Most proteins are long, chain-like molecules that have two ends respectively called the N-terminus and C-terminus. However, certain proteins can close on themselves to become circular. This requires a chemical reaction between the N- and C-termini, which creates a strong bond between the two extremities.

To go through this ‘cyclization’ process, a straight protein attaches to a certain type of protease, a class of enzyme that usually cuts proteins into smaller pieces. In plants that are distantly related, the same group of enzymes – called AEPs – has been selected to perform cyclization. Here, Haywood et al. study an AEP enzyme from sunflowers: they identify what about this enzyme’s structure is important to drive the complex chemical reaction that results in the protein being cyclized rather than simply cut.

Using a technique called X-ray crystallography to see the positions of individual atoms in the enzyme, Haywood et al. caught a snapshot of the enzyme. Its structure explained how the enzyme’s shape can guide cyclization. In particular, the part of the enzyme that binds to the proteins, the active site, was relatively flat and open, but also flexible: this helped the N and C-termini react with each other and close the protein. Further experiments artificially mutated specific areas of the enzyme, which helped determine exactly which elements guide this succession of chemical reactions.

The activity of AEPs is influenced by their local environment, such as acidity. In fact, Haywood et al. showed that certain AEPs, which do not normally carry out cyclization, can start performing this role when exposed to a different level of acidity.

The pharmaceutical industry is increasingly interested in circular proteins, as these are stable, easily used by the body, and can be genetically customized to act only on specific targets. If the cyclization process is better understood, and then harnessed, new drug compounds could be produced.

Asparaginyl endopeptidases (AEPs) are a group of asparagine/aspartic acid (Asx) specific proteases that have been classified as belonging to the C13 family of cysteine proteases based on the presence of a His-Gly-spacer-Ala-Cys motif (Hara-Nishimura et al., 1993; Chen et al., 1997; Mathieu et al., 2002; Shafee et al., 2015). First described in plants as vacuolar processing enzymes based on their propensity for processing seed proteins stored in vacuoles, AEPs have since been described in a variety of organisms and shown to be involved in a wide range of processes including, cell death antigen processing and hemoglobin degradation (Hara-Nishimura et al., 1993; Manoury et al., 1998; Hatsugai et al., 2004; Kuroyanagi et al., 2005; Yamada et al., 2005; Sojka et al., 2007). In addition to the proteolytic function observed in these processes, AEP has become well known for its curious ligation reactions (Min and Jones, 1994; Sheldon et al., 1996; Mylne et al., 2012; Nguyen et al., 2014; Zhao et al., 2014; Dall et al., 2015).

The ability of endoproteases to perform ligation reactions was first observed by Bergmann and Fruton in 1938 with chymotrypsin (Bergmann and Fruton, 1938). Later, in vitro ligation reactions were performed with AEP from jack bean (Canavalia ensiformis) seeds (Bowles et al., 1986; Min and Jones, 1994). The recent discovery that evolutionarily distinct plant families have repeatedly recruited AEPs to catalyze the formation of ribosomally synthesized and post-translationally modified peptides (RiPPs), through the macrocyclization of linear precursor sequences, has caught the attention of drug designers keen to overcome the current inefficiencies in native chemical ligation that limit the therapeutic use of cyclic peptides (Pattabiraman and Bode, 2011; Mylne et al., 2012; Arnison et al., 2013). Such therapeutic cyclic peptides are viewed by many to have the potential to capitalize on a niche in the current pharmaceutical market by virtue of their intermediate size between small molecule drugs and large protein structures, and their unique capacity to combine favorable bioavailability and stability characteristics with high target specificity facilitated by tolerance to site-directed mutagenesis (Clark et al., 2005, 2010; Gould et al., 2011; Ji et al., 2013; Poth et al., 2013; Truman, 2016). Moreover, as computational techniques for the discovery of RiPPs improve and the number of cyclic peptides described continues to expand, an ever-wider array of scaffolds might be exploited to tailor molecules to specific drug targets (Bhardwaj et al., 2016; Truman, 2016; Hetrick and van der Donk, 2017) (Figure 1).

Figure 1

Download asset Open asset

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14-residue, bicyclic peptide with a cyclic backbone and an internal disulfide bond (Luckett et al., 1999). Its biosynthesis is rather unusual as its sequence is buried within a precursor that also encodes seed storage albumin. Seed storage albumins are a major class of seed storage protein that constitute over 50% of total seed protein and become a source of nitrogen and sulfur during seed germination (Youle and Huang, 1978; Shewry and Halford, 2002). The common sunflower (Helianthus annuus) has many genes encoding precursors for these napin-type or 2S seed storage albumins that are synthesized in the rough endoplasmic reticulum before undergoing cleavage maturation by AEP and localizing to storage vacuoles (Bollini and Chrispeels, 1979; Franke et al., 2016; Jayasena et al., 2016). Along with an adjacent albumin, SFTI-1 is post-translationally processed by AEP from within a unique seed storage albumin precursor called Preproalbumin with SFTI-1 (PawS1) (Mylne et al., 2011). SFTI-1 is a potent inhibitor of serine proteases, and its intrinsic stability and cellular penetration capabilities have led to its application as a bioactive scaffold (White and Craik, 2016; Swedberg et al., 2017).

