Alanine Scanning Mutagenesis of the Prototypic Cyclotide Reveals a Cluster of Residues Essential for Bioactivity

Abstract

The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.Cyclotides are a fascinating family of plant-derived mini-proteins of around 30 residues that are characterized by the topologically unique combination of a head-to-tail cyclized backbone and a cystine knot. The original discovery of the prototypic member kalata B1 (1) was guided by the indigenous medicinal use in Africa of a tea made from the plant Oldenlandia affinis (Rubiaceae) to accelerate childbirth. Kalata B1 was identified as a uterotonic component of the plant, but its unusual macrocyclic nature (2) and the fact that it was just one member of a large family of related macrocyclic peptides were elucidated only recently (3). Cyclotides have now been reported in several other species of the Rubiaceae (coffee) family (4, 5) and in all tested members of the Violaceae (violet) plant family (3, 6, 7) bringing the number of known cyclotides to over 100.

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

“…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.…”

Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes

“…This is in agreement with previous studies with kB1 analogues with a mutation in this residue showing that Trp23 is important for kB1 membrane binding properties and activity. [8][9][10]25 The way in which membrane conduction (Gm) and imperfect capacitance (Qm) are altered as a result of adding kB1 is depicted in Figure 3B&C (Supplementary Material Fig S2 B&C for kB2). Membrane conduction increases in response to addition of 10 µM kB1, then returns to baseline levels following a washing step.…”

“…[7][8] Interestingly, following a subsequent PBS wash step, any effect of the cyclotides on the membrane impedance spectra disappeared and the membrane appeared to return to its former state. To gain further evidence of the specificity of kB1 for PE containing membranes, [W23A]kB1, a kB1 analogue with a mutation in the hydrophobic face (see Figure 1C) and previously shown to be inactive in the activities tested 9 was here included as a control. As expected, [W23A]kB1 exhibited no response in either POPE containing membranes or POPConly membranes (Supplementary Material Figures S3 & S4).…”

“…C, Surface representation of kalata B1 in two views showing the location of the amendable face (blue), hydrophobic face (green) and bioactive face (red). [9][10] The residues that have been shown to be important for the reported activities of kalata B1 are located in the hydrophobic and bioactive faces. Point mutations in these regions might render the peptide inactive and also unable to bind to membranes.…”

“…4 kB1 mutant, [W23A]kB1, was synthesised and purified as before. 9 The concentration of kB1 and kB2 in various experiments was quantified by absorbance at 280 nm assuming an extinction coefficient of 5875 M -1 cm -1 , as determined from the contribution of aromatic residues and disulfide bonds. As the analogue [W23A]kB1 does not contain aromatic residues, the concentration of [W23A]kB1 samples was quantified by weight of lyophilised peptide.…”