The name cyclotides
synthesized peptides from plants that are
characterized by a head
tail cyclic peptide backbone and a cystine knot arrangement of three
conserved disulfide bonds
They were originally discovered in plants of the Rubiaceae (c
and Violaceae (vi
olet) families but have since
been found i
n the Cucurbitaceae
(legume) families. They are expressed in many plant tissues, including leaves, stems, flowers and
roots. Dozens to hundreds of different cyclotides are
in an individual plant
appears to be
crossovers of cyclotides between different
, i.e., most plants have
unique set of cyclotides
Figure 1 shows the structure of the prototypic cyclotide
kalata B1, from the African herb
It comprises 29 amino acids, including the six conserved Cys
residues that form the signature cyclic cystine knot (CCK) motif of the family. The backbone
segments between successive Cys residues are referred to as loops and the sequence variations of
otides occur within these loops.
Cyclotides have been classified into two main subfamilies,
Möbius or bracelet, based on the presence or absence of a cis X
Pro peptide bond in loop 5 of the
sequence but there is
a smaller subfamily that is referred to
as the trypsin inhibitor
subfamily. This third subfamily has high sequence homology to some members of the knottin
family of proteins from
are also referred to as cyclic knotti
So far more than 200
of cyclotides have been reported and
are documented in a
database dedicated to circular
proteins called CyBa
They range in size
37 amino acids and are typically not highly charged peptides.
Aside from their disulfide
tail cyclised backbone, there appear to be no other post
modifications in cyclotides.
Cyclotides appear to be ubiquitous in
family plants but
, occurring in about 5% of
the hundreds of
screened so far.
The reports of cyclotides in other plant families are more recent and there is
limited information available on the distribution of cyclotides in these families.
appears that cyclot
a very large family of plant proteins
ly numbering in the tens
Cyclotides are gene
encoded peptides that are processed from
precursors of Rubiaceae and Violaceae cyclotid
es are dedicated proteins, whose purpose appears
to be only to produce cyclotides. By contrast, cyclotides in
, a member of the
are produced from a chimeric precursor protein that also encodes an albumin
a situation similar to that recently
a small cyclic peptide trypsin inhibitor from
Thus, there appear to be multiple
types of precursors leading
In the case of Rubiaceae and Violaceae family plants the leader
considered to comprise a pro
region and an N
terminal region (NTR) that is
repeated in some
genes along with the
adjacent core peptide region, as illustrated in Figure 1.
re of kalata B1 and cyclotide
representation of three
is shown at the top of the figure. The
n endoplasmic reticulum
signal sequence labeled ER, a
(comprising a pro
a conserved repeated fragmen
t labeled NTR
and either one or
multiple copies of the
peptides; for example,
B3 and B6. A s
terminal recognition sequence
present in each precursor
At the bottom of the
he amino acid sequence of kalata B1
is shown, with the cysteine residues labeled with
Roman numerals. The cleavage sites
excision of a
domain with an N
terminal Asn, which
subsequently linked by an asparaginyl endopeptidase,
are indicated by arrows.
The location of the ligation point which forms loop 6 of the mature
cyclic peptide is shown on the right. Parts of the precursor protein flanking the mature domain
are shown in
lighter shading. Figure adapted from Daly et al.
Biological activities of cyclotides
are thought to be plant def
ence molecules, given their potent
they also have a broad range of other
activities, including anti
Some of these activities are of potential
pharmaceutical interest and because of their exceptional stability cyclotides have also attracted
attention as potential protein engineering or drug design templates
diverse range of activities
seems to have a common mechanism that involves
binding to and disruption of biological membranes.
larvae fed a diet containing cyclotide
similar concentration to that whi
ch occurs naturally in
plants for example
marked swelling and blebbing of mid
Larvae that have
ingested cyclotides are markedly stunted in their growth and development, presumably as a
result of this disr
A range of biophysical studies
embrane interactions for
both Möbius and bracelet cyclotides
information is available on the residues involved in making contact
with membrane surfaces.
electrophysiological and vesicle
studies have confirmed the
treated with cyclotide
It appears that cyclotides are able
to self associate in th
environment and form
large pores in membranes.
As indicated in Figure x, c
yclotides are derived from precursor proteins that encode one or more
copies of the core (cyclotide)
For example the
gene) encodes an 11 kDa precursor
that contains an
signal, leader peptide, kalata B1
, and a C
terminal peptide region,
gene, encodes a precursor containing three copies of ka
The single or multiple
copies of the cyclotide domain
terminal and C
sequences that are thought to be implicated in the processing reactions.
of the presence of the ER signal it is thought that cyclotide
in the ER prior to processing (excision and cyclization) of the cyclotide domain. Protein
disulfide isomerases are o
he folding of disulfide
shown increased yiel
in the presence of PDI,
although so far there h
ave been no
of PDI for
. A r
dy has sh
are targeted to vacuoles in plant cells
and this is where the
excision and cyclization processes are
terminal repeat (NTR) region
of cyclotide precursors
to adopt an α
but the significance, if any, of this is not yet known.
It has been proposed that it might
sequence for cyclotide folding
but interestingly cyclotide precursors from
lack the NTR region
seem in Violaceae or Rubiaceae precursors. The
nature of the processing reaction
terminal end of the cyclotide domain is
here is strong evidence that asparaginyl endopr
otease (AEP) activity is responsible for
processing at the C
terminal end of
With just one exception (circulin F) all cyclotides have an Asn or Asp
residue as the C
terminal end of the core peptide
an AEP is principle able to cleave after this
residue. It has been proposed that
cleavage reaction occurs contemporaneously with
to the earlier
of the core
peptide region. Mutage
transgenic tobacco and
plants have shown a
as well as other key fl
show decreased production
peptide in transgenic
Overall, the processing of cyclotides can be
as involving excision and
from a pre
Specific recommendations for the family
At present there is no common gene nomenclature for
given the limited
knowledge of the full complement of associated
biosynthetic genes encoding proteins involved
processing it see
premature to propose one. A naming scheme for the mature
that involves a fo
ur letter pronounceable acronym
based on the Latin name of the plant species in
which the cyclotid
e was first discovered has been proposed
but has not been uniformly
lowed so far
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knotted proteins that defines the cyclic cystine knot structural motif.
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epithelial cells in the midgut of lepidopteran larvae.
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properties of plant cyclotides: the cyclic knotted proteins from
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activity of kalata B2, a circular protein with a twist: do Mobius strips exist in nature?
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peptides containing end
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the National Academy of Sciences of the United States of America
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testinal nematode parasites of sheep.
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of the cyclotides: natural variants with enhanced activity.
Barry, D.G.; Daly, N.L.; Clark, R.J.; Sando, L.; Craik, D.J. Linearization of a naturally
occurring circular protein maintains structure but eliminates hemolytic activity.
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miniproteins: nature's combinatorial peptide template.
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biological activity of the prototypic cyclotide Kalata B1 is modulated by the formation of
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the cyclotides kalata B1 and kalata B6 on model membrane systems by surface plasmon
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spatial structure of ternary complex Kalata B7/Mn2+/DPC micelle.
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Broussalis, A.M.; Göransson, U.; Coussio, J.D.; Ferraro, G.; Martino, V.; Claeson, P. First