An Orbitide from Ratibida columnifera Seed Containing 16 Amino Acid

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An Orbitide from Ratibida columnifera Seed Containing 16 Amino Acid Residues Mark F. Fisher,† Colton D. Payne,‡ K. Johan Rosengren,‡ and Joshua S. Mylne*,† †

School of Molecular Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia Faculty of Medicine, School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia

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S Supporting Information *

ABSTRACT: Cyclic peptides are abundant in plants and have attracted interest due to their bioactivity and potential as drug scaffolds. Orbitides are head-to-tail cyclic peptides that are ribosomally synthesized, post-translationally modified, and lack disulfide bonds. All known orbitides contain 5−12 amino acid residues. Here we describe PLP-53, a novel orbitide from the seed of Ratibida columnifera. PLP-53 consists of 16 amino acids, four residues larger than any known orbitide. NMR structural studies showed that, compared to previously characterized orbitides, PLP53 is more flexible and, under the studied conditions, did not adopt a single ordered conformation based on analysis of NOEs and chemical shifts.

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PLP or PDP and cyclizes it via a cleavage-coupled transpeptidation reaction.6 During our work on the PDPs and PLPs, we discovered a transcript in the species Ratibida columnifera (family Asteraceae) that appeared to encode a PLP larger than any orbitide previously known, which we named PLP-53. Here we use a transcriptomics-led approach together with liquid chromatography coupled to mass spectrometry (LC-MS) and NMR spectroscopy to characterize this novel PLP containing 16 amino acid residues, four more residues than the two largest known orbitides and double the size of typical orbitides.

yclic peptides are abundant within the Viridiplantae (green plants). Many cyclic peptides, such as the cyclotides,1 cyclic knottins,2−4 and PawS-Derived Peptides (PDPs),5,6 are head-to-tail cyclic and stabilized by one or more disulfide bonds. Another large family of plant peptides, the orbitides, consists of smaller cyclic peptides of 5−12 amino acid residues, which lack disulfide bonds.7 Orbitides have a number of interesting bioactivities: antifungal, antibacterial, and cytotoxic activities for example.8 They are generally thought to be ribosomally synthesized, although this has only been substantiated for a few examples, namely, the segetalins from Vaccaria hispanica,9 the cyclolinopeptides from flax (Linum usitatissimum),10 two curcacyclines from Jatropha curcas,11 and some orbitides in Citrus species.9,12 The PawL-derived Peptides (PLPs) form the largest known group of orbitides; there are 52 PLPs, ranging in size from five to 12 amino acid residues.13−15 As their discovery was transcriptomics-led, the genetic origin of every PLP is known.14 We have previously studied the PLPs and PDPs which are found in seeds of the Asteroideae subfamily of the daisy family (Asteraceae).14 PLPs and PDPs, unusually, evolved within precursors of seed storage proteins; these seed storage albumins, as they are known, are abundant and ubiquitous among the seeds of flowering plants. Albumin precursors are processed by a cysteine protease known as asparaginyl endopeptidase (AEP), which cleaves at Asp or Asn residues, releasing the large and small subunits of the albumin, which are held together by disulfide bonds. When one or more buried peptide-generating sequences are present upstream of an albumin within their precursor protein, AEP also releases the © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Transcriptomic data from seed of the prairie coneflower, R. columnifera, were searched for precursors of albumin that might possess buried PDPs or PLPs. A transcript encoding a 152-residue protein appeared to encode an unusually large PLP (Figure 1). Based on homology to related genes,14,16,17 the large and small subunits of the seed storage albumin and their maturation points after Asn residues could be predicted (Figure 1). Buried PLPs and PDPs are often trailed by a fourresidue “tail”, typically Gly-Leu-Asp-Asn, which we observed. Each PLP or PDP is preceded by an Asn residue.14 Using these markers for protein processing, the transcript appeared to encode a 16-residue PLP with the sequence GSRIWVPGLGPVYEED (Figure 1), four residues longer than any known orbitide. To confirm the nucleotide sequence encoding the peptide, the 2275 trimmed RNA-seq reads used to discover the Received: February 5, 2019

