Tài liệu Báo cáo khoa học: Purification and structural characterization of a D-amino acid-containing conopeptide, conomarphin, from Conus marmoreus docx

Purification and structural characterization of a D-amino
acid-containing conopeptide, conomarphin, from
Conus marmoreus
Yuhong Han1,2,*, Feijuan Huang3,*, Hui Jiang1,4, Li Liu1, Qi Wang1, Yanfang Wang1, Xiaoxia Shao1,
Chengwu Chi1,2, Weihong Du3,* and Chunguang Wang1
1
2
3
4

Institute of Protein Research, Tongji University, Shanghai, China
Institute of Biochemistry and Cell Biology, Shanghai Institute of Biology Sciences, Chinese Academy of Sciences, China
Department of Chemistry, Renmin University of China, Beijing, China
Research Institute of Pharmaceutical Chemistry, Beijing, China

Keywords
conomarphin; Conus marmoreus; D-Phe;
M-superfamily; NMR structure
Correspondence
C. Wang, Institute of Protein Research,
Tongji University, 1239 Siping Road,
Shanghai 200092, China
Fax: 86 21 65988403
Tel: 86 21 65984347
E-mail:
W. Du, Department of Chemistry, Renmin
University of China, 59 Zhong Guan Cun
Street, Beijing 100872, China
Fax: +86 10 62516444
Tel: +86 10 62512660
E-mail:

*These authors contribute equally to this
paper
(Received 7 November 2007, revised 24
January 2008, accepted 22 February 2008)
doi:10.1111/j.1742-4658.2008.06352.x

Cone snails, a group of gastropod animals that inhabit tropical seas, are
capable of producing a mixture of peptide neurotoxins, namely conotoxins,
for defense and predation. Conotoxins are mainly disulfide-rich short peptides that act on different ion channels, neurotransmitter receptors, or
transporters in the nervous system. They exhibit highly diverse compositions, structures, and biological functions. In this work, a novel Cys-free
15-residue conopeptide from Conus marmoreus was purified and designated
as conomarphin. Conomarphin is unique because of its d-configuration
Phe at the third residue from the C-terminus, which was identified using
HPLC by comparing native conomarphin fragments and the corresponding
synthetic peptides cleaved by different proteases. Surprisingly, the cDNAencoded precursor of conomarphin was found to share the conserved signal
peptide with other M-superfamily conotoxins, clearly indicating that conomarphin should belong to the M-superfamily, although conomarphin
shares no homology with other six-Cys-containing M-superfamily conotoxins. Furthermore, NMR spectroscopy experiments established that conomarphin adopts a well-defined structure in solution, with a tight loop in
the middle of the peptide and a short 310-helix at the C-terminus. By contrast, no loop in l-Phe13-conomarphin was found, which suggests that
d-Phe13 is essential for the structure of conomarphin. In conclusion, conomarphin may represent a new conotoxin family, whose biological activity
remains to be identified.

Conus snails are a group of predatory mollusks living
in tropical oceans all over the world. They can produce highly diversified conotoxins for predation and
defense. Conotoxins are believed to number about
50 000, and could serve as a rich source of active
compounds for neuroscience research and nervous
system disease therapy [1]. Conotoxins are mainly
disulfide bond-rich peptides of 10–40 residues. A
small number of conotoxins have zero (Table 1) or
only one disulfide bond. All conotoxins are classified

into different families on the basis of the Cys frame1976

work in the primary sequence and their different targets [1].
It is known that each conotoxin is encoded by an
individual mRNA. The original translation products of
conotoxin genes are in most cases composed of a signal peptide, a propeptide, and mature conotoxins at
the C-terminus. On the basis of the conserved signal
peptide sequences, conotoxins of different families are
grouped into several major superfamilies: A, M, O, I,
T, P, L, and S [2]. For example, most of the M-superfamily conotoxins have the ‘-CC-C-C-CC-’ pattern,

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS


A D-amino acid-containing conomarphin

Y. Han et al.

Table 1. The identified cysteine-free conopeptide families. c, c-carboxylated glutamate; *, amidated C-terminus;  , glycosylation; O, hydroxyproline; V, D-Val; F, D-Phe; NMDA, N-methyl-D-aspartate.
Family

Example

Sequence

Target

Reference

Conantokin

Contulakin
Conorfamide
Conomap
Conophan
Conomarphin

Conantokin-G
Contulakin-G
Conorfamide
Conomap-Vt
Conophan gld-V
Conomarphin

GEccLQcNQcLIRcKSN*
ZSEEGGSNAT KKPYIL
GPMGWVPVFYRF*
AFVKGSAQRVAHGY*
AOANSVWS
DWEYHAHPKONSFWT

NMDA receptor
Neurotensin receptor
FR amide receptor
Unknown
Unknown
Unknown

McIntosh et al. [51]
Craig et al. [52]
Maillo et al. [53]