AEP-catalyzed macrocyclization of SFTI-1 is hypothesized to proceed via a cleavage-coupled intramolecular transpeptidation reaction whereby catalysis begins with the deprotonation of the active site Cys by a localized His of the catalytic center, facilitating the nucleophilic attack on the carbonyl carbon of the Asp by the activated Cys thiol of AEP (Bernath-Levin et al., 2015). This attack culminates in the formation of a thioacyl intermediate between the substrate and AEP, and the removal of the C-terminus. The reaction is proposed to be subsequently concluded by the nucleophilic attack of this intermediate by a Gly at the N-terminus of the substrate, resulting in the macrocyclization of SFTI-1 in a head-to-tail manner (Mylne et al., 2011; Bernath-Levin et al., 2015). Notably, this reaction proceeds in competition with nucleophilic attack upon the thioacyl intermediate by any nearby water molecules which would produce hydrolyzed, acyclic-SFTI. In vitro studies revealed the ratio of acyclic to cyclic SFTI-1 to be in the order of 5.8:1, with the less stable acyclic products hypothesized to be quickly degraded in vivo (Bernath-Levin et al., 2015). Evidence for AEP-mediated and hydrolysis-independent transpeptidation was demonstrated through the exclusion of a heavy atom O¹⁸ in the cyclic SFTI-1 product from an in vitro jack bean AEP (CeAEP1) catalyzed reaction (Bernath-Levin et al., 2015). The formation of a much larger cyclic peptide by an AEP from Oldenlandia affinis (OaAEP1) was also shown to lack O¹⁸ incorporation, suggesting a conserved mechanism of macrocyclization despite differences in substrate sequences (Harris et al., 2015). However, the suggestion that an AEP from butterfly pea (Clitoria ternatea) termed butelase one functions only as a ligase (Nguyen et al., 2014), combined with the proposal of a succinimide-driven, cleavage-independent ligation event based on the crystal structure of human AEP (hAEP) and its ability to ligate substrate in the absence of the catalytic Cys, has cast uncertainty on the mechanism of AEP-catalyzed macrocyclization (Dall et al., 2015).

In order to elucidate a structural explanation why plant AEPs have been recruited by distinct plant lineages to perform macrocyclization and to understand the catalytic and structural nuances that might allow preferences towards cleavage or ligation reactions, we sought the crystal structure of a sunflower AEP.

Herein, we describe the first structure of an active plant AEP; one capable of performing peptide macrocyclization. This AEP, the most abundant AEP of five AEPs in the common sunflower (HaAEP1), displays structural similarity to previously published active AEPs from mammals, with subtle differences at residues involved in substrate recognition. Our characterization by site-directed mutagenesis of HaAEP1 residues integral to macrocyclization will facilitate the bioengineering of plant AEPs for improved macrocyclization efficiency, diversifying the scaffolds usable as cyclic therapeutic leads.

Herein, we have described the structure of an active plant AEP containing a peptide ligand with partial occupancy bound to the active site catalytic Cys as a tetrahedral intermediate, illustrating conformational flexibility in the AEP catalytic dyad. This structure has enabled us to predict a model for SFTI-1 macrocyclization by HaAEP1 where the GLDN tail of the SFTI-1 precursor orients in a manner analogous to cystatin binding to hAEP, over the catalytic His to a hydrophobic patch on the β_II region, and where the N-terminus attack occurs between the diminutive β_I sheet and β5-β_IV loop in a manner analogous to the attack of thioacyl intermediates by peptidoglycan precursors in sortase-catalyzed transpeptidation reactions (Suree et al., 2009; Clancy et al., 2010). This model complements the requirement for a small amino acid followed by a hydrophobic residue at P1′ and P2′ due to their orientation over the catalytic His and towards the hydrophobic β_II-β_III region, respectively.

For several Cys proteases, including AEPs, a minor role for a third Asx/Arg residue in catalysis has previously described (Vernet et al., 1995; Dodson and Wlodawer, 1998; Dall and Brandstetter, 2016; Clancy et al., 2010; Buller and Townsend, 2013; Dall and Brandstetter, 2013). This third residue has been suggested to function in stabilization and orientation of the His imidazole ring. Interestingly, functional analysis of HaAEP1 catalytic triad residue N73 revealed that an N73A mutation resulted in the production of a higher ratio of cyclic SFTI-1. The observation of potential conformational shifts in the crystal structure of HaAEP1 during catalysis suggests that the loss of an orientating N73 side chain allows for further conformational flexibility of H178. This increased flexibility of His in a relatively open pocket that might accommodate a range of rotamers could reduce steric hindrance of an N-terminus entry towards the acyl intermediate and also facilitate deprotonation of the substrate N-terminus.

The requirement for space and flexibility between the catalytic dyad favors the convergence for peptide macrocyclization upon Cys proteases over Ser proteases due to the close proximity between Ser-His residues in these proteases, an inherent requirement based upon the reduced nucleophilic properties of Ser (Buller and Townsend, 2013). Indeed, a recent structure of a macrocyclizing Ser protease, PCY1, suggested that a shift in the catalytic His away from Ser is required for macrocyclization, based on a comparison to their hydrolytic relatives, and suggested a role for the catalytic His in deprotonation of the attacking peptide N-terminus (Chekan et al., 2017). Furthermore, comparison with other Cys proteases suggests that AEPs have been converged upon for macrocyclization based on their relatively flat and open catalytic site. In contrast to a recent hypothesis for efficient macrocyclization based on a comparison of AEP structures focused on a region closer to the S4 pocket, here we suggest that differences in AEP catalytic efficiency and substrate specificity will be defined by subtle amino acid differences in the β_II-β_III region, β_I sheet and β5-β_IV loop and the S1-S5 pocket (Yang et al., 2017). Moreover, this orientation of the tail away from the β_I sheet and β5-β_IV loop is hindered in other caspases which exhibit a relatively straight substrate channel (Figure 5) and thus prevent simultaneous attack of the thioacyl intermediate by the N-terminus loop upon scissile bond cleavage.

Following activation, AEP functionality has been illustrated to be dependent on a delicate balance between pH and stability, where endopeptidase activity is favored at a low pH (~pH 4) and ligase activity is favored at a higher pH (~pH 6) (Dall et al., 2015). A pH-dependent activity has been well documented for cysteine proteases and is a function of the formation of a thiolate and imidazolium ions on the catalytic dyad of Cys and His residues (Dall and Brandstetter, 2016; Frankel et al., 2005). Given the hypothesized role of His in deprotonating the attacking N-terminal peptide chain in peptide macrocyclization it would therefore be expected that at a low pH the catalytic His would be more readily protonated and hence this reaction might be less frequent resulting in more hydrolysis at low pH. This hypothesis is supported by the low level of endopeptidase activity observed with Ala mutations of the catalytic His in HaAEP1 and other proteases, illustrating that thiolates might form and that hydrolytic resolution of the thioacyl intermediate might occur in the absence of His (Frankel et al., 2007; Zhao et al., 2014). Previous reports have also suggested that a local Gly amide backbone might facilitate catalysis via a transfer of a proton to the leaving group of the tetrahedral intermediate from a hydrogen bonded water molecule; analysis of the HaAEP1 structure reveals Gly179 could potentially perform this role in the absence of His (Brady et al., 1999). Moreover, mutation of the acidic residue following the catalytic Cys to a basic residue in hAEP has previously been shown to enhance catalytic efficiency by decreasing the local pKa of the Cys residue (Dall and Brandstetter, 2013). Interestingly, we found an equivalent mutation in HaAEP1 to result in the loss of peptide macrocyclization indicating that E221 could also aid in deprotonation of the incoming N-terminus or that the residue’s larger side chain and expected orientation in activating the catalytic Cys could impart steric hindrance upon the N-terminus attack of the thioacyl intermediate (Figure 4, Figure 4—figure supplement 1). However, given the finding that E221K mutation is unable to prevent re-ligation of the cap domain to the active core domain upon pH shift in HaAEP1, there is likely to be redundancy between the local residues in creating a nucleophilic amine group to complete a transpeptidation reaction.

