Purification and structural characterization of a D-amino
acid-containing conopeptide, conomarphin, from
Conus marmoreus
Yuhong Han
1,2,
*, Feijuan Huang
3,
*, Hui Jiang
1,4
,LiLiu
1
, Qi Wang
1
, Yanfang Wang
1
, Xiaoxia Shao
1
,
Chengwu Chi
1,2
, Weihong Du
3,
* and Chunguang Wang
1
1 Institute of Protein Research, Tongji University, Shanghai, China
2 Institute of Biochemistry and Cell Biology, Shanghai Institute of Biology Sciences, Chinese Academy of Sciences, China
3 Department of Chemistry, Renmin University of China, Beijing, China
4 Research Institute of Pharmaceutical Chemistry, Beijing, China
Conus snails are a group of predatory mollusks living
in tropical oceans all over the world. They can pro-
duce highly diversified conotoxins for predation and

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 pep-
tides that act on different ion channels, neurotransmitter receptors, or
transporters in the nervous system. They exhibit highly diverse composi-
tions, 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 cDNA-
encoded precursor of conomarphin was found to share the conserved signal
peptide with other M-superfamily conotoxins, clearly indicating that cono-
marphin should belong to the M-superfamily, although conomarphin
shares no homology with other six-Cys-containing M-superfamily conotox-
ins. Furthermore, NMR spectroscopy experiments established that cono-
marphin adopts a well-defined structure in solution, with a tight loop in
the middle of the peptide and a short 3
10

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 dermo-
phin, an opiate-like peptide from the skin of the
American frog Phyllomedusa [9]. Later, this was
found in other toxins and peptides, such as the spi-
der toxin x-agatoxin [10], C-type natriuretic peptide
from the Australian platypus [11], fulicin from Afri-
can giant snails [12], and contryphan from cone
snails (Table 2). The first d-residue containing cono-
toxin, contryphan-R, was purified from Conus radia-
tus [13]. To date, a series of contryphans has been
identified [14–17]. A group of I-superfamily conotox-
ins 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 post-
translational modifications, such as C-terminal amida-
tion, Pro hydroxylation, or Glu c-carboxylation,
which exist in many conotoxin families, residue epi-
merization from the l-configuration to the d-configu-
ration is relatively rare [8].
In this work, we purified a novel 15 amino acid pep-
tide 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 indi-
cates that it belongs to the M-superfamily, albeit it

Conophan gld-V AOANS
VWS )3 Pisarewicz et al. [20]
Conomap-Vn A
FVKGSAQRVAHGY* +2 Dutertre et al. [19]
Snail Fulicin F
NEFV* +2 Ohta et al. [12]
Frog Dermorphin Y
AFGYPS* +2 Montecucchi et al. [9]
Spider x-Agatoxin EDNCIAEDYGKCTWGGTKCCRGRPCRCSMIGTNCECTPRLIMEGL
SFA )3 Kuwada et al. [10]
Australian
platypus
C-type natriuretic
peptide
L
LHDHPNPRKYKPANKKGLSKGCFGLKLDRIGSTSGLGC +2 Torres et al. [11]
Defensin-like peptide I
MFFEMQACWSHSGVCRDKSERNCKPMAWTYCENRNQKCCEY +2 Torres et al. [39]
Y. Han et al. A
D-amino acid-containing conomarphin
FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 1977
effect of the d-Phe on structure, was also studied. The
unusual structure of conomarphin further demon-
strates 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

thetic peptide DWEYHAHPKONSFWT. The diges-
tion of natural conomarphin gave the expected results:
two fragments and the intact peptide with relative
molecular masses identical to the corresponding calcu-
lated 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. Com-
parison between the two digestion products demon-
strated 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 natu-
ral conomarphin and the synthetic peptide DWEY-
HAHPKONSFWT. 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 cono-
marphin and the synthetic peptide exhibited different
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,

were clearly not the same as the retention time of
the C-terminal tetrapeptide fragment from natural
conomarphin. However, S
FWT and SFWT did elute
at the same time as the natural fragment (supplemen-
tary 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 pep-
tide with d-Phe13 but not the one with d-Trp14 coe-
luted 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 cono-
toxins was carried out from C. marmoreus in our labo-
ratory. Besides the clones for other conventional
M-superfamily conotoxins with six Cys residues, one
clone encoded a precursor comprising the exact cono-
marphin sequence at the C-terminus (Fig. 2). This was
entirely unexpected, as conomarphin shares no
sequence homology with other M-superfamily cono-
toxins.
Nevertheless, the cDNA structure of conomarphin
was similar to those of other conotoxin cDNAs. The
cDNA-encoded precursor of conomarphin consisted of

