Background: Cyclotides are a family of plant-expressed pesticidal cyclic peptides. Results: A broad range of cyclotides specifically interact with membranes containing phosphatidylethanolamine (PE)-phospholipids. Conclusion: Cyclotide bioactivity correlates with an ability to target, insert into, and disrupt lipid membranes containing PE-phospholipids. Significance: Cyclotides constitute a new lipid-binding protein family that has potential as a scaffold to target tumor cells.

The cyclotides are a large family of circular mini-proteins containing a cystine knot motif. They are expressed in plants as defense-related proteins, with insecticidal activity. Here we investigate their role in membrane interaction and disruption. Kalata B1, a prototypic cyclotide, was found to induce leakage of the self-quenching fluorophore, carboxyfluorescein, from phospholipid vesicles. Alanine-scanning mutagenesis of kalata B1 showed that residues essential for lytic activity are clustered, forming a bioactive face. Kalata B1 was sequestered at the membrane surface and showed slow dissociation from vesicles. Electrophysiological experiments showed that conductive pores were induced in liposome patches on incubation with kalata B1. The conductance calculated from the current-voltage relationship indicated that the diameter of the pores formed in the bilayer patches is 41-47 Å . Collectively, the findings provide a mechanistic explanation for the diversity of biological functions ascribed to this fascinating family of ultrastable macrocyclic peptides.The cyclotides are a family of topologically unique macrocyclic peptides abundant in plants of the Rubiaceae (coffee) and Violaceae (violet) families. They possess unusual structural and biophysical properties and are composed of a head-to-tail cyclic backbone and a cystine knot (1). The cystine knot is formed by a disulfide bond that penetrates a ring made by two other disulfide bonds and their connecting backbone segments. The structure of kalata B1, the prototypic cyclotide, is illustrated in Fig. 1 (2). The cyclic cystine knot at the core of three-dimensional structure contributes to the exceptional chemical and biological stability of cyclotides (3) and underpins their exciting potential for pharmaceutical and agricultural applications (4).Cyclotides display a diverse range of biological activities, including anti-human immunodeficiency virus (5-8), neurotensin antagonism (9), hemolytic (10), antimicrobial (11), antifouling (12), and pesticidal activities (13-19). Cyclotides have been postulated to be defense-related proteins on the basis of their pesticidal activity and the suite of natural isoforms present in individual plants (20). Little is known about their mechanism of action, but their observed activities potentially might be associated with membrane interactions. Studies utilizing analytical ultracentrifugation (21) have shown that the cyclotide kalata B2 forms specific oligomers in solution, which could potentially have a role in the formation of membrane-spanning pores. A membrane-based mechanism of action is supported by a recent surface plasmon resonance study, which demonstrated that several kalata-like cyclotides bind to phosphatidylethanolamine-containing membranes (22). More recently, the cyclotide cycloviolacin O2 was shown to be cytotoxic to a human lymphoma cell line and induce leakage of calceinloaded HeLa cells (23). NMR studies showed that the binding of kalata B1, and other analogues, to dodecylphosphocholine micelles is modulated by...

Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide-lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.

Cyclotides are a family of macrocyclic peptides that combine the unique features of a head-to-tail cyclic backbone and a cystine knot motif, the combination of which imparts them with extraordinary stability. The prototypic cyclotide kalata B1 is toxic against two economically important gastrointestinal nematode parasites of sheep, Haemonchus contortus and Trichostrongylus colubriformis. A lysine scan was conducted to examine the effect of the incorporation of positive charges into the kalata B1 cyclotide framework. Each of the non-cysteine residues in this 29-amino acid peptide was successively substituted with lysine, and the nematocidal and hemolytic activities of the suite of mutants were determined. Substitution of 11 residues within kalata B1 decreased the nematocidal activity dramatically. On the other hand, six other residues that are clustered on the surface of kalata B1 were tolerant to Lys substitution, and indeed the introduction of positively charged residues into this region increased nematocidal activity. This activity was increased further in double and triple lysine mutants, with a maximal increase (relative to the native kalata B1) of 13-fold obtained with a triple lysine mutant (mutated at positions Thr-20, Asn-29, and Gly-1). Hemolytic activity correlated with the nematocidal activity of all lysine mutants. 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.Gastrointestinal nematodes cause major losses to livestock industries worldwide. The control of these pests, until now, has been via synthetic anthelmintics, including benzimidazoles (e.g. thiabendazole), nicotinic acetylcholine receptor agonists (e.g. levamisole), and macrocyclic lactones (e.g. ivermectin and moxidectin). However, resistance of sheep and cattle nematodes to these broad-spectrum anthelmintics is a serious issue for livestock production globally (1-3). Thus, the development of novel agents with potent anthelmintic activity is of great economic and agricultural importance.Cyclotides are circular miniproteins discovered originally in plants of the Rubiaceae, Violaceae, and Cucurbitaceae families (4, 5). Kalata B1, the first cyclotide to be structurally characterized, is abundant in the tropical African plant Oldenlandia affinis (6, 7) but has also been reported in the European Sweet Violet (Viola odorata) (8) and several other Viola species (9 -13). Members of the cyclotide family possess a cyclic peptide backbone and a cystine knot motif, which is formed by three disulfide bonds at the core of their three-dimensional structure. The sequence and structure of kalata B1 is illustrated in Fig. 1, which highlights the six backbone loops between the six conserved cysteine residues that make up the cystine knot (14). The cyclic cystine knot motif is thought to be responsible for the extraordinary enzymatic and chemical stability (15) In a recent study (30), the anthel...