as the DLPs. Six b-defensins and four a-defensins haveso far been identified from platypus genome analysis [18]. The dis-covery of the a-defensins in the genome suggests that this type ofdefensin has evolved from b-defensins and that they emerged phy-logenetically prior to the divergence of the three extant mamma-lian lineages, 210 mya. Besides this phenomenon, a new 44amino acid residue peptide designated DEFB-VL (venom-like b-defensin) or intermediate-DLP (Int-DLP) was discovered in a geneanalysis of the platypus genome [18]. Int-DLP has an amino acidsequence similar to both mammalian (including the platypus) b-defensins and the DLPs (see Fig. 1). It was suggested that this poly-peptide evolved from platypus defensins to finally form the DLPs ofthe platypus venom, and hence we posit that it is ‘transitional’ be-tween the two types of polypeptides. Thus, it was expected thatInt-DLP would display folding characteristics that are similar tob-defensins and/or the platypus venom DLPs. Preliminary struc-tural modeling showed that Int-DLP had a similar fold to the DLPsand may have antimicrobial activity [18].

In the present work we investigated in greater details the ter-tiary structure of Int-DLP in solution and its antimicrobial activity.The results obtained add to a more general understanding of theevolution of defensins and the possible role of DLPs in the platypusand potentially higher mammals.

2. Methods

2.1. Sample preparation and synthesis

The 44-residue Int-DLP was synthesized on a 0.50 mmol scaleusing HBTU activation of Boc-amino acids with in situ neutraliza-tion chemistry, as described previously [19]. The 40-residue Int-DLPa was purchased from GL Biochem Ltd. (Shanghai, China).

Int-DLP RRRRRRPPCEDVNGQCQPRGN-PC-LRLRGAC-PRGSRCCMPTVAAH CC RRCCCCMMCCCC GG

PVPMRQIGTCFGRPVKCCRCGRNGGVCIPIRCβ-defensin-12 GPLS---- - ----SW

DLP-1 FVQHRPRDCESINGVCRHKDTVNCREIFLADCYNDGQKCC -----RK10 20 30 40

939

2361

0442

Fig. 1. Primary structures of DLP-1, Int-DLP, and b-defensin-12. The amino acidresidues were aligned using Kalign [35] to show maximum correspondencebetween the structures. Shaded regions indicate identical residues. The disulfidebonding pairs are shown below the numbered sequences with dashed lines.

NMR samples were prepared by dissolving 1–2 mmol of peptidesamples in 0.350 mL of H2O/D2O (9:1, v/v) in a 5-mm magneticsusceptibility 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 ob-tained from Oxoid (Australia) in 1 L of deionised H2O. The final pHof the medium was 7.0. Nutrient gel was prepared with bacterio-logical agar (1 g L�1), bacteriological peptone (10 g L�1), yeast ex-tract (5 g L�1), and sodium chloride (5 g L�1). The bacteria used inthe antimicrobial activity assay were Staphylococcus aureus (ATCC12600), Escherichia coli (ATCC 11775), and Pseudomonas aeruginosa(ATCC 19582) which were obtained from the University of NewSouth Wales [20]. Among these, S. aureus is a Gram-positive bacte-rium while E. coli and P. aeruginosa are Gram-negative bacteria. Li-quid cultures of nutrient broth (20 mL) for each microorganismwere inoculated with a single colony from the stock agar platesand grown overnight (18 h) with shaking at 150 rpm before dilu-tion to 0.2 of OD595. Cultures were inoculated with the peptidesat the final concentrations (lM) of 78, 39, 19.5, and 0, in 96-wellmicrotitre plates with shaking at 600 rpm. Initial readings weremade at the start of incubations, and the final growth was readat 24 h, using a 96-well plate reader (Multiskan EX, Thermo Elec-tron). The screenings were carried out in duplicate. Growth inhibi-tion 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, and500 spectrometers with 5-mm triple resonance inverse probes,with operating temperatures of 15, 25, 30, and 35 �C. The two-dimensional homonuclear proton (2D) experiments that were per-formed 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 Overhauserenhancement spectroscopy (NOESY) [24] with mixing times of200 and 250 ms. Solvent-signal suppression was achieved byapplying either presaturation or WATERGATE [25] pulse se-quences. H-D exchange experiments were carried-out by addingD2O to freeze-dried NMR samples and acquiring a series of 1Dspectra for at least 1 h, followed by a 5 h TOCSY spectrum. All spec-tra were processed using TOPSPIN software (Bruker) and were ana-lysed using the standard protocol in the program SPARKY (T.D.Goddard and D.G. Kneller, SPARKY 3, University of California, SanFrancisco).

