CYCLOTIDES (Craik)
The name cyclotides was introduced for ribosomally-synthesized peptides from plants that are
characterized by a head-to-tail cyclic peptide backbone and a cystine knot arrangement of three
conserved disulfide bonds1. They were originally discovered in plants of the Rubiaceae (coffee)
and Violaceae (violet) families but have since been found in the Cucurbitaceae and Fabaceae
(legume) families. They are expressed in many plant tissues, including leaves, stems, flowers and
roots. Dozens to hundreds of different cyclotides are expressed in an individual plant and there
appears to be little crossovers of cyclotides between different species, i.e., most plants have a
unique set of cyclotides.
Figure 1 shows the structure of the prototypic cyclotide, kalata B1, from the African herb
Oldenlandia affinis (Rubiaceae).2 It comprises 29 amino acids, including the six conserved Cys
residues that form the signature cyclic cystine knot (CCK) motif of the family. The backbone
segments between successive Cys residues are referred to as loops and the sequence variations of
cyclotides occur within these loops. Cyclotides have been classified into two main subfamilies,
Möbius or bracelet, based on the presence or absence of a cis X-Pro peptide bond in loop 5 of the
sequence but there is also a smaller subfamily that is referred to as the trypsin inhibitor
subfamily. This third subfamily has high sequence homology to some members of the knottin
family of proteins from squash plants and its members are also referred to as cyclic knottins3.
So far more than 200 sequences of cyclotides have been reported and they are documented in a
database dedicated to circular proteins called CyBase (www.cybase.org.au).4 They range in size
from 28-37 amino acids and are typically not highly charged peptides. Aside from their disulfide
bonds5 and head-to-tail cyclised backbone, there appear to be no other post-translational
modifications in cyclotides. Cyclotides appear to be ubiquitous in Violaceae family plants but
are more sparsely distributed in the Rubiaceae, occurring in about 5% of the hundreds of plants
screened so far. The reports of cyclotides in other plant families are more recent and there is
limited information available on the distribution of cyclotides in these families. Nevertheless, it
appears that cyclotides are a very large family of plant proteins, potentially numbering in the tens
of thousands.6
Cyclotides are gene-encoded peptides that are processed from larger precursor proteins. The
precursors of Rubiaceae and Violaceae cyclotides are dedicated proteins, whose purpose appears
to be only to produce cyclotides. By contrast, cyclotides in Clitoria ternatea, a member of the
Fabaceae family, are produced from a chimeric precursor protein that also encodes an albumin,7,
8
a situation similar to that recently reported for a small cyclic peptide trypsin inhibitor from
sunflower seeds.9 Thus, there appear to be multiple types of precursors leading to circular
proteins in plants. In the case of Rubiaceae and Violaceae family plants the leader sequence is
considered to comprise a pro-region and an N-terminal region (NTR) that is repeated in some
genes along with the adjacent core peptide region, as illustrated in Figure 1.
Figure x. Structure of kalata B1 and cyclotide precursor proteins. A schematic
representation of three precursor proteins from O. affinis is shown at the top of the figure. The
proteins comprise an endoplasmic reticulum signal sequence labeled ER, a leader sequence
(comprising a pro-region and a conserved repeated fragment labeled NTR) and either one or
multiple copies of the core peptides; for example, kalata B1, B2, B3 and B6. A short
hydrophobic C-terminal recognition sequence is present in each precursor. At the bottom of the
figure the amino acid sequence of kalata B1 is shown, with the cysteine residues labeled with
Roman numerals. The cleavage sites for excision of a core cyclotide domain with an N-terminal
Gly and a C-terminal Asn, which may be subsequently linked by an asparaginyl endopeptidase,
are indicated by arrows. The location of the ligation point which forms loop 6 of the mature
cyclic peptide is shown on the right. Parts of the precursor protein flanking the mature domain
are shown in lighter shading. Figure adapted from Daly et al.10
Biological activities of cyclotides
Cyclotides are thought to be plant defence molecules, given their potent insecticidal activity
against Helicoverpa species11-14. However, they also have a broad range of other biological
activities, including anti-HIV15, antimicrobial16, cytotoxic17, molluscicidal18, anti-barnacle19,
nematocidal20, 21 and haemolytic activities22. Some of these activities are of potential
pharmaceutical interest and because of their exceptional stability cyclotides have also attracted
attention as potential protein engineering or drug design templates.23, 24
The diverse range of activities of cyclotides seems to have a common mechanism that involves
binding to and disruption of biological membranes.25 Electron micrographs of Helicoverpa
larvae fed a diet containing cyclotides at a similar concentration to that which occurs naturally in
plants for example show marked swelling and blebbing of mid-gut cells11. Larvae that have
ingested cyclotides are markedly stunted in their growth and development, presumably as a
result of this disruption of their mid-gut membranes. A range of biophysical studies26-28 have
confirmed membrane interactions for both Möbius and bracelet cyclotides, and detailed
information is available on the residues involved in making contact with membrane surfaces.