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DOI: 10.1021/acs.jnatprod.9b00111 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

To verify the presence of PLP-53, peptides were extracted from the seeds of R. columnifera and subjected to LC-MS/MS. Searching the corresponding extracted ion chromatogram for the predicted mass of PLP-53, a peak at m/z 878.439 ([M + 2H]2+) was identified that was in agreement with the predicted monoisotopic mass for PLP-53 (1754.863 Da). The mass spectrum at this peak showed an isotopic profile consistent with it being a peptide having the predicted sequence (Figure 3), based upon the expected 13C content. When the protonated molecule with m/z 878.439 was fragmented by collision-induced dissociation, the MS/MS spectrum revealed peaks supporting the presence of the predicted cyclic peptide (Figure 4). The main sequence of ions found in the MS/MS spectrum was due to ring cleavage between the Val6 and Pro7 residues, followed by fragmentation into a and b ions. There were a number of secondary ion sequences created by cleavage at other peptide bonds in the ring, the most prominent being a break between the Tyr13 and Glu14 residues. No y ions were seen in the MS/MS data, indicating that the peptide was cyclic. The main ion series contains ions identifying 12 of the 16 residues; in combination with transcriptomic data, this provides evidence supporting the PLP-53 sequence. All previously known orbitides contain between five and 12 amino acid residues.7 The two largest orbitides known are cycloleonurinin (cyclo-GPTQYPPYYTPA) from the seeds of Leonurus artemesia18 and PLP-30 (cyclo-YIDPAIGKRFGD) from the seeds of Cosmos bipinnatus,13 both containing 12 residues. Other, larger cyclic peptides, such as cyclotides and the PDP family, are stabilized by one or more disulfide bonds. PLP-53 is the largest cyclic peptide that does not contain such a stabilizing bond and, at 16 residues, is larger than almost half of the (10 of the 22 known) PDP family of peptides, which have a single stabilizing disulfide bond. Seeds of R. columnifera are tiny, and the low abundance of PLP-53 in the extracts made it impossible to isolate PLP-53 in any significant amount and purify it to homogeneity. Instead, for further characterization, PLP-53 was made by solid-phase peptide synthesis. We assume all the amino acids of PLP-53 are the L-isomer, as is almost universally the case among orbitides, due to their ribosomal mode of synthesis. Synthetic PLP-53 was analyzed by LC-MS/MS, which confirmed it to be identical to the native peptide by comparison of the tandem mass spectrum of the synthetic peptide (Figure 5) with the native compound (Figure

Figure 1. Translated sequence of the PawL1a transcript from R. columnifera. The endoplasmic reticulum targeting sequence is shown in pink, the predicted sequence of the seed storage albumin is green (small subunit) and orange (large subunit), and the 16-residue PLP sequence, called PLP-53, is in aqua. The asterisk denotes a stop codon.

sequence were mapped onto the assembled open reading frame (ORF). The minimum read coverage was one, but only for the first two bases of the ORF. With the exception of the first four nucleotides of the ORF that encode the start Met1 and Ala2 of the ER signal sequence, the minimum coverage was 20 reads and the maximum coverage was 815 reads (Figure 2A). There

Figure 2. (A) Coverage of trimmed RNA-seq reads over the length of the PawL1a ORF from R. columnifera. (B) Contigs mapped across the sequence coding for PLP-53. Red lines are forward reads; green are reverse reads. The PLP-53 encoding sequence is shown as a darker shade.

were 137 reads that mapped to the sequence encoding PLP-53 (Figure 2B), with most of these reads encoding the complete peptide. This gave us confidence in the peptide sequence encoded by R. columnifera PawL1a.