2.4. Structure calculations and analysis

Distance constraints for structure calculations were obtainedfrom cross-peak volumes in the NOESY spectra, recorded at25 �C. Additional distant constraints from H-bonding were ob-tained from a hydrogen–deuterium exchange experiment afteranalysis of medium resolution structures. Automatic structure cal-culations were performed with the program CYANA [26]. The ‘best’20 structures with the lowest target function values were selectedfrom the 1000 structures that were generated; these were consid-ered to be representative of the structure of Int-DLP. The 3D struc-tures were visualized and analyzed using the program MOLMOL[27].

Table 1Structural statistics of the 20 Int-DLP structures.

Quantity Value

Distance restraintsIntraresidue (i–j = 0) 104Sequential (|i–j| = 1) 138Medium-range (|i–j| 6 5) 45Long-range (|i–j| > 5) 130Hydrogen bonds 24Disulfide bonds 9Total 450

Compliance with restraintsCYANA average target function value 0.66Number of NOE violations > 0.30 Å 0

Atomic rms difference with the mean (Å)Backbone atoms (10–40) 0.38 ± 0.10Heavy atoms (10–40) 0.90 ± 0.12

Ramachandran plot statisticsMost favourable region (%) 58.8Additionally allowed region (%) 38.6Generously allowed region (%) 2.2Disallowed region (%) 0.5

A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826 1823

3. Results and discussion

3.1. NMR experiments

The primary structure of Int-DLP is peculiar among those of b-defensins in that it incorporates six consecutive arginine residuesat the N-terminus. This unusual property was expected to compli-cate the process of 1H NMR resonance (peak) assignment as it wasanticipated to cause extensive peak overlap of the arginine peaks.Preliminary NMR experiments on Int-DLP indeed yielded spectrawith many overlapping broad peaks (see Supplemental Fig. 1).

Fig. 2. The calculated Int-DLP structure. (A) Ensemble of 20 Int-DLP structures aligned bysecondary structures and disulfide connectivities. (C) Similar to (B) but rotated �90� abouelectrostatic potential. Surfaces with positive, negative and neutral electrostatic potential(B). (E) Similar to (D) but rotated �90� about the assigned vertical axis. (F) Similar to (D) bMOLMOL [27].

Besides this, it was also seen that Int-DLP NMR peaks were broaderthan those obtained earlier for DLP-1 and DLP-2 [13,14]. To addressthese problems, we performed additional NMR experiments on anInt-DLP analogue that we called Int-DLPa, a shorter peptide withfour arginine residues deleted from the N-terminus. It was antici-pated that the deletion of these four consecutive residues wouldsimplify and improve the quality of the NMR spectra, but not alterthe overall fold of the polypeptide.

NMR spectra of Int-DLPa had peaks with linewidths that werenot significantly narrower than those of Int-DLP; however the dele-tion of the four arginine residues to make the Int-DLP analoguesimplified resonance assignment by lessening the peak overlap inthe pertinent regions of the spectra. Also, there were no significantchanges in the chemical shifts of the remaining peaks in the Int-DLPa spectra, suggesting that Int-DLPa and Int-DLP had very simi-lar overall structural folds.

By comparing the NMR spectra of Int-DLP and Int-DLPa, we ableto assign most the resonances of the two polypeptides. As both setsof NMR spectra were very similar, structure calculations were per-formed only on the full-length Int-DLP. The summaries of thestructural data from the calculated Int-DLP structures are givenin Table 1.

3.2. Int-DLP structures

The 20 structures of Int-DLP of the ensemble are shown inFig. 2A. Despite the relatively broad 1H NMR peaks, the resolutionof the obtained structure was good, showing a root mean squaredeviation (RMSD) from the mean structure of 0.38 Å, when back-bone atoms of residues 10–40 were superimposed. The N- and C-termini of Int-DLP were also seen to be disordered in a mannersimilar to the DLPs [13,14,28].

superimposing the backbone atoms of residues 10–40. (B) Ribbon diagram showingt the assigned vertical axis. (D–F), Molecular surface of Int-DLP highlighted to show

s are drawn in blue, red and white, respectively. (D) Molecular orientation similar tout rotated �180� about the assigned vertical axis. The figures were generated using

β-defensin-12DLP-1 Int-DLP

Fig. 3. Comparison of three-dimensional structural folds of DLP-1, Int-DLP and b-defensin-12. The figures were generated using MOLMOL [27].

