cystine knot motifs


Summary: Amino acid sequence in which two disulfide bonds (DISULFIDES) and their connecting backbone form a ring that is penetrated by a third disulfide bond. Members include CYCLOTIDES and agouti-related protein.

Top Publications

  1. Gruber C, Cemazar M, Anderson M, Craik D. Insecticidal plant cyclotides and related cystine knot toxins. Toxicon. 2007;49:561-75 pubmed
  2. Silverman A, Levin A, Lahti J, Cochran J. Engineered cystine-knot peptides that bind alpha(v)beta(3) integrin with antibody-like affinities. J Mol Biol. 2009;385:1064-75 pubmed publisher
    ..Collectively, these data validate AgRP as a scaffold for protein engineering and demonstrate that modification of a single loop can lead to AgRP-based peptides with antibody-like affinities for their target. ..
  3. Cemazar M, Joshi A, Daly N, Mark A, Craik D. The structure of a two-disulfide intermediate assists in elucidating the oxidative folding pathway of a cyclic cystine knot protein. Structure. 2008;16:842-51 pubmed publisher
  4. Kolmar H. Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J. 2008;275:2684-90 pubmed publisher
  5. Heitz A, Avrutina O, Le Nguyen D, Diederichsen U, Hernandez J, Gracy J, et al. Knottin cyclization: impact on structure and dynamics. BMC Struct Biol. 2008;8:54 pubmed publisher
    ..Unfolding simulations showed however that cyclization is a stabilizing factor in strongly denaturing conditions. This information should be useful if one wants to use the squash inhibitor scaffold in drug design. ..
  6. Simonsen S, Sando L, Rosengren K, Wang C, Colgrave M, Daly N, et al. Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. J Biol Chem. 2008;283:9805-13 pubmed publisher
    ..Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities. ..
  7. Wang C, Colgrave M, Gustafson K, Ireland D, Goransson U, Craik D. Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis. J Nat Prod. 2008;71:47-52 pubmed
  8. Leta Aboye T, Clark R, Craik D, Goransson U. Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2. Chembiochem. 2008;9:103-13 pubmed
    ..The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure. ..
  9. Huang Y, Colgrave M, Daly N, Keleshian A, Martinac B, Craik D. The biological activity of the prototypic cyclotide kalata b1 is modulated by the formation of multimeric pores. J Biol Chem. 2009;284:20699-707 pubmed publisher
    ..Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides...

More Information


  1. Gracy J, Le Nguyen D, Gelly J, Kaas Q, Heitz A, Chiche L. KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007. Nucleic Acids Res. 2008;36:D314-9 pubmed
    ..The KNOTTIN database is available at ..
  2. Kolmar H. Biological diversity and therapeutic potential of natural and engineered cystine knot miniproteins. Curr Opin Pharmacol. 2009;9:608-14 pubmed publisher
  3. Krause S, Schmoldt H, Wentzel A, Ballmaier M, Friedrich K, Kolmar H. Grafting of thrombopoietin-mimetic peptides into cystine knot miniproteins yields high-affinity thrombopoietin antagonists and agonists. FEBS J. 2007;274:86-95 pubmed
    ..This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand-induced dimerization. ..
  4. Reinwarth M, Nasu D, Kolmar H, Avrutina O. Chemical synthesis, backbone cyclization and oxidative folding of cystine-knot peptides: promising scaffolds for applications in drug design. Molecules. 2012;17:12533-52 pubmed publisher
    ..Herein we describe the topology of cystine-knot peptides, their synthetic availability and briefly discuss potential applications of engineered variants in diagnostics and therapy. ..
  5. Gunasekera S, Daly N, Anderson M, Craik D. Chemical synthesis and biosynthesis of the cyclotide family of circular proteins. IUBMB Life. 2006;58:515-24 pubmed
    ..In this review the current chemical, recombinant and biosynthetic routes to the production of cyclotides are discussed. ..
