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All of the cyclotides discovered to date share the absolute conservation of the six cystines that comprise the cystine knot motif. When combined with the cyclic backbone a cyclotide can be conceptualised as a series of six loops connecting consecutive cystine residues. Following the nomenclature of Craik and co-workers , the cystines are numbered with roman numerals from one to six in the order in which they appear in the linear precursor. As discussed below the ligation of the N and C-termini occurs in loop 6, consequently the residue corresponding to residue one in the linear precursor varies according to the number of residues in loop 6 that originate from the N-terminal of the linear cyclotide. Accordingly, in this work, the convention of numbering CysI as residue one is followed as this allows the meaningful comparison of loop 6. The details of this nomenclature can be seen in Figure 1.1.
Figure 1.2 presents an alignment of the reported cyclotides, and it can be seen that they fall into two major groups that vary in sequence conservation and loop size. These two groups have been termed the Möbius and bracelet cyclotide families. The naming of the groups arises from the presence of a conserved cis-Pro residue in loop 5 of the Möbius cyclotides. This cis-Pro residue gives the Möbius subfamily a conceptual backbone twist, similar to that seen in the Möbius strips studied by the German mathematician August Ferdinand Möbius. Lacking the cis peptide bond the bracelet cyclotides were named to reflect the lack of a conceptual twist and hence their resemblance is more to a bracelet than the Möbius strip. Although useful for classification purposes, it should be stressed that a backbone twist is a purely conceptual idea arising when the cyclic backbone is considered as a ribbon, and does not reflect the actual topology of the peptide backbone.
Considered as a whole the cyclotides exhibit a great deal of sequence variation with only the six Cys residues and a Glu acid residue in loop 1 absolutely conserved. Loops 1 and 4 share the greatest amount of sequence conservation with loop 1 having the consensus sequence of (G/A)E(T/S) and loop 4, comprising only one residue in all cyclotides, namely Thr, Ser, or, in three cases, a Lys residue. The presence of a Thr residue in the third position of loop 1 was initially an additional characteristic of the Möbius subfamily and a Ser in this position characteristic of the bracelet subfamily, however the discovery of palicourein and vodo M, in which these residues are reversed, has blurred this distinction. It is worth noting that loops 1 and 4 are the backbone loops that form the ring of the cystine knot and their strong conservation across the cyclotides as a whole may reflect this fact. Other residues that appear to be highly conserved include a Asn/Asp in loop 6 and a Pro residue, also found in loop 6, which is conserved in all cyclotides except cycloviolin B. A Gly residue is the last residue of loop 3 in all cyclotides, except vodo M, and there is a strong conservation of a hydroxyl bearing residue in the second or third position of loop 3. As discussed below some of these conserved residues appear to have a role in the compact structure of a cyclotide. However, of particular interest are the conserved residues in loop 6, apart from loops 1 and 4 this loop is the most conserved loop within the cyclotides  and this may reflect the fact that processing occurs in this loop. For example the point at which ligation of the N and C-termini of the linear cyclotide occurs is after the conserved Asn/Asp residue, consequently this residue may have some importance in the cleavage of the C-terminal tail from the precursor, the subsequent ligation of the termini or it may be important for both reactions.
When considered as separate subfamilies it is interesting to note that the Möbius subfamily exhibits a much greater degree of conservation in loop size and sequence. The sequence logo of a Möbius cyclotide is set out in Figure 1.3 and it can be seen that, apart from the Cys and Glu residues, five residues are absolutely conserved. These include include a Gly residue in the second to last position of loop 2, a Thr residue in the second position of loop 3, the defining Pro in the second to last position of loop 5, another Gly residue is conserved in the fourth last position of loop 6 and a Pro is conserved as the second last residue in this same loop. The Arg residue in loop 6 is conserved in all the Möbius cyclotides except violapeptide 1 in which this residue was reported as an X , it is likely, therefore, that this Arg is also completely conserved. Some of these conserved residues, including Glu3 and the Thr in loop 3, appear to have a structural function, however the role of the conserved Gly residues and the, most likely, conserved Arg is not known at this time. Apart from these six invariant residues another 10 residues are strongly conserved, making approximately 60% of the residues in the Möbius subfamily highly conserved. Apart from sequence conservation it can also be seen in Figure 1.3 that there is generally a conservation of hydrophobic residues (coloured green) especially in loops 2, 5 and 6, and these residues are thought to constitute a surface exposed hydrophobic patch that is characteristic of the cyclotides.
The bracelet subfamily contrasts with the Möbius in exhibiting a much greater degree of variation in loop size and sequence. Figure 1.3 sets out the sequence logo for this subfamily and it can be seen that when compared to the logo for the Möbius family a much greater degree of variation is evident. Apart from the absolute conservation of the Cys residues and the loop 1 Glu residue there is only one other absolutely conserved residue - the terminal Pro of loop 2. Even residues strongly conserved in the cyclotides as a whole show a greater amount of variation in this family. For example the most conserved loops, 1 and 4, show a number of substitutions in this family that are not present in the Möbius subfamily. The most variable of the loops is loop 3, however, it is interesting to note that despite a great deal of substitution the hydrophobic nature of the loop is retained, particularly in the third and second last positions. Likewise loop 2 shows a consistent hydrophobic character despite the presence of occasional polar residues or in one instance an Arg, although the Arg residue is found in palicourein which is an unusual cyclotide exhibiting characteristics of both subfamilies. This subfamily also displays a greater amount of charged residues. These residues are generally found in loops 5 and 6 where they are usually positively charged Lys residues, although other residues and other charges are found. These extra charges give the bracelet subfamily a net charge between -1 and +2, with +2 being the most common. The functional or structural significance of these conserved charges is not known although it is interesting to compare with the Möbius subfamily which, apart from the Glu residue in Loop 1 and the Arg residue in loop 6, has fewer conserved charges. Indeed over half of the Möbius cyclotides have a net charge of 0. Given this difference the charged residues are unlikely to be crucial for the cyclotide structure and may be linked to the natural activity of the bracelet subfamily.
Although the cyclotides have been divided into two classes based on sequence homology and loop size, recent discoveries have suggested that a clear demarcation between the two families may not exist. The sequence of palicourein possesses a loop 1 and 6 that share a greater similarity with the Möbius subfamily while loops 2 and 3 show characteristics common to the bracelet subfamily. In this work it has been placed in the bracelet subfamily on the basis of the lack of a cis-Pro in loop 5, although it can properly be considered a hybrid of the two families. Another possible hybrid is vodo M which shows greater overall similarity to the Möbius subfamily but contains the Gly-Glu-Ser motif characteristic of the bracelet subfamily in loop 1. Although this makes this molecule a hybrid it is perhaps less compelling than palicourein as a Thr to Ser substitution involves only a single codon change and is a highly conservative substitution. Hence, it is likely that this cyclotide may represent a minor mutation to what was originally an unambiguous Möbius cyclotide.
Next: Structural Characteristics of the Up: The Cyclotides Previous: Discovery and Initial Characterisation Jason Mulvenna