Host-defense activities of cyclotides

Abstract

Cyclotides are plant mini-proteins whose natural function is thought to be to protect plants from pest or pathogens, particularly insect pests. They are approximately 30 amino acids in size and are characterized by a cyclic peptide backbone and a cystine knot arrangement of three conserved disulfide bonds. This article provides an overview of the reported pesticidal or toxic activities of cyclotides, discusses a possible common mechanism of action involving disruption of biological membranes in pest species, and describes methods that can be used to produce cyclotides for potential applications as novel pesticidal agents.

Keywords: circular protein; cyclic peptide; cyclotide; cystine knot; insecticide; kalata B1.

Figure 1

Schematic illustration of (a) the sequence and (b) the structure of the prototypical cyclotide, kalata B1 showing the head-to-tail cyclic backbone and six conserved cysteine residues (numbered using Roman numerals) that are connected together in a I–IV, II–V and III–VI connectivity leading to a cystine knot [15]. This connectivity has been established by a range of chemical and NMR methods [16,17]. The structure folds up into a compact three-dimensional shape that contains a β-sheet motif (indicated by the broad arrows) as well as a series of turns. The backbone segments between cysteine residues are referred to as loops and are numbered 1–6. Some loops, e.g. loops 1 and 4, are relatively highly conserved, whereas others are typically hypervariable in cyclotides from different plants, leading to the description of cyclotides as a natural combinatorial template [18]; (c) Space-filling view of the surface of kalata B1 showing the location of a surface-exposed patch of hydrophobic residues.

Figure 2

Schematic illustration of the architecture of cyclotide precursor proteins from the Violaceae and Rubiaceae. They comprise an ER signal sequence, an N-terminal prodomain, the mature peptide sequence and a C-terminal pro-peptide. Some precursors contain multiple copies (i.e., up to three copies) of the cyclotide domain flanked by short pro-peptide regions. Recent studies of cyclotides from the Fabaceae family have suggested that they are produced from what appears to be an ancestral albumin gene [20,52], suggesting that plants have evolved several alternative mechanisms for the biosynthesis of cyclotides.

Figure 3

Schematic illustration of studies done to establish membrane binding of cyclotides. Panel (a) shows preferred binding mode of kalata B1 to DPC micelles as deduced from NMR studies, indicating binding via the hydrophobic patch [78,80]; (b) shows vesicle leakage studies in which the addition of cyclotides to phospholipid vesicles causes leakage of vesicle contents in a dose dependent manner, with differential effects for different lipid compositions made up of palmitoyloleoylethanolamine (POPC), palmitoyloleoylethanolamine (POPE), cholesterol (Chol) and sphingomyelin (SM) [83]; (c) shows surface plasmon resonance studies indicating preferential binding to PE compared to PC lipids [83].

Figure 4

Schematic illustration of synthetic chemical approach for the production of cyclotides. The peptide chain is assembled using solid phase peptide chemistry (SPPS) with the C-terminal residue (aa₁) linked to the resin using a thioester linker. Amino acids are added sequentially and the chain completed with an N-terminal cysteine. Since cyclotides contain six cysteines there are thus six possible linear cyclotide designs that could be used to produce a given cyclotide [87]. The linear cyclotide is cleaved from the solid-phase resin under acidic conditions. Cyclization occurs under basic conditions through a trans-esterification reaction and spontaneous S, N-acyl shift.