  6. Pi L, Shenoy A, Liu J, Kim S, Nelson N, Xia H, et al. CCN2/CTGF regulates neovessel formation via targeting structurally conserved cystine knot motifs in multiple angiogenic regulators. FASEB J. 2012;26:3365-79 pubmed publisher
    ..approach and demonstrated a broad affinity of connective tissue growth factor (CCN2/CTGF) to C-terminal cystine knot motifs present in key angiogenic regulators Slit3, von Willebrand factor, platelet-derived growth factor-B, and ..
  7. Chen B, Colgrave M, Daly N, Rosengren K, Gustafson K, Craik D. Isolation and characterization of novel cyclotides from Viola hederaceae: solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide. J Biol Chem. 2005;280:22395-405 pubmed
    ..Met(7) was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC(50) value of 0.87 microm...
  8. Daly N, Craik D. Bioactive cystine knot proteins. Curr Opin Chem Biol. 2011;15:362-8 pubmed publisher
  9. Reiss S, Sieber M, Oberle V, Wentzel A, Spangenberg P, Claus R, et al. Inhibition of platelet aggregation by grafting RGD and KGD sequences on the structural scaffold of small disulfide-rich proteins. Platelets. 2006;17:153-7 pubmed
    ..Differences that were observed between the engineered proteins indicate the importance of the structural scaffold and the amino acids neighbouring the grafted peptide sequences. ..
  10. Ireland D, Colgrave M, Nguyencong P, Daly N, Craik D. Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins. J Mol Biol. 2006;357:1522-35 pubmed
    ..Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule...
  11. Clark R, Daly N, Craik D. Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design. Biochem J. 2006;394:85-93 pubmed
    ..This finding confirms the tolerance of the cyclotide framework to residue substitutions and opens up possibilities for the substitution of biologically active peptide epitopes into the framework. ..
  12. Herrmann A, Svangård E, Claeson P, Gullbo J, Bohlin L, Goransson U. Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2. Cell Mol Life Sci. 2006;63:235-45 pubmed
    ..In addition, we show that the activity is dependent on an intact disulfide network and that the short sequences between the six cysteine residues, that is, the backbone loops, are devoid of cytotoxic activity. ..
  13. Craik D, Cemazar M, Wang C, Daly N. The cyclotide family of circular miniproteins: nature's combinatorial peptide template. Biopolymers. 2006;84:250-66 pubmed
    ..Cyclotides are exceptionally stable and are resistant to denaturation via thermal, chemical, or enzymatic treatments. The structural features that contribute to their remarkable stability are described in this review. ..
  14. Getz J, Rice J, Daugherty P. Protease-resistant peptide ligands from a knottin scaffold library. ACS Chem Biol. 2011;6:837-44 pubmed publisher
    ..More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. ..
  15. Wang C, Hu S, Martin J, Sjögren T, Hajdu J, Bohlin L, et al. Combined X-ray and NMR analysis of the stability of the cyclotide cystine knot fold that underpins its insecticidal activity and potential use as a drug scaffold. J Biol Chem. 2009;284:10672-83 pubmed publisher
    ..As well as being important for their bioactivity, the structural rigidity of cyclotides makes them very suitable as a stable template for peptide-based drug design. ..
  16. Kolmar H. Engineered cystine-knot miniproteins for diagnostic applications. Expert Rev Mol Diagn. 2010;10:361-8 pubmed publisher
    ..Promising candidates for tumor imaging have been generated recently and evaluated in animal models, and more applications are expected in the near future. ..
  17. Huang Y, Colgrave M, Clark R, Kotze A, Craik D. Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity. J Biol Chem. 2010;285:10797-805 pubmed publisher
    ..Our data clearly highlight the residues crucial for nematocidal and hemolytic activity in cyclotides, and demonstrate that the nematocidal activity of cyclotides can be increased by incorporation of basic amino acids. ..
  18. Shen K, Krakora S, Cunningham M, Singh M, Wang X, Hu F, et al. Medical treatment of craniosynostosis: recombinant Noggin inhibits coronal suture closure in the rat craniosynostosis model. Orthod Craniofac Res. 2009;12:254-62 pubmed publisher
    ..Topical application of rhNoggin protein prevents craniosynostosis in the weanling nude rat xenotransplantation model of syndromic craniosynostosis...