The finding that AEPs from species that lack cyclic peptides may be coaxed into performing peptide macrocyclization of a linear peptide under favorable conditions significantly expands the potential use of AEPs for the production of cyclic peptides (Figure 6). Moreover, further investigation into the subtle nuances that define substrate specificity and catalytic activity between AEPs is warranted, with differences in the β_II-β_III region, β_I sheet, β5-β_IV loop and around the S1-5 pocket including the variable α5-β6 and β_IV-β_V loops likely to be key (James et al., 2017). Indeed, in comparison to HaAEP1 and OaAEP1, the efficient peptide macrocyclizing AEP butelase one displays several different amino acids that could be responsible for this AEPs efficacy. These differences include shorter sidechains in the β_I sheet and β5-β_IV loops that may reduce steric hindrance on a peptides N-terminus during attack on a thioacyl intermediate (HaAEP1: P181, Q245, N247 OaAEP1: A178, T242, S244 Butelase-1: A168, G232, S234) and differences in the β_IV-β_V region that could generate substrate specificity (Figure 5—figure supplement 1).

Active HaAEP1 exhibits a succinimide at the same position as hAEP which has previously been postulated to perform a Cys-independent ligation reaction through cyclic rearrangement with the P1 Asx side chain (Dall et al., 2015). Functional analysis reveals that HaAEP1 is able to perform peptide macrocyclization despite D177G/A mutations that cannot form a succinimide group (Figure 4B–C). Moreover, these mutants continued to perform re-ligation of the cap domain when activated at pH >5.5 (Figure 4—figure supplement 4), a result that has previously been shown with mAEP (Zhao et al., 2014). In the absence of a critical requirement for succinimide formation in macrocyclization the question remains as to why this relatively unstable aspartimide appears stable in AEP crystal structures and is largely conserved in the C13 proteases. Succinimides have been shown to form more readily when adjacent to His residues as the His Nδ abstracts a proton from the His backbone NH allowing the deprotonated main chain amine to attack the Asx side chain (Brennan and Clarke, 1995; Takahashi et al., 2016). Once formed this succinimide could enhance the activity of the catalytic His by virtue of reducing stabilizing hydrogen bonding interactions with the carboxyl terminus and be involved in the proper positioning of the catalytic His. A further potential role for succinimides could present upon their hydrolysis through racemization and favored formation of iso-Asp (Geiger and Clarke, 1987; Reissner and Aswad, 2003). This iso-Asp could potentially represent a subtle mode of AEP regulation as the orientation of this side chain towards the S1 pocket is likely to disrupt substrate binding. Such iso-Asp residues have previously been proposed to regulate protein activity by a time-dependent molecular switch (Geiger and Clarke, 1987; Ritz-Timme and Collins, 2002).

In the absence of caspases, plants have evolved a wide range of cysteine proteases to ensure functional redundancy in a myriad of highly regulated programmed cell death pathways in response to environmental and developmental cues (Fagundes et al., 2015). Furthermore, plants have developed a variety of strategies to control the destructive prowess of these proteases including the expression of proteases as inactive zymogens with cofactor dependency, compartmentalization and the production of protease inhibitors (Martínez et al., 2012). Of these cysteine proteases the AEPs have recently attracted considerable interest due to their ability to carry out peptide macrocyclization and their potential application in the synthesis of pharmacoactive cyclic peptides. Herein, the structural and functional analysis of HaAEP1 has revealed residues that are able to favor the production of cyclic or acyclic products from SFTI(D14N)-GLDN. Moreover, we have modeled a binding mode for productive macrocyclization of the HaAEP1 natural ligand SFTI-1, based on a comparison with related cysteine proteases that is likely to be conserved between AEPs, where substrate specificity is defined by the amino acids around the binding site for respective AEPs. Furthermore, we have shown that AEPs from diverse species lacking cyclic peptides are able to perform macrocyclization under favorable pH conditions. These findings provide the foundation for further optimization of AEPs, potentially widening the array of peptide substrates that could be cyclized by AEPs.

1. 1. Bond CS

   (2017) Asparaginyl endopeptidase 1 bound to AAN peptide, a tetrahedral intermediate

   Publicly available at Protein Data Bank (accession no. 6AZT).

   https://www.rcsb.org/structure/6AZT

1. 1. Arnison PG
   2. Bibb MJ
   3. Bierbaum G
   4. Bowers AA
   5. Bugni TS
   6. Bulaj G
   7. Camarero JA
   8. Campopiano DJ
   9. Challis GL
   10. Clardy J
   11. Cotter PD
   12. Craik DJ
   13. Dawson M
   14. Dittmann E
   15. Donadio S
   16. Dorrestein PC
   17. Entian KD
   18. Fischbach MA
   19. Garavelli JS
   20. Göransson U
   21. Gruber CW
   22. Haft DH
   23. Hemscheidt TK
   24. Hertweck C
   25. Hill C
   26. Horswill AR
   27. Jaspars M
   28. Kelly WL
   29. Klinman JP
   30. Kuipers OP
   31. Link AJ
   32. Liu W
   33. Marahiel MA
   34. Mitchell DA
   35. Moll GN
   36. Moore BS
   37. Müller R
   38. Nair SK
   39. Nes IF
   40. Norris GE
   41. Olivera BM
   42. Onaka H
   43. Patchett ML
   44. Piel J
   45. Reaney MJ
   46. Rebuffat S
   47. Ross RP
   48. Sahl HG
   49. Schmidt EW
   50. Selsted ME
   51. Severinov K
   52. Shen B
   53. Sivonen K
   54. Smith L
   55. Stein T
   56. Süssmuth RD
   57. Tagg JR
   58. Tang GL
   59. Truman AW
   60. Vederas JC
   61. Walsh CT
   62. Walton JD
   63. Wenzel SC
   64. Willey JM
   65. van der Donk WA

