Discovery and characterization of pseudocyclic
cystine-knot a-amylase inhibitors with high resistance to
heat and proteolytic degradation
Phuong Q. T. Nguyen, Shujing Wang, Akshita Kumar, Li J. Yap, Thuy T. Luu, Julien Lescar and
James P. Tam
School of Biological Sciences, Nanyang Technological University, Singapore
Keywords
cis-proline; cystine knot; pseudocyclics;
wrightide; a-amylase inhibitors
Correspondence
J. P. Tam, School of Biological Sciences,
Nanyang Technological University,
Singapore
Fax: +65 6515 1632
Tel: +65 6316 2833
E-mail: [email protected]
(Received 22 February 2014, revised 19
June 2014, accepted 15 July 2014)
doi:10.1111/febs.12939
Obesity and type 2 diabetes are chronic metabolic diseases, and those
affected could benefit from the use of a-amylase inhibitors to manage
starch intake. The pseudocyclics, wrightides Wr-AI1 to Wr-AI3, isolated
from an Apocynaceae plant show promise for further development as
orally active a-amylase inhibitors. These linear peptides retain the stability
known for cystine-knot peptides in the presence of harsh treatment. They
are resistant to heat treatment and endopeptidase and exopeptidase degradation, which is characteristic of cyclic cystine-knot peptides. Our NMR
and crystallography analysis also showed that wrightides, which are currently the smallest proteinaceous a-amylase inhibitors reported, contain the
backbone-twisting cis-proline, which is preceded by a nonaromatic residue
rather than a conventional aromatic residue. The modeled structure and a
molecular dynamics study of Wr-AI1 in complex with yellow mealworm
a-amylase suggested that, despite having a similar structure and cystine-knot fold, the knottin-type a-amylase inhibitors may bind to insect
a-amylase via a different set of interactions. Finally, we showed that the
precursors of pseudocyclic cystine-knot a-amylase inhibitors and their biosynthesis in plants follow a secretory protein synthesis pathway. Together,
our findings provide insights for the use of the pseudocyclic a-amylase
inhibitors as useful leads for the development of orally active peptidyl bioactives, as well as an alternative scaffold for cyclic peptides for engineering
metabolically stable human a-amylase inhibitors.
Database
The nucleotide sequences for Wr-AI1 to Wr-AI3 have been deposited in the GenBank database
under GenBank accession numbers KF679826, KF679827, and KF679828, respectively. The
Wr-AI1 solution structure solved for 10 ensembles with the lowest target function is available
in the Protein Data Bank under accession code 2MAU. The coordinates of Wr-AI1 crystal
structure are available in the Protein Data Bank database under accession code 4BFH.
Introduction
Small disulfide-rich, proteinaceous bioactives are
prominent in toxins, hormones, growth factors, and
protease inhibitors [1]. Many contain a cystine-knot
(CK) motif, with a three-disulfide knotted structure
formed by two disulfide bonds, together with the
connecting backbones, forming an embedded ring
Abbreviations
AAI, amaranth a-amylase inhibitor; CCK, cyclic cystine-knot; CK, cystine-knot; ER, endoplasmic reticulum; HSA, human salivary a-amylase;
PCK, pseudocylic cystine-knot; PDB, Protein Data Bank; TMA, Tenebrio molitor a-amylase; UPLC, ultra-performance liquid chromatography.
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P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
through which the third bond penetrates [2]. Of particular interest in drug development is the knottin
family of CK peptides containing 25–45 residues, and
often possessing protease inhibitory functions, from
which the name was derived [3]. Knottins form compact and defined structures with extensive internal
hydrogen bonding, endowing them with resistance to
proteolytic degradation by endopeptidases and denaturation by heat or chemicals, as shown by numerous
studies, including those using sequencing experiments
to determine their primary structures. Certain CK
peptides of the knottin family have further evolved
as macrocycles such as cyclotides, harboring cyclic
CKs (CCKs) with no termini, a feature that has
made them resistant to degradation by exopeptidases
[4]. Cyclotides, generally consisting of 28–37 residues,
are known be ultrastable to proteolytic and heat degradation, and possess robust qualities comparable to
those of small-molecule drug candidates. All of these
features bode well for the development of orally
active peptidyl bioactives.
In a program to identify potentially orally active
peptidyl bioactives for the treatment of metabolic diseases such as diabetes, we have initiated MS profiling
to identify cysteine-rich peptidyl a-amylase inhibitors
in traditional medicines. Plants and microorganisms
produce a diverse group of proteinaceous a-amylase
inhibitors that function in defense pathways. These
inhibitors vary greatly in structure and size, ranging
from small peptides (3 kDa), such as amaranth a-amylase inhibitor (AAI) [5], to large proteins, such as
a-AI1, a 23-kDa a-amylase inhibitor from kidney bean
(Phaseolus vulgaris) [6]. They are structurally classified
into seven groups: knottin-type, c-thionin-like, CMproteins, Kunitz-type, thaumatin-like, legume-lectinlike, and microbial [7]. These classes of a-amylase
inhibitor have attracted attention as tools in agriculture and for antidiabetes management.
The smallest proteinaceous a-amylase inhibitor
known to date, the 3-kDa AAI, is currently the only
member of the knottin-type group to be reported. AAI
comprises 32 residues harboring a CK core. This
inhibitor specifically inhibits the yellow mealworm
Tenebrio molitor a-amylase (TMA), but is inactive
against human and bovine a-amylases [5]. Although
detailed structural study of the inhibition mechanism
of AAI on TMA has been reported, little is known
about its knottin-type homologs or their genetic precursors.
Here, we report on the discovery and characterization of a group of linear knottins with characteristics
and a potential for use in drug development comparable to those of CCK peptides. Using a combination of
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proteomic and genomic methods, we identified three
AAI-like a-amylase inhibitors, wrightide-amylaseinhibitors Wr-AI1 to Wr-AI3, from the medicinal
plant Wrightia religiosa (Apocynaceae family). We
showed that they are resistant not only to heat treatment and endopeptidase degradation, but also to exopeptidase. The structure of Wr-AI1 was analyzed in
both solution and crystal form by NMR and X-ray
resolution), respectively.
crystallography (to 1.25-A
Modeling the Wr-AI1–TMA complex with docking
and molecular dynamics suggests that a-amylase inhibition by knottins occurs via an overall shape-fitting
mechanism rather than through a particular set of
polar or ionic interactions in the TMA active site
pocket. We also showed that the precursors of knottin-type a-amylase inhibitors contain a three-domain
structure common to CK peptides. Taken together,
our findings provide new insights into the sequence,
structure and biosynthesis of CK a-amylase inhibitors,
which could be used as stable scaffolds in engineering
human a-amylase inhibitors.
Results
Isolation of a-amylase inhibitors from
W. religiosa
Our preliminary MS profiling of crude extracts of
W. religiosa leaves and flowers revealed strong positive signals in the mass range of 3–5 kDa, indicative
of cysteine-rich peptides (Fig. 1). We therefore performed extraction of the putative cysteine-rich peptides from fresh W. religiosa leaves from Vietnam
and Singapore in 50% ethanol, and purified them
through several rounds of RP-HPLC and strong cation-exchange HPLC. The most abundant peptides
from Vietnam and Singapore leaves were named
wrightide-amylase-inhibitors Wr-AI1 and Wr-AI2,
respectively. Each purified wrightide was fully
reduced by dithiothreitol, and then digested with
trypsin and chymotrypsin. The resulting fragments
were sequenced by tandem MS, and their sequences
were deduced by analyzing b-ions and y-ions (Fig. 2).
By genetic analysis, we also obtained the sequence of
wrightide Wr-AI3, which could not be detected in the
MS profile.
Wrightides Wr-AI1 to Wr-AI3, all 30 residues in
length, contain six cysteines, three glycines, and two
prolines. Together, these three residues account for
> 35% of the sequences. Wrightides share high
sequence homology with each other (93–96%), differing by one or two residues (Fig. 3), and high sequence
homology with AAI (48%).
