Kalata B5

Cyclotides are a family of plant defense proteins with a unique cyclic backbone and cystine knot. Their remarkable stability under harsh thermal, enzymatic, and chemical conditions, combined with their range of bioactivities, including anti-HIV activity, underpins their potential as protein drug scaffolds. The vast majority of cyclotides possess a conserved glutamate residue in loop 1 of the sequence that is involved in a structurally important network of hydrogen bonds to an adjacent loop (loop 3). A single native cyclotide sequence, kalata B12, has been discovered that has an aspartic acid in this otherwise conserved position. Previous studies have determined that methylation of the glutamate or substitution with alanine abolishes the membrane disrupting activity that is characteristic of the family. To further understand the role of this conserved structural feature, we studied the folding, structure, stability, and activity of the natural aspartic acid variant kalata B12 and compared it to the prototypical cyclotide kalata B1, along with its glutamate to alanine or aspartate mutants. We show that the overall fold of kalata B12 is similar to the structure of other cyclotides, confirming that the cyclotide framework is robust and tolerant to substitution, although the structure appears to be more flexible than other cyclotides. Modification of the glutamate in kalata B1 or replacing the aspartate in kalata B12 with a glutamate reduces the efficiency of oxidative folding relative to the native peptides. The bioactivity of all modified glutamate cyclotides is abolished, suggesting an important functional role of this conserved residue. Overall, this study shows that the presence of a glutamic acid in loop 1 of the cyclotides improves stability and is essential for the membrane disrupting activity of cyclotides.

Cyclotides are a family of bioactive plant peptides that are characterized by a circular protein backbone and three conserved tightly packed disulfide bonds. The antimicrobial and hemolytic properties of cyclotides, along with the relative hydrophobicity of the peptides, point to the biological membrane as a target for cyclotides. To assess the membrane-induced conformation and orientation of cyclotides, the interaction of the Möbius cyclotide, kalata B1, from the African perennial plant Oldenlandia affinis, with dodecylphosphocholine micelles was studied using NMR spectroscopy. Under conditions where the cyclotide formed a well-defined complex with micelles, the spatial structure of kalata B1 was calculated from NOE and J couplings data, and the model for the peptide-micelle complex was built using 5- and 16-doxylstearate relaxation probes. The binding of divalent cations to the peptide-micelle complex was quantified by Mn2+ titration. The results show that the peptide binds to the micelle surface, with relatively high affinity, via two hydrophobic loops (loop 5, Trp19-Val21; and loop6, Leu27-Val29). The charged residues (Glu3 and Arg24), along with the cation-binding site (near Glu3) are segregated on the other side of the molecule and in contact with polar head groups of detergent. The spatial structure of kalata B1 is only slightly changed during incorporation into micelles and represents a distorted triple-stranded beta-sheet cross-linked by a cystine knot. Detailed structural analysis and comparison with other knottins revealed structural conservation of the two-disulfide motif in cyclic and acyclic peptides. The results thus obtained provide the first model for interaction of cyclotides with membranes and permit consideration of the cyclotides as membrane-active cationic antimicrobial peptides.

Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.