A Synthetic Mirror Image of Kalata B1 Reveals that Cyclotide Activity Is Independent of a Protein Receptor

Abstract

Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti-cancer, anti-HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid-bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all-D enantiomer and assessed its bioactivities. After the all-D enantiomer had been confirmed by (1)H NMR to be the structural mirror image of the native kalata B1, it was tested for anti-HIV activity, cytotoxicity, and hemolytic properties. The all-D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association-dissociation equilibrium.

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

“…Interestingly, the all-D-kB2 is hemolytic but less so than the native kB2. This result is in agreement with previous studies with D-kB1 and its native form (28). At 4°C, no hemolytic activity was found for any of the cyclotides in the concentration range tested.…”

“…D-kB2 bound to membranes containing PE-phospholipids, but with a lower affinity than the mirror image native form (Fig. 3A), consistent with previous studies on D-kB1 (28). Disulfide bond-reduced kB1 and cyO2 did not bind to membranes, even when PE-phospholipids were present (Fig.…”

“…Although D-kB1 (28) and D-kB2 were active and had affinity for PE-containing membranes, they were less hemolytic and had correspondingly lower affinity for model membranes than the cognate L-peptides. As there are no differences in charge, hydrophobicity, or three-dimensional structure (apart from mirror symmetry) between D-kB1 and the native enantiomer (28), the lower membrane affinity for the D-enantiomers can only be explained by the membrane chiral environment.…”

“…Peptide Synthesis-All-D-kB2 was synthesized using manual solid-phase peptide synthesis as described previously for all-DkB1 (28). The purity of the peptide (Ն95%) was evaluated using analytical reverse phase-HPLC, and the folding was confirmed by 1 H NMR spectroscopy as before (29).…”

“…The amount of lipid deposited at the chip surface was determined by converting the response units (RU), at the end of lipid deposition, into pg⅐mm Ϫ2 (1 RU ϭ 1 pg⅐mm Ϫ2 ) (32) and into moles by considering the average mass of the lipid mixture. The amount of peptide bound to the lipid bilayer was calculated at the end of association phase (170 s after peptide injection starts) by converting the RU into pg⅐mm Ϫ2 and then into moles (21,28). P/L versus injected peptide concentration was plotted and fitted with a nonlinear regression equation dose-response binding…”