Structural Characteristics of the cyclotides
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The 3D structure of five members of the cyclotide family have been solved using solution NMR spectroscopy. The bracelet cyclotides circulin A [34], cycloviolacin O1 [32,26] and vhr1 [35], the Möbius and prototypic cyclotide kalata B1 [19,32] and the hybrid palicourein [36] have all been shown to adopt a similar fold that appears to be characteristic of the cyclotides. Figure 1.4 sets out ribbon diagrams of these structures and it can be seen that in all cases they are characterised by a cystine knot arrangement of disulfide bonds and a -sheet secondary structure. The knotted topology involves the formation of disulfides between CysI-CysIV and CysII-CysV which, along with loops 1 and 4, form the ring that is penetrated by the third disulfide between CysIII-CysVI (Figure 1.4). Superimposition of the structures calculated to date shows a mean pairwise RMSD over the C of the cystine residues that comprise the cystine knot of 0.46 Å, clearly demonstrating the structural conservation of the cystine knot core of the cyclotides.
Figure 1.4: A comparison of cyclotide structures and the cystine knot motif. A. Ribbon diagrams of the cyclotide structures determined to date. Clockwise from top left is kalata B1, circulin A, palicourein and cycloviolacin O1. The numbering of the Cys residues and loops have been indicated on kalata B1. B. Magnified view of the cystine knot motif showing the ring formed by loops 1 and 4 and the disulphide bonds between CysI-CysIV and CysII-CysV. The ring is penetrated by the disulphide between CysIII-CysVI. These loops and Cys residues have been indicated on the diagram. All diagrams were produced using the programs MOLMOL [37] and POV-ray.
To facilitate the tight folding of the backbone the cyclotides show the formation of a -hairpin that is centred in loop 5. A third -strand can usually be inferred from analysis of NOE's, slow exchanging amide protons and coupling constants, however this strand is distorted from the typical geometry and is consequently not recognised by structure analysis programs. Consistent with the comparative loop sizes the single Möbius cyclotide characterised, kalata B1, shows no other secondary structure in solvent exposed loops. However, the bracelet cyclotides possess a short helical segment in loop 3, a loop which is expanded in size in this subfamily.
A magnified view of the cystine knot ring is shown in Figure 1.4 along with the penetrating disulphide. Combined with the cyclic backbone, the cystine knot is the defining characteristic of the cyclotides. However, the cystine knot motif is also found in a large range of linear proteins. The first cystine knots described were those found in several growth factors, including nerve growth factor, transforming growth factor , and platelet derived growth factor BB [38]. Subsequently it was also realised that the motif was found in a group of small, toxic peptides from a variety of organisms that had been named the knottins [39]. In both groups of proteins the cystine knot disulphide connectivity is the same (I-IV, II-V and III-VI) but the topology of the knot differs and to distinguish the two groups they were named the inhibitor cystine knot (ICK) and the growth factor cystine knot (GFCK) [40,41]. In the GFCKs the penetrating disulphide is CysI-IV while in the ICKs the penetrating disulphide is CysIII-VI and this topological difference leads to a situation where the two folds are not superimposable. The presence of an ICK combined with backbone cyclisation in the cyclotides has since led to a third classification denoted the cyclic cystine knot (CCK) [26].
The cystine knot appears to be essential for the stability of the CCK fold. Studies involving acyclic permutants, in which synthetic variants with the backbone broken in each of the loops were structurally characterised, indicate that the knot is crucial for maintaining the overall fold of the cyclotides [42]. In this study it was shown that only breaks in loops 1 and 4 prevented folding into a native conformation suggesting that it is the cystine knot rather than the cyclic backbone that is essential in stabilising the overall fold. However the activity of the cyclotides, as measured by haemolysis in this study, was abolished by linearisation in any loop suggesting that the extra conformational rigidity caused by backbone cyclisation is important for this activity. The ICK possessed by the cyclotides is also distinguished by possessing the smallest possible loop that will allow the passage of a disulphide [36] -- further emphasising the compactness of the cyclotide structure.
Figure 1.5: Hydrogen bonding network formed by Glu3 in kalata B1. Orientation of the conserved Glu residue and the network of hydrogen bonds in kalata B1. Atoms involved in hydrogen bonding have been indicated with a shaded circle and for the stick representations oxygen atoms are coloured red, hydrogen atoms grey, nitrogen atoms blue and carbon atoms green.
As discussed above several conserved residues appear to be crucial for stabilising the CCK fold. Glu3 is the only non-Cys residue absolutely conserved in the cyclotides and temperature and pH titrations of kalata B1 and cycloviolacin O1 have revealed the important role that Glu3 plays in stabilising the CCK fold [32]. This residue appears to link the cystine knot core of the molecules to loop 3 through hydrogen bonding interactions. In particular, Glu3 and other backbone amide hydrogens form network of hydrogen bonds that includes the hydroxyl containing residue, conserved as the second or third residue of loop 3. This network, which seems to stabilise the compact fold, is illustrated in Figure 1.5 for kalata B1. Other conserved residues thought to be important for the structure of the cyclotides include the hydroxyl-bearing (Thr or Ser) residue immediately following the Glu residue in loop 1, which has been implicated in intra-residual hydrogen bonding [32]. Additionally, the Gly residue, conserved as the last residue in loop 3, generally possesses a positive angle, thought to be necessary for linking loop 3 to the cystine knot [32].
Figure 1.6: Surface hydrophobicity and hydrophobic interactions important for cyclotide structure. Surface renderings of A. kalata B1 and B. cycloviolacin O1 showing the unusual hydrophobicity of the cyclotides. Hydrophobic residues are coloured green, residues with a negative charge are coloured red and residues with a positive charge are depicted in blue. Selected loops are also indicated. In both cases the rendering on the right is related to the left by a 180 rotation about the Y-axis. C. shows ribbon diagrams of kalata B1 (left) and cycloviolacin O1 (right) with residues from loops 2 and 5 highlighted. These residues are thought to stabilise the CCK fold via hydrophobic interactions. Surface renderings were produced using the program PyMOL [43] and the ribbon diagrams were produced using the programs MOLMOL and POV-ray.
In the cyclotides that have been structurally characterised the formation of the cystine knot at the core of the molecules seems to force the surface exposure of hydrophobic residues. In effect cyclotides can be considered as ``inside out'' proteins in which the normally hydrophobic core of the peptide is forced to the surface to form hydrophobic patches. This unusual hydrophobicity, illustrated in Figure 1.6, is of great interest as it has been speculated that the unusual hydrophobicity of the cyclotides may mediate their various biological activities [31]. In the Möbius cyclotides, as typified by kalata B1, the side chain of Val21 forms the centre of the patch by interacting with Val6, Pro20, and Pro28. In the bracelet cyclotides, such as circulin A and cycloviolacin O1, hydrophobic residues from loops 2 and 3 form a solvent exposed hydrophobic patch with additional hydrophobic residues from loops 5 and 6 exposed on the other face of the molecule. The bracelet subfamily possesses an extra charged residue that is also solvent exposed, producing a surface characterised by a hydrophobic patch on one face of the molecule and charges, and additional hydrophobic residues, on the other. It is known from structural studies of kalata B1 that hydrophobic interactions between the sidechains of Trp19, Pro20, Val21 and Val6 from loops 2 and 5 may act to stabilise the CCK fold [32]. In the bracelet cyclotides, as typified by cycloviolacin O1, similar interactions between these two loops, in particular the side chains of Tyr7, Val6, Val24 and the hydrophobic portions of Arg23 and Asn22, may also function to stabilise the fold in the bracelet cyclotides [32].