Bibliography




Next: Cyclic Bacteriocins Up: Other Cyclic Peptides Previous: Other Cyclic Peptides
Knottins and Cyclic trypsin inhibitors from Cucurbitaceae
As discussed above small linear, cystine-rich proteins that possess
the typical cystine knot topology of I-IV, II-V and III-VI have
been categorised as knottins. This structural class is presently
thought to contain at least 12 different protein families, with
virtually no sequence identity, including conotoxins from cone snails,
spider toxins, potato inhibitors and the antifungal peptide from
Phytolacca americana (PAFP-S). The widespread occurrence of
the knottin fold within nature suggests that there was either an
ancestral knottin or that the fold has evolved independently in many
different lineages. The squash trypsin inhibitors (TI) are a class of
knottin that were discovered in the late 1970's in the seeds of the
winter squash [114]. Before the discovery of
SFTI-1 they were the smallest known inhibitors of serine proteinases
and also one of the most potent with association constants as high as
M
[115]. Almost 30 homologs have
since been identified, exclusively from plants of the Cucurbitaceae
family, and all these peptides contain six Cys residues arranged into
three disulfides connected in the typical cystine knot arrangement
[114,117,115,116].
Until recently the cyclotides were thought to be the only example of the knottin fold that included macrocyclisation of the peptide backbone, however this changed with the discovery of the 34 residue trypsin inhibitors, MCoTI-I and II, from the dormant seeds of Momordica cochinchinensis [30]. These TIs combine the typical knottin fold and disulphide connectivity with backbone cyclisation. Although sequence homology with the cyclotides is low and the loop lengths differ, the cyclic proteins from Momordica cochinchinensis have been classed as cyclotides on the basis of the conserved CCK motif [7]. Intriguingly, unlike the cyclotides, the seeds of Momordica cochinchinensis contain not only the macrocyclic MCoTI-I and II but also a range of other species including a linear version of the TI [30].
![]() |
It can be seen in Figure 1.16 that the sequence of both MCoTI-I and II are similar to other linear squash TI's apart from the addition of a short linker, consisting predominantly of Ser and Gly residues, that completes the cyclic backbone of the peptides. The linear precursor of these peptides is unknown, however the presence of a conserved Gly after the final Cys residue, common to other squash TIs, would seem to indicate that this may be the C-terminal processing point. Additionally, the MCoTI-II linker region (SGSDGGV) shares strong sequence identity with the pro-sequence of the linear precursor of the squash TI TGT-II (SGRHGGI) [118], suggesting that the cyclic variant is produced via a typical squash TI precursor protein. It is interesting to note that the two residues at either end of this putative linear precursor of MCoTI-II are the same residues that have been shown to be critical for the cyclising reaction that produces the cyclic pilin.
Other species of the squash TIs have been isolated from
Momordica cochinchinesis including species of MCoTI-II
possessing a -Asp-Gly bond in the linker region and a
succinimide cyclic intermediate of the same bond formed during the
conversion of the
-Asp-Gly to the
form. Similarly a
species of MCoTI-I has been identified that would appear to also
possess a
-Asp-Gly in the same position
[30,7]. MCoTI-III was also isolated and
it appears to be a regular linear member of the squash TI family, with
the exception of a N-terminal pyroglutamate residue
[30]. The presence of a linear species is of great
interest. It has been pointed out that processing of the squash TIs
appears to be variable [117]. It can be seen in Figure
1.16 that examples from the same species often display
almost complete sequence identity apart from the addition of a
N-terminal segment that, significantly, possesses multiple Glu
residues. Whether the existence of two species of TI is a result of
two distinct genes or the result of post-translational modifications
is not known, although in the peptides from Cucurbita maxima
and Momordica charantis there are enough substitutions, apart
from the N-terminal, to suggest separate genes. It is interesting,
therefore, to note that MCoTI-III could represent a version of the
expanded peptide in Momordica cochinchinensis and the apparent
presence of precursor sequence in the cyclic versions is a consequence
of mutations in the N-terminal region of the precursor protein for the
shorter version of the TI -- mutations resulting in cyclisation.
Although this is an interesting parallel, resolution of these
questions relies on the determination of a range of squash TI
precursor sequences, particularly the linear precursors of the cyclic
peptides from Momordica cochinchinensis, and these data are
eagerly awaited.
![]() |
The 3D structure of MCoTI-II has been determined by NMR spectroscopy
[7,119] and it displays a
similar fold to the linear versions of the squash TIs, EETI-II
[121,120] and CMTI-I
[123,122] (see Figure 1.17). The
structure of MCoTI-II is characterised by the presence of a
-hairpin and the molecule contains several turns. Compared to
the cystine knot of the cyclotides the squash TIs exhibit a looser
ring, with eleven backbone residues making up the ring through which
the third disulphide threads. Nonetheless the cyclotides and MCoTI-II
both contain the identical structural elements of a cystine knot and a
triple stranded
-sheet. Although the overall structure of
MCoTI-II is well defined the linker region, unlike the cyclotides,
shows considerable disorder. One possible advantage of cyclisation is
reduced conformational flexibility leading to increased binding
efficiency, however this does not appear to be the case in MCoTI-II as
the linker region does not seem to impart conformational rigidity. It
has been suggested that cyclisation has evolved in Momordica
cochinchinensis to impart resistance to proteolytic activity or to
increase stability [7]. Although the latter is
feasible, it should be noted that linear squash inhibitors possess a
very stable framework, with the melting temperature of EETI-I reported
as 140
C [72] and consequently the most likely
advantage that would be conferred would be resistance to
exo-peptidases. Given the existence of a linear homologue in
Momordica cochinchinensis and if other Cucurbitaceae species do
not contain cyclic TIs then it is also quite possible that cyclisation
in Momordica is a relatively recent event, possibly brought about via
serendipitous mutations in the precursor sequence.
Although the possibility exists that cyclic squash TIs are present in other Cucurbitaceae species but have been overlooked due to sequencing failure brought about by backbone cyclisation, the existence of cyclic peptides in one isolated species follows the trend discussed elsewhere within this chapter. If the Momordica peptides are the result of a fortuitous mutation than it may be illustrating a more general trend in the evolution of cyclic proteins. It is possible that cyclisation relies only on the close proximity of the N and C-termini and the presence of key residues in the precursor sequence -- cyclisation then proceeds using regular proteases or other processing enzymes typical to the biosynthetic pathway of the particular species. Interestingly it has been pointed out that the cysteine topology of the cyclotides and the squash TIs is conducive to the positioning of the N and C-termini in close proximity, as these two classes are the only knottins to have CysIV closer to CysV than to CysIII (see Figure 1.16), hence increasing the probability of cyclisation.




Next: Cyclic Bacteriocins Up: Other Cyclic Peptides Previous: Other Cyclic Peptides Jason Mulvenna
2005-04-24