The Biological Activity of the Prototypic Cyclotide Kalata B1 Is Modulated by the Formation of Multimeric Pores

Abstract

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...

Decoding the Membrane Activity of the Cyclotide Kalata B1

“…Such a mechanism of action is consistent with their hemolytic properties (6) and ability to disrupt gut epithelial cells in lepidopteron larvae (3) and is further supported by a range of biophysical studies (12)(13)(14)(15). The prototypic cyclotide kalata B1 (kB1) 7 is the most well studied cyclotide, and it has been shown to bind to (12) and disrupt phospholipid bilayers by a pore-forming mechanism (15).…”

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.…”

“…Kalata B2 Kalata B8 vhl-2 A relationship between membrane binding and cytotoxic activity is also supported by several other chemical or biophysical studies, [14,[37][38][39] but all of the features involved in membrane binding have not yet been elucidated. 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.…”

“…The mechanism of the cytotoxic activities of cyclotides, including their haemolytic and insecticidal activities, is thought to involve membrane binding, [14,[37][38][39] but all of the features involved in that binding have yet to be elucidated. The hydrophobicity of cyclotides is likely to play a role in their biological activity, as suggested by comparisons of cyclotides of vastly different hydrophobicity.…”

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.…”