Dutertre et al. [19]
Pisarewicz et al. [20]
This work

but some of them have been found to act on the
Na+-channel, K+-channel, and nicotinic acetylcholine
receptor [3–5]. Furthermore, different disulfide bond
linkages are formed with this same Cys framework
[3,6,7]. The mechanisms that lead to conservation of
signal peptides, particularly as compared to the
sequence hyperdivergence of mature conotoxins,
remain a subject for further study.
Another striking feature of conotoxins is the high
content of different post-translational modifications
[8]. A variety of post-translational modifications have
been found in conotoxins, such as C-terminal amidation, hydroxylation of Pro, Val or Lys, c-carboxylation
of Glu, glycosylation of Ser or Thr, bromination of
Trp, sulfation of Tyr, cyclization of N-terminal Gln,
and epimerization of several different residues. It is
quite rare for such a variety of post-translational modifications to take place in a defined cluster of gene
products. Cone snails have probably developed a complicated but delicate machinery to carry out these
modifications, which might be critical for the structure
and function of conotoxins.
Epimerization, namely converting an amino acid
residue in a peptide chain from the l-configuration
to the d-configuration, was first identified in dermophin, an opiate-like peptide from the skin of the
Table 2. D-Amino acid-containing peptides from different organisms.
hydroxyproline; c, c-carboxylated glutamate.

American frog Phyllomedusa [9]. Later, this was

found in other toxins and peptides, such as the spider toxin x-agatoxin [10], C-type natriuretic peptide
from the Australian platypus [11], fulicin from African giant snails [12], and contryphan from cone
snails (Table 2). The first d-residue containing conotoxin, contryphan-R, was purified from Conus radiatus [13]. To date, a series of contryphans has been
identified [14–17]. A group of I-superfamily conotoxins has also been found to contain a d-residue [18].
Recently, two other families of d-residue-containing
conopeptides, conophan and conomap, were identified
biochemically [19,20]. In comparison with other posttranslational modifications, such as C-terminal amidation, Pro hydroxylation, or Glu c-carboxylation,
which exist in many conotoxin families, residue epimerization from the l-configuration to the d-configuration is relatively rare [8].
In this work, we purified a novel 15 amino acid peptide from the venom of Conus marmoreus. This peptide
was found to be particularly unique because it contains
no Cys residues, a hydroxylated Pro at position 10,
and a d-Phe at position 13. Its cDNA sequence indicates that it belongs to the M-superfamily, albeit it
shares no sequence homology with other M-superfamily conotoxins. Its solution structure, including the
D-Amino

acid residues are underlined. *, amidated C-terminus; O,

Organism

Example

Sequence

Position

Reference

Cone snail

Conomarphin

r11a
Contryphan-R
Glacontryphan-M
Conophan gld-V
Conomap-Vn
Fulicin
Dermorphin
x-Agatoxin
C-type natriuretic
peptide
Defensin-like peptide

DWEYHAHPKONSFWT
GOSFCKADEKOCEYHADCCNCCLSGICAOSTNWILPGCSTSSFFKI
GCOWEPWC*
NcScCPWHPWC*
AOANSVWS
AFVKGSAQRVAHGY*
FNEFV*
YAFGYPS*
EDNCIAEDYGKCTWGGTKCCRGRPCRCSMIGTNCECTPRLIMEGLSFA
LLHDHPNPRKYKPANKKGLSKGCFGLKLDRIGSTSGLGC

)3
)3
)5
)5
)3
+2
+2

+2
)3
+2

This work
Buczek et al. [35]
Jimenez et al. [13]
Hansson et al. [16]
Pisarewicz et al. [20]
Dutertre et al. [19]
Ohta et al. [12]
Montecucchi et al. [9]
Kuwada et al. [10]
Torres et al. [11]

IMFFEMQACWSHSGVCRDKSERNCKPMAWTYCENRNQKCCEY

+2

Torres et al. [39]

Snail
Frog
Spider
Australian
platypus

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1977

A D-amino acid-containing conomarphin

Y. Han et al.

effect of the d-Phe on structure, was also studied. The
unusual structure of conomarphin further demonstrates the high diversity of conotoxins.

Results
Purification and primary sequence of
conomarphin
As previously reported, the crude venom of
C. marmoreus was separated into two peaks on a gel
filtration column (Fig. 1A); the second one contained
mainly peptides [6]. Separation using an RP-HPLC

C-18 column provided at least 19 major peaks as well
as many minor peaks from the peptide fraction
(Fig. 1B). Every major peak was collected, repurified,
and sequenced. The peak that eluted at 53 min corresponded to a novel 15 amino acid peptide with the
sequence DWEYHAHPKONSFWT, where O is
hydroxylated Pro. The Mr of the peptide, 1931.1,
matched perfectly with the calculated one, 1931.05,
indicating that this was the complete sequence of this
peptide. This sequence shares no homology with that
of any other known conotoxin. This peptide was
named conomarphin, indicating that it is a Cys-free
conotoxin and originated from C. marmoreus.
D-Phe13