Figure 3. Extracted ion chromatogram for the predicted m/z of PLP-53. Inset left: Predicted amino acid sequence of PLP-53 and predicted (m/ zexp) and observed m/z (m/zobs). Inset right: Mass spectrum at the peak of the extracted ion chromatogram showing an isotope profile consistent with a peptide of the given sequence. B



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Figure 4. Tandem mass spectrum of PLP-53. Two ion series are marked: the a/b series due to initial breakage between Val6 and Pro7 residues and the a′/b′ series created by breakage between the Tyr13 and Glu14 residues. Immonium ions are denoted by the corresponding single-letter amino acid code. Within the inset, X represents the ambiguity between the isobaric Ile and Leu residues. Abundance is shown as a percentage relative to the highest peak at 155.079.

Figure 5. Tandem mass spectrum of synthetic PLP-53. The most abundant ion series is marked: the a/b series due to initial cleavage between Val6 and Pro7 residues. Immonium ions are denoted by the corresponding single-letter amino acid code. X represents the ambiguity between the isobaric Ile and Leu residues. Abundance is shown as a percentage relative to the highest peak at 155.081.

but the dispersion of the resonances was limited compared to the disulfide-containing PDPs.19 As seen in some PDPs, evidence for minor conformations of PLP-53 was present. However, a single main conformation could be readily assigned using sequential assignment strategies. Analysis of 1Hα secondary shifts (Figure 6), which are sensitive indicators of secondary structure, showed distinct random coil features for the N-terminal part of the sequence,

4). The most prominent b ion series of the two peptides are compared in more detail in Table S1. To compare the structural features of PLP-53 with other orbitides, the synthetic peptide was then dissolved in water and analyzed by NMR spectroscopy at 288 K and 700 MHz. The data (Table S1, Figures S1 and S2, Supporting Information) were of good quality in terms of line shape and signal-to-noise, C



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included in the structure calculations. TALOS-N did not make predictions for the remaining residues. The predicted order parameters (S2) were 0.69−0.72 for Glu14-Gly1, with the remaining residues predicted to have an order parameter of 0.48−0.55, again confirming flexibility throughout most of the backbone.29 The final structure calculated based on the NMR data is shown in Figure 7, highlighting the ordered and disordered regions.

Figure 6. Secondary 1Hα chemical shifts for PLP-53. The observed chemical shifts minus random coil shifts are indicative of secondary structure, with negative values consistent with helical or turn structure.

while the C-terminal region did reveal deviations from random coil chemical shifts suggesting some local turn-like structure.20 To identify potential hydrogen bonds, lyophilized peptide was dissolved in D2O to monitor amide exchange; however all amides exchanged within minutes, indicating there were no strong hydrogen bonds within the peptide. This is in contrast to the PDPs, which generally contain a number of slow exchanging amide protons.19,21 An analysis of the NOEs could essentially only detect sequential contacts and no medium- or long-range interactions, indicating the structure in solution is highly dynamic at 288 K. This lack of ordered structure is in contrast to previous NMR studies of smaller orbitides, which typically possess stable structural features such as β and γ turns.22−25 A recent structural study of several orbitides by Xray crystallography has also shown well-ordered structures.26 Additional NMR data were then recorded at 280 K, in order to further slow the correlation time in solution and thereby maximize the nuclear Overhauser effect. Strikingly this relatively small drop in temperature significantly improved the NOESY spectrum, revealing a substantial number of additional nonsequential NOEs and also revealing some minor differences in non-amide chemical shifts, which suggested a more ordered fold at the lower temperature. Using the NOESY data recorded at 280 K, sufficient distance constraints could be generated and used to compute the 3D structure of PLP-53. As predicted from secondary shifts, residues 1−3 and 13−16 are well ordered, while residues 4−12 are disordered. Key interactions include side chain packing of Arg3 and Tyr13, which was evident from a number of NOEs. Structure calculations also suggest a hydrogen bond between the amide of Gly1 and the backbone carbonyl of Glu14. Data recorded at different temperatures were analyzed to determine temperature coefficients, which are indicative of hydrogen bonding. Although the subtle spectral changes observed with increased temperature were consistent with a degree of thermal unfolding already below 25 °C (298 K), the Gly1 amide proton chemical shift was linearly dependent on the temperature throughout the range 280−298 K, with a temperature coefficient of −3.1 ppb/K, confirming this hydrogen bond does exist.27 13C NMR chemical shifts were determined from a natural abundance HSQC, allowing a TALOS-N28 (Torsion Angle Likelihood Obtained from Shift and sequence similarity) analysis of backbone angles and prediction of S2 order parameters. For Asp16 and Gly1, backbone ϕ and ψ angles could be predicted with high confidence, and these were