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Pseudocyclic cystine-knot a-amylase inhibitors
Fig. 1. Tissue-specific and region-specific
expression profiles of wrightides from
flower and leaf of W. religiosa. A 50%
ethanol extract of 1 g of each plant
sample was purified with a C18 solidphase extraction column. The eluate with
80% acetonitrile was profiled with MALDITOF MS to determine the occurrence of
putative CK peptides in different
W. religiosa plant parts from Singapore
and Vietnam.
Solution structure of Wr-AI1 determined by
1
H-NMR
With the distance, dihedral angle and hydrogen bond
restraints derived from 1H-NMR experiments
(Table 1), the solution structures of Wr-AI1 showed
that it adopts a similar CK scaffold as AAI, with the
same three disulfide linkages: CysI–IV, CysII–V, and
CysIII–VI, where CysIII–VI is the penetrating disulfide
bond (Fig. 3A,B). The structure contains three short
b-strands: Tyr7–Cys8, His19–Cys20, and Gly27–Ala30;
His19–Ala30 forms a b-hairpin (Fig. 3C). The
b-strands are connected by four b-turns, two pointing
towards the N-terminal and C-terminal ends on one
side of the molecule, and the other two towards the
opposite side. This compact fold is also stabilized by
an abundance of intramolecular hydrogen bonds
(Fig. 3C), as reported in other cysteine-rich peptides
such as plant defensin PhD1 and cyclotide kalata B5
[8,9]. Moreover, the structure is devoid of N-terminal
or C-terminal tails that would extend away from the
CK core stabilized by three disulfide bonds. Approximately 30% of the amide proton signals remained in
the 1D spectra after 18 h of H/D exchange in D2O.
These amide protons are identified as hydrogen bond
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donors on the basis of the structures. Wr-AI1 contains
two prolines, whose conformations were identified as
cis-Pro17 and trans-Pro23. The proline cis and trans
conformations were confirmed by the observation of
NOE crosspeaks HNAsp16–HaPro17 and HNGln22–
HdPro23, respectively, as the Ha strips of these four residues were not identified from the noise region of H2O
around 4.7 p.p.m.
Structure of Wr-AI1 determined by X-ray
crystallography
Wr-AI1 showed a high propensity to form fiber-like
precipitates at neutral pH. Crystals of Wr-AI1 suitable
for X-ray crystallography were successfully obtained
resafter 1 day of incubation, and diffracted to 1.25-A
olution at a synchrotron beamline. The complete peptide chain comprising 30 residues was unambiguously
traced (Table 2 and Movie S1), clearly confirming the
disulfide connectivity of CysI–IV, CysII–V and CysIII–VI as determined by NMR. In addition, the crystal
structure of Wr-AI1 agreed with its solution structure
ensemble (Fig. 3D), with an average rmsd of 0.93 A
for backbone atoms. Minor differences were observed,
mainly in the loop region and side chain orientations,
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Fig. 2. Tandem MALDI-TOF/TOF MS/MS profiles of two tryptic fragments (m/z 2072 and and m/z 1198) provide the full wrightide Wr-AI1
sequence. Ile/Leu assignment was determined from the genetic sequence and X-ray and NMR structures.
including the disulfide bonds. This could be attributable to the flexibility in solution of the loop region
and side chains. This observation is consistent with the
overlapping of the chemical shifts of HbCys, which created ambiguities in clearly defining the orientation of
disulfide bonds by NMR.
A systematic search for homologous structures
deposited in the Protein Data Bank (PDB) by use of
the DALI server (http://www.ebi.ac.uk/) returned four
homologous structures with a Z-factor > 3.0 (Fig. 3A).
The structure of Wr-AI1 is most similar to that of
AAI (PDB code: 1QFD in its free form, and 1CLV as
a complex with the a-amylase from the yellow mealworm): a superposition of 29 a-carbon atoms returns
with strict conservation observed
an rmsd of 1.10 A,
for the inhibitor core and disulfide bridges. Variations
between the two structures are confined to the two
turns connecting the individual inhibitor strands that
come into contact with the a-amylase upon complex
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formation. The structure of Wr-AI1 also resembles
those of several spider toxins (Fig. 3F): Hainan toxins III and IV (PDB codes: 2JTB and 1RYV, respectively), which are neuronal sodium channel inhibitors
comprising 33 and 35 residues [10], and the GXTX-1E
high-affinity tarantula toxin (PDB code: 2WH9),
which is a potassium channel inhibitor [11].
Further analysis of the crystal structure unambiguously established the two prolins as cis-Pro17 and
trans-Pro23 (Fig. 4). In the structure of Wr-AI1, the
cis peptide bond between Asp16 and Pro17 causes a
local backbone twist (Mobius-like structure similar to
Mobius cyclotides). This energetically unfavorable
twist is partly stabilized by a strong hydrogen bond
between main chain atoms of Cys15 and Tyr18
(Table 3 shows the list of intramolecular hydrogen
bonds). Previous studies showed that the cis conformation occurs at a higher frequency in X–Pro peptides,
where X is an aromatic residue [12]. This high
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Pseudocyclic cystine-knot a-amylase inhibitors
Thus, the interaction between the
phenol ring is 4.9 A.
aromatic side chain and the pyrrolidone ring could
contribute to the stabilization of cis-Pro17 form.
A
Modeled complex between Wr-AI1 and TMA
B
C
D
E
F
Fig. 3. The 3D structure of Wr-AI1. (A) Secondary structure
illustration for the sequence of Wr-AI1. The sequence of Wr-AI1 is
aligned with sequences of: AAI, Hainan toxin III (HT-III), Hainan
toxin IV (HT-IV), and GxTX-1E Guangxiensis toxin 1E (GxTX-1E)
(PDB codes: 1QFD, 2JTB, 1RYV, and 2WH9, respectively). b
stands for b-strand; yellow bridges indicate disulfide bonds; and
the red turn depicts the b-hairpin. (B) Solution structure of Wr-AI1
(PDB code: 2MAU). (C) Illustration of intramolecular hydrogen
bonds. (D) Backbone trace alignment of the crystal structure of WrAI1 (blue) (PDB code: 4BFH), with the 10 solution structure
ensemble (tan). (E) Superposition of the solution structures of WrAI1 and AAI. (F) Structure alignment of Wr-AI1 with its structural
homologs. The figure was prepared with PYMOL.
frequency of occurrence is explained by the interaction
between the aromatic side chain and the proline, which
gives rise to ring-current-induced shifts for the cis conformers but not for the trans conformers in NMR
experiments. Here, we observed parallel stacking
between the phenol ring of Tyr18 and the pyrrolidine
ring of Pro17. Significant shifts from the average
chemical shifts of Hd (2.39 with a reference
average value of 3.63) and Hc (0.71 with a reference
average value of 2.02) were also observed on the basis
of the NMR assignments. The distance between the
centers of the Pro17 pyrrolidine ring and the Tyr18
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The complex between TMA and AAI was previously
characterized by Pereira et al., using X-ray crystallography [13]. In this structure, AAI inserts into a
V-shaped crevice located at the interface of TMA
domains A and B that forms the active site accommodating the carbohydrate residues. A total of 18
residues of AAI are in contact (distance of < 4.0 A)
with 24 residues of TMA. Among them, several residues of the AAI inhibitor occupy or block the
entrance to the six carbohydrate-binding subsites at
the TMA active site cleft. These residues include
Lys4–Arg7, Met12, Tyr27, and Tyr28, all of which
are located at the hydrophilic accessible surface of
the inhibitor molecule. In particular, Arg7 forms a
salt bridge with the catalytic residue Asp287 from
TMA. This residue is also involved in a water-mediated hydrogen-bonding network with two other catalytic residues, Glu222 and Asp185, from TMA. To
better understand how Wr-AI1 can inhibit the amylase activity of TMA, we built an atomic model for
their interaction by using the AAI–TMA complex as
a template (Fig. 5) and assuming an overall conservation of the molecular orientation of the inhibitors in
the TMA active site pocket. With the exception of
Gly27 (Wr-AI1 numbering), no residue located at the
interface with the enzyme is strictly conserved
between Wr-AI1 and AAI. Upon complex formation,
2, which is comparathe buried surface area is 1831 A
2) [13].