   (2013) Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

   Nat. Prod. Rep. 30:108–160.

   https://doi.org/10.1039/C2NP20085F

   • PubMed
   • Google Scholar
2. 1. Battye TG
   2. Kontogiannis L
   3. Johnson O
   4. Powell HR
   5. Leslie AG

   (2011) iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM

   Acta Crystallographica. Section D, Biological Crystallography 67:271–281.

   https://doi.org/10.1107/S0907444910048675

   • PubMed
   • Google Scholar
3. 1. Bergmann M
   2. Fruton JS

   (1938)

   Some synthetic and hydrolytic experiments with chymotrypsin

   The Journal of Biological Chemistry 124:321–329.

   • Google Scholar
4. 1. Bernath-Levin K
   2. Nelson C
   3. Elliott AG
   4. Jayasena AS
   5. Millar AH
   6. Craik DJ
   7. Mylne JS

   (2015) Peptide macrocyclization by a bifunctional endoprotease

   Chemistry & Biology 22:571–582.

   https://doi.org/10.1016/j.chembiol.2015.04.010

   • PubMed
   • Google Scholar
5. 1. Bhardwaj G
   2. Mulligan VK
   3. Bahl CD
   4. Gilmore JM
   5. Harvey PJ
   6. Cheneval O
   7. Buchko GW
   8. Pulavarti SV
   9. Kaas Q
   10. Eletsky A
   11. Huang PS
   12. Johnsen WA
   13. Greisen PJ
   14. Rocklin GJ
   15. Song Y
   16. Linsky TW
   17. Watkins A
   18. Rettie SA
   19. Xu X
   20. Carter LP
   21. Bonneau R
   22. Olson JM
   23. Coutsias E
   24. Correnti CE
   25. Szyperski T
   26. Craik DJ
   27. Baker D

   (2016) Accurate de novo design of hyperstable constrained peptides

   Nature 538:329–335.

   https://doi.org/10.1038/nature19791

   • PubMed
   • Google Scholar
6. 1. Bollini R
   2. Chrispeels MJ

   (1979) The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

   Planta 146:487–501.

   https://doi.org/10.1007/BF00380865

   • PubMed
   • Google Scholar
7. 1. Bowles DJ
   2. Marcus SE
   3. Pappin DJ
   4. Findlay JB
   5. Eliopoulos E
   6. Maycox PR
   7. Burgess J

   (1986) Posttranslational processing of concanavalin A precursors in jackbean cotyledons

   The Journal of Cell Biology 102:1284–1297.

   https://doi.org/10.1083/jcb.102.4.1284

   • PubMed
   • Google Scholar
8. 1. Brady KD
   2. Giegel DA
   3. Grinnell C
   4. Lunney E
   5. Talanian RV
   6. Wong W
   7. Walker N

   (1999) A catalytic mechanism for caspase-1 and for bimodal inhibition of caspase-1 by activated aspartic ketones

   Bioorganic & Medicinal Chemistry 7:621–631.

   https://doi.org/10.1016/S0968-0896(99)00009-7

   • PubMed
   • Google Scholar
9. 1. Brennan TV
   2. Clarke S

   (1995) Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides

   International Journal of Peptide and Protein Research 45:547–553.

   https://doi.org/10.1111/j.1399-3011.1995.tb01318.x

   • PubMed
   • Google Scholar
10. 1. Buller AR
    2. Townsend CA

    (2013) Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad

    PNAS 110:E653–E661.

    https://doi.org/10.1073/pnas.1221050110

    • PubMed
    • Google Scholar
11. 1. Chekan JR
    2. Estrada P
    3. Covello PS
    4. Nair SK

    (2017) Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants

    PNAS 114:6551–6556.

    https://doi.org/10.1073/pnas.1620499114

    • PubMed
    • Google Scholar
12. 1. Chen JM
    2. Dando PM
    3. Rawlings ND
    4. Brown MA
    5. Young NE
    6. Stevens RA
    7. Hewitt E
    8. Watts C
    9. Barrett AJ

    (1997) Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase

    Journal of Biological Chemistry 272:8090–8098.

    https://doi.org/10.1074/jbc.272.12.8090

    • PubMed
    • Google Scholar
13. 1. Chen VB
    2. Arendall WB
    3. Headd JJ
    4. Keedy DA
    5. Immormino RM
    6. Kapral GJ
    7. Murray LW
    8. Richardson JS
    9. Richardson DC

    (2010) MolProbity: all-atom structure validation for macromolecular crystallography

    Acta Crystallographica Section D Biological Crystallography 66:12–21.

    https://doi.org/10.1107/S0907444909042073

    • PubMed
    • Google Scholar
14. 1. Chu MH
    2. Liu KL
    3. Wu HY
    4. Yeh KW
    5. Cheng YS

    (2011) Crystal structure of tarocystatin-papain complex: implications for the inhibition property of group-2 phytocystatins

    Planta 234:243–254.

    https://doi.org/10.1007/s00425-011-1398-8

    • PubMed
    • Google Scholar
15. 1. Clancy KW
    2. Melvin JA
    3. McCafferty DG

    (2010) Sortase transpeptidases: insights into mechanism, substrate specificity, and inhibition

    Biopolymers 94:385–396.

    https://doi.org/10.1002/bip.21472

    • PubMed
    • Google Scholar
16. 1. Clark AC

    (2016) Caspase allostery and conformational selection

    Chemical Reviews 116:6666–6706.