Figure 7. NMR structure of the 16-residue PLP-53 (cycloGSRIWVPGLGPVYEED). Superposition of the 20 lowest energy structures calculated by CYANA based on 53 short-, eight medium-, and ten long-range distance restraints, four backbone dihedral angles, and one hydrogen bond. No violations of the experimental data were seen for any of the conformations. Structures are superimposed on the backbone of residues 1−3 and 13−16. Side chains of the ordered residues are shown in red and labeled with residue numbers.

PLP-53 was tested for antibacterial and antifungal activity. In a disc diffusion assay PLP-53 showed no activity against either the Gram-negative bacterial strain Escherichia coli K-12 or the Gram-positive strain Bacillus subtilis Marburg No. 168 (Figure S3). A positive control disc containing 50 μg of kanamycin did, however, clear bacteria from the area around the disc. In an assay against the fungus Aspergillus f umigatus, no difference could be seen between cultures containing up to 500 μg/mL of PLP-53 in DMSO and water and control cultures containing an equivalent amount of DMSO only. Backbone cyclization contributes to peptide stability by protecting the proto-termini, but also by thermodynamically favoring a folded state, as the link between the termini significantly reduces the favorable entropy gain resulting from unfolding. For smaller ring structures, such as previously studied orbitides, the number of possible conformations is limited, contributing to the common occurrence of a highly favored shape. PLP-53 is considerably larger, and it is likely that the added flexibility is a direct result of this. The head-totail cyclic type IIc bacteriocins contain >55 residues and a cyclic backbone but no disulfide bond, yet they are able to adopt highly ordered and stable structures.30,31 These are however large enough to be more protein-like, adopting a tertiary helical bundle that is significantly stabilized by an extensive hydrophobic core. The functional significance of PLP-53, like most other PLPs and PDPs, remains an enigma. Prototypical orbitides have been shown to have multiple activities including antiplasmodial,32 antifungal,33 and cytotoxic activity against several cell lines.34,35 These functions are likely, at least in part, related to an effect on biological membranes, as observed in other types of cyclic peptide.36−38 As a whole, orbitides do not have a common sequence, but do share a common residue composition. Orbitides are rich in aromatic residues and D



