Isolation, sequencing, and structure-activity relationships of cyclotides.

Cyclotides are a topologically fascinating family of miniproteins discovered over the past decade that have expanded the diversity of plant-derived natural products. They are approximately 30 amino acids in size and occur in plants of the Violaceae, Rubiaceae, and Cucurbitaceae families. Despite their proteinaceous composition, cyclotides behave in much the same way as many nonpeptidic natural products in that they are resistant to degradation by enzymes or heat and can be extracted from plants using methanol. Their stability arises, in large part, due to their characteristic cyclic cystine knot (CCK) structural motif. Cystine knots are present in a variety of proteins of insect, plant, and animal origin, comprising a ring formed by two disulfide bonds and their connecting backbone segments that is threaded by a third disulfide bond. In cyclotides, the cystine knot is uniquely embedded within a head-to-tail cyclized peptide backbone, leading to the ultrastable CCK structural motif. Apart from the six absolutely conserved cysteine residues, the majority of amino acids in the six backbone loops of cyclotides are tolerant to variation. It has been predicted that the family might include up to 50,000 members; although, so far, sequences for only 140 have been reported. Cyclotides exhibit a variety of biological activities, including insecticidal, nematocidal, molluscicidal, antimicrobial, antibarnacle, anti-HIV, and antitumor activities. Due to their diverse activities and common structural core from which variable loops protrude, cyclotides can be thought of as combinatorial peptide templates capable of displaying a variety of amino acid sequences. They have thus attracted interest in drug design as well as in crop protection applications.

Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes.

Cyclotides are cyclic proteins produced by plants for defense against pests. Because of their remarkable stability and diverse bioactivities, they have a range of potential therapeutic applications. The bioactivities of cyclotides are believed to be mediated through membrane interactions. To determine the structural basis for the biological activity of the two major subfamilies of cyclotides, we determined the conformation and orientation of kalata B2 (kB2), a Möbius cyclotide, and cycloviolacin O2 (cO2), a bracelet cyclotide, bound to dodecylphosphocholine micelles, using NMR spectroscopy in the presence and absence of 5- and 16-doxylstearate relaxation probes. Analysis of binding curves using the Langmuir isotherm indicated that cO2 and kB2 have association constants of 7.0 x 10(3) M(-1) and 6.0 x 10(3) M(-1), respectively, consistent with the notion that they are bound near the surface, rather than buried deeply within the micelle. This suggestion is supported by the selective broadening of micelle-bound cyclotide NMR signals upon addition of paramagnetic Mn ions. The cyclotides from the different subfamilies exhibited clearly different binding orientations at the micelle surface. Structural analysis of cO2 confirmed that the main element of the secondary structure is a beta-hairpin centered in loop 5. A small helical turn is present in loop 3. Analysis of the surface profile of cO2 shows that a hydrophobic patch stretches over loops 2 and 3, in contrast to the hydrophobic patch of kB2, which predominantly involves loops 2 and 5. The different location of the hydrophobic patches in the two cyclotides explains their different binding orientations and provides an insight into the biological activities of cyclotides.

Distribution and evolution of circular miniproteins in flowering plants.

Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. Cyclotides have been found only in two plant families: in every analyzed species of the violet family (Violaceae) and in few species of the coffee family (Rubiaceae). In this study, we analyzed >200 Rubiaceae species and confirmed the presence of cyclotides in 22 species. Additionally, we analyzed >140 species in related plant families to Rubiaceae and Violaceae and report the occurrence of cyclotides in the Apocynaceae. We further report new cyclotide sequences that provide insights into the mechanistic basis of cyclotide evolution. On the basis of the phylogeny of cyclotide-bearing plants and the analysis of cyclotide precursor gene sequences, we hypothesize that cyclotide evolution occurred independently in various plant families after the divergence of Asterids and Rosids ( approximately 125 million years ago). This is strongly supported by recent findings on the in planta biosynthesis of cyclotides, which involves the serendipitous recruitment of ubiquitous proteolytic enzymes for cyclization. We further predict that the number of cyclotides within the Rubiaceae may exceed tens of thousands, potentially making cyclotides one of the largest protein families in the plant kingdom.