  19. Alvarez E, Cahoreau C, Combarnous Y. Comparative structure analyses of cystine knot-containing molecules with eight aminoacyl ring including glycoprotein hormones (GPH) alpha and beta subunits and GPH-related A2 (GPA2) and B5 (GPB5) molecules. Reprod Biol Endocrinol. 2009;7:90 pubmed publisher
    ..Consequently, GPA2 and GPB5 could exert separate endocrine roles either during development and/or during adult life of both vertebrates and invertebrates. ..
  20. Sommerhoff C, Avrutina O, Schmoldt H, Gabrijelcic Geiger D, Diederichsen U, Kolmar H. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta. J Mol Biol. 2010;395:167-75 pubmed publisher
    ..These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors. ..
  21. Schmoldt H, Wentzel A, Becker S, Kolmar H. A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle. Protein Expr Purif. 2005;39:82-9 pubmed
    ..The ICK peptides are released from the barnase expression and purification handle either by cyanogen bromide or by protease cleavage to give pure and correctly folded cystine knot peptides. ..
  22. Durek T, Zhang J, He C, Kent S. Synthesis of photoactive analogues of a cystine knot trypsin inhibitor protein. Org Lett. 2007;9:5497-500 pubmed
    ..Site-specific labeling of proteins with this amino acid analogue through chemical synthesis provides valuable probes for photoaffinity cross-linking studies. ..
  23. Craik D, Clark R, Daly N. Potential therapeutic applications of the cyclotides and related cystine knot mini-proteins. Expert Opin Investig Drugs. 2007;16:595-604 pubmed
    ..The cystine knot framework is tolerant to a wide range of residue substitutions and is showing great promise as a scaffold in drug design and protein engineering. ..
  24. Craik D, Daly N. Oxidative folding of the cystine knot motif in cyclotide proteins. Protein Pept Lett. 2005;12:147-52 pubmed
    ..This mini-review reports on the current understanding of the folding process in cyclotides. The synthesis and folding of these molecules paves the way for their application as stable molecular templates. ..
  25. Werle M, Kafedjiiski K, Kolmar H, Bernkop Schnurch A. Evaluation and improvement of the properties of the novel cystine-knot microprotein McoEeTI for oral administration. Int J Pharm. 2007;332:72-9 pubmed
    ..In conclusion, this study indicates that McoEeTI represents a promising candidate as a novel scaffold for oral peptide drug delivery. ..
  26. Honegger H, Dewey E, Ewer J. Bursicon, the tanning hormone of insects: recent advances following the discovery of its molecular identity. J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 2008;194:989-1005 pubmed publisher
    ..melanogaster. Although much remains to be learned, the elucidation of its molecular identity and that of its receptor has provided the breakthrough needed for investigating the diverse actions of this critical insect neurohormone. ..
  27. Ireland D, Clark R, Daly N, Craik D. Isolation, sequencing, and structure-activity relationships of cyclotides. J Nat Prod. 2010;73:1610-22 pubmed publisher
    ..They have thus attracted interest in drug design as well as in crop protection applications. ..
  28. Burman R, Gunasekera S, Strömstedt A, Goransson U. Chemistry and biology of cyclotides: circular plant peptides outside the box. J Nat Prod. 2014;77:724-36 pubmed publisher
  29. Wegener H, Paulsen H, Seeger K. The cysteine-rich region of type VII collagen is a cystine knot with a new topology. J Biol Chem. 2014;289:4861-9 pubmed publisher
    ..The conserved motif of the cystine knot is CX3CP. The cystine knot is N-terminal to the collagen triple helix in both collagens and therefore probably impedes unfolding of the collagen triple helix from the N terminus. ..
