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Next: Biosynthesis Up: The Cyclotides Previous: Nomenclature and Sequence Conservation
Structural Characteristics of the cyclotides
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.
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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.
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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].
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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].




Next: Biosynthesis Up: The Cyclotides Previous: Nomenclature and Sequence Conservation Jason Mulvenna
2005-04-24