1.A.118. The Plant Cyclotide (Cyclotide) Family

Cyclotides (Craik et al. 1999) are topologically unique plant proteins that are exceptionally stable. They comprise approximately 30 amino acids arranged in a head-to-tail cyclized peptide backbone that additionally is restrained by a cystine knot motif. The cystine knot is built from two disulfide bonds, and their connecting backbone segments form an internal ring in the structure that is threaded by a third disulfide bond to form an interlocked and cross-braced structure. Superimposed on this cystine knot core are a β-sheet and a series of turns displaying surface-exposed loops (Poth et al. 2011).

Cyclotides have a diversity of peptide sequences in their backbone loops and have a broad range of biological activities, including uterotonic, anti-HIV, antimicrobial (Tam et al. 1999), and anticancer activities (Svangård et al. 2007). Accordingly, they are of great interest for pharmaceutical applications. Some plants from which they are derived are used in indigenous medicines, including kalata-kalata, a tea from the plant Oldenlandia affinis, which is used for accelerating childbirth in Africa and contains the prototypic cyclotide kalata B1, kB1 (Henriques et al. 2011). This ethnobotanical use and recent biophysical studies illustrate the remarkable stability of cyclotides; i.e., they survive boiling and ingestion, observations unprecedented for conventional peptides. Their exceptional stability has led to their use as templates in peptide-based drug design applications, where the grafting of bioactive peptide sequences into a cyclotide framework offers a new approach to stabilize peptide-based therapeutics, thereby overcoming one of the major limitations of peptides as drugs.

The natural function of cyclotides appears to be in plant defense, based on their pesticidal activities, including insecticidal, nematocidal, and molluscicidal activities. These activities appear to be mediated by selective membrane binding and disruption (Barbeta et al. 2008; Huang et al. 2009) that occurs as a result of cyclotides having a surface-exposed patch of hydrophobic residues. Individual plants typically contain dozens of cyclotides, expressed in multiple tissues, including flowers, leaf, and seeds, leading to their description as a natural combinatorial template. Plants presumably use this combinatorial strategy to target multiple pests or to reduce the possibility of an individual pest species developing resistance to the protective cyclotide armory. More than 170 cyclotides have been sequenced, although it is estimated that the family probably comprises around 50,000 members, making it a particularly large family of plant proteins (Gruber et al. 2008).

Cyclotides have been found in the Rubiaceae (coffee), Violaceae (violet), Cucurlitaceae (Cucurbit or gourd) and Fabaceae (legume) families. The stability of cyclic peptides in harsh biological milieu may be responsible for their multiple biosynthetic pathways. These proteins form structurally conserved alpha-helical motifs (Dutton et al. 2004) and insert into phospholipid bilayeres to form pores and destabilize the membrane (Wang et al. 2012).



Barbeta, B.L., A.T. Marshall, A.D. Gillon, D.J. Craik, and M.A. Anderson. (2008). Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc. Natl. Acad. Sci. USA 105: 1221-1225.

Craik, D.J., N.L. Daly, T. Bond, and C. Waine. (1999). Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 294: 1327-1336.

Dutton, J.L., R.F. Renda, C. Waine, R.J. Clark, N.L. Daly, C.V. Jennings, M.A. Anderson, and D.J. Craik. (2004). Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins. J. Biol. Chem. 279: 46858-46867.

Gruber, C.W., A.G. Elliott, D.C. Ireland, P.G. Delprete, S. Dessein, U. Göransson, M. Trabi, C.K. Wang, A.B. Kinghorn, E. Robbrecht, and D.J. Craik. (2008). Distribution and evolution of circular miniproteins in flowering plants. Plant Cell 20: 2471-2483.

Henriques, S.T., Y.H. Huang, K.J. Rosengren, H.G. Franquelim, F.A. Carvalho, A. Johnson, S. Sonza, G. Tachedjian, M.A. Castanho, N.L. Daly, and D.J. Craik. (2011). Decoding the membrane activity of the cyclotide kalata B1: the importance of phosphatidylethanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities. J. Biol. Chem. 286: 24231-24241.

Huang, Y.H., M.L. Colgrave, N.L. Daly, A. Keleshian, B. Martinac, and D.J. Craik. (2009). The biological activity of the prototypic cyclotide kalata b1 is modulated by the formation of multimeric pores. J. Biol. Chem. 284: 20699-20707.

Poth, A.G., M.L. Colgrave, R.E. Lyons, N.L. Daly, and D.J. Craik. (2011). Discovery of an unusual biosynthetic origin for circular proteins in legumes. Proc. Natl. Acad. Sci. USA 108: 10127-10132.

Svangård, E., R. Burman, S. Gunasekera, H. Lövborg, J. Gullbo, and U. Göransson. (2007). Mechanism of action of cytotoxic cyclotides: cycloviolacin O2 disrupts lipid membranes. J Nat Prod 70: 643-647.

Tam, J.P., Y.A. Lu, J.L. Yang, and K.W. Chiu. (1999). An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc. Natl. Acad. Sci. USA 96: 8913-8918.

Wang, C.K., H.P. Wacklin, and D.J. Craik. (2012). Cyclotides insert into lipid bilayers to form membrane pores and destabilize the membrane through hydrophobic and phosphoethanolamine-specific interactions. J. Biol. Chem. 287: 43884-43898.


TC#NameOrganismal TypeExample

Cyclotide, kalata-B1, kB1. Targets membranes in a process that depends on lipid interactions; hemolytic; can disrupt HIV membranes (Henriques et al., 2011).


Kalata-B1 of Oldenlandia affinis (P56254)


Cyclotide, Cycloviolacin O8


Cycloviolacin O8 of Viola odovata (P58440)


The Varv peptide A/Kalata-B1


Varv of Viola odorata (Q5USN7)


Cyclotide Oak6


Oak6 of Oldenlandia affinis (D8WS37)