  30. Jiang L, Liu C, Duan Z, Deng M, Tang X, Liang S. Transcriptome analysis of venom glands from a single fishing spider Dolomedes mizhoanus. Toxicon. 2013;73:23-32 pubmed publisher
    ..The results may contribute to discovering novel potential drug leads from spider venoms and a better understanding of the evolutionary relationship of the spider toxin. ..
  31. Moore S, Leung C, Norton H, Cochran J. Engineering agatoxin, a cystine-knot peptide from spider venom, as a molecular probe for in vivo tumor imaging. PLoS ONE. 2013;8:e60498 pubmed publisher
    ..This work validates AgTx as a viable molecular scaffold for protein engineering, and further demonstrates the promise of using tumor-targeting knottins as probes for in vivo molecular imaging. ..
  32. Niño Barrera J, Garzón Alvarado D. Does the geometric location of odontoblast differentiation and dentinal tubules depend on a reaction-diffusion system between BMP2 and Noggin? A mathematical model. J Endod. 2012;38:1635-8 pubmed publisher
    ..This mathematic approach allows us to understand biological phenomena and biochemical activity during the period of pulp differentiation. ..
  33. Wilczynski A, Wang X, Bauzo R, Xiang Z, Shaw A, Millard W, et al. Structural characterization and pharmacology of a potent (Cys101-Cys119, Cys110-Cys117) bicyclic agouti-related protein (AGRP) melanocortin receptor antagonist. J Med Chem. 2004;47:5662-73 pubmed
    ..J. Med. Chem. 2004, 47, 2194) in attempts to identify a "bioactive" conformation of the bicyclic hAGRP peptide and account for the 80-fold decreased ligand potency compared to hAGRP(87-132). ..
  34. Muthukuru M. Bone morphogenic protein-2 induces apoptosis and cytotoxicity in periodontal ligament cells. J Periodontol. 2013;84:829-38 pubmed publisher
    ..BMP-induced tooth ankylosis is controversial and is poorly understood. Disruption of PDL homeostasis by BMP-induced apoptosis could play a role in tooth ankylosis. ..
  35. Eliasen R, Andresen T, Conde Frieboes K. Handling a tricycle: orthogonal versus random oxidation of the tricyclic inhibitor cystine knotted peptide gurmarin. Peptides. 2012;37:144-9 pubmed publisher
    ..Importantly, native gurmarin was obtained by random oxidation, which was confirmed by a synthetic approach for the first time. ..
  36. Lopez M, Seoane L, Tovar S, Garcia M, Nogueiras R, Dieguez C, et al. A possible role of neuropeptide Y, agouti-related protein and leptin receptor isoforms in hypothalamic programming by perinatal feeding in the rat. Diabetologia. 2005;48:140-8 pubmed
  37. Gould A, Ji Y, Aboye T, Camarero J. Cyclotides, a novel ultrastable polypeptide scaffold for drug discovery. Curr Pharm Des. 2011;17:4294-307 pubmed
    ..The present review reports recent findings in the biological diversity and therapeutic potential of natural and engineered cyclotides. ..
  38. Tang J, Wang C, Pan X, Yan H, Zeng G, Xu W, et al. Isolation and characterization of cytotoxic cyclotides from Viola tricolor. Peptides. 2010;31:1434-40 pubmed publisher
    ..Homology modeling of the structures was used in deriving structure-activity relationships...
  39. Tjernberg P, Vos H, Spaargaren van Riel C, Luken B, Voorberg J, Bertina R, et al. Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease. Thromb Haemost. 2006;96:717-24 pubmed
  40. Kamolkijkarn P, Prasertdee T, Netirojjanakul C, Sarnpitak P, Ruchirawat S, Deechongkit S. Synthesis, biophysical, and biological studies of wild-type and mutant psalmopeotoxins--anti-malarial cysteine knot peptides from Psalmopoeus cambridgei. Peptides. 2010;31:533-40 pubmed publisher
    ..Correlation of biophysical parameters and biological activities showed that both PcFKs may have different mechanisms of actions for antiplasmodial activity. ..
