Despite a Conserved Cystine Knot Motif, Different Cyclotides Have Different Membrane Binding Modes

Abstract

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.

Discovery of Linear Cyclotides in Monocot Plant Panicum laxum of Poaceae Family Provides New Insights into Evolution and Distribution of Cyclotides in Plants

“…Bioactivities-The diverse biological activities of cyclotides have been proposed to arise from their ability to target, interact, and disrupt cell membranes (43)(44)(45)(46). In this work, we indirectly assessed the membrane-active properties of the novel panitides by using antimicrobial and cytotoxicity assays.…”

Structure and Activity of the Leaf-Specific Cyclotide vhl-2

“…Studies with dodecylphosphocholine (DPC) micelles [47][48][49] showed that the hydrophobic regions of cyclotides are generally involved in membrane binding, but it has also been suggested that self-association of cyclotides (as well as membrane binding) is probably required to elicit a biological response. [37,39] Recent electrophysiological studies have confirmed that kalata B1 forms pores of ∼45 Å in diameter in membranes, consistent with a self-association mechanism.…”

“…It has also been reported that different cyclotides have different membrane-binding modes. [49] As plants typically express a suite of cyclotides, it is possible that cyclotide assemblies that form pores in the gut membranes of insect pests might comprise mixtures of different cyclotides working synergistically. If that is the case, then the mechanisms that regulate the relative expression of different cyclotides are of interest.…”

Anionic Host Defence Peptides from the Plant Kingdom: Their Anticancer Activity and Mechanisms of Action

“…These residues and patches make the major hydrophobic contribution to the membrane partitioning of cyclotides and are mainly centred on loops 5 and 6 of Möbius peptides and loops 2 and 3 of bracelet peptides [80], as shown for kalata B2 and cycloviolacin Y4 in Fig. 4.…”

“…However, the electrostatic contribution to the membrane partitioning of both subfamilies appears to involve hydrophilic patches formed primarily by the presence of positively charged residues in loops 5 and 6 of these peptides (Fig. 4) [80,81]. Examples of this residue distribution can be seen in most cyclotide anticancer AHPDs, including: kalata B2, which has an arginine residue in loop 6 [78], and cycloviolacin Y5, which has a lysine residue in this loop [67].…”