    https://doi.org/10.1021/acs.chemrev.5b00540

    • PubMed
    • Google Scholar
17. 1. Clark RJ
    2. Fischer H
    3. Dempster L
    4. Daly NL
    5. Rosengren KJ
    6. Nevin ST
    7. Meunier FA
    8. Adams DJ
    9. Craik DJ

    (2005) Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII

    PNAS 102:13767–13772.

    https://doi.org/10.1073/pnas.0504613102

    • PubMed
    • Google Scholar
18. 1. Clark RJ
    2. Jensen J
    3. Nevin ST
    4. Callaghan BP
    5. Adams DJ
    6. Craik DJ

    (2010) The engineering of an orally active conotoxin for the treatment of neuropathic pain

    Angewandte Chemie International Edition 49:6545–6548.

    https://doi.org/10.1002/anie.201000620

    • PubMed
    • Google Scholar
19. 1. Dall E
    2. Brandstetter H

    (2013) Mechanistic and structural studies on legumain explain its zymogenicity, distinct activation pathways, and regulation

    PNAS 110:10940–10945.

    https://doi.org/10.1073/pnas.1300686110

    • PubMed
    • Google Scholar
20. 1. Dall E
    2. Brandstetter H

    (2016) Structure and function of legumain in health and disease

    Biochimie 122:126–150.

    https://doi.org/10.1016/j.biochi.2015.09.022

    • PubMed
    • Google Scholar
21. 1. Dall E
    2. Fegg JC
    3. Briza P
    4. Brandstetter H

    (2015) Structure and mechanism of an aspartimide-dependent peptide ligase in human legumain

    Angewandte Chemie International Edition 54:2917–2921.

    https://doi.org/10.1002/anie.201409135

    • PubMed
    • Google Scholar
22. 1. Dodson G
    2. Wlodawer A

    (1998) Catalytic triads and their relatives

    Trends in Biochemical Sciences 23:347–352.

    https://doi.org/10.1016/S0968-0004(98)01254-7

    • PubMed
    • Google Scholar
23. 1. Dolinsky TJ
    2. Czodrowski P
    3. Li H
    4. Nielsen JE
    5. Jensen JH
    6. Klebe G
    7. Baker NA

    (2007) PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations

    Nucleic Acids Research 35:W522–W525.

    https://doi.org/10.1093/nar/gkm276

    • PubMed
    • Google Scholar
24. 1. Emsley P
    2. Cowtan K

    (2004) Coot: model-building tools for molecular graphics

    Acta Crystallographica Section D Biological Crystallography 60:2126–2132.

    https://doi.org/10.1107/S0907444904019158

    • PubMed
    • Google Scholar
25. 1. Fagundes D
    2. Bohn B
    3. Cabreira C
    4. Leipelt F
    5. Dias N
    6. Bodanese-Zanettini MH
    7. Cagliari A

    (2015) Caspases in plants: metacaspase gene family in plant stress responses

    Functional & Integrative Genomics 15:639–649.

    https://doi.org/10.1007/s10142-015-0459-7

    • PubMed
    • Google Scholar
26. 1. Franke B
    2. Colgrave ML
    3. Mylne JS
    4. Rosengren KJ

    (2016) Mature forms of the major seed storage albumins in sunflower: A mass spectrometric approach

    Journal of Proteomics 147:177–186.

    https://doi.org/10.1016/j.jprot.2016.05.004

    • PubMed
    • Google Scholar
27. 1. Franke B
    2. James AM
    3. Mobli M
    4. Colgrave ML
    5. Mylne JS
    6. Rosengren KJ

    (2017) Two proteins for the price of one: Structural studies of the dual-destiny protein preproalbumin with sunflower trypsin inhibitor-1

    Journal of Biological Chemistry 292:12398–12411.

    https://doi.org/10.1074/jbc.M117.776955

    • PubMed
    • Google Scholar
28. 1. Frankel BA
    2. Kruger RG
    3. Robinson DE
    4. Kelleher NL
    5. McCafferty DG

    (2005) Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

    Biochemistry 44:11188–11200.

    https://doi.org/10.1021/bi050141j

    • PubMed
    • Google Scholar
29. 1. Frankel BA
    2. Tong Y
    3. Bentley ML
    4. Fitzgerald MC
    5. McCafferty DG

    (2007) Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA

    Biochemistry 46:7269–7278.

    https://doi.org/10.1021/bi700448e

    • PubMed
    • Google Scholar
30. 1. Geiger T
    2. Clarke S

    (1987)

    Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation

    The Journal of Biological Chemistry 262:785–794.

    • PubMed
    • Google Scholar
31. 1. Gould A
    2. Ji Y
    3. Aboye TL
    4. Camarero JA

    (2011) Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery

    Current Pharmaceutical Design 17:4294–4307.

    https://doi.org/10.2174/138161211798999438

    • PubMed
    • Google Scholar
32. 1. Hara-Nishimura I
    2. Kinoshita T
    3. Hiraiwa N
    4. Nishimura M

    (1998) Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles

    Journal of Plant Physiology 152:668–674.

    https://doi.org/10.1016/S0176-1617(98)80028-X

    • Google Scholar
33. 1. Hara-Nishimura I
    2. Takeuchi Y
    3. Nishimura M

    (1993) Molecular characterization of a vacuolar processing enzyme related to a putative cysteine proteinase of Schistosoma mansoni

    The Plant Cell Online 5:1651–1659.

    https://doi.org/10.1105/tpc.5.11.1651

    • PubMed
    • Google Scholar
34. 1. Harris KS
    2. Durek T
    3. Kaas Q
    4. Poth AG
    5. Gilding EK
    6. Conlan BF
    7. Saska I
    8. Daly NL
    9. van der Weerden NL
    10. Craik DJ
    11. Anderson MA

    (2015) Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase

    Nature Communications 6:10199.

    https://doi.org/10.1038/ncomms10199

    • PubMed
    • Google Scholar
35. 1. Hatsugai N
    2. Kuroyanagi M
    3. Yamada K
    4. Meshi T
    5. Tsuda S
    6. Kondo M
    7. Nishimura M
    8. Hara-Nishimura I

    (2004) A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death

    Science 305:855–858.

