Kalata B2

Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.

The recently discovered cyclotides kalata B1 and kalata B2 are miniproteins containing a head-to-tail cyclized backbone and a cystine knot motif, in which disulfide bonds and the connecting backbone segments form a ring that is penetrated by the third disulfide bond. This arrangement renders the cyclotides extremely stable against thermal and enzymatic decay, making them a possible template onto which functionalities can be grafted. We have compared the hydrodynamic properties of two prototypic cyclotides, kalata B1 and kalata B2, using analytical ultracentrifugation techniques. Direct evidence for oligomerization of kalata B2 was shown by sedimentation velocity experiments in which a method for determining size distribution of polydisperse molecules in solution was employed. The shape of the oligomers appears to be spherical. Both sedimentation velocity and equilibrium experiments indicate that in phosphate buffer kalata B1 exists mainly as a monomer, even at millimolar concentrations. In contrast, at 1.6 mm, kalata B2 exists as an equilibrium mixture of monomer (30%), tetramer (42%), octamer (25%), and possibly a small proportion of higher oligomers. The results from the sedimentation equilibrium experiments show that this self-association is concentration dependent and reversible. We link our findings to the three-dimensional structures of both cyclotides, and propose two putative interaction interfaces on opposite sides of the kalata B2 molecule, one involving a hydrophobic interaction with the Phe6, and the second involving a charge-charge interaction with the Asp25 residue. An understanding of the factors affecting solution aggregation is of vital importance for future pharmaceutical application of these molecules.

The cyclotides are a family of small head-to-tail cyclic plant defense proteins. In addition to their cyclic backbone, cyclotides comprise three disulfide bonds in a knotted arrangement, resulting in a highly cross-braced structure that provides exceptional chemical and proteolytic stability. A number of bioactivities have been associated with cyclotides, including insecticidal, antimicrobial, anti-viral and cytotoxic, and these activities are related to an ability to target and disrupt biological membranes. Kalata B2 and to a lesser extent kalata B1, isolated from Oldenlandia affinis, self-associate to tetramers and octamers in aqueous buffers, and this oligomerization has been suggested to be relevant for their ability to form pores in membranes. Here we demonstrate by solution NMR spectroscopy analysis that the oligomerization of kalata B2 is concentration dependent and that it involves the packing of hydrophobic residues normally exposed on the surface of kalata B2 into a multimeric hydrophobic core. Interestingly, the hydrophobic surface that is "quenched" has previously been shown to be responsible for the ability of kalata B2 to insert into membranes. Thus, it seems unlikely that the oligomers observed in aqueous solution are related to any multimeric state present in a membrane environment, and responsible for the formation of pores. The ability to self-associate might alternatively provide a mechanism for preventing self-toxicity when stored at high concentrations in intracellular compartments.