ble to that of the AAI–TMA complex (2085 A
The network of interactions that stabilizes the WrAI1 complex is detailed in Table 3 and Fig. 6
(Movie S2). Lys4, Glu6, Tyr7 and Thr21 from WrAI1 form hydrogen bonds with several negatively
charged residues from TMA (Fig. 6). This set of
hydrogen bonds are preserved in > 95% of all
configurations sampled along the last 250 ns of the
molecular dynamics simulation. The total DG is
40.56 1.07 kcalmol1 for this complex. An
analysis of the various components contributing to
molecular complex stabilization gives the following
values: van der Waals, –88.88 0.93 kcalmol1;
electrostatics, 138.44 4.47 kcalmol1; and nonpolar
solvation, –77.49 3.79 kcalmol1. This analysis
suggests that the enthalpic contribution of the association between TMA and Wr-AI1 is mainly driven by
van der Waals interactions, with a smaller contribution of electrostatic interactions.
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A
C
D
B
Heat and proteolytic stability
To determine whether wrightides would resist degradation by boiling or protease treatment, which are
important for administering decoctions in traditional
medicines, Wr-AI1 was heated at 100 °C for 1 h or
incubated with chymotrypsin or carboxypeptidase A
for 4 h. More than 95% of Wr-AI1 remaining intact
was observed at the same retention time in the ultraperformance liquid chromatography (UPLC) profiles
after heat treatment (Fig. 7A). The MS profiles of corresponding peaks showed that both peaks contained
native Wr-AI1 (m/z 3246) with a small amount of
degraded products.
To confirm that the CK structure is important for
its proteolytic stability, Wr-AI1 was fully reduced by
dithiothreitol, and served as the control in a chymotrypsin stability assay. A nine-residue linear peptide
was used as the control in a carboxypeptidase A assay.
The control peptides were almost completely hydrolyzed after 4 h of incubation with chymotrypsin or 1 h
of treatment with carboxypeptidase A at 37 °C. Under
similar conditions, the native peptide Wr-AI1 was
resistant to protease degradation, with > 95% of peptides remaining intact (Fig. 7B,C). Our results provide
strong evidence for the stability of wrightides against
thermal, endopeptidase and exopeptidase treatments.
a-Amylase inhibitory activity
We performed inhibition assays with TMA and a-amylases from human saliva, porcine pancreas, and fungus
(Aspergillus oryzae), by using the Bernfeld method
[14]. Preliminary results showed that both Wr-AI1 and
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Fig. 4. Ribbon diagram and structural
features of Wr-AI1. (A and B) Stereoview
of the electron density (2Fobs Fcalc) and
schematic presentation of the backbone
twist caused by cis Pro17. The electron
density was contoured at 1.0 r. (C and D)
Pro17 and Pro23 adopt the cis and trans
conformations, respectively. The three
beta strands are shown in blue and
disulfide bonds highlighted in yellow. The
figure was prepared with the program
PYMOL.
Wr-AI2 had inhibitory activities against TMA in a
dose-dependent manner, with IC50 values of 1.9 and
2.3 lM, respectively (Fig. 7D). Like AAI, Wr-AI1 and
Wr-AI2 did not inhibit a-amylases from fungus or
mammals at concentrations up to 100 lM.
Biological activity of amylase inhibitors
The cytotoxic, hemolytic and antibacterial activities of
Wr-AI1 and Wr-AI2 were tested. In our experiments,
wrightides did not show appreciable toxic, hemolytic
or antibacterial activity at concentrations up to
100 lM.
Cloning of wrightide-encoding genes
Using 30 -RACE and 50 -RACE PCRs, we obtained the
Wr-AI2 full-length gene from an RNA extract from a
Singapore plant. Subsequently, we used two primers
derived from the 50 -UTR and 30 -UTR of a Wr-AI2
clone, and successfully amplified DNA sequences of
Wr-AI1, Wr-AI2, and a novel wrightide, Wr-AI3,
which was not found at the protein level.
Figure 8 shows the deduced 87-residue precursors of
Wr-AI1 to Wr-AI3 and their alignment with previously characterized CK trypsin inhibitor and x-conotoxin precursors. In general, wrightide precursors
contain a 21-residue endoplasmic reticulum (ER) signal sequence followed by a 36-residue prodomain and
a 30-residue wrightide domain at the C-terminus.
Comparison between RACE and DNA PCRs showed
that wrightide genes contain a phase 1 intron in the
middle of the ER signal. The signal sequence and
prodomain of wrightide precursors are almost
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Pseudocyclic cystine-knot a-amylase inhibitors
A
B
C
D
E
F
Fig. 5. Cartoon and surface view of superimposition of AAI and Wr-AI1 in complex with TMA. (A) Model of the complex of AAI (magenta)
and Wr-AI1 (cyan) with TMA (gray) derived from the AAI–TMA complex (PDB code: 1CLV). (B, C) Zoom-view of the binding region between
AAI (B)/Wr-AI1 (C) and TMA. Residues of AAI/Wr-AI1 with atoms within 6 A of TMA are colored in magenta/cyan, and residues of TMA
with atoms within 6 A of AAI/Wr-AI1 are colored in blue/red. Residues that are > 6 A away from TMA in both AAI and Wr-AI1 are colored
orange. (D) Superimposition of AAI and Wr-AI1 at the active site of TMA. The three CysI–IV, CysII–V and CysIII–VI disulfide bonds arranged
in a CK motif are highlighted in yellow. (E, F) Close-ups of AAI (E) and Wr-AI1 (F) residues at the TMA active site. The figure was prepared
with PYMOL.
identical, except for a three-residue difference in the
prodomain and several silent mutations at the gene
level, as highlighted in Fig. 8A.
Discussion
In this study, we used proteomic, genomic and structural methods to characterize the 30-residue knottintype a-amylase inhibitors Wr-AI1 to Wr-AI3 from
W. religiosa of the Apocynaceae family. Since the discovery of AAI in 1994, the 32-residue AAI has
remained the only representative of the knottin group
that shows a-amylase inhibitory activity [5]. The discovery of wrightides thus extends the list of the family
of knottin a-amylase inhibitors. With two fewer residues than AAI, the wrightide family represents the
smallest proteinaceous a-amylase inhibitors reported.
Interestingly, these wrightides are resistant to both
heat denaturation and proteolytic degradation, including exopeptidase treatment. Thus, wrightides have the
favorable stability features of cyclic CK peptides such
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as cyclotides. In wrightides, the N-terminus and C-terminus are protected by disulfide bonds at the ultimate
or penultimate residues. Our structural analysis
showed that this arrangement enables the termini to
loop back to the peptide chain via disulfide bonds like
‘pseudocyclics’, particularly at the N-terminus of
wrightides. These pseudocylic CK (PCK) peptides,
with or without one extra residue flanking the disulfide-looping terminus, would probably escape degradation by exopeptidases.
The backbone-twisting cis-proline in PCK
inhibitors
The presence of cis-proline in naturally occurring cysteine-rich peptides generally causes a twist in the peptide backbone. This was used as a benchmark to
classify cyclotides into M€
obius (with cis-proline) and
the bracelet (without cis-proline) subfamilies [4]. In this
study, we found that the PCK wrightide Wr-AI1 also
contains one backbone-twisting cis-proline. Also, in
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Pseudocyclic cystine-knot a-amylase inhibitors
H189
D225
D185
E6
study of local interactions that stabilize the cis-prolines
in Wr-AI1 and other PCK a-amylase inhibitors may
reveal diverse mechanisms of cis-proline formation in
cis-proline-rich peptides.