1824 A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826

As shown in Fig. 2B and C, Int-DLP has a compact tertiary foldcontaining a helix and a b-sheet, laced together by three disulfidebonds. This overall fold was analysed using PDBefold structuresearch algorithm [29] and was found to be very similar with thatof b-defensins and the DLPs, as illustrated in Fig. 3. The disulfideconnectivities of 1–5, 2–4 and 3–6 (revealed by NMR spectroscopy)are identical with that of the DLPs. The presence and locations ofthe short sections of secondary structures in Int-DLP and the DLPswere also very similar. The structures of DLP-1, -2 and -4 incorpo-rate anti-parallel b-sheets from residues 15–18 and 37–40, and ahelix-like structure from residues 10–12 [13,14,28]. In comparison,the Int-DLP structure contained a definite anti-parallel b-sheet thatencompassed residues 15–17 and 35–38 and it also contained ana-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 twob-strands, many anti-microbial b-defensins from mammaliansources, such as hBD-1, hBD-2 and b-defensin 12, incorporate threeb-strands [4,9,30,31]. Int-DLP displayed a third b-strand similar tothe b-defensins encompassing residues 24–27 with a b-bulge atresidues 26–27 (see Fig. 3). The chemical shift deviations from ran-dom coil values, as shown in Supplemental Fig. 2, also support thepresence of this b-strand but suggest that that this strand could belonger, perhaps spanning residues 21–26.

05

101520

Int-DLPa(78 µM)

Int-DLPa(39 µM)

Int-DLPa(19.5 µM)

Int-DLPa(0 µM)

05

101520

Int-DLPa(78 µM)

Int-DLPa(39 µM)

Int-DLPa(19.5 µM)

Int-DLPa(0 µM)

05

101520

Int-DLPa(78 µM)

Int-DLPa(39 µM)

Int-DLPa(19.5 µM)

Int-DLPa(0 µM)

E. coli (ATCC 11775)

S. aureus (ATCC 12600)

P. aeruginosa (ATCC 19582)

Fig. 4. Antimicrobial activities of Int-DLP and Int-DLPa. The antimicrobial activities ofaeruginosa (ATCC 19582) are expressed as a percentage of growth inhibition. Data represhorizontal bars.

It is clear that the third b-strand in Int-DLP makes its overallstructure more similar to b-defensins than to DLPs. Although thecysteine spacing in Int-DLP is more similar to other b-defensinsin the platypus, the amino acid sequence of Int-DLP is more similarto DLPs than to b-defensins [18]. The N-terminus that incorporatesfour arginine residues was found to be disordered (and could beflexible) due to the dearth of medium and long-range NOESYcross-peaks corresponding to this part of the molecule. These N-terminal residues did not appear to have a significant role in theoverall fold of Int-DLP as the chemical shifts of the 1H atoms ofArg 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 testedagainst three bacterial species viz., S. aureus, E. coli, and P. aerugin-osa. Fig. 4 shows the antimicrobial activity of the Int-DLP polypep-tide with various concentrations, expressed as a percentage ofgrowth 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 andthe least activity against E. coli. For E. coli, the very slight activity(at less than 5% growth inhibition at 39 lM polypeptide) observedfor both polypeptides suggests insignificant or no activity againstthis microorganism. For S. aureus, a consistent activity of �10%growth inhibition occurred with the two peptides at concentra-tions of 20–78 lM. Int-DLPa showed uniform activity of �8–9%at concentrations of 20–78 lM, while Int-DLP showed slightlyhigher activity of �12% at 39 and 78 lM; but its activity decreasedby �30% when its concentration was decreased to 20 lM.

With P. aeruginosa, there were significant differences in theantimicrobial activities of the two polypeptides. With 78 lM Int-DLP the growth inhibition was 18%, which was �3 times higherthan the 7% obtained with Int-DLPa. Unlike that seen with S. aur-eus, growth inhibitions in P. aeruginosa for the two polypeptidesdecreased almost linearly with concentration.