    https://doi.org/10.1126/science.1099859

    • PubMed
    • Google Scholar
36. 1. Hetrick KJ
    2. van der Donk WA

    (2017) Ribosomally synthesized and post-translationally modified peptide natural product discovery in the genomic era

    Current Opinion in Chemical Biology 38:36–44.

    https://doi.org/10.1016/j.cbpa.2017.02.005

    • PubMed
    • Google Scholar
37. 1. Hiraiwa N
    2. Nishimura M
    3. Hara-Nishimura I

    (1999) Vacuolar processing enzyme is self-catalytically activated by sequential removal of the C-terminal and N-terminal propeptides

    FEBS Letters 447:213–216.

    https://doi.org/10.1016/S0014-5793(99)00286-0

    • PubMed
    • Google Scholar
38. 1. Holm L
    2. Rosenström P

    (2010) Dali server: conservation mapping in 3D

    Nucleic Acids Research 38:W545–W549.

    https://doi.org/10.1093/nar/gkq366

    • PubMed
    • Google Scholar
39. 1. James AM
    2. Haywood J
    3. Mylne JS

    (2017) Macrocyclization by asparaginyl endopeptidases

    New Phytologist.

    https://doi.org/10.1111/nph.14511

    • PubMed
    • Google Scholar
40. 1. Jayasena AS
    2. Franke B
    3. Rosengren J
    4. Mylne JS

    (2016) A tripartite approach identifies the major sunflower seed albumins

    Theoretical and Applied Genetics 129:613–629.

    https://doi.org/10.1007/s00122-015-2653-3

    • PubMed
    • Google Scholar
41. 1. Ji Y
    2. Majumder S
    3. Millard M
    4. Borra R
    5. Bi T
    6. Elnagar AY
    7. Neamati N
    8. Shekhtman A
    9. Camarero JA

    (2013) In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide

    Journal of the American Chemical Society 135:11623–11633.

    https://doi.org/10.1021/ja405108p

    • PubMed
    • Google Scholar
42. 1. Kuroyanagi M
    2. Yamada K
    3. Hatsugai N
    4. Kondo M
    5. Nishimura M
    6. Hara-Nishimura I

    (2005) Vacuolar processing enzyme is essential for mycotoxin-induced cell death in Arabidopsis thaliana

    Journal of Biological Chemistry 280:32914–32920.

    https://doi.org/10.1074/jbc.M504476200

    • PubMed
    • Google Scholar
43. 1. Laskowski RA
    2. Swindells MB

    (2011) LigPlot+: multiple ligand-protein interaction diagrams for drug discovery

    Journal of Chemical Information and Modeling 51:2778–2786.

    https://doi.org/10.1021/ci200227u

    • PubMed
    • Google Scholar
44. 1. Lee T-W
    2. James MNG

    (2008) 1.2Å-resolution crystal structures reveal the second tetrahedral intermediates of streptogrisin B (SGPB)

    Biochimica Et Biophysica Acta (BBA) - Proteins and Proteomics 1784:319–334.

    https://doi.org/10.1016/j.bbapap.2007.11.012

    • Google Scholar
45. 1. Liebschner D
    2. Afonine PV
    3. Moriarty NW
    4. Poon BK
    5. Sobolev OV
    6. Terwilliger TC
    7. Adams PD

    (2017) Polder maps: improving OMIT maps by excluding bulk solvent

    Acta Crystallographica Section D Structural Biology 73:148–157.

    https://doi.org/10.1107/S2059798316018210

    • PubMed
    • Google Scholar
46. 1. Lu H
    2. Chandrasekar B
    3. Oeljeklaus J
    4. Misas-Villamil JC
    5. Wang Z
    6. Shindo T
    7. Bogyo M
    8. Kaiser M
    9. van der Hoorn RA

    (2015) Subfamily-specific fluorescent probes for cysteine proteases display dynamic protease activities during seed germination

    Plant Physiology 168:1462–1475.

    https://doi.org/10.1104/pp.114.254466

    • PubMed
    • Google Scholar
47. 1. Luckett S
    2. Garcia RS
    3. Barker JJ
    4. Konarev AV
    5. Shewry PR
    6. Clarke AR
    7. Brady RL

    (1999) High-resolution structure of a potent, cyclic proteinase inhibitor from sunflower seeds

    Journal of Molecular Biology 290:525–533.

    https://doi.org/10.1006/jmbi.1999.2891

    • PubMed
    • Google Scholar
48. 1. Manoury B
    2. Hewitt EW
    3. Morrice N
    4. Dando PM
    5. Barrett AJ
    6. Watts C

    (1998) An asparaginyl endopeptidase processes a microbial antigen for class II MHC presentation

    Nature 396:695–699.

    https://doi.org/10.1038/25379

    • PubMed
    • Google Scholar
49. 1. Martínez M
    2. Cambra I
    3. González-Melendi P
    4. Santamaría ME
    5. Díaz I

    (2012) C1A cysteine-proteases and their inhibitors in plants

    Physiologia Plantarum 145:85–94.

    https://doi.org/10.1111/j.1399-3054.2012.01569.x

    • PubMed
    • Google Scholar
50. 1. Mathieu MA
    2. Bogyo M
    3. Caffrey CR
    4. Choe Y
    5. Lee J
    6. Chapman H
    7. Sajid M
    8. Craik CS
    9. McKerrow JH

    (2002) Substrate specificity of schistosome versus human legumain determined by P1-P3 peptide libraries

    Molecular and Biochemical Parasitology 121:99–105.

    https://doi.org/10.1016/S0166-6851(02)00026-9

    • PubMed
    • Google Scholar
51. 1. Mazmanian SK
    2. Liu G
    3. Ton-That H
    4. Schneewind O

    (1999) Staphylococcus aureus sortase, an enzyme that anchors surface proteins to the cell wall

    Science 285:760–763.

    https://doi.org/10.1126/science.285.5428.760

    • PubMed
    • Google Scholar
52. 1. McLuskey K
    2. Rudolf J
    3. Proto WR
    4. Isaacs NW
    5. Coombs GH
    6. Moss CX
    7. Mottram JC

    (2012) Crystal structure of a Trypanosoma brucei metacaspase

    PNAS 109:7469–7474.