V151
Y11
Y7
K4
D287
T21
N331
Fig. 6. Stable hydrogen bond interactions between Wr-AI1
(residues shown as sticks in cyan) and TMA (residues shown as
green sticks) following molecular dynamics simulation (see text).
our unreported work, we found PCK peptides in three
other Apocynaceae plants, each of which contained
three or four prolines, and in four determined by
NMR spectroscopy to have one or two cis-prolines.
Together, these results suggested that the occurrence
of cis-proline bond could be underestimated in proline-rich cysteine-rich peptides.
Surveys of protein databases revealed approximately
35% and 6–8% cis-proline in small polypeptides and
native proteins, respectively [15]. The percentage of
cis-proline amide bonds increases to as high as 12–
16% when proline is preceded by an aromatic residue
in protein primary sequences. The steric repulsion
between the pyrrolidine rings of a proline and the two
neighboring Ca atoms generally renders the cis configuration energetically less favorable than the trans configuration. In peptides with cis-proline preceded by an
aromatic residue, clustering of the aromatic side chain
and the pyrrolidine ring provides stability to the sterically constrained cis-proline, which is manifested in
part by the selective ring-current-induced shifts of proline Ha and Hb in NMR spectroscopy [12]. Our analysis of the Wr-AI1 structure demonstrated the
occurrence of one cis-proline in X–Pro amide bonds,
where X is a nonaromatic residue. The backbone twist
caused by this cis-proline is probably stabilized by the
hydrogen bond between the neighboring residues
Cys15 and Tyr18 rather than by direct stacking of the
preceding aromatic side chain and proline. Thus, the
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Shape-fitting inhibition mechanism between PCK
a-amylase inhibitors and TMA
A model of the interaction between Wr-AI1 and TMA
was constructed on the assumption of overall conservation of molecular orientation in the TMA active site
pocket as compared with AAI. Interestingly, despite
the lack of sequence conservation between both peptide inhibitors, several side chains that project from
the surfaces of the two inhibitors are placed in similar
positions in the active site crevice of TMA, and small
movements would allow them to make equivalent contacts with the enzyme. Molecular dynamics study of
the modeled complex suggests that Wr-AI1 binds to
the TMA active site depression via an interaction network composed largely of nonpolar interactions and
completely lacking the critical salt bridge observed for
AAI (Table 3). A crystal structure for the TMA–WrAI1 complex is needed to confirm this hypothesis.
Wrightides follow the biosynthesis pathway for
secretory proteins
Our genetic analysis showed that wrightide precursors
consist of an ER signal domain with a phase 1 intron,
a prodomain, and a single wrightide domain at the
C-terminus. The gene organization, starting with a
signal peptide, provides hints on the biosynthesis
pathway of wrightides, which are gene-encoded and
ER-targeted following the conventional pathway for
secretory proteins (Fig. 8B), as suggested for many
cysteine-rich peptides [16]. The signal peptide is generally removed by SPase I from the precursor to release
the propeptide. A single cleavage between the 36-residue prodomain and the 30-residue functional domain
subsequently produces the native wrightide. These
characteristics distinguish wrightides as ribosomally
synthesized peptides from smaller peptides of 5–12
residues that are synthesized by nonribosomal multienzyme complexes [17,18].
The three-domain precursor structure is commonly
found in many CK peptides, both cyclic and linear.
Examples of such cyclic plant CK peptide precursors
include cyclotides from the Rubiaceae, Violaceae and
Solanaceae families [17,19], and squash trypsin inhibitors from Momordica cochinchinensis [20]; selected
examples of linear plant CK peptide precursors are
acyclic cyclotides from the Violaceae, Rubiaceae and
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A
B
C
D
Fig. 7. Stabilities and a-amylase inhibitory activity of wrightides. (A) Thermal stability of Wr-AI1 and Wr-AI2. The minor peak after heat
treatment contained mainly Wr-AI1/Wr-AI2, with a small amount of degraded products, as determined from the MS profile. (B)
Chymotrypsin stability of wrightide Wr-AI1. (C) Carboxypeptidase A stability of Wr-AI1. (D) Inhibition of T. molitor a-amylase by wrightides.
Peptides were preincubated with TMA for 20 min at 37 °C. Hydrolysis was started by addition of 1% starch. The reaction was allowed to
proceed for 5 min, and stopped by addition of a color reagent containing 3,5-dinitrosalicylic acid. The IC50 values are 1.9 and 2.3 lM for WrAI1 and Wr-AI2, respectively. The error bars show standard deviations.
Poaceae families [21–23], and towel gourd trypsin
inhibitors [24]. This precursor organization is also used
by animals such as cone snails to produce ion channel
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
blockers: x-conotoxins [25] and d-conotoxins [26]. It
should be noted that, within the plant kingdom,
diverse structures are used to organize CK peptide
4359
P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
Fig. 8. Precursor structure of Wr-AI
wrightides. (A) Alignment of wrightide
precursors with precursors of other CK
peptides, including x-conotoxin SO-3 and
towel gourd trypsin inhibitor TGTI-II. The
ER signal peptide was assigned to
x-conotoxin SO-3 by SIGNALP 3.0, whereas
this domain was not recognized by
SIGNALP 3.0 for TGTI-II. (B) Secretory
protein synthesis pathway for wrightides.
precursors. One example is provided by precursors
with multiple repeats of cyclic or linear CK peptides,
such as those reported for cyclotides from the Rubiaceae and Violaceae families [17,19], and TIPTOP
squash trypsin inhibitors [20]. Other examples are chimeric precursors of both CK peptides and other types
of protein, such as cliotide precursors of both cyclotides and legume albumin PA1a in Clitoria ternatea
[27,28]. Given such a diversity of precursor organization even within a CK peptide family, understanding
the genetic sequences of each CK peptide family, here
PCK a-amylase inhibitors, is thus beneficial for their
applications in crop protection, and also provides
insights into their biosynthesis.
Knottin-type a-amylase inhibitors with
applications in engineering peptidyl bioactives
The CK structure has been employed in nature as a
scaffold for a variety of unrelated protein families
found in microorganisms, animals, and plants. In particular, a-amylase inhibitors adopting CK folds are
small, extraordinarily stable against heat and endopeptidase and exopeptidase degradation, and highly tolerant to sequence variation [29]. Thus, small CK
peptides such as wrightides with molecular masses of
3–5 kDa possess appealing features as potential peptide therapeutics [30,31]. First, the small size makes
wrightides more amenable to chemical synthesis
[32,33]. Second, the CK peptides in general are highly
tolerant to sequence variations and the spacing of the
half-cystines, allowing a-amylase inhibitors to potentially serve as scaffolds for protein engineering to
attain new functions, such as in the successful grafting
of the bradykinin antagonist peptides DALK or DAK
onto the cyclotide kalata B1 scaffold [34].
4360
A potential application of considerable interest in
drug development is the engineering of wrightides to
be orally active mammalian a-amylase inhibitors for
the treatment of obesity and type 2 diabetes mellitus.
The literature shows that extended hydrophobic interactions could be important for AAI inhibition of
mammalian a-amylases [13]. Human salivary a-amylase (HSA) (PDB code: 3DHP) and TMA share high
sequence homology (65%) and structural homology
(Z-score of 57.1 with 468 equivalent residues at an
by the DALI server). Superimposing the
rmsd of 1 A,
HSA–Wr-AI1 complex on the TMA–Wr-AI1 complex
reveals four additional loops present in HSA at the
interface of the active site, including loops Asn53–
Phe55, Asn137–Gly146, Gly304–Ala310, and Trp344–
Val358. The conformational flexibility of these loops
might be responsible for the low-affinity binding of
Wr-AI1 to HSA. Our docking experiments suggested
that it is possible that careful incorporation of aromatic and positively charged residues into wrightide
templates could improve their contact with the negatively charged enzyme active sites, to make wrightides
active against mammalian a-amylases. In this regard,
our work showing the interaction of Wr-AI1 and
TMA provides new insights for a structure-guided
approach to designing potentially useful orally active
a-amylase inhibitors for managing type 2 diabetes
mellitus and obesity.
