The KNOTTIN database

Historical landmarks

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2011

The first knottin... that is not a knottin !

A two-disulfide toxin from the venom of scorpion Liocheles australasiae, LaIT1, has been shown to display a knottin fold [Horita et al., 2011]. Although two-disulfide knottin-like domains were predicted to be stable many years ago [Le-Nguyen et al., 1993; Heitz et al., 1999], this is however, to our knowledge, the first evidence of a wild two-disullfide knottin-like miniprotein without additional domains or disulfide bridges.
As there is no cystine knot in this toxin, it has not been included in the KNOTTIN database. The PDB ID for LaIT1 is 2lds.

2010

A new class of "double-knot" spider toxins

Two groups have independently reported new spider toxins made of two successive knottin domains. The principal toxic component, CpTx 1, in the venom of the yellow sac spider consists of two different, yet homologous, knottin domains. [Vassilevski et al., 2010;]. Similarly, a bivalent toxin was observed in the venom of the earth tiger tarantula. [Bohlen et al., 2010]. The synergy provided by the tandemly repeated knottin domains results in a ligand with an exceedingly high avidity. These reports suggest new strategies for engineering multivalent ligands from the knottin scaffold. See [Krause et al., 2007] for previous engineering works along similar directions.


Diselenium knottins

Knottins in which disulfide bridges have been replaced with diselenium bonds between selenocysteines were shown to retain the native folding and to display similar or even greater biological activities [Raffa 2010; Gowd et al., 2010]. The use of diselenium bridges was also shown useful to map disulfide bridges in knottins with unknown cystine connectivities by using 77Se NMR scalar coupling [Mobli et al., 2009].


Yet another knottin translocates accross cell membranes

Imperatoxin A, a toxin isolated from the venom of the African scorpion Pandinus imperator, has been shown to be capable of crossing cell membranes to alter the release of Ca2+ in vivo. Moreover, the toxin is able to carry a large membrane-impermeable cargo across the plasma membrane [Gurrola et al., 2010]. Previous knottins shown to translocate accross membranes were maurocalcine [Esteve et al., 2005; Ram et al., 2008] and the cyclic squash inhibitor MCoTI-II [Greenwood et al., 2007]. A paper on Cyclic Cell Penetrating Peptides (CCPP) has appeared in 2011 [Cascales et al., 2011].
All these findings open new exciting routes for knottin-based drug delivery.

2009

Knottin-based peptides as tools for cancer therapy and diagnosis

A new class of integrin-binding agents was engineered through directed evolution of the EETI-II knottin (Serine protease inhibitor1), or of the AgRP knottin (Agouti-related) [Kimura et al., 2009; Lahti et al., 2009; Silverman et al., 2009]. These were successfully tested as cancer imaging agents in living subjects [Jiang et al., 2010; Nielsen et al., 2010] or for targeted contrast-enhanced ultrasound imaging of tumor angiogenesis when coupled to the surface of ultrasound contrast agents [Willmann et al., 2010].

2008

Spider toxin Tx2-6: a tool for the development of new therapeutics for erectile dysfunction.

Tx2-6, a toxin purified from the Phoneutria nigriventer spider venom was shown to induce and facilitate the ganglionic-stimulated penile erection in rats. It also displayed a potentiation effect on penile erection of animals presenting severe erectile dysfunction such as the deoxycorticosterone-acetate-salt hypertensive rats. The potentiating effect of Tx2-6 on erection seems to be mediated by relaxation of the vasculature and smooth muscle in the corpus cavernosum induced by the release of NO. [Nunes et al., 2008].


Primary culture of venom glands as an in vitro alternative to extraction of crude material from venom ?

A first step toward the functional cultures of venom glands of Phoneutria spiders has been reported. The presence of venom components was evidenced in supernatant of primary cultures of venom glands of Phoneutria nigriventer young adults. [Silva et al., 2008].