aN(i,i +1)
connectivities found in the C
a
H-NH fingerprint region
of the NOESY spectra for conomarphin and l-Phe13-
conomarphin are represented in supplementary
Fig. S6A,B, respectively. The NOESY data acquired
at 300 K and pH 3 for conomarphin and l-Phe13-
conomarphin showed a large number of NOEs, which
suggested that the structures of the two peptides were
sufficiently constrained for distance–geometry calcula-
tions.
Structural calculation, refinement, and evaluation
The NMR experimental data were converted into dis-
tance and angle constraints as usual, providing enough
constraints for the structure calculation of conomar-
phin. The three-dimensional structure of conomarphin
was determined from NMR data using the same strat-
egy previously used for structural studies of conotoxins
and their analogs [24–27].
Most NOESY crosspeaks were assigned and inte-
grated, with concomitant cycles of structure calcula-
tions 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

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

tained 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 cono-
marphin 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 3
10
-
helix. The helix was supported by O
i
-HN
i +3
hydro-

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
3
J
HN-Ha
cou-
pling constants for residues Asn11 and Ser12 and the
d
NN(i,i +2)
, d
aN(i,i +2)
[Ser12(C
a
H)–Trp14(NH) and
Asn11(C
a
H)–Phe13(NH)] and d
aN(i,i +3)
[Asn11
(C
a
H)–Trp14(NH)] NOEs in the region of resi-
dues 11–14 are in agreement with the presence of a
short 3
10
-helix. A random coil rather than a compact
loop in the region of residues 1–10 existed in

except for one Cys-free fraction having a novel
sequence of DWEYHAHPKONS
FWT with Pro10
hydroxylated and Phe13 in the d-conformation. Sev-
eral 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 con-
served 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 super-
family. 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 resi-
dues 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 pre-
viously. The enzyme responsible for this cleavage and

minus (+2), and positions 3 and 5 at the C-terminus
()3 and )5) (Table 2). It is noteworthy that epimeriza-
tion 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, epimeriza-
tion happens mainly at the +2 position in other
organisms. Probably, cone snails have developed a
more advanced system to achieve this difficult modifi-
cation at different positions. This is not surprising,
because of the well-known high content of post-trans-
lational modifications in conotoxins [8]. It is notewor-
thy that from the single species C. marmoreus, two
epimerization positions have been found, the )3 posi-
tion for conomarphin and the )5 position for glacon-
tryphan-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 natri-
uretic peptides from the Australian platypus [11].
With the help of such a developed post-translational
modification system, conotoxins exhibit amazing struc-
tural diversity. In this work, we found that conomar-
phin, 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 signifi-
cant effect on the structure of the peptide; a tight loop
around Pro8 and a short 3

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 con-
omarphins 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 Tech-
nologies (Santa Clara, CA, USA), and trifluoroacetic acid
and acetonitrile used for HPLC were from Merck (Darms-
tadt, Germany). Trypsin and tosyl phenylalanyl chloro-
methyl 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).

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 CaCl
2
buffer to a
concentration of 1 l gÆlL
)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 CaCl
2
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

and TOCSY spectra [44] of samples in 99.99% D
2
O and
90 : 10 H
2
O ⁄ D
2
O, 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 soft-
ware. Phase-shifted sine-squared window functions were
applied before Fourier transformation. To identify the slow
exchange of backbone amide protons, the hydrogen–deute-
rium exchange experiments were carried out by dissolving
the lyophilized sample in D
2
O and recording a series of one-
dimensional 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-dimen-
sional 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 dem-
onstrated a leveling of the intensity of the NOE at mixing
times greater than 200 ms. Peak picking, spin system identi-

1984 FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS
dihedral constraints were not applied to
3
J
NHa
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
NH
i
–O
j
bonds and 2.8–3.2 A
˚
for N
i
–O
j
bonds, respectively.
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

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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 d
aN(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 struc-
tures between conomarphin and l-Phe13-conomar-
phin.
Table S1. Proton resonance assignments (p.p.m.) for
conomarphin.