Furthermore, electrophysiological and vesicle leakage studies have confirmed the leakage of
marker molecules through membranes treated with cyclotides. It appears that cyclotides are able
to self associate in the membrane environment and form large pores in membranes.
Cyclotide biosynthesis
As indicated in Figure x, cyclotides are derived from precursor proteins that encode one or more
copies of the core (cyclotide) peptide sequence. For example the Oak1 (Oldenlandia affinis
kalata B1 gene) encodes an 11 kDa precursor protein that contains an endoplasmic reticulum
(ER) signal, leader peptide, kalata B1 core peptide, and a C-terminal peptide region, whereas the
Oak4 gene, encodes a precursor containing three copies of kalata B213. The single or multiple
copies of the cyclotide domains are flanked in each case by N-terminal and C-terminal
recognition sequences that are thought to be implicated in the processing reactions.
Because of the presence of the ER signal it is thought that cyclotide precursors are probably
folded in the ER prior to processing (excision and cyclization) of the cyclotide domain. Protein
disulfide isomerases are often involved in the folding of disulfide-rich proteins29 and in vitro
experiments have shown increased yields of folded cyclotide kalata B1 in the presence of PDI,
although so far there have been no definitive studies on the involvement of PDI for in planta
cyclotide folding. A recent study has shown that cyclotides are targeted to vacuoles in plant cells
and this is where the excision and cyclization processes are thought to occur30.
The N-terminal repeat (NTR) region of cyclotide precursors has been found to adopt an α-helical
structure as an isolated synthetic peptide,31 but the significance, if any, of this is not yet known.
It has been proposed that it might represent a recognition sequence for cyclotide folding by PDI,
but interestingly cyclotide precursors from the Fabaceae plant C. ternatea lack the NTR region
seem in Violaceae or Rubiaceae precursors. The nature of the processing reaction(s) occurring at
the N-terminal end of the cyclotide domain is still essentially unknown.
However, there is strong evidence that asparaginyl endoprotease (AEP) activity is responsible for
processing at the C-terminal end of the cyclotide domain including involvement in the
cyclization process.32, 33 With just one exception (circulin F) all cyclotides have an Asn or Asp
residue as the C-terminal end of the core peptide, and an AEP is principle able to cleave after this
residue. It has been proposed that this cleavage reaction occurs contemporaneously with ligation
to the earlier-released N-terminus of the core peptide region. Mutagenesis experiments in
transgenic tobacco and Arabidopsis plants have shown a vital role for the N-terminal Asn
residue, as well as other key flanking residues.32 Similarly, gene silencing experiments in which
AEP activity is knocked down show decreased production of cyclic peptide in transgenic
plants.33 Overall, the processing of cyclotides can be summarized as involving excision and
cyclization from a pre-folded precursor.
Specific recommendations for the family
At present there is no common gene nomenclature for cyclotide precursors and given the limited
knowledge of the full complement of associated biosynthetic genes encoding proteins involved
processing it seems premature to propose one. A naming scheme for the mature cyclic peptides
that involves a four letter pronounceable acronym based on the Latin name of the plant species in
which the cyclotide was first discovered has been proposed34 but has not been uniformly
followed so far.
References
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11.
12.
13.
14.
15.
16.
17.
Craik, D.J.; Daly, N.L.; Bond, T.; Waine, C. Plant cyclotides: A unique family of cyclic and
knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 1999,
294, 1327-1336.
Saether, O.; Craik, D.J.; Campbell, I.D.; Sletten, K.; Juul, J.; Norman, D.G. Elucidation of
the primary and three-dimensional structure of the uterotonic polypeptide kalata B1.
