The cyclotide family is the the largest class of circular proteins with as many as 50,000 predicted members. They are currently only found in the Violaceae, Cucurbitaceae, Rubiaceae and recently (1) Fabaceae family of the plant kingdom (violets, gourds, coffee and legumes, respectively ). Cyclotides are a prime example of typical circular proteins; they are small (~30 residues), they maintain internal stabilizing covalent bonds between amino acid side chains, and they have a seamless topologically circular peptide backbone that is processed from mature mRNA transcripts to connect the free N- and C-terminal ends. The evolutionary origin of the mechanism of cyclization is subject to speculation, because peptide bond formation is thermodynamically unfavorable. Unfavorable reactions typically do not take place unless the expended energy results in an increased survival advantage. But how could the advantage be realized and passed on if the reaction theoretically shouldn’t take place?
It has been proposed that cyclotides are processed from endogenous pre-existing asparaginyl endopeptidases (AEPs) that adapted a secondary function aside from their normal protease activity to assist in cyclization (2). Circular proteins exist in all kingdoms of life, and a multitude of mechanisms for processing circular proteins have arisen, which suggests that cyclization confers organismal fitness. Interestingly, despite the various origins of cyclization mechanisms, the activity of circular proteins is commonly implicated in host defense via membrane binding and disruption.
The unique characteristics of circular proteins are also obstacles to their discovery and characterization. They are immune to sequencing by Edman degradation, for example. As the technology to characterize circular proteins advances, their significance is being realized creating a feedback loop of increasing interest. The prototypic protein for cyclotides, kalata B1, was discovered on a 1960’s expedition to the Democratic Republic of Congo. A Norwegian doctor noted that African women used a medicinal tea, kalata-kalata, made from the leaves of the plant Oldenlandia affinis to induce labor and facilitate childbirth (uteronic activity verified via rat experiments). The newly discovered protein was aptly named kalata B1, but it wasn’t until the mid-1990’s when NMR analysis determined the presence of a circular backbone and the cyclic cysteine knot (CCK) motif nearly 30 years later! There are six conserved cysteine residues found in all cyclotides that form the CCK, which results in six variable loops formed in between the crosslinks.
Figure 1 denotes the glycine and asparagine where cyclization occurs; which are both conserved and necessary for most cyclotides. Loops L2, L3, L5, and L6 are excluded from the core of the protein, while L1 and L4 are embedded. This allows for hydrophobic residues on the excluded loops to be exposed to the aqueous environment. This characteristic makes X-ray crystallography analysis difficult due to poor crystal formation.
The CCK motif fixes the conserved internal structure and confers cyclotides with extreme stability in addition to the circular backbone (cyclotides can maintain activity after being subjected to boiling temperatures or extreme pH levels). The variability in loop length and residue composition earned cyclotides the status of being a natural combinatorial template, hence, the largest family of circular proteins.
Figure 2 shows examples from the two classes of cyclotides, which differ based upon the presence or absence of a proline residue in L5. However, their existence is a contradiction to the generally accepted school of thought that structure begets function, and that the primary structure determines the secondary structure. As for cyclotides, there is little variation in the secondary structure, and their activity is mainly determined by the variation in loop composition. This unique characteristic can help researchers provide insight into protein engineering by observing trends in activity based on changes in residue composition while still maintaining the same general structure.
Cyclotides were found to bind with specificity to various compositions of membrane lipids. Site directed mutagenesis studies can be utilized to manipulate engineered cyclotides to specifically target membranes of interest, or to specifically reduce certain activities (reducing hemolytic activity of potential drug therapies, for example). Cyclotides have shown potential in preventing HIV transmission and reducing pesticide use by transgenically introducing exogenous host defenses to crop plants. They also show high potential as a peptide based drug scaffold due to their extreme stability (which may help overcome previous limitations to peptide based therapies).
To summarize this blog I want to reiterate three main points. Cyclotides have unique characteristics and intriguing properties. These characteristics have been obstacles to their discovery and characterization. As the ability to screen, purify, engineer and characterize circular proteins improves, the discovery of significant applications will follow.
I would like to thank Dr. David J. Craik for being a bona fide circular protein enthusiast that is spearheading a lot of the research in the budding circular protein field.
Poth A.G., Colgrave M.L., Lyons R.E., Daly N.L. & Craik D.J. (2011). From the Cover: Discovery of an unusual biosynthetic origin for circular proteins in legumes, Proceedings of the National Academy of Sciences, 108 (25) 10127-10132. DOI: 10.1073/pnas.1103660108
Saska I., Gillon A.D., Hatsugai N., Dietzgen R.G., Hara-Nishimura I., Anderson M.A. & Craik D.J. (2007). An Asparaginyl Endopeptidase Mediates in Vivo Protein Backbone Cyclization, Journal of Biological Chemistry, 282 (40) 29721-29728. DOI: 10.1074/jbc.M705185200