Decoding the Membrane Activity of the Cyclotide Kalata B1

Abstract

Cyclotides, a large family of cyclic peptides from plants, have a broad range of biological activities, including insecticidal, cytotoxic, and anti-HIV activities. In all of these activities, cell membranes seem likely to be the primary target for cyclotides. However, the mechanistic role of lipid membranes in the activity of cyclotides remains unclear. To determine the role of lipid organization in the activity of the prototypic cyclotide, kalata B1 (kB1), and synthetic analogs, their bioactivities and affinities for model membranes were evaluated. We found that the bioactivity of kB1 is dependent on the lipid composition of target cell membranes. In particular, the activity of kB1 requires specific interactions with phospholipids containing phosphatidylethanolamine (PE) headgroups but is further modulated by nonspecific peptide-lipid hydrophobic interactions, which are favored in raft-like membranes. Negatively charged phospholipids do not favor high kB1 affinity. This lipid selectivity explains trends in antimicrobial and hemolytic activities of kB1; it does not target bacterial cell walls, which are negatively charged and lacking PE-phospholipids but can insert in the membranes of red blood cells, which have a low PE content and raft domains in their outer layer. We further show that the anti-HIV activity of kB1 is the result of its ability to target and disrupt the membranes of HIV particles, which are raft-like membranes very rich in PE-phospholipids.Cyclotides are plant-derived peptides characterized by a cyclic backbone and three disulfide bonds forming a cystine knot motif (1) (Fig. 1) that confers them with exceptional stability (2). Their natural function appears to be as host defense insecticidal agents (3), but many other activities have also been reported, including uterotonic (4), anti-HIV (5), hemolytic (6), antibacterial (7), anti-cancer (8), and pesticidal action (9). These activities reflect the ability of cyclotides to target cells of different compositions. More than 150 cyclotides have been characterized (10), and their diverse sequences, remarkable stability, and various bioactivities suggest that they have exciting potential as molecular frameworks in drug design (11).It is believed that the activity of cyclotides is mediated by their ability to target and disrupt cell membranes. 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 (12-15). 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). Furthermore, in a previous study we showed that the all-D enantiomer (D-kB1) is bioactive, suggesting that activity does not require recognition by a chiral protein receptor (9). In combination, these reports suggest that cyclotides target cell membranes by interacting directly with lipids.Differences in bioactivity are...

Backbone cyclization of a recombinant cystine‐knot peptide by engineered Sortase A

“…1A) to isomerize, leading to the formation of succinimide and b-aspartyl isomers as observed in earlier reports [12,13]. Regardless, both isomers undergo backbone cyclization in the presence of m 4 SrtA, as observed by the shift in their retention times and molecular weights ( Fig. 2B and D, peaks 1 ⁄ and 2 ⁄ ).…”

“…This strategy led to the identification of four amino acid mutations P94S/D160N/D165A/K196T within the catalytic domain of Sortase A (Q60-K206) that, when combined, resulted in a 140-fold increase in k cat /K m . We generated this mutant Sortase A (referred to as m 4 SrtA) and confirmed that the reaction kinetics were indeed significantly improved over the wild-type enzyme (Supp. Fig.…”

“…1). Therefore, the use of m 4 SrtA would be advantageous for backbone cyclization of cystine-knot peptides due to potentially higher reaction yields compared to the wild-type enzyme. To our knowledge, engineered Sortase A has not been used for backbone cyclization of cyclotides.…”

“…We monitored the progress of the backbone cyclization reaction mediated by m 4 SrtA by using liquid chromatography-mass spectrometry (LC-MS). Treatment of oxidized rMCoTI-II substrate (prepared above) with m 4 SrtA under conditions which were optimized as described in detail below led to a loss of 132 atomic mass units as expected, owing to cleavage of two glycine residues on rMCoTI-II and loss of a water molecule from amide bond formation ( Fig.…”

“…The presence of the cyclized backbone and the cystine-knot framework in part make cyclotides remarkably stable to chemical degradation and proteolytic digestion [1][2][3], yet stability might be influenced by other factors such as shape and amino acid sequence. Initially discovered in plants, cyclotides were shown to exhibit a wide range of biological functions including oxytocic, insecticidal and antibacterial activities, to name a few [1,4]. Yet, how cyclotides are produced and circularized in nature has remained elusive.…”