Experimental procedures
Isolation of a-amylase inhibitors
W. religiosa leaves (800 g) were homogenized and extracted
twice in 50% (v/v) ethanol. After centrifugation
(8500 g, 10 min), the supernatant was partitioned with
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
P. Q. T. Nguyen et al.
dichloromethane. The aqueous upper layer was concentrated,
filtered, and loaded onto a C18 flash column (Grace Vydac,
Hesperia, CA, USA). Elution was performed with increasing
concentrations of ethanol. The presence of cysteine-rich peptides in all fractions was monitored by MALDI-TOF MS. To
purify individual peptides, several dimensions of strong cation
exchange and RP-HPLC were employed.
De novo sequencing with MALDI-TOF MS/MS
Approximately 40 lg of each purified peptide was dissolved
in 50 mM ammonium bicarbonate buffer (pH 7.8) containing 50 mM dithiothreitol, and incubated at 37 °C for 2 h.
Digestion with endoproteinase Glu-C, trypsin or chymotrypsin was carried out at room temperature for 5 min, and
this was followed by MALDI-TOF MS/MS sequencing, as
previously described [27]. Isobaric residues were assigned
on the basis of gene sequences for Wr-AI1 and Wr-AI2,
and confirmed on the basis of the X-ray or NMR structure
for Wr-AI1. Wr-AI3 was sequenced only at the gene level.
Solution structure determination with NMR
spectroscopy
The NMR sample was prepared by dissolving lyophilized
Wr-AI1 in 95% H2O/5% D2O or 99.9% D2O directly
(~ 1 mM protein and pH/pD 3.3). All NMR experiments
were carried out on a Bruker 600-MHz NMR spectrometer
equipped with a cryogenic probe. Two-dimensional (2D)
TOCSY and NOESY experiments were performed with mixing times of 80 and 200 ms, respectively [35]. The 2D data
were acquired at 298 K. Water suppression was achieved
with modified WATERGATE pulse sequences [36]. The
NMR spectra were processed with NMRPIPE [37]. The amides
involved in hydrogen bonding were identified by hydrogen–
deuterium exchange (1D 1H) experiments [38].
Sequence-specific assignments were achieved with 2D
TOCSY and NOESY, and NOEs were assigned from 2D
NMRSPY
(http://yangdw.sci
NOESY
results
with
ence.nus.edu.sg/Software&Scripts/NMRspy/index.htm). The
chemical shifts are deposited in BioMagResBank (accession
number: 18983). Distance restraints were derived from the
peak intensities of the assigned NOEs. Dihedral angles (φ)
were obtained from 3JHN-Ha coupling constants measured
from the 1D 1H-spectrum. Hydrogen bond restraints were
incorporated on the basis of the observation of amide protons in the 1D 1H-spectra recorded after resuspension of the
lyophilized Wr-AI1 in D2O for up to 18 h at 25 °C.
Structure was calculated with a simulated annealing
approach with CYANA 2.0 [39]. Distance restraints are
(strong NOEs),
divided into three classes: 1.8 < d < 3.4 A
1.8 < d < 4.2 A (medium NOEs), and 1.8 < d < 5.5 A
(weak NOEs). Disulfide bond restraints of 2.0 < d
3.0 < d(Cbi, Scj) < 3.1 A
and 3.0 < d
(Sci, Scj) < 2.1 A,
c
b
were employed for structure calculation.
(S i, C j) < 3.1 A
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
Pseudocyclic cystine-knot a-amylase inhibitors
During the structure calculation, hydrogen bond restraints
for the NH–O distance and 2.2–3.2 A
for the
of 1.8–2.2 A
N–O distance were applied on nine identified hydrogen
bonds according to the slowly exchanging amide protons.
Φ angles were constrained to the range of –150° to –90° for
3
JHN-Ha > 8 Hz. Structures were displayed and analyzed
with PYMOL and PROCHECK-NMR, respectively [40]. The
experimental and structural statistics are summarized in
Table 1.
Crystal structure determination
With the sitting drop vapor diffusion method, the native
crystals were obtained from the mixture of 1 lL of Wr-AI1
solution (4.8 mgmL1) and 1 lL of precipitant solution
(3.6 M sodium formate, 10% glycerol) after 1 day of incubation at 16 °C. The crystals were stabilized in the precipitant solution supplemented with 40% (v/v) glycerol, and
flash-frozen in liquid nitrogen. Diffraction intensities to
resolution were collected at 100 K at the Swiss
1.25-A
Light Source Beamline PXIII with a Pilatus 6M detector
(Dectris, Baden, Switzerland). Integration, scaling and
merging of intensities were carried out with XDS [41] and
SCALA [42] from the CCP4 suite [43]. Data collection statistics
are summarized in Table 2.
The structure was determined by molecular replacement
with PHASER [44]. The search probe was the structure of
AAI (PDB code: 1CLV [13]). ARP-WARP [45] was used for
chain tracing and map improvement, and the resulting
Table 1. NMR experimental and structural statistics of Wr-AI1.
NOE constraints
Intraresidue (|i j| = 0)
Sequential (|i j| = 1)
Medium-range (1 < |i j| < 5)
Long-range (|i j| ≥ 5)
Dihedral angle restraints
Hydrogen bonds
PROCHECK-NMR Ramachandran plot (%)
Most favored region
Additionally allowed region
Generously allowed region
Disallowed region
Average maximum violations per structure
Distance (
A)
Van der waals (
A)
Torsion angles (°)
CYANA target function value (
A2)
Average rmsd to mean structure (
A)
All backbone atoms (1–30)
All heavy atoms (1–30)
681
332
193
36
120
10
7
70.9
28.7
0.4
0
0.02 0.002
3.7 0.4
0.25 0.11
1.35 0.15
0.38 0.08
0.85 0.12
model was corrected manually (Table 2). The Ramachandran plot calculated with PROCHECK [46] revealed that 87%
of the residues were in the most favored region and 13%
were in the additional allowed region.
4361
P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
Table 2. Crystallography data collection and refinement statistics
of Wr-AI1.
Data collection
Space group
Cell parametersa
a, b, c (
A)
a, b, c (°)
Resolution range (
A)
Observed reflections
Unique reflections
Completeness (%)
Multiplicity
Rmergeb
Mean I/r(I)
Refinement
Resolution range (
A)
Number of reflections
used for refinement
Number of reflections used
for Rfree calculation (5%)
Rfactor (%)c, Rfree (%)d
Number of nonhydrogen atoms
Number of water molecules
Mean B-factor (
A2)
Protein whole chain
Water
rmsd from ideality
Bond lengths (
A)
Bond angles (°)
Ramachandran plot (%)
Most favored regions
Additional allowed regions
G-factore
P212121
a = 16.19, b = 29.13, c = 47.70
a = b = c = 90
29.1–1.25 (1.28–1.25)
21 115 (2864)
6362 (856)
95.4 (90.3)
3.3 (3.3)
0.029 (0.071)
23.2 (11.9)
24.88–1.25 (1.28–1.25)
6017 (410)
317 (21)
15.8, 15.9
223
41
5.4
32.9
0.020
1.85
87
13
0.07
a
The numbers in parentheses refer to the last (highest) resolution
shell. b Rmerge = ΣhΣi|Ihi <Ih>|/Σh,I Ihi, where Ihi is the ith observation of the reflection h, and <Ih> is its mean intensity.
c
Rfactor = Σh||Fobs(h)| |Fcalc(h)||/Σ|Fobs(h)|. d Rfree was calculated
with 5% of reflections excluded from the whole refinement procedure. e G-factor is the overall measure of structure quality from
procheck.