Fig. 1. Purification of conomarphin from the venom of
C. marmoreus. (A) The crude venom was separated into two main
peaks on a Sephadex G-25 column (100 · 2.6 cm). (B) The second
peak was further separated on an HPLC C-18 column
(9.4 · 250 mm) with an elution gradient of 0–10 min 100%
Buffer A, 10–20 min 0–27% Buffer B, 20–25 min 27% Buffer B,
25–58 min 27–42.5% Buffer B, and 58–63 min 42.5–100%
Buffer B. Buffer A is 0.1% trifluoroacetic acid and Buffer B is 0.1%
trifluoroacetic acid in 70% (v ⁄ v) acetonitrile. The flow rate was
2 mLỈmin)1. The peak labeled with an asterisk is conomarphin.

1978

in conomarphin

In order to obtain more material for structural studies,
conomarphin was chemically synthesized using standard Fmoc–l-amino acids. However, to our great surprise, the synthesized peptide did not have the same
retention time on a C-18 HPLC column as the natural
one (supplementary Fig. S1), although the peptides
have identical sequences and relative molecular masses.
The only explanation for this is that natural conomarphin must have one or more d-amino acids.
The strategy of protease digestion was employed to
locate the d-amino acid(s) in conomarphin. As Lys9 is
the only basic residue in this peptide, trypsin was first
used to cleave both natural conomarphin and the synthetic peptide DWEYHAHPKONSFWT. The digestion of natural conomarphin gave the expected results:
two fragments and the intact peptide with relative
molecular masses identical to the corresponding calculated ones (supplementary Fig. S2A). However, for the
synthetic all-l-amino acid conomarphin, trypsin
cleaved at two sites, Lys9-Hyp10 and Asn11-Ser12

(Fig. 2B). The second cleavage site was unexpected, as
trypsin usually only cleaves after a basic residue. Comparison between the two digestion products demonstrated that the difference between natural and
synthetic conomarphin came from the C-terminal
fragment ONSFWT, which had an identical relative
molecular mass but a different retention time (P2 in
supplementary Fig. S2A and P3 in supplementary
Fig. S2B).
To narrow the range of the possible position of the
d-amino acid, chymotrypsin was used to digest natural conomarphin and the synthetic peptide DWEYHAHPKONSFWT. The cleaved fragments were
analyzed on a C-18 HPLC column and were assigned
on the basis of their relative molecular masses. The
shorter C-terminal fragment SFWT of natural conomarphin and the synthetic peptide exhibited different

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS


A D-amino acid-containing conomarphin

Y. Han et al.

Fig. 2. cDNA and the deduced precursor
sequence of conomarphin. The signal peptide is shadowed and the mature peptide is
underlined. The polyA signal AATAAA in the
3¢-UTR is also underlined. The cDNA of conomarphin has been deposited in the Genbank database with the accession number
EU048276.

retention times, which indicated that one or more of
the four C-terminal amino acids of natural conomarphin should be in the d-configuration (supplementary
Fig. S3).
As chymotrypsin cleaved the Asn11-Ser12 peptide

bond of natural conomarphin, it was deduced that
Ser12 of the SFWT fragment was not in the d-configuration. The C-terminal Trp14-Thr15 peptide bond
could be cleaved with a large amount of chymotrypsin
(data not shown), which suggested that Thr15 was an
l-amino acid. Thus, the possible positions for the
d-amino acid residue in conomarphin were determined
to be Phe13, Trp14, or both.
To check these three possibilities, four peptides
were synthesized, all l-configuration SFWT, SFWT
with d-Phe, SFWT with d-Trp, and SFWT with
d-Phe and d-Trp. Under the same HPLC elution
conditions, the retention times of SFWT and SFWT
were clearly not the same as the retention time of
the C-terminal tetrapeptide fragment from natural
conomarphin. However, SFWT and SFWT did elute
at the same time as the natural fragment (supplementary Fig. S4), which suggested that there is only one
d-amino acid residue in natural conomarphin, d-Phe13
or d-Trp14.
Finally, two full-length conomarphin sequences
with either d-Phe13 or d-Trp14 were chemically syn-

thesized and compared to natural conomarphin. The
coelution results unambiguously demonstrated that
conomarphin contains d-Phe13, as the synthetic peptide with d-Phe13 but not the one with d-Trp14 coeluted with natural conomarphin (supplementary
Fig. S5).
cDNA structure of conomarphin
The cDNA encoding conomarphin was obtained by
chance when gene cloning of the M-superfamily conotoxins was carried out from C. marmoreus in our laboratory. Besides the clones for other conventional
M-superfamily conotoxins with six Cys residues, one
clone encoded a precursor comprising the exact conomarphin sequence at the C-terminus (Fig. 2). This was

entirely unexpected, as conomarphin shares no
sequence homology with other M-superfamily conotoxins.
Nevertheless, the cDNA structure of conomarphin
was similar to those of other conotoxin cDNAs. The
cDNA-encoded precursor of conomarphin consisted of
a conserved M-superfamily signal peptide of 25 residues, a proregion of 29 residues, the conomarphin
mature peptide, and the two additional residues, Leu
and Val, at the C-terminus, which are cleaved during
maturation.

