Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides (similar to 30 amino acids), with diverse biol ogical activities, have been isolated from the Rubiaceae and Violaceae plan t families over recent years. We have significantly expanded the range of k nown macrocyclic peptides with the discovery of 16 novel peptides from extr acts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represe nt novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of thes e novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structu re consists of a distorted triple-stranded beta-sheet and a cystine-knot ar rangement of the disulfide bonds. This structure is similar to kalata B1 an d circulin A, the only two macrocyclic peptides for which a structure was a vailable, suggesting that despite the sequence variation throughout the pep tides they form a family in which the overall fold is conserved. We refer t o these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of t hese molecules makes them a fascinating example of topologically complex pr oteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively c harged residues and has a bracelet-like circularization of the backbone. Th e second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we defi ne the structural features of the two apparent subfamilies of the CCK pepti des which may be significant for the likely defense related role of these p eptides within plants. (C) 1999 Academic Press.