  41. Rey J, Ellies D. Wnt modulators in the biotech pipeline. Dev Dyn. 2010;239:102-14 pubmed publisher
    ..e., Sclerostin, Dan, Sostdc1, Vwf, Norrin, Pdgf, Mucin) and discusses how this motif plays a role in mediating Wnt signaling through interactions with LRP. ..
  42. Van Loy T, Van Hiel M, Vandersmissen H, Poels J, Mendive F, Vassart G, et al. Evolutionary conservation of bursicon in the animal kingdom. Gen Comp Endocrinol. 2007;153:59-63 pubmed
    ..Our results illustrate the remarkable conservation of bursicon in invertebrate species and provide an avenue for functional analyses of this hormone in a wide range of animal species. ..
  43. Craik D, Daly N. NMR as a tool for elucidating the structures of circular and knotted proteins. Mol Biosyst. 2007;3:257-65 pubmed
    ..In this review the role of NMR in defining the structures of cyclotides is described. ..
  44. Gruber C, Cemazar M, Clark R, Horibe T, Renda R, Anderson M, et al. A novel plant protein-disulfide isomerase involved in the oxidative folding of cystine knot defense proteins. J Biol Chem. 2007;282:20435-46 pubmed
    ..The nucleotide sequence(s) reported in this paper have been submitted to the GenBank/EBI Data Bank with accession number(s) 911777...
  45. Suzuki A, Takayama T, Suzuki N, Kojima T, Ota N, Asano S, et al. Daily low-intensity pulsed ultrasound stimulates production of bone morphogenetic protein in ROS 17/2.8 cells. J Oral Sci. 2009;51:29-36 pubmed
    ..Noggin markedly inhibited the daily LIPUS-induced phosphorylation of Smad1. Our findings demonstrate that the osteogenic activity of daily LIPUS may be mediated by BMPs in ROS 17/2.8 cells. ..
  46. Avrutina O, Schmoldt H, Gabrijelcic Geiger D, Wentzel A, Frauendorf H, Sommerhoff C, et al. Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis. Chembiochem. 2008;9:33-7 pubmed
  47. Maxson R, Ishii M. The Bmp pathway in skull vault development. Front Oral Biol. 2008;12:197-208 pubmed publisher
  48. Chen J, Deng M, He Q, Meng E, Jiang L, Liao Z, et al. Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao. Cell Mol Life Sci. 2008;65:2431-44 pubmed publisher
    ..The gating modifier families contain advanced toxins, usually have many members and interact with diverse regulatory components of channels. ..
  49. Leulliot N, Quevillon Cheruel S, Graille M, Schiltz M, Blondeau K, Janin J, et al. Crystal structure of yeast YER010Cp, a knotable member of the RraA protein family. Protein Sci. 2005;14:2751-8 pubmed
    ..A tunnel at the interface between two monomers, lined with conserved charged residues, has unassigned residual electron density and may constitute an active site for a yet unknown activity. ..
  50. Kimura R, Jones D, Jiang L, Miao Z, Cheng Z, Cochran J. Functional mutation of multiple solvent-exposed loops in the Ecballium elaterium trypsin inhibitor-II cystine knot miniprotein. PLoS ONE. 2011;6:e16112 pubmed publisher
    ..However, reduced serum and metabolic stability were observed compared to an engineered integrin-binding EETI-II knottin peptide containing only one mutated loop. ..
  51. Saez N, Senff S, Jensen J, Er S, Herzig V, Rash L, et al. Spider-venom peptides as therapeutics. Toxins (Basel). 2010;2:2851-71 pubmed publisher
  52. Craik D. Host-defense activities of cyclotides. Toxins (Basel). 2012;4:139-56 pubmed publisher
  53. Gunasekera S, Foley F, Clark R, Sando L, Fabri L, Craik D, et al. Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides. J Med Chem. 2008;51:7697-704 pubmed publisher
    ..In general, the stabilization of bioactive peptide epitopes is a significant problem in medicinal chemistry and in the current study we have provided insight into one approach to stabilize these peptides in a biological environment. ..