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1979


A D-amino acid-containing conomarphin

Y. Han et al.

Sequence-specific resonance assignments

Table 3. Structural statistics for the family of 20 structures of
conomarphin and L-Phe13-conomarphin.

Complete proton resonances for both conomarphin
and l-Phe13-conomarphin were assigned by wellestablished methods [21], which were pioneered by
Wuthrich and successfully applied to various animal
conotoxins [22,23]. The spin systems were identified
on the basis of both DQF-COSY and TOCSY spectra. For conomarphin, 14 of the 15 spin systems were
found in the ‘fingerprint’ region of a 120 ms TOCSY

spectrum (supplementary Table S1), which were verified in a relevant DQF-COSY spectrum. For l-Phe13conomarphin, sequence-specific resonance assignments
(supplementary Table S2) were performed using the
same strategy. The results of sequential daN(i,i + 1)
connectivities found in the CaH-NH fingerprint region
of the NOESY spectra for conomarphin and l-Phe13conomarphin are represented in supplementary
Fig. S6A,B, respectively. The NOESY data acquired
at 300 K and pH 3 for conomarphin and l-Phe13conomarphin showed a large number of NOEs, which
suggested that the structures of the two peptides were
sufficiently constrained for distance–geometry calculations.
Structural calculation, refinement, and evaluation
The NMR experimental data were converted into distance and angle constraints as usual, providing enough
constraints for the structure calculation of conomarphin. The three-dimensional structure of conomarphin
was determined from NMR data using the same strategy previously used for structural studies of conotoxins
and their analogs [24–27].
Most NOESY crosspeaks were assigned and integrated, with concomitant cycles of structure calculations for evaluation of distance and angle constraint
violations as well as assignments of additional peaks
based on the preliminary structure. For conomarphin,
the process study led to 172 NOE-based distance
restraints, of which 105 were derived from intraresidue
NOEs, 50 from sequential backbone NOEs, 14 from
medium-range NOEs, and three from long-range
NOEs (Table 3). Eight dihedral angle constraints were
used from J coupling constants. For l-Phe13-conomarphin, 160 NOE-based distance restraints (44 sequential
NOEs, 104 intraresidue NOEs, and 12 medium NOEs)
and six dihedral angle constraints were used to build
up the structure (Table 3).
At this stage of the structure elucidation process, the
cyana program was used to provide hydrogen bond
information. Hydrogen–deuterium exchange-out experiments indicated the hydrogen bonds that might exist

1980

L-Phe13-

Structural statistics

Conomarphin

conomarphin

Assigned NOE crosspeaks
172
160
Intraresidue
105
104
Sequential (|i – j| = 1)
50
44
Medium range
14
12
Long range
3
0
AMBER energies (kcalỈmol)1)
Bond
4.98 ± 0.15
4.87 ± 0.18
Angle

62.05 ± 1.11
64.28 ± 1.14
Dihedral
131.15 ± 1.88
124.25 ± 1.45
Van der Waals
)80.48 ± 2.64
)71.66 ± 3.44
Electrostatic energy
)1064.45 ± 59.17 )992.47 ± 66.95
Egb (generalized born energy) )470.50 ± 64.27 )543.59 ± 69.59
Constraints
3.07 ± 0.41
2.22 ± 0.16
Total
)766.95 ± 7.20 )785.47 ± 6.77
˚
rmsd to mean coordinates (A)
Mean global backbone rmsd 0.80 ± 0.36
0.82 ± 0.36
Mean global heavy rmsd
1.78 ± 0.53
1.95 ± 0.43
Mean global backbone rmsd 0.54 ± 0.23
0.63 ± 0.30
(2–15)
Mean global heavy
1.59 ± 0.46
1.88 ± 0.44
rmsd (2–15)

Ramachandran statistics from PROCHECK-NMR
Most favored regions (%)
87.8
68.9
Additional allowed
12.2
31.1
regions (%)
Generously allowed
0
0
regions (%)
Disallowed regions (%)
0
0

between the slow-exchange amide protons and their
nearby oxygen or nitrogen atoms. Thus, two hydrogen
bonds related to four distance constraints were
Trp14(HN)–Asn11(CO) and Thr15(HN)–Ser12(CO)
for conomarphin. The l-Phe13-conomarphin had the
same hydrogen bonds as conomarphin. With the additional hydrogen bond distance constraints, another
round of minimization was performed as previously
described [22].
The simulated annealing calculations were carried
out starting with 100 random structures, and the 20
final structures selected were in good agreement with
the NMR experimental constraints, for which the
NOE distance and torsion angle violations were smal˚
ler than 0.2 A and 3°, respectively. The atomic rmsd

values about the mean coordinate positions of cono˚
marphin were 0.54 ± 0.24 A for the backbone atoms
˚
(N, CR, and C) and 1.25 ± 0.30 A for all heavy
atoms, and the values for l-Phe13-conomarphin were
˚
˚
0.44 ± 0.16 A and 1.22 ± 0.22 A, respectively.
Finally, the 20 best models with the lowest residual
target function and lowest rmsd values were further