Biochemistry 1995, 34, 4147-4158.
Chiche, L.; Heitz, A.; Gelly, J.C.; Gracy, J.; Chau, P.T.; Ha, P.T.; Hernandez, J.F.; Le-Nguyen,
D. Squash inhibitors: from structural motifs to macrocyclic knottins. Curr Protein Pept Sci
2004, 5, 341-349.
Wang, C.K.; Kaas, Q.; Chiche, L.; Craik, D.J. CyBase: a database of cyclic protein
sequences and structures, with applications in protein discovery and engineering.
Nucleic Acids Res 2008, 36, D206-210.
Göransson, U.; Craik, D.J. Disulfide mapping of the cyclotide kalata B1. Chemical proof of
the cyclic cystine knot motif. J. Biol. Chem. 2003, 278, 48188-48196.
Gruber, C.W.; Elliott, A.G.; Ireland, D.C.; Delprete, P.G.; Dessein, S.; Goransson, U.; Trabi,
M.; Wang, C.K.; Kinghorn, A.B.; Robbrecht, E.; Craik, D.J. Distribution and evolution of
circular miniproteins in flowering plants. Plant Cell 2008, 20, 2471-2483.
Nguyen, G.K.; Zhang, S.; Nguyen, N.T.; Nguyen, P.Q.; Chiu, M.S.; Hardjojo, A.; Tam, J.P.
Discovery and characterization of novel cyclotides originated from chimeric precursors
consisting of albumin-1 chain a and cyclotide domains in the fabaceae family. J Biol
Chem 2011, 286, 24275-24287.
Poth, A.G.; Colgrave, M.L.; Lyons, R.E.; Daly, N.L.; Craik, D.J. From the Cover: Discovery
of an unusual biosynthetic origin for circular proteins in legumes. Proceedings of the
National Academy of Sciences, USA 2011, 108, 10127-10132.
Mylne, J.S.; Colgrave, M.L.; Daly, N.L.; Chanson, A.H.; Elliott, A.G.; McCallum, E.J.; Jones,
A.; Craik, D.J. Albumins and their processing machinery are hijacked for cyclic peptides
in sunflower. Nature Chemical Biology 2011, 7, 257-259.
Daly, N.L.; Rosengren, K.J.; Craik, D.J. Discovery, structure and biological activities of
cyclotides. Adv Drug Deliv Rev 2009, 61, 918-930.
Barbeta, B.L.; Marshall, A.T.; Gillon, A.D.; Craik, D.J.; Anderson, M.A. Plant cyclotides
disrupt epithelial cells in the midgut of lepidopteran larvae. Proceedings of the National
Academy of Sciences of the United States of America 2008, 105, 1221-1225.
Gruber, C.W.; Cemazar, M.; Anderson, M.A.; Craik, D.J. Insecticidal plant cyclotides and
related cystine knot toxins. Toxicon 2007, 49, 561-575.
Jennings, C.; West, J.; Waine, C.; Craik, D.; Anderson, M. Biosynthesis and insecticidal
properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis.
Proceedings of the National Academy of Sciences of the United States of America 2001,
98, 10614-10619.
Jennings, C.V.; Rosengren, K.J.; Daly, N.L.; Plan, M.; Stevens, J.; Scanlon, M.J.; Waine, C.;
Norman, D.G.; Anderson, M.A.; Craik, D.J. Isolation, solution structure, and insecticidal
activity of kalata B2, a circular protein with a twist: do Mobius strips exist in nature?
Biochemistry 2005, 44, 851-860.
Gustafson, K.R.; McKee, T.C.; Bokesch, H.R. Anti-HIV cyclotides. Current Protein &
Peptide Science 2004, 5, 331-340.
Tam, J.P.; Lu, Y.A.; Yang, J.L.; Chiu, K.W. An unusual structural motif of antimicrobial
peptides containing end-to-end macrocycle and cystine-knot disulfides. Proceedings of
the National Academy of Sciences of the United States of America 1999, 96, 8913-8918.
Svangard, E.; Göransson, U.; Hocaoglu, Z.; Gullbo, J.; Larsson, R.; Claeson, P.; Bohlin, L.
Cytotoxic cyclotides from Viola tricolor. J. Nat. Prod. 2004, 67, 144-147.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
Plan, M.R.; Saska, I.; Cagauan, A.G.; Craik, D.J. Backbone cyclised peptides from plants
show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple
snail). J Agric Food Chem 2008, 56, 5237-5241.