Before the dynamic simulations, the solvated system was
relieved of any unfavorable interactions by subjecting it
to 100 steps of energy minimization. Harmonic restraints
during the equilibration were placed on Ca atoms with
2 to the energy-minimized coordinates. The
1 kcalmol1 A
system was heated to 300 K in steps of 100 K, and this
was followed by gradual removal of the positional
restraints and a 10-ns unrestrained equilibration at
300 K. Analysis of the resulting trajectories revealed that
the simulated complex reached stability after 50 ns, with
The first 10 ns of simulation were
an RMSD of < 1.8 A.
performed in NPT, and the production run of 500 ns was
performed in NVT. The simulation temperature of 300 K
was set with Langevin dynamics, with a collision frequency of 0.1 ps1. The pressure was maintained at
1 atm by the use of weak coupling with a pressure relaxation time of 1 ps. During the simulation, all long-range
electrostatic interactions were treated with particle mesh
Ewald methods [49], with a real space cut-off distance of
Bonds involving hydrogen atoms were constrained
9 A.
with the M-SHAKE algorithm [50]. A time step of 4 fs with
hydrogen mass repartitioning was used, and coordinates
were saved every 100 ps. Hydrogen bond analysis was
performed with the PTRAJ module in AMBER for the last
250 ns of the stabilized trajectory, with a cut-off distance
Binding energy analysis based on molecular
of 3.5 A.
mechanics/generalized Born surface area [51] was performed on the simulated trajectory to calculate the free
energy of binding of Wr-AI1 to TMA. For binding
energy calculation, a total of 100 structures were
extracted at regular intervals from the last 250 ns of the
trajectory. A salt concentration of 150 mM and a Born
implicit solvent model of 2 (igb = 2) [52] was used. The
binding surface area was calculated with NACCESS [53].
Simulation trajectories were visualized in VMD [54], and
figures were generated with PYMOL.
Heat stability test
Molecular docking and molecular dynamics study
of the Wr-AI1–TMA complex
To investigate the stability of the TMA–Wr-AI1 complex
obtained from docking (which was initially obtained by
simply superimposing Wr-AI1 onto AAI in the TMA–
AAI crystal structure), we performed three molecular
dynamics simulation of 500 ns each, using ACEMD [47]
and all-atom ff12SB forcefield parameters. Hydrogen
atoms were added to this initial complex with the XLEAP
module of AMBER [48]. The system was solvated with
TIP3P water molecules to form a box with at least 10 A
separating the solute atoms and the edge of the box. A
total of 92 sodium ions and 70 chloride ions, corresponding to a salt concentration of 150 mM, were added to the
system by replacing water molecules random positions.
4362
Purified Wr-AI1 was heated in boiling water for 1 h and
then subjected to UPLC. Wr-AI1 without heat treatment
was used as a control. Peaks collected from UPLC were
monitored by MALDI-TOF MS.
Proteolytic stability test
Purified Wr-AI1 was incubated with or without chymotrypsin (at a final peptide/enzyme ratio of 10 : 1 mol/mol) in
20 mM ammonium bicarbonate (pH 7.8) at 37 °C for 4 h.
Purified Wr-AI1 that had been completely reduced with
50 mM dithiothreitol (2 h, 37 °C) was treated in the same
way, and used as a control. Treated samples or controls
were subjected to UPLC, and the collected peaks were
monitored by MALDI-TOF MS.
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
Table 3. Potential intramolecular hydrogen bonds in Wr-AI1 and intermolecular interactions between PCK inhibitors and TMA (distance of
< 3.4 A). Intramolecular distances were determined by X-ray crystallography. Intermolecular distances between AAI and TMA were derived
from the crystal complex of AAI and TMA (PDB code: 1CLV), and the distances between Wr-AI1 and TMA were calculated from the
molecular dynamics simuation of the TMA–Wr-AI1 complex for the last 250 ns of the trajectory.
Wr-AI1
Wr-AI1
Residue/atom
Residue/atom
Distance (
A)
Residue/atom
Residue/atom
Distance (
A)
A2 N
A2 O
Q3 N
Q3 O
Q3 O
K4 NZ
G5 N
E6 O
C8 N
C8 O
S9 N
S9 O
S9 O
S9 O
S9 OG
Q13 O
C15 N
E6 OE1
G5 N
E6 N
A30 OXT
C29 O
C29 N
G27 O
G27 N
L12 O
Y11 N
L12 N
L12 O
Y11 N
2.91
2.84
2.87
3.30
3.00
2.88
2.76
2.93
2.99
3.01
3.02
3.15
3.32
3.03
3.06
S9 OG
V10 N
C15 O
C15 O
P17 O
H19 N
H19 O
C20 O
T21 N
T21 OG1
Q22 O
Q22 O
Q22 OE1
Q22 OE1
L12 N
P23 O
Y18 N
P17 N
H19 NE2
A30 O
A30 N
Q22 N
I28 O
I28 N
G26 N
G27 N
V24 N
I25 N
3.06
2.97
2.80
3.31
3.21
2.91
3.01
3.35
2.92
3.01
2.79
3.00
2.94
3.01
AAI
Residue/atom
Distance (
A)
TMA
Residue/atom
Distance (
A)
Wr-AI1
Residue/atom
Occupancy (%)
C1 N
K4 NZ
K4 NZ
N6 OD1
D13 O
T24 O
2.80
2.73
3.27
2.81
2.93
3.18
N137 O
Q295 OE1
Q295 NE1
K188 NZ
E135 OE1
N331 ND2
D332 OD2
D332 OD1
D287 O
R290 O
G292 N
V151 O
D185 OD2
H189 NE2
D225 O
D225 OD1
D287 OD2
S25 OG
N30 ND2
N30 ND2
S32 OG
2.76
3.19
3.06
3.09
2.75
2.80
2.75
2.81
2.81
2.64
Y11 OH
Y7 OH
E6 OE1
K4 NZ
K4 NZ
T21 OG1
49.89
99.09
97.04
99.07
48.70
98.44
In the carboxypeptidase A stability assay, Wr-AI1 was
incubated with or without enzyme (at a final peptide/enzyme
ratio of 40 : 1 mol/mol) in 50 mM NaCl/Tris and 1 M NaCl
(pH 7.5) at room temperature for up to 24 h. A linear nineresidue peptide was used as a control. Degradation products
were monitored by UPLC and MALDI-TOF MS.
Assay for a-amylase activity
a-Amylase was isolated from lavvae of the yellow mealworm,
T. molitor, with the procedure described previously [55].
Assays for a-amylase were carried out in 96-well plates with
the Bernfeld method [14]. TMA with or without treatment
with peptides (20 min, 37 °C) was incubated with 1% starch
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
(in 20 mM sodium phosphate buffer, pH 6.7) for 5 min.
Color reagent (3,5-dinitrosalicylic acid and sodium
potassium tartrate; Sigma, St. Louis, MO, USA) [56] was dispensed into each well, and color was allowed to develop for
20 min at 100 °C. Absorbance at 540 nm was read to determine the a-amylase activity. Similar inhibition experiments
were performed for human salivary, porcine pancreatic and
A. oryzae a-amylases (Sigma).
Hemolysis assay
Fresh type AB blood was donated by a healthy volunteer.
The hemolysis assay was performed as described elsewhere
[27].
4363
P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
Cytotoxicity assay
Author contributions
The cytotoxicity of the purified wrightides was tested with
PrestoBlue Cell Viability Reagent (Invitrogen, Carlsbad,
CA, USA). African green monkey kidney (Vero) cells
seeded onto 96-well plates were incubated with Wr-AI1 and
Wr-AI2 at 1–100 lM for 24 h at 37 °C. After incubation,
the PrestoBlue reagent (Invitrogen, Carlsbad, CA, USA)
was dispensed into the wells and left at 37 °C for 2 h. The
fluorescence was subsequently read as instructed by the
manufacturer. Triton X-100 solution (1%) was used as a
positive control.
P. Q. T. Nguyen performed or was involved in all of
the experiments except for NMR experiments.
S. Wang performed NMR spectroscopy experiments
and sequence calculation. A. Kumar analyzed molecular dynamics results and enzyme alignment. L. J. Yap
performed X-ray crystallization experiments. T. T.
Luu contributed to peptide and enzyme extraction. J.