The anthelmintic activity of the cyclotides: natural variants with enhanced activity.

The cyclotides are a family of backbone-cyclised cystine-knot-containing peptides from plants that possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematode parasites of sheep. In the current study, we investigated the in vitro effects of newly discovered natural cyclotides on the viability of larval and adult life stages of these pests. The natural variants cycloviolacin O2, cycloviolacin O3, cycloviolacin O8, cycloviolacin O13, cycloviolacin O14, cycloviolacin O15, and cycloviolacin O16 extracted from Viola odorata showed up to 18-fold greater potency than the prototypic cyclotide kalata B1 in nematode larval development assays. Cycloviolacin O2 and cycloviolacin O14 were significantly more potent than kalata B1 in adult H. contortus motility assays. The lysine and glutamic acid residues of cycloviolacin O2, the most potent anthelmintic cyclotide, were chemically modified to investigate the role of these charged residues in modulating the biological activity. The single glutamic acid residue, which is conserved across all known cyclotides, was shown to be essential for activity, with a sixfold decrease in potency of cycloviolacin O2 following methylation. The three lysine residues present in cycloviolacin O2 were acetylated to effectively mask the positive charge, resulting in a 18-fold decrease in anthelmintic activity. The relative anthelmintic activities of the natural variants assayed against nematode larvae correlated with the number of charged residues present in their sequence.

Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis.

Cyclotides are macrocyclic plant peptides characterized by a knotted arrangement of three disulfide bonds. They display a range of interesting bioactivities, including anti-HIV and insecticidal activities. More than 100 different cyclotides have been isolated from two phylogenetically distant plant families, the Rubiaceae and Violaceae. In this study we have characterized the cyclotides from Viola yedoensis, an important Chinese herb from the Violaceae family that has been reported to contain potential anti-HIV agents. From V. yedoensis five new and three known cyclotides were identified and shown to have anti-HIV activity. The most active of these is cycloviolacin Y5, which is one of the most potent of all cyclotides tested so far using in vitro XTT-based anti-HIV assays. Cycloviolacin Y5 is the most hydrophobic of the cyclotides from V. yedoensis. We show that there is a positive correlation between the hydrophobicity and the anti-HIV activity of the new cyclotides and that this trend tracks with their ability to disrupt membranes, as judged from hemolytic assays on human erythrocytes.

Cyclotides as natural anti-HIV agents.

Cyclotides are disulfide rich macrocyclic plant peptides that are defined by their unique topology in which a head-to-tail cyclized backbone is knotted by the interlocking arrangement of three disulfide bonds. This cyclic cystine knot motif gives the cyclotides exceptional resistance to thermal, chemical, or enzymatic degradation. Over 100 cyclotides have been reported and display a variety of biological activities, including a cytoprotective effect against HIV infected cells. It has been hypothesized that cyclotides from one subfamily, the Möbius subfamily, may be more appropriate than bracelet cyclotides as drug candidates given their lower toxicity to uninfected cells. Here, we report the anti-HIV and cytotoxic effects of three cyclotides, including two from the Möbius subfamily. We show that Möbius cyclotides have comparable inhibitory activity against HIV infection to bracelet cyclotides and that they are generally less cytotoxic to the target cells. To explore the structure activity relationships (SARs) of the 29 cyclotides tested so far for anti-HIV activity, we modeled the structures of the 21 cyclotides whose structures have not been previously solved. We show that within cyclotide subfamilies there is a correlation between hydrophobicity of certain loop regions and HIV inhibition. We also show that charged residues in these loops impact on the activity of the cyclotides, presumably by modulating membrane binding. In addition to providing new SAR data, this report is a mini-review that collates all cyclotide anti-HIV information reported so far and provides a resource for future studies on the therapeutic potential of cyclotides as natural anti-HIV agents.

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability.

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins.

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. 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.

A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae).

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.