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times of 80 and 300 ms, respectively, were recorded at 280−298 K on a 700 MHz Bruker Avance III spectrometer equipped with a cryoprobe. In addition to this, 13C HSQC data were recorded at natural abundance. Data processing was done using Topspin 4.0.3 (Bruker), and the spectra were referenced to the solvent signal. Sequential assignment strategies43 were used to analyze the TOCSY and NOESY data with the program CARA.44 The secondary structure was identified through determination of secondary 1Hα shifts via comparison to the equivalent values in random coil peptides.20 TALOS-N was used to predict backbone torsion angles and S2 values.29 For structure calculations, cross-peaks in the NOESY spectrum were integrated and assigned manually. Structures were calculated using torsion angle dynamics within CYANA-3.85.45 For the final round 50 structures were calculated, of which the 20 with the lowest CYANA target function were chosen as representative of the solution structure of PLP-53. Liquid Chromatography−Mass Spectrometry. LC-MS was carried out on a 1200 HPLC system (Agilent Technologies) coupled to an Agilent 6550 Q-TOF mass spectrometer, following methods previously described.13,14 Briefly, a gradient elution was run over 15 min from 5% to 95% solvent B, where solvent A was 0.1% (v/v) formic acid in water and solvent B was 0.1% (v/v) formic acid in MeCN, using a high-capacity nano-LC chip (Agilent Technologies; part no. G4240-62010). MS data were collected at one scan per second, and MS/MS data at two spectra per second. Fragmentation for MS/MS was targeted to the m/z values identified by the transcriptomic peptide prediction. The candidate PLP-53 ion was fragmented with a voltage set to 38.1 V. Peptides were sequenced by visual examination of MS/MS spectra, aided by fragment predictions from the program mMass46 for cyclic peptides. The parameters used for mMass allowed for a, b, and immonium ions. Permitted neutral losses were water (when S, T, E or D was present) or ammonia (when R, K, Q or N was present). Other parameters were set at their default values. Antibacterial Assay. Synthetic PLP-53 was tested for activity against the Gram-negative bacterium Escherichia coli K-12 and the Gram-positive bacterium Bacillus subtilis Marburg 168. Bacteria were grown on LB agar plates overnight at 37 °C. One colony was picked from each plate and used to inoculate a 5 mL liquid culture. Each culture was incubated overnight at 37 °C with shaking. Fresh 5 mL aliquots of LB were inoculated with 25 μL of overnight culture and incubated at 37 °C. These cultures were diluted to an OD600 (optical density at 600 nm) of 0.1 and spread onto LB agar plates with a sterile swab. Synthetic PLP-53 was dissolved to a concentration of 10 μg/μL in DMSO and dispensed onto sterile 8 mm diameter filter paper discs in the following amounts: 3.1, 6.25, 12.5, 25, 50, and 100 μg. A disc containing 50 μg of kanamycin in water was used as a positive control, and a disc with 5 μL water as a negative control. All discs were left to dry completely before placing them onto the inoculated LB agar plates. The plates were incubated overnight at 37 °C and inspected in the morning for bacterial growth. Antifungal Assay. Synthetic PLP-53 was tested for activity against the fungus Aspergillus f umigatus. The fungus was grown on a yeast extract, peptone, dextrose (YPD) agar plate. Spores were harvested from the plate with 3 mL of 0.1% (v/v) Tween 80 and dispensed into YPD medium. This culture was adjusted to an OD600 of 0.05, and 180 μL of it was dispensed into 20 wells of a 96-well plate. PLP-53 solution (20 μL) was added to 10 of these wells at the following concentrations: 5000, 2500, 1250, 625, 313, 156, 78, 39, 19, and 9 μg/μL. These solutions were made by serial dilution with water from an initial solution of 5000 μg/μL synthetic PLP-53 in DMSO. Since DMSO is known to be toxic to fungi at high concentrations, in the other 10 wells we made a series of DMSO controls by adding 20 μL of the same dilutions of DMSO without PLP-53. The plate was incubated at 37 °C for 24 h and visually inspected for fungal growth.

proline as well as leucine and isoleucine; this leads to the characteristic hydrophobicity presented by these peptides.11,13 Additionally, these peptides rarely contain a charged residue, either positive or negative. PLP-53, although containing prolines and aromatics, differs from typical orbitides in both sequence length and composition. PLP-53 contains an Arg as well as three sequential negatively charged residues, Glu-GluAsp, giving it an overall negative charge and making it a unique member of the family. The proximity of the negative charges gives PLP-53 an overall amphipathic character, which is often seen in membrane-active antimicrobial peptides. However, amphipathic peptides acting on membranes are generally positively charged.39 Given these features it is unlikely that PLP-53 shares a biological mechanism and activity with the previously studied orbitides. Cyclic peptides are intriguing natural products that have been widely explored for biotechnological and pharmaceutical applications. In particular the prototypic PDP, sunflower trypsin inhibitor-1 (SFTI-1), has been extensively trialed for such purposes.40 With the discovery of PLP-53 we further significantly expand on the size and types of features reported for these classes of peptides derived from seed storage albumin precursors.