    https://doi.org/10.1073/pnas.1200885109

    • PubMed
    • Google Scholar
53. 1. Min W
    2. Jones DH

    (1994) In vitro splicing of concanavalin A is catalyzed by asparaginyl endopeptidase

    Nature Structural Biology 1:502–504.

    https://doi.org/10.1038/nsb0894-502

    • PubMed
    • Google Scholar
54. 1. Mylne JS
    2. Chan LY
    3. Chanson AH
    4. Daly NL
    5. Schaefer H
    6. Bailey TL
    7. Nguyencong P
    8. Cascales L
    9. Craik DJ

    (2012) Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis

    The Plant Cell 24:2765–2778.

    https://doi.org/10.1105/tpc.112.099085

    • PubMed
    • Google Scholar
55. 1. Mylne JS
    2. Colgrave ML
    3. Daly NL
    4. Chanson AH
    5. Elliott AG
    6. McCallum EJ
    7. Jones A
    8. Craik DJ

    (2011) Albumins and their processing machinery are hijacked for cyclic peptides in sunflower

    Nature Chemical Biology 7:257–259.

    https://doi.org/10.1038/nchembio.542

    • PubMed
    • Google Scholar
56. 1. Nguyen GK
    2. Wang S
    3. Qiu Y
    4. Hemu X
    5. Lian Y
    6. Tam JP

    (2014) Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis

    Nature Chemical Biology 10:732–738.

    https://doi.org/10.1038/nchembio.1586

    • PubMed
    • Google Scholar
57. 1. Otto HH
    2. Schirmeister T

    (1997) Cysteine proteases and their inhibitors

    Chemical Reviews 97:133–172.

    https://doi.org/10.1021/cr950025u

    • PubMed
    • Google Scholar
58. 1. Pattabiraman VR
    2. Bode JW

    (2011) Rethinking amide bond synthesis

    Nature 480:471–479.

    https://doi.org/10.1038/nature10702

    • PubMed
    • Google Scholar
59. 1. Poth AG
    2. Chan LY
    3. Craik DJ

    (2013) Cyclotides as grafting frameworks for protein engineering and drug design applications

    Biopolymers 100:480–491.

    https://doi.org/10.1002/bip.22284

    • PubMed
    • Google Scholar
60. 1. Radisky ES
    2. Lee JM
    3. Lu CJ
    4. Koshland DE

    (2006) Insights into the serine protease mechanism from atomic resolution structures of trypsin reaction intermediates

    PNAS 103:6835–6840.

    https://doi.org/10.1073/pnas.0601910103

    • PubMed
    • Google Scholar
61. 1. Rawlings ND
    2. Barrett AJ
    3. Finn R

    (2016) Twenty years of the MEROPS database of proteolytic enzymes, their substrates and inhibitors

    Nucleic Acids Research 44:D343–D350.

    https://doi.org/10.1093/nar/gkv1118

    • PubMed
    • Google Scholar
62. 1. Reissner KJ
    2. Aswad DW

    (2003) Deamidation and isoaspartate formation in proteins: unwanted alterations or surreptitious signals?

    Cellular and Molecular Life Sciences 60:1281–1295.

    https://doi.org/10.1007/s00018-003-2287-5

    • PubMed
    • Google Scholar
63. 1. Ritz-Timme S
    2. Collins MJ

    (2002) Racemization of aspartic acid in human proteins

    Ageing Research Reviews 1:43–59.

    https://doi.org/10.1016/S0047-6374(01)00363-3

    • PubMed
    • Google Scholar
64. 1. Roy A
    2. Kucukural A
    3. Zhang Y

    (2010) I-TASSER: a unified platform for automated protein structure and function prediction

    Nature Protocols 5:725–738.

    https://doi.org/10.1038/nprot.2010.5

    • PubMed
    • Google Scholar
65. 1. Schechter I
    2. Berger A

    (1967) On the size of the active site in proteases. I. Papain

    Biochemical and Biophysical Research Communications 27:157–162.

    https://doi.org/10.1016/S0006-291X(67)80055-X

    • PubMed
    • Google Scholar
66. Software

    1. Schrodinger L

    (2010)

    The PyMOL Molecular Graphics System, version 1.4

    The PyMOL Molecular Graphics System.
67. 1. Shafee T
    2. Harris K
    3. Anderson M

    (2015)

    Advances in Botanical Research

    227–269, Biosynthesis of cyclotides, Advances in Botanical Research, Academic Press, 10.1016/bs.abr.2015.08.005.

    • Google Scholar
68. 1. Sheldon PS
    2. Keen JN
    3. Bowles DJ

    (1996) Post-translational peptide bond formation during concanavalin A processing in vitro

    Biochemical Journal 320:865–870.

    https://doi.org/10.1042/bj3200865

    • PubMed
    • Google Scholar
69. 1. Shewry PR
    2. Halford NG

    (2002) Cereal seed storage proteins: structures, properties and role in grain utilization

    Journal of Experimental Botany 53:947–958.

    https://doi.org/10.1093/jexbot/53.370.947

    • PubMed
    • Google Scholar
70. 1. Sojka D
    2. Hajdusek O
    3. Dvorák J
    4. Sajid M
    5. Franta Z
    6. Schneider EL
    7. Craik CS
    8. Vancová M
    9. Buresová V
    10. Bogyo M
    11. Sexton KB
    12. McKerrow JH
    13. Caffrey CR
    14. Kopácek P

    (2007) IrAE: an asparaginyl endopeptidase (legumain) in the gut of the hard tick Ixodes ricinus

    International Journal for Parasitology 37:713–724.

    https://doi.org/10.1016/j.ijpara.2006.12.020

    • PubMed
    • Google Scholar
71. 1. Suree N
    2. Liew CK
    3. Villareal VA
    4. Thieu W
    5. Fadeev EA
    6. Clemens JJ
    7. Jung ME
    8. Clubb RT

    (2009) The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized

    Journal of Biological Chemistry 284:24465–24477.

    https://doi.org/10.1074/jbc.M109.022624

    • PubMed
    • Google Scholar
72. 1. Swedberg JE
    2. Li CY
    3. de Veer SJ
    4. Wang CK
    5. Craik DJ