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS


A D-amino acid-containing conomarphin

Y. Han et al.

refined for simulated annealing and restrained energy
minimization [24,28], using the sander module of the
amber 9.0 package. The resulting conformers contained no significant violations of any constraint with
lower energy, better Ramachandran plots were chosen
to represent the three-dimensional solution structure of
conomarphin, and the mean structure was generated
by molmol.
Structural characterization and comparison
The program procheck was used to analyze the family
of 20 structures (Table 3). Figure 3 shows an overlay
of the backbone atoms for the 20 structures of conomarphin and l-Phe13-conomarphin (Protein Data
Bank codes: 2YYF and 2JQC). The overall rmsd

reported for the final 20 structures was influenced by
the disorder of the N-terminal residue Asp1. When
Asp1 was eliminated and the molecule consisted of
only residues 2–15, the mean global backbone rmsd
dropped markedly. Unlike the C-terminal portion,
the N-terminal portion of the molecule was poorly
resolved.
The three-dimensional structure of conomarphin was
characterized by one compact loop of five residues
from Ala6 to Hyp10 with a loop center at residue 8,

and another secondary structure region at the peptide
C-terminus from residues Asn11 to Trp14 with a 310helix. The helix was supported by Oi-HNi + 3 hydrogen bonds for Asn11(CO)–Trp14(NH) and Ser12(CO)–
Thr15(NH), which were confirmed by the slow solvent
exchange kinetics of the Trp14 and Thr15 amide
protons. The two observed small 3JHN-Ha coupling
constants for residues Asn11 and Ser12, and the
dNN(i,i + 2), daN(i,i + 2) [Phe13(CaH)–Thr15(NH)] and
daN(i,i + 3) NOEs in the region of residues 11–14
support the presence of a short 310-helix.
Similar to conomarphin, l-Phe13-conomarphin contained a short 310-helix near its C-terminus, from
Asn11 to Trp14, supported by Oi-HNi + 3 hydrogen
bonds for Asn11(CO)–Trp14(HN) and Ser12(CO)–
Thr15(HN), and confirmed by the slow solvent
exchange kinetics of the amide protons of Trp14
and Thr15. The observation of two small 3JHN-Ha coupling constants for residues Asn11 and Ser12 and the
dNN(i,i + 2), daN(i,i + 2) [Ser12(CaH)–Trp14(NH) and
Asn11(CaH)–Phe13(NH)] and daN(i,i + 3) [Asn11
(CaH)–Trp14(NH)] NOEs in the region of residues 11–14 are in agreement with the presence of a
short 310-helix. A random coil rather than a compact

loop in the region of residues 1–10 existed in
l-Phe13-conomarphin.

Fig. 3. The overlay of the backbone atoms
for the 20 converged structures of conomarphin (A) and L-Phe13-conomarphin (C),
respectively. The N-terminal Asp1 is in a
poorly resolved region of the molecule. The
backbone peptide folding of conomarphin
(B) and L-Phe13-conomarphin (D) is also
shown. A short 310-helix at the C-terminus
is shown in red.

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A D-amino acid-containing conomarphin

Y. Han et al.

Discussion
Cone snails have developed a collection of highly
diversified peptide toxins over 50 million years of
evolution, so even after research for more than
20 years, conotoxin classification needs to be updated.
In this work, we purified a novel Cys-free 15-residue
peptide containing a d-amino acid from C. marmoreus.
This peptide is not homologous to any other identified conotoxin, and thus represents a new conotoxin
family.