As clearly shown by our results, Int-DLP and Int-DLPa hadantimicrobial properties, unlike the DLPs which had none[13,14]. Int-DLP was seen to be more potent than Int-DLPa against

05

101520

Int-DLP(78 µM)

Int-DLP(39 µM)

Int-DLP(19.5 µM)

Int-DLP(0 µM)

05

101520

Int-DLP(78 µM)

Int-DLP(39 µM)

Int-DLP(19.5 µM)

Int-DLP(0 µM)

05

101520

Int-DLP(78 µM)

Int-DLP(39 µM)

Int-DLP(19.5 µM)

Int-DLP(0 µM)

E. coli (ATCC 11775)

S. aureus (ATCC 12600)

P. aeruginosa (ATCC 19582)

both of the peptides against E. coli (ATCC 11775), S. aureus (ATCC 12600), and P.ent the average of two replicates with very small standard deviations shown by the

A.M. Torres et al. / FEBS Letters 588 (2014) 1821–1826 1825

P. aeruginosa. It is surmised that the deletion of the four arginineresidues in Int-DLPa caused the observed decrease in activity andthat these arginine residues are important for Int-DLP anti-microbial potency. This is consistent with the known activity ofstretches of arginine and lysine residues that mediate cell entryof membrane-crossing peptides like penetratin [32].

The antimicrobial activity of Int-DLP is not surprising as it canbe readily explained by the obtained 1H NMR-based structure. Asshown in the surface diagrams in Fig. 2D–F, there is a clear separa-tion between various types of amino acid in the Int-DLP molecule.The membrane-active cationic arginine residues (blue surface) arelargely situated on one side of the molecule while clusters ofhydrophobic residues (white surface) are situated on the otherside. The amphipathic nature of Int-DLP is similar to that of defen-sins, such as b-defensin-12. It is believed that this structural fea-ture is responsible for defensins antimicrobial activities as itfacilitates interaction with targets such as bacterial membranes[5].

An interesting feature of the Int-DLP structure is the clusterof cationic residues. In human b-defensins, hBD-1, hBD-2, andhBD-3, these membrane active basic residues are located at theC-terminus [3] while in Int-DLP they are at the N-terminus. Notethat in antimicrobial studies of hBD-3 analogues [33] a linearpeptide fragment corresponding to the C-terminus gave en-hanced activity against E. coli, a Gram-negative bacterium, whilethe linear fragment corresponding to the N-terminus was activeagainst S. aureus, a Gram-positive bacterium. Since the positionof the basic residues in Int-DLP is reversed, in contrast to thatof human b-defensins, it might be expected that the N-terminusof Int-DLP is important for ‘attacking’ Gram-negative bacteria.While no significant activity against E. coli was detected forInt-DLP, it was evident that the four arginine residues at its N-terminus were important for activity against P. aeruginosa, aGram-negative bacterium.

In addition to the previously mentioned structural feature, wesuggest that the proline residues in Int-DLP play a significant partin the overall fold and hence the antibacterial activity. As shown inFig. 1, b-defensin-12 and Int-DLP have four and five prolineresidues, respectively, but DLP-1 has only one. The presence oftwo consecutive proline residues near the N-terminus of Int-DLPprobably gives a specific orientation to the six cationic argininecharged groups so that they interact with the cell membrane lead-ing to enhanced antimicrobial activity. In the same manner, thepresence of other proline residues at the same, or nearly same,position in the sequence of Int-DLP and b-defensin-12 makes theirstructures more similar and hence a similar antimicrobial activityor spectrum.

4. Concluding remarks

Overall, we determined the structural fold and antimicrobialactivity of another polypeptide from the platypus genome: Int-DLP displays the robust structural fold of the b-defensin family ofpolypeptides, incorporating a helix and a triple stranded b-sheet.It also exhibited antibacterial activity although gene and aminoacid sequence analyses show that Int-DLP is more similar to DLPs,which are devoid of antimicrobial activity, than to b-defensins. It ispossible that Int-DLP constitutes an evolutionary link between theb-defensins and the DLPs. Further phylogenetic study similar tothat performed on fungal defensin-like peptides [34] will helpdetermine if this ‘intermediate’ molecule has indeed evolved fromb-defensins and is the precursor of the DLPs. It would also be inter-esting to characterise the other unique defensins that have beenidentified in the platypus genome [18] and to compare their struc-tures and antimicrobial activities with those of other mammaliandefensins, and with Int-DLP. Such future studies may yield

effective antimicrobials that can be used to treat newly emergentdrug-resistant bacteria that have been inadvertently selected byoveruse of conventional antibiotics.

Acknowledgements

The work was supported by a Discovery Project Grant from theAustralian Research Council to PWK. We thank Dr Ann Kwan forimplementing some of the 2D NMR experiments. We also thankProfessor Kathy Belov and Dr Camilla Whittington for valuable dis-cussions on platypus genomics.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.febslet.2014.03.044.

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