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EXPERIMENTAL SECTION

Procurement of Materials. Seeds of R. columnifera were purchased from Chiltern Seeds (Wallingford, UK), and the synthetic PLP-53 peptide cyclo-GSRIWVPGLGPVYEED was purchased from GenScript (Piscataway, NJ, USA). The peptide was supplied at a purity of ≥95%. Seed Transcriptome and Peptide Prediction. We used an existing transcriptome of R. columnifera seeds assembled de novo from which PLP peptide predictions were made, as described in a previous paper.14 In that study, RNA-seq was carried out on an Illumina HiSeq 1500 instrument resulting in 137 764 288 raw 100 bp paired-end reads. The transcriptome was assembled de novo with the program CLC Genomics Workbench (QIAGEN Aarhus A/S) with word size 60 and other parameters set to their defaults, resulting in 163 253 contigs. The raw RNA-seq data are available from the Sequence Read Archive under accession number SRX2544345. Seed Peptide Extraction. Seed peptides were extracted as described previously.14 Briefly, seeds were ground under liquid nitrogen, and the resultant fine powder was vortexed in 400 μL of MeOH, 400 μL of CH2Cl2, and 100 μL of 0.05% (v/v) trifluoroacetic acid (TFA). Phases were separated by alternate addition of 0.05% TFA and CHCl3 followed by centrifugation. The upper (aqueous) phase was dried by vacuum centrifugation (Labconco). Purification of Seed Extracts. Seed peptide extracts were purified using solid-phase extraction. The dried extract was redissolved in 400 μL of 5% MeCN/0.1% formic acid. A Strata-X polymeric solid-phase extraction column (Phenomenex) was conditioned with MeCN (1 mL) and equilibrated twice with 5% MeCN/ 0.1% formic acid (1 mL). The column was then washed in 5% MeCN/0.1% formic acid followed by 10% MeCN/0.1% formic acid (1 mL each) before peptides were eluted with 400 μL of 85% MeCN/ 0.1% formic acid. A 100g centrifugation was performed for 1 min between each step. The sample was dried by vacuum centrifuge and dissolved in 200 μL of 5% MeCN/0.1% formic acid for LC-MS analysis. Preparation of Synthetic Peptide Solution. One milligram of synthetic PLP-53 was dissolved in 1 mL of HPLC-grade water; then 5 μL of the solution was diluted to a final concentration of 5 μg/mL with 5% MeCN/0.1% formic acid. Aliquots of this solution were used for LC-MS analysis. NMR Spectroscopy. PLP-53 was prepared for NMR analysis by dissolving 1 mg of synthetic peptide in 500 μL of (90:10) H2O/D2O. Two-dimensional TOCSY41 and NOESY42 experiments, with mixing E



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00111. Additional information (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.S.M.). Tel: +61 8 6488 4415. ORCID

Mark F. Fisher: 0000-0002-6971-4285 K. Johan Rosengren: 0000-0002-5007-8434 Joshua S. Mylne: 0000-0003-4957-6388 Author Contributions

M.F.F. and J.S.M. conceived the study; M.F.F. performed all the experiments, except NMR, which was done by C.D.P. and K.J.R.; all authors analyzed data. M.F.F., K.J.R., and J.S.M. wrote the manuscript with the help of all authors. All authors have approved the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M.F.F. was supported by the Australian Research Training Program and a Bruce and Betty Green Postgraduate Research Scholarship. C.D.P. was supported by a University of Queensland Postgraduate Research Award. This work was supported by Australian Research Council grant DP190102058 to J.S.M. and K.J.R. The authors thank Y. H. Chooi and H. Li for assistance with antifungal assays and N. L. Taylor for providing advice and assistance in mass spectrometry aspects of this project.

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REFERENCES

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An Orbitide from Ratibida columnifera Seed Containing 16 Amino Acid

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