Lescar analyzed X-ray crystallography data, built the
structure, and modeled the complex. J. P. Tam analyzed the data. P. Q. T. Nguyen, J. Lescar and J. P.
Tam contributed mainly to manuscript preparation.
All of the authors discussed the results and contributed to the writing of the manuscript.
Antibacterial assay
The antibacterial activity of wrightides was assessed with a
radial diffusion assay, as described previously [57], on
Gram-negative Escherichia coli (FDA strain Seattle 1946)
and Gram-positive Staphylococcus aureus. D4R, an inhouse peptide dendrimer with potent antibacterial activity,
was used as a positive control. The experiments were performed in duplicate.
Cloning of a-amylase inhibitor genes
Total RNA extraction was performed with the PureLink
Mini RNA purification kit (Invitrogen, Carlsbad, CA, USA),
with addition of 3% 2-mercaptoethanol and 4% polyvinylpyrrolidone to the lysis buffer. A total RNA extract of Singapore W. religiosa leaves was subsequently converted to
30 -RACE and 50 -RACE cDNA libraries with the 30 -RACE
System for Rapid Amplification of cDNA Ends (Invitrogen)
and the SMARTer RACE cDNA Amplification Kit (Clontech, Takara Biotechnology, Dalian, China), respectively.
30 -RACE PCR products obtained with the degenerate primer
targeting the sequence CAQKGE (50 -TGTGCTCArAArGGnGA-30 ) were gel-purified, cloned into pGEM-T
Easy Vector (Promega Madison, WI, USA), and sequenced.
A reverse primer based on the newly obtained partial
sequence was designed to reveal the remaining encoding gene
in 50 -RACE PCR. To determine the DNA sequences of
wrightide genes, we performed PCR on the W. religiosa
DNA extract with two primers: Wr2speF (50 -TAGGCGCAAACAACATGGCTAAGC-30 ) and Wr2speR (50 The
CCACATAGCTCG-TAGAACAAGCTTACAG-30 ).
ER signal peptides were predicted with SIGNALP 3.0 (http://
www.cbs.dtu.dk/services/-SignalP-3.0/).
Acknowledgements
We thank P. Q. T. Nguyen, C. H. Teo and Y. S. Lam
for technical assistance with this project. This research
was supported in part by the Competitive Research
Grant from the National Research Foundation in Singapore (NRF-CRP8-2011-05).
4364
References
1 Cheek S, Krishna SS & Grishin NV (2006) Structural
classification of small, disulfide-rich protein domains.
J Mol Biol 359, 215–237.
2 Pallaghy PK, Nielsen KJ, Craik DJ & Norton RS
(1994) A common structural motif incorporating a
cystine knot and a triple-stranded beta-sheet in toxic
and inhibitory polypeptides. Protein Sci 3, 1833–1839.
3 Le Nguyen D, Heitz A, Chiche L, Castro B, Boigegrain
RA, Favel A & Coletti-Previero MA (1990) Molecular
recognition between serine proteases and new bioactive
microproteins with a knotted structure. Biochimie 72,
431–435.
4 Craik DJ, Daly NL, Bond T & Waine C (1999) Plant
cyclotides: a unique family of cyclic and knotted
proteins that defines the cyclic cystine knot structural
motif. J Mol Biol 294, 1327–1336.
5 Chagolla-Lopez A, Blanco-Labran A & Patthy A (1994)
A novel a-amylase inhibitor from amaranth (Amaranthus
hypocondriacus) seeds. J Biol Chem 269, 23675–23680.
6 Le Berre-Anton V, Bompard-Gilles C, Payan F &
Rouge P (1997) Characterization and functional
properties of the alpha-amylase inhibitor (alpha-AI)
from kidney bean (Phaseolus vulgaris) seeds. Biochim
Biophys Acta 14, 31–40.
7 Svensson B, Fukuda K, Nielsen PK & Bønsager BC
(2004) Proteinaceous alpha-amylase inhibitors. Biochim
Biophys Acta 1696, 145–156.
8 Plan MR, Rosengren KJ, Sando L, Daly NL & Craik
DJ (2010) Structural and biochemical characteristics of
the cyclotide kalata B5 from Oldenlandia affinis. Pept
Sci 94, 647–658.
9 Janssen BJC, Schirra HJ, Lay FT, Anderson MA &
Craik DJ (2003) Structure of Petunia hybrida
defensin 1, a novel plant defensin with five disulfide
bonds. Biochemistry 42, 8214–8222.
10 Li D, Xiao Y, Xu X, Xiong X, Lu S, Liu Z, Zhu Q,
Wang M, Gu X & Liang S (2004) Structure–activity
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
P. Q. T. Nguyen et al.
11
12
13
14
15
16
17
18
19
20
21
22
relationships of hainantoxin-iv and structure
determination of active and inactive sodium channel
blockers. J Biol Chem 279, 37734–37740.
Lee S, Milescu M, Jung HH, Lee JY, Bae CH, Lee
CW, Kim HH, Swartz KJ & Kim JI (2010) Solution
structure of GxTX-1E, a high-affinity tarantula toxin
interacting with voltage sensors in Kv2.1 potassium
channels. Biochemistry 49, 5134–5142.
Wu WJ & Raleigh DP (1998) Local control of peptide
conformation: stabilization of cis proline peptide bonds by
aromatic proline interactions. Biopolymers 45, 381–394.
Pereira PJ, Lozanov V, Patthy A, Huber R, Bode W,
Pongor S & Strobl S (1999) Specific inhibition of insect
alpha-amylases: yellow meal worm alpha-amylase in
complex with the amaranth alpha-amylase inhibitor at
2.0 A resolution. Structure 7, 1079–1088.
Bernfeld P (1955) Amylases a and b. Methods Enzymol
1, 149–158.
Milner-White EJ, Bell LH & Maccallum PH (1992)
Pyrrolidine ring puckering in cis and trans-proline
residues in proteins and polypeptides. Different puckers
are favoured in certain situations. J Mol Biol 228, 725–
734.
Mergaert P, Nikovics K, Kelemen Z, Maunoury N,
Vaubert D, Kondorosi A & Kondorosi E (2003) A
novel family in Medicago truncatula consisting of more
than 300 nodule-specific genes coding for small,
secreted polypeptides with conserved cysteine motifs.
Plant Physiol 132, 161–173.
Jennings C, West JL, Waine C, Craik DJ & Anderson
MZ (2001) Biosynthesis and insecticidal properties of
plant cyclotides: the cyclic knotted proteins from
Oldenlandia affinis. Proc Natl Acad Sci USA 98,
10614–10619.
Marahiel MA (2009) Working outside the proteinsynthesis rules: insights into non-ribosomal peptide
synthesis. J Pept Sci 15, 799–807.
Dutton JL, Renda RF, Waine C, Clark RJ, Daly NL,
Jennings CV, Anderson MA & Craik DJ (2004)
Conserved structural and sequence elements implicated
in the processing of gene-encoded circular proteins.
J Biol Chem 279, 46858–46867.
Mylne JS, Chan LY, Chanson AH, Daly NL, Schaefer
H, Bailey TL, Nguyencong P, Cascales L & Craik DJ
(2012) Cyclic peptides arising by evolutionary
parallelism via asparaginyl-endopeptidase-mediated
biosynthesis. Plant Cell 24, 2765–2778.
Nguyen GK, Zhang S, Wang W, Wong CT, Nguyen
NT & Tam JP (2011) Discovery of a linear cyclotide
from the bracelet subfamily and its disulfide mapping
by top-down mass spectrometry. J Biol Chem 286,
44833–44844.
Ireland DC, Colgrave ML & Craik DJ (2006) A novel
suite of cyclotides from Viola odorata: sequence
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS
Pseudocyclic cystine-knot a-amylase inhibitors
23
24
25
26
27
28
29
30
31
32
33
34
35
variation and the implications for structure, function
and stability. Biochem J 400, 1–12.