C. marmoreus is a molluscivorous cone snail, the
major part of whose venom consists of peptide toxins
(Fig. 1). These toxins are mainly Cys-rich peptides [6],
except for one Cys-free fraction having a novel
sequence of DWEYHAHPKONSFWT with Pro10
hydroxylated and Phe13 in the d-conformation. Several families of Cys-free conopeptides from different
cone snails have been reported (Table 1). However,
this novel peptide does not exhibit obvious homology
with the others, so it was designated conomarphin.
It is very surprising that, on the basis of the conserved signal peptide sequence, conomarphin belongs
to the M-superfamily, a major conotoxin superfamily.
All of the conventional M-superfamily conotoxins have
three disulfide bonds, although their disulfide linkages
and targets are significantly different from each other
[3–7]. Now, with conomarphin, the M-superfamily
may become the most diversified conotoxin superfamily. It is interesting that conotoxin precursor signal
peptides are rather conserved, whereas mature peptides
are very diversified. Gene structure exploration of this
conotoxin superfamily would give some hints, as has
been done for the A-superfamily of conotoxins [29].
Obviously, conomarphin maturation involves several
different post-translational modifications. Apart from
cleavage of the signal peptide and the propeptide,
which happens in the maturation process for every
conotoxin [8], the removal of the two additional residues Leu and Val at the C-terminus is rather unique
to conomarphin. To our knowledge, the cleavage of
the C-terminal Leu and Val has not been reported previously. The enzyme responsible for this cleavage and
the recognition sequence for post-translational modifications, namely hydroxylation of Pro10 and epimerization of l-Phe13 to d-Phe13, remain to be clarified.
Hydroxyproline has been found in many conotoxins
with or without disulfide bonds [7,20,30]. It has also

been found that Pro hydroxylation happens with high
specificity; that is, only one Pro residue is hydroxylated
in many conotoxins. However, the physiological role
of specific Pro hydroxylation in conotoxins is still
elusive.
1982

The epimerized d-Phe13 is another striking feature
of conomarphin. The l-amino acid to d-amino acid
epimerization in a polypeptide chain is quite rare and
not well understood, although some d-amino acids
have been known for a long time to act as neurotransmitters. d-Amino acid-containing peptides or toxins
have been found in mollusks [12,18–20], arthropods
[10], amphibians [9] and even mammals [11]. They are
produced by a ribosome protein translation pathway
based on their mRNAs [17,18,31], so epimerization
must occur on an incorporated l-amino acid of a peptide chain. So far, epimerization enzymes have been
found in frog skin [32], spider venom [33], and the
venom of the Australian platypus [34], but they are
completely different with respect to sequence and
mechanism.
Although the detailed mechanism of epimerization is
unclear, epimerization in short peptides has been
found to have a ‘position rule’; namely, epimerization
occurs only at three positions: position 2 at the N-terminus (+2), and positions 3 and 5 at the C-terminus
()3 and )5) (Table 2). It is noteworthy that epimerization at each of these three positions has been found in
cone snails, such as the +2 position in conomap [19],
the )3 position in conomarphin and r11a [35], and the
)5 position in contryphan [16]. However, epimerization happens mainly at the +2 position in other
organisms. Probably, cone snails have developed a

more advanced system to achieve this difficult modification at different positions. This is not surprising,
because of the well-known high content of post-translational modifications in conotoxins [8]. It is noteworthy that from the single species C. marmoreus, two
epimerization positions have been found, the )3 position for conomarphin and the )5 position for glacontryphan-M [16]. It is not known whether they are
modified by the same enzyme system but with different
recognition sequences. It is also worth pointing out
that the l-amino acid to d-amino acid epimerization
seems to be complete for conopeptides, whereas both
isoforms coexist in defensin-like peptides and natriuretic peptides from the Australian platypus [11].
With the help of such a developed post-translational
modification system, conotoxins exhibit amazing structural diversity. In this work, we found that conomarphin, despite being a short peptide of 15 residues, is
well structured in solution (Fig. 3 and supplementary
Fig. S7). The d-Phe13 of conomarphin has a significant effect on the structure of the peptide; a tight loop
around Pro8 and a short 310-helix at the C-terminus
were identified. However, for l-Phe13-conomarphin,
there was no loop in the middle and the peptide chain
seemed to be rather straight, whereas the C-terminal

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS


A D-amino acid-containing conomarphin

Y. Han et al.

Fig. 4. Stereo view of the superimposed
structures of conomarphin (red) and
L-Phe13-conomarphin (gray). The side chain
of L-Phe13 might cause spatial hindrance to
the side chain of Lys9 and Hyp10 in forming
the tight loop as in conomarphin. The side

chain of Lys9, Hyp10 and D-Phe13 of conomarphin is shown in stick mode, and the
side chain of L-Phe13 in L-Phe13-conomarphin is shown as a gray stick.

helix was two residues longer. Superimposition of the
C-terminal helices of conomarphin and l-Phe13-conomarphin showed that the relatively large side chain of
l-Phe13 might cause spatial hindrance to Hyp10 and
Lys9 forming a loop (Fig. 4), so that the rest of the
peptide chain of l-Phe13-conomarphin extends roughly
along the axis orientation of the C-terminal helix.
The structures of several d-amino acid-containing
peptides have been determined, including glacontryphan-M [36], contraphan-R [37], contrypan-Vn [38],
DLP-2 and DLP-4 [39], and the C-terminal peptide of
x-agatoxin [10]. The NMR structure of excitatory
r11a was reported very recently [40]. There are one or
more disulfide bonds in all of these peptides, which
supports their rigid structures. Consequently, the terminal d-amino acid has only a minimal effect on the
overall structure. However, in Cys-free conomarphin,
d-Phe13 has a critical role in maintaining the peptide
conformation. Converting d-Phe13 to l-Phe13 dramatically changes the structure (Fig. 4). It is plausible
that this d-Phe13 and the well-maintained structure of
conomarphin could be very important for its function,
the exploration of which will certainly be of great
interest.
In summary, a new conotoxin family, conomarphin,
was identified and structurally studied in this work.
Furthermore, the critical influence of a d-amino acid
on the conformation of a peptide was demonstrated. It