    (2017) Design of potent and selective cathepsin g inhibitors based on the sunflower trypsin inhibitor-1 scaffold

    Journal of Medicinal Chemistry 60:658–667.

    https://doi.org/10.1021/acs.jmedchem.6b01509

    • PubMed
    • Google Scholar
73. 1. Takahashi O
    2. Manabe N
    3. Kirikoshi R

    (2016) A computational study of the mechanism of succinimide formation in the asn-his sequence: intramolecular catalysis by the his side chain

    Molecules 21:327.

    https://doi.org/10.3390/molecules21030327

    • PubMed
    • Google Scholar
74. 1. Truman AW

    (2016) Cyclisation mechanisms in the biosynthesis of ribosomally synthesised and post-translationally modified peptides

    Beilstein Journal of Organic Chemistry 12:1250–1268.

    https://doi.org/10.3762/bjoc.12.120

    • PubMed
    • Google Scholar
75. 1. Tsiatsiani L
    2. Van Breusegem F
    3. Gallois P
    4. Zavialov A
    5. Lam E
    6. Bozhkov PV

    (2011) Metacaspases

    Cell Death & Differentiation 18:1279–1288.

    https://doi.org/10.1038/cdd.2011.66

    • PubMed
    • Google Scholar
76. 1. Vercammen D
    2. van de Cotte B
    3. De Jaeger G
    4. Eeckhout D
    5. Casteels P
    6. Vandepoele K
    7. Vandenberghe I
    8. Van Beeumen J
    9. Inzé D
    10. Van Breusegem F

    (2004) Type II metacaspases Atmc4 and Atmc9 of Arabidopsis thaliana cleave substrates after arginine and lysine

    Journal of Biological Chemistry 279:45329–45336.

    https://doi.org/10.1074/jbc.M406329200

    • PubMed
    • Google Scholar
77. 1. Vernet T
    2. Tessier DC
    3. Chatellier J
    4. Plouffe C
    5. Lee TS
    6. Thomas DY
    7. Storer AC
    8. Ménard R

    (1995) Structural and functional roles of asparagine 175 in the cysteine protease papain

    Journal of Biological Chemistry 270:16645–16652.

    https://doi.org/10.1074/jbc.270.28.16645

    • PubMed
    • Google Scholar
78. 1. White AM
    2. Craik DJ

    (2016) Discovery and optimization of peptide macrocycles

    Expert Opinion on Drug Discovery 11:1151–1163.

    https://doi.org/10.1080/17460441.2016.1245720

    • PubMed
    • Google Scholar
79. 1. Wilmouth RC
    2. Edman K
    3. Neutze R
    4. Wright PA
    5. Clifton IJ
    6. Schneider TR
    7. Schofield CJ
    8. Hajdu J

    (2001) X-ray snapshots of serine protease catalysis reveal a tetrahedral intermediate

    Nature Structural Biology 8:689–694.

    https://doi.org/10.1038/90401

    • PubMed
    • Google Scholar
80. 1. Winn MD
    2. Ballard CC
    3. Cowtan KD
    4. Dodson EJ
    5. Emsley P
    6. Evans PR
    7. Keegan RM
    8. Krissinel EB
    9. Leslie AG
    10. McCoy A
    11. McNicholas SJ
    12. Murshudov GN
    13. Pannu NS
    14. Potterton EA
    15. Powell HR
    16. Read RJ
    17. Vagin A
    18. Wilson KS

    (2011) Overview of the CCP4 suite and current developments

    Acta Crystallographica. Section D, Biological Crystallography 67:235–242.

    https://doi.org/10.1107/S0907444910045749

    • PubMed
    • Google Scholar
81. 1. Yamada K
    2. Shimada T
    3. Nishimura M
    4. Hara-Nishimura I

    (2005) A VPE family supporting various vacuolar functions in plants

    Physiologia Plantarum 123:369–375.

    https://doi.org/10.1111/j.1399-3054.2005.00464.x

    • Google Scholar
82. 1. Yang R
    2. Wong YH
    3. Nguyen GKT
    4. Tam JP
    5. Lescar J
    6. Wu B

    (2017) Engineering a catalytically efficient recombinant protein ligase

    Journal of the American Chemical Society 139:5351–5358.

    https://doi.org/10.1021/jacs.6b12637

    • PubMed
    • Google Scholar
83. 1. Youle RJ
    2. Huang AH

    (1978) Albumin storage proteins in the protein bodies of castor bean

    Plant Physiology 61:13–16.

    https://doi.org/10.1104/pp.61.1.13

    • PubMed
    • Google Scholar
84. 1. Zacks MA
    2. Garg N

    (2006) Recent developments in the molecular, biochemical and functional characterization of GPI8 and the GPI-anchoring mechanism [review]

    Molecular Membrane Biology 23:209–225.

    https://doi.org/10.1080/09687860600601494

    • PubMed
    • Google Scholar
85. 1. Zhao L
    2. Hua T
    3. Crowley C
    4. Ru H
    5. Ni X
    6. Shaw N
    7. Jiao L
    8. Ding W
    9. Qu L
    10. Hung LW
    11. Huang W
    12. Liu L
    13. Ye K
    14. Ouyang S
    15. Cheng G
    16. Liu ZJ

    (2014) Structural analysis of asparaginyl endopeptidase reveals the activation mechanism and a reversible intermediate maturation stage

    Cell Research 24:344–358.

    https://doi.org/10.1038/cr.2014.4

    • PubMed
    • Google Scholar

Author details

1. Joel Haywood

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Jason W Schmidberger

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. Amy M James

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

4. Samuel G Nonis

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

5. Kirill V Sukhoverkov

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

6. Mikael Elias

   Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, United States

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

7. Charles S Bond

   School of Molecular Sciences, The University of Western Australia, Perth, Australia

   Contribution

   Formal analysis, Supervision, Validation, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

8. Joshua S Mylne

   1. School of Molecular Sciences, The University of Western Australia, Perth, Australia
   2. The ARC Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley, Australia

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   joshua.mylne@uwa.edu.au

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4957-6388

• 3,060

  views

• 500

  downloads

• 49

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.