2007

MCoTI-II has the potential to transport bioactive peptides to intracellular targets

It has been shown that the cyclic knottin MCoTI-II is internalized into mammalian cells by macropinocytosis. Thanks to its low cytotoxicity, its cell internalization, and its sequence tolerance, MCoTI-II might be used to carry bioactive epitopes to intracellular targets. [Greenwood et al., 2007].


Synthetic dimeric knottin-based molecules as receptor agonists ?

Thrombopoietin is the primary regulator of platelet production. Peptides shown to act as high-affinity thrombopoietin antagonists were grafted onto knottins (EETI-II and the minimized Agouti-related protein). Interestingly, covalent dimerization through lysine cross-linking yielded potent bivalent receptor agonists [Krause et al., 2007].

2006

Discovery of the first 'linear cyclotide' !

A linear cyclotide has been isolated from Viola odorata [Ireland et al., 2006]. The absence of cyclization is due to the presence of a stop codon that prevents translation of a key residue required for cyclization. Despite the linearization, the overall stability is conserved, in agreement with what is observed in other knottin families.


Spider toxins activate the capsaicin receptor to produce inflammatory pain

Three knottins named 'vanillotoxins' and isolated from tarantula Psalmopoeus cambridgei target the TRPV1 capsaicin receptor involved in the pain pathway. Animals and plants (e.g. chilli peppers), avert predators by activating TRP channels to elicit pain and inflammation [Siemens et al., 2006].


Marine sponges: a new source of knottins

The knottin asteropine A has been discovered in marine sponge Asterpus simplex [Takada et al., 2006]. Asteropine A is a conotoxin-like knottin that inhibits bacterial sialidases.

2005

PRIALT : A knottin for the treatment of severe chronic pain

Omega-conotoxin MVIIA (ziconotide) has been approved by the FDA for the intrathecal treatment of severe chronic pain [Wallace, 2006]. It is distributed under the commercial name PRIALT by Azur Pharma Inc (USA).


New cyclotides in maize

Putative cyclotides were first discovered in Zea mays [Basse, 2005], then in several monocot species including graminaceae [Mulvenna et al., 2006]. This raises the question of old cyclotide ancestors.

2003

New knottins from plants with hormone or insecticidal activity

Two new homologous knottins, Leginsulin [Yamazaki et al., 2003] and PA1b [Jouvensal et al., 2003], have been found in soybean and pea seeds, respectively. Leginsulin is probably involved in cellular signal transduction and thus displays a hormone-like action, similarly to what is observed in animal systems. PA1b, on the other hand, has been shown to display accute and specific toxicity on cereal weevils.

2002

New family of chitin-binding peptides with the Knottin fold

 Tachystatin A, an antimicrobial peptide isolated from the Japanese horseshoe crab (Tachypleus tridentatus), displays a typical Knottin fold and is a chitin-binding peptide [Fujitani et al., 2002]. Omega-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A displays sequence similarity with spider toxins [Osaki et al., 1999].


The disulfide connectivity in plant cyclotides is discussed

A new solution structure of kalata B1 suggests that the disulfide connectivity would be I-VI, II-V, III-IV [Skjeldal et al., 2002]. This connectivity is different from the previously determined connectivity and would push kalata B1 out of the Knottin structural family. This also questions the connectivity in homologous plant cyclotides. However, still newer high-resolution solution structures of kalata B1 and of cycloviolacin O1 confirm the knotted I-IV, II-V, III-VI connectivity of the disulfide bridges in the plant cyclotides [Rosengren et al., 2003]. Chemical proofs of the knotted connectivity in cyclotide were also provided by Goransson & Craik..


A new spider toxin is found NOT to be a knottin

Huwentoxin-II is an insecticidal peptide purified from the venom of spider Selenocosmia huwena. The solution structure (PDB ID: 1i25) has revealed a unique disulfide bond linkage I-III, II-V, and IV-VI in contrast to the knotted I-IV, II-V, III-VI connectivity found in all other elucidated spider toxins [Shu et al., 2002].

2001

Insect toxins are found to belong to the Knottin structural class

 Ptu1, a toxin extracted from the saliva of the assassin bug Peirates turpis is structurally similar to omega-conotoxins. [Bernard et al., 2001]. Homologous peptides Ado1 and Iob1 probably fold similarly.