is noteworthy that this conotoxin family exists in all
three major feeding types of cone snails. Apart from

conomarphin purified from the mollusk-hunting
C. marmoreus, a similar sequence was identified on the
cDNA level from the worm-hunting Conus litteratus
[41], and a homologous peptide was purified from the
fish-hunting Conus achatinus (H. Jiang & C. X. Fan,
unpublished data). The widespread occurrence of conomarphins in fish, mollusks and worm-hunting cone
snails suggests that this family of peptides may have a
specific function.

Experimental procedures
Materials
Specimens of C. marmoreus were collected from Sanya near
the South China Sea. Sephadex G-25 was purchased from
Amersham Biosciences (Uppsala, Sweden), a ZORBAX
300SB-C18 semipreparative column was from Agilent Technologies (Santa Clara, CA, USA), and trifluoroacetic acid
and acetonitrile used for HPLC were from Merck (Darmstadt, Germany). Trypsin and tosyl phenylalanyl chloromethyl ketone-treated chymotrypsin were from Sigma
(St Louis, MO, USA). The 3¢-RACE kit and TRIzol
reagent were purchased from Invitrogen (Carlsbad, CA,
USA), and Taq DNA polymerase and the pGEM-T Easy
vector system were from Promega (Madison, WI, USA).

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1983


A D-amino acid-containing conomarphin

Y. Han et al.

resulting PCR products were inserted directly into the
pGEM-T Easy vector for sequencing.

Purification
The purification procedure and conditions were exactly the
same as previously described [6]. Briefly, the crude venom
was first separated on a Sephadex G-25 column and the
second peak was then applied to a ZORBAX 300SB-C18
semipreparative column (9.4 · 250 mm) connected to an
HPLC instrument. The peptides were eluted with a gradient
of acetonitrile.

Peptide synthesis
Peptides were synthesized by solid-phase methods on an
ABI 433A peptide synthesizer using standard Fmoc chemistry and side-chain protection.

N-terminal sequencing and MS
N-terminal amino acid sequence analysis was performed by
automated Edman degradation on an ABI model 491A
Procise Protein Sequencing System (Applied Biosystems,
Foster City, CA, USA). A 20 pmol sample was loaded onto
a glass fiber filter previously conditioned with BioBrene
Plus (Applied Biosystems).
All purified and synthetic peptides were analyzed in the
scan type of Enhanced MS by Qtrap (Applied Biosystems).
The mass spectrometer, equipped with a TurboIonSpray
Source, was operated in positive ionization mode.

Protease digestion
The native and synthesized peptides were dissolved in

50 mm Tris ⁄ HCl (pH 7.8) and 20 mm CaCl2 buffer to a
concentration of 1 lgỈlL)1. Trypsin was added to a ratio of
1 : 20. The digestion was carried out at 25 °C for 18 h, and
then quenched with 50% trifluoroacetic acid before HPLC
analysis.
The chymotrypsin digestion was performed in 50 mm
Tris ⁄ HCl (pH 7.8) and 20 mm CaCl2 buffer with the same
peptide concentration and enzyme ratio. The reactions were
kept at 25 °C for 18 h, and analyzed by HPLC after being
quenched with 50% trifluoroacetic acid.

cDNA cloning
The cDNA of conomarphin was obtained unexpectedly
when the cDNA cloning of M-superfamily conotoxins was
performed from cDNAs reverse transcribed from total
RNAs of the venom duct of C. marmoreus, as previously
described [6]. The 5¢-primer corresponded to the highly
conserved
M-superfamily
signal
peptide
sequence
(5¢-ATGTTGAAAATGGGAGT(G ⁄ A)GTG-3¢), and the
3¢-primer was the abridged universal amplification primer
devoid of the poly(dT) tail from the 3¢-RACE kit. The