Nguyen GK, Lian Y, Pang EW, Nguyen PQ, Tran TD
& Tam JP (2013) Discovery of linear cyclotides in
monocot plant Panicum laxum of Poaceae family
provides new insights into evolution and distribution of
cyclotides in plants. J Biol Chem 288, 3370–3380.
Ling MH, Qi HY & Chi CW (1993) Protein, cDNA,
and genomic DNA sequences of the towel gourd
trypsin inhibitor. A squash family inhibitor. J Biol
Chem 268, 810–814.
Colledge CJ, Hunsperger JP, Imperial JS & Hillyard
DR (1992) Precursor structure of omega-conotoxin
GVIA determined from a cDNA clone. Toxicon 30,
1111–1116.
Woodward SR, Cruz LJ, Olivera BM & Hillyard DR
(1990) Constant and hypervariable regions in conotoxin
propeptides. EMBO J 9, 1015–1020.
Nguyen GKT, Zhang S, Nguyen NTK, Nguyen PQT,
Chiu MS, Hardjojo A & Tam JP (2011) Discovery and
characterization of novel cyclotides originated from
chimeric precursors consisting of albumin-1 chain a and
cyclotide domains in the Fabaceae family. J Biol Chem
286, 24275–24287.
Poth AG, Colgrave ML, Lyons RE, Daly NL & Craik
DJ (2011) Discovery of an unusual biosynthetic origin
for circular proteins in legumes. Proc Natl Acad Sci
USA 108, 10127–10132.
Norton RS & Pallaghy PK (1998) The cystine knot
structure of ion channel toxins and related
polypeptides. Toxicon 36, 1573–1583.
Tam JP & Lu Y-A (1997) Synthesis of large cyclic
cystine-knot peptide by orthogonal coupling strategy
using unprotected peptide precursor. Tetrahedron Lett
38, 5599–5602.
Taichi M, Hemu X, Qiu Y & Tam JP (2013) A
thioethylalkylamido (TEA) thioester surrogate in the
synthesis of a cyclic peptide via a tandem acyl shift.
Org Lett 15, 2620–2623.
Wong CTT, Taichi M, Nishio H, Nishiuchi Y & Tam
JP (2011) Optimal oxidative folding of the novel
antimicrobial cyclotide from Hedyotis biflora requires
high alcohol concentrations. Biochemitry 50, 7275–7283.
Tam JP, Lu Y-A & Yu Q (1999) Thia zip reaction for
synthesis of large cyclic peptides: mechanisms and
applications. J Am Chem Soc 121, 4316–4324.
Wong CTT, Rowlands DK, Wong C-H, Lo TWC,
Nguyen GKT, Li H-Y & Tam JP (2012) Orally active
peptidic bradykinin b1 receptor antagonists engineered
from a cyclotide scaffold for inflammatory pain
treatment. Angew Chem Int Ed 51, 5620–5624.
Kumar A, Ernst RR & Wuthrich K (1980) A twodimensional nuclear Overhauser enhancement
(2D NOE) experiment for the elucidation of complete
4365
P. Q. T. Nguyen et al.
Pseudocyclic cystine-knot a-amylase inhibitors
36
37
38
39
40
41
42
43
44
45
46
47
48
proton–proton cross-relaxation networks in biological
macromolecules. Biochem Biophys Res Commun 95, 1–6.
Piotto M, Saudek V & Sklenar V (1992) Gradienttailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2, 661–665.
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J
& Bax A (1995) NMRPipe: a multidimensional spectral
processing system based on UNIX pipes. J Biomol
NMR 6, 277–293.
Saether O, Craik DJ, Campbell ID, Sletten K, Juul J &
Norman DG (1995) Elucidation of the primary and
three-dimensional structure of the uterotonic
polypeptide kalata B1. Biochemistry 34, 4147–4158.
Guntert P, Mumenthaler C & Wuthrich K (1997)
Torsion angle dynamics for NMR structure calculation
with the new program DYANA. J Mol Biol 273,
283–298.
Laskowski RA, Rullmannn JA, MacArthur MW,
Kaptein R & Thornton JM (1996) AQUA and
PROCHECK-NMR: programs for checking the quality
of protein structures solved by NMR. J Biomol NMR
8, 477–486.
Kabsch W (2001) Integration, scaling, space-group
assignment and post refinement. In International Tables
for Crystallography, Volume F: Crystallography of
Biological Macromolecules (Rossmann MG & Arnold
E, eds), pp. 218–225. Springer, Dordrecht, Netherlands.
Evans P (2006) Scaling and assessment of data quality.
Acta Crystallogr D 62, 72–82.
Winn MD, Ballard CC, Cowtan KD, Dodson EJ,
Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie
AG, McCoy A et al. (2011) Overview of the CCP4
suite and current developments. Acta Crystallogr D 67,
235–242.
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn
MD, Storoni LC & Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658–674.
Langer G, Cohen SX, Lamzin VS & Perrakis A (2008)
Automated macromolecular model building for X-ray
crystallography using ARP/wARP version 7. Nat
Protoc 3, 1171–1179.
Laskowski RA, MacArthur MW, Moss DS &
Thornton JM (1993) PROCHECK: a program to check
the stereochemical quality of protein structures. J Appl
Crystallogr 26, 283–291.
Harvey MJ, Giupponi G & De Fabritiis G (2009)
ACEMD: accelerating biomolecular dynamics in the
microsecond time scale. J Chem Theory Comput 5,
1632–1639.
Jorgensen WL, Chandrasekhar J, Madura JD, Impey
RW & Klein ML (1983) Comparison of simple
4366
49
50
51
52
53
54
55
56
57
potential functions for simulating liquid water. J Chem
Phys 79, 926–935.
Darden T, York D & Pedersen L (1993) Particle mesh
Ewald – an N.Log(N) method for Ewald sums in large
systems. J Chem Phys 98, 10089–10092.
Krautler V, Van Gunsteren WF & Hunenberger PH
(2001) A fast SHAKE: algorithm to solve distance
constraint equations for small molecules in molecular
dynamics simulations. J Comput Chem 22, 501–508.
Bashford D & Case DA (2000) Generalized Born
models of macromolecular solvation effects. Annu Rev
Phys Chem 51, 129–152.
Onufriev A, Bashford D & Case DA (2000)
Modification of the generalized Born model suitable for
macromolecules. J Phys Chem B 104, 3712–3720.
Hubbard SJ & Thornton JM (1993) NACCESS.
Department of Biochemistry and Molecular Biology,
University College, London.
Humphrey W, Dalke A & Schulten K (1996) VMD:
visual molecular dynamics. J Mol Graph Model 14, 33–38.
Strobl S, Gomis-R€
uth F-X, Maskos K, Frank G,
Huber R & Glockshuber R (1997) The a-amylase from
the yellow meal worm: complete primary structure,
crystallization and preliminary X-ray analysis. FEBS
Lett 409, 109–114.
Miller GL (1959) Use of dinitrosalicylic acid reagent
for determination of reducing sugar. Anal Chem 31,
426–429.
Lehrer RI, Rosenman M, Harwig SSSL, Jackson R &
Eisenhauer P (1991) Ultrasensitive assays for
endogenous antimicrobial polypeptides. J Immunol
Methods 137, 167–173.
Supporting information
Additional supporting information may be found in
the online version of this article at the publisher’s web
site:
Movie S1.Stick representation of Wr-AI1 with 2Fo Fc
map overlaid. The complete chain was unambiguously
traced, and contains three disulfide bonds in a
cystine-knot motif.
Movie S2. Movie of molecular dynamics simulation
trajectory of TMA bound to Wr-AI1. The TMA protein (white) and Wr-AI1 (blue) are shown as ‘cartoons’. Interacting residues from TMA (Asp225,
His189, Asp185, Val151, and Asp287) and Wr-AI1
(Lys4, Glu6, Tyr7, Tyr11, and Thr21) are shown as
sticks. See also Table 3. For clarity, hydrogen atoms
are not shown.
FEBS Journal 281 (2014) 4351–4366 ª 2014 FEBS