Göransson, U.; Sjogren, M.; Svangard, E.; Claeson, P.; Bohlin, L. Reversible Antifouling
Effect of the Cyclotide Cycloviolacin O2 against Barnacles. J Nat Prod 2004, 67, 12871290.
Colgrave, M.L.; Kotze, A.C.; Huang, Y.H.; O'Grady, J.; Simonsen, S.M.; Craik, D.J.
Cyclotides: natural, circular plant peptides that possess significant activity against
gastrointestinal nematode parasites of sheep. Biochemistry 2008, 47, 5581-5589.
Colgrave, M.L.; Kotze, A.C.; Ireland, D.C.; Wang, C.K.; Craik, D.J. The anthelmintic activity
of the cyclotides: natural variants with enhanced activity. ChemBioChem 2008, 9, 19391945.
Barry, D.G.; Daly, N.L.; Clark, R.J.; Sando, L.; Craik, D.J. Linearization of a naturally
occurring circular protein maintains structure but eliminates hemolytic activity.
Biochemistry 2003, 42, 6688-6695.
Craik, D.J.; Cemazar, M.; Wang, C.K.; Daly, N.L. The cyclotide family of circular
miniproteins: nature's combinatorial peptide template. Biopolymers 2006, 84, 250-266.
Jagadish, K.; Camarero, J.A. Cyclotides, a promising molecular scaffold for peptide-based
therapeutics. Biopolymers 2010, 94, 611-616.
Huang, Y.H.; Colgrave, M.L.; Daly, N.L.; Keleshian, A.; Martinac, B.; Craik, D.J. The
biological activity of the prototypic cyclotide Kalata B1 is modulated by the formation of
multimeric pores. J Biol Chem 2009, 284, 20699-20707.
Kamimori, H.; Hall, K.; Craik, D.J.; Aguilar, M.I. Studies on the membrane interactions of
the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon
resonance. Anal Biochem 2005, 337, 149-153.
Shenkarev, Z.O.; Nadezhdin, K.D.; Lyukmanova, E.N.; Sobol, V.A.; Skjeldal, L.; Arseniev,
A.S. Divalent cation coordination and mode of membrane interaction in cyclotides: NMR
spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle. J Inorg Biochem 2008,
102, 1246-1256.
Shenkarev, Z.O.; Nadezhdin, K.D.; Sobol, V.A.; Sobol, A.G.; Skjeldal, L.; Arseniev, A.S.
Conformation and mode of membrane interaction in cyclotides. Spatial structure of
kalata B1 bound to a dodecylphosphocholine micelle. FEBS Journal 2006, 273, 26582672.
Gruber, C.W.; Cemazar, M.; Mechler, A.; Martin, L.L.; Craik, D.J. Biochemical and
biophysical characterization of a novel plant protein disulfide isomerase. Biopolymers
2009, 92, 35-43.
Conlan, B.F.; Gillon, A.D.; Barbeta, B.L.; Anderson, M.A. Subcellular targeting and
biosynthesis of cyclotides in plant cells. Am J Bot 2011, 98, 2018-2026.
Dutton, J.L.; Renda, R.F.; Waine, C.; Clark, R.J.; Daly, N.L.; Jennings, C.V.; Anderson, M.A.;
Craik, D.J. Conserved Structural and Sequence Elements Implicated in the Processing of
Gene-encoded Circular Proteins. J. Biol. Chem. 2004, 279, 46858-46867.
Gillon, A.D.; Saska, I.; Jennings, C.V.; Guarino, R.F.; Craik, D.J.; Anderson, M.A.
Biosynthesis of circular proteins in plants. Plant Journal 2008, 53, 505-515.
Saska, I.; Gillon, A.D.; Hatsugai, N.; Dietzgen, R.G.; Hara-Nishimura, I.; Anderson, M.A.;
Craik, D.J. An asparaginyl endopeptidase mediates in vivo protein backbone cyclisation.
J. Biol. Chem. 2007, 282, 29721-29728.
Broussalis, A.M.; Göransson, U.; Coussio, J.D.; Ferraro, G.; Martino, V.; Claeson, P. First
cyclotide from Hybanthus (Violaceae). Phytochemistry 2001, 58, 47-51.