1984

NMR experiments
Samples of conomarphin and l-Phe13-conomarphin for

NMR studies were prepared in either 99.99% D2O (Cambridge Isotopes Lab) or 9 : 1 (v ⁄ v) H2O ⁄ D2O with 0.01%
trifluoroacetic acid, at pH 3 (uncorrected for the isotope
effect), with a final sample concentration of approximately
2 mm. For experiments in D2O, the peptide was lyophilized
and redissolved in 99.99% D2O.
NMR spectra were collected on a Bruker-DRX 600 MHz
spectrometer using standard pulse sequences and phase
cycling at 300 K. Proton DQF-COSY [42], NOESY [43]
and TOCSY spectra [44] of samples in 99.99% D2O and
90 : 10 H2O ⁄ D2O, respectively, were acquired with the
transmitter set at 4.70 p.p.m. and a spectral window of
6000 Hz, as described previously [22].
Spectra were processed with topspin or xwinnmr software. Phase-shifted sine-squared window functions were
applied before Fourier transformation. To identify the slow
exchange of backbone amide protons, the hydrogen–deuterium exchange experiments were carried out by dissolving
the lyophilized sample in D2O and recording a series of onedimensional spectra every 5 min for 1 h, and subsequently
every hour for 10 h. Chemical shifts were referenced to the
methyl resonance of 4,4-dimethyl-4-silapentane-1-sulfonic
acid as an internal standard. Complete sets of two-dimensional spectra for both samples of conomarphin and
l-Phe13-conomarphin were recorded at 300 K and pH 3.

Restraint set generation
An initial survey of distance constraints was performed on
a series of NOESY spectra acquired at mixing times of 100,
200 and 350 ms. Buildup curves were produced that demonstrated a leveling of the intensity of the NOE at mixing
times greater than 200 ms. Peak picking, spin system identification and volume integration of the NOESY crosspeaks
were performed with the interactive program sparky
(v. 1.113). Non-stereospecifically assigned atoms were treated as pseudo-atoms and given correction distances. A set
of distance restraints was generated from these data and
used as input for cyana (v. 2.1).

Six u dihedral angles were determined on the basis of the
3
JNHa coupling constants derived by analysis of a high-resolution one-dimensional proton spectrum of the conotoxin
conomarphin. For peaks that did not show a splitting
pattern, the 3JNHa value was derived from a measure of
the line width at half the height of the signal. For 3JNHa
values < 5.5 Hz, the u angle was constrained in the
range )65 ± 25°, and for 3JNHa values > 8.0 Hz, it was
constrained in the range )120 ± 40° [21,45]. Backbone

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS


Y. Han et al.

dihedral constraints were not applied to 3JNHa values
between 5.5 and 8.0 Hz.
The hydrogen bond acceptors for the slowly exchanged
amide protons were identified by analysis of the preliminary
calculated structures [46,47]. The hydrogen bond distance
˚
restraints were added as target values of 1.8–2.2 A for
˚
NHi–Oj bonds and 2.8–3.2 A for Ni–Oj bonds, respectively.

A D-amino acid-containing conomarphin

5

6

Structural computation and refinement
The experimentally derived distance constraints, torsion
angle constraints and hydrogen bond constraints were
input for the molecular modeling protocol. One hundred
calculations with the program cyana were started with
random polypeptide conformations, and the 20 resulting
conformers with the lowest residual target function values
were analyzed.
The 20 structures with the lowest target functions were
submitted to molecular dynamics refinement with the
sander module of the amber 9 program as the starting
structure [47]. The molecular dynamics simulations were
performed using the parm03 force field and the GB ⁄ SA
implicit solvation system [48]. The visual analysis of conomarphin was done using molmol [49] software, and the
geometric qualities of the obtained structures were assessed
with procheck-nmr software [50].

7

8

9

10

Acknowledgements
This work was supported by the National Basic
Research Program of China (2004CB719900), the
National Natural Science Foundation of China

(20473013) and the Chinese Academy of Sciences for
Key Topics in Innovation Engineering (KSCX2-YWR-104). Chunguang Wang is supported by the Program for young excellent talents in Tongji University
(2006KJ063) and Dawn Program of Shanghai Educational Development Foundation (06SG26).

11

12

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Supplementary material
The following supplementary material is available
online:
Fig. S1. Natural and synthetic conomarphin with all
l-amino acids on HPLC.
Fig. S2. The fragments of natural conomarphin (A)
and the synthetic l-Phe13-conomarphin (B) cleaved by
trypsin on an HPLC C-18 column.
Fig. S3. The fragments of the natural conomarphin
(A) and the synthetic conomarphin (B) cleaved by
chymotrypsin on an HPLC C-18 column.
Fig. S4. SFWT tetrapeptides with one or two d-amino
acids and the natural one on an HPLC C-18 column.
Fig. S5. The coelution of the synthetic conomarphin
with d-Trp14 (A) or d-Phe13 (B) with the natural
conomarphin.
Fig. S6. Sequential daN(i,i + 1) connectivities in the
CaH-NH fingerprint region of the NOESY spectrum
of conomarphin (A) and l-Phe13-conomarphin (B).
Fig. S7. Comparison of the three-dimensional structures between conomarphin and l-Phe13-conomarphin.
Table S1. Proton resonance assignments (p.p.m.) for
conomarphin.
Table S2. Proton resonance assignments (p.p.m.) for
l-Phe13-conomarphin.
This material is available as part of the online article
from
Please note: Blackwell Publishing are not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corresponding author for the article.

FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS

1987