Antifungal peptides with the Knottin fold.

PAFP-S, an antifungal peptide from the seeds of Phytolacca americana is shown to display a Knottin fold [Gao et al., 2001a, 2001b]. Homologous antimicrobial peptides Mj-AMP1 and Mj-AMP2, isolated from seeds of the four-o'clock plant (Mirabilis jalapa L.), probably fold similarly [Cammue et al., 1992].


The knottin scaffold has been discovered in insect polydnaviruses.

Some insect parasitoids inject resident polydnaviruses to their host. The viruses encode genes that must be expressed in the host for successful parasitization among which the VHv1 genes encoding for proteins with one or several knottin repeats. These are thought to alter the host immune system [Einerwold et al., 2001; Dupas et al., 2003].

2000

A new class for macrocyclic Knottins

 Cyclic squash trypsin inhibitors are isolated from seeds of Momordica Cochinchinensis: MCoTI-I and MCoTI-II [Hernandez et al, 2000].

1999

The elementary two-disulfide Cystine-Stabilized Beta-sheet (CSB) motif in Knottins

 It is shown that Knottins are built from a possibly ancestral two disulfide bridge elementary motif. The 23-residue Min-23 peptide corresponds to the CSB motif isolated from the squash trypsin inhibitor EETI-II. It contains only two disulfide bridges but retains significant stability (Tm 100°C) [Heitz et al, 1999].


More macrocyclic Knottins

 More than 40 new macrocyclic Knottins are found in plant from the Rubiaecae and Violaceae families and called plant cyclotides [Craik, 1999].


First Knottin discovered from a mamalian source

 The NMR structure of a minimized human agouti-related protein shows that this protein belongs to the Knottin fold [Bolin et al., 1999; McNulty et al., 2001; Jackson et al., 2002].

1994

First 3D structure of a macrocyclic Knottin

 Kalata B1 is a cyclic peptide isolated from leaves of an african plant and used in traditional medicine. Its three-dimensional structure reveals the presence of a Knottin scaffold in which the N- and C-termini are connected by a peptide bond [Pallaghy et al., 1994; Saether et al., 1995].

1993

Toxins from marine cone snail (conotoxins) and from spider (agatoxins) join the Knottin club

 The three-dimensional structures of omega-conotoxin GIVA [Davis et al, 1993] and of omega-agatoxin IVB [Yu et al, 1993] show that these toxins belong to the Knottin structural family.
Structural divergences appear however: the 310 helix present in PCI and in the squash inhibitors between the 2nd and the 3rd cysteines is absent in omega-conotoxin GIVA and in omega-agatoxin IVB. Moreover, an additional fourth disulfide bridge is observed in omega-agatoxin IVB.

1990

The "Knottin" structure.

 The particular disulfide bridge arrangement, in which one disulfide crosses the macrocycle formed by the two other disulfides and interconnecting backbone segments, is recognized as a general structural scaffold. Proteins sharing this scaffold are called "Knottins" [Le-Nguyen et al, 1990].


First use of the "Knottin" structure in protein engineering

 The active site of PCI is grafted onto EETI-II to give a bis-headed peptide able to inhibit Trypsin and Carboxypeptidase A, either separately or concomitantly [Le-Nguyen et al, 1990; Chiche et al, 1993].

1989

The squash family of trypsin inhibitors share the knotted topology of PCI

 The three-dimensionnal structure of the trypsin inhibitors EETI-II [Heitz et al. 1989; Chiche et al., 1989] and CMTI-I [Bode et al., 1989] isolated from squash seeds evidence disulfide bridge connectivities and topologies similar to those observed in PCI.

1982

Discovery of the first protein with knotted disulfide bridges

 The three-dimensionnal structure of the Carboxypeptidase A inhibitor from Potato (PCI) revealed for the first time "the threading of the disulfide bond between CysP18 and CysP34 through a loop closed by the other two disulfide bridges" [Rees & Lipscomb, 1982].