For example, most of the M-super-family conotoxins have the ‘-CC-C-C-CC-’ pattern, Keywords conomarphin; Conus marmoreus; D -Phe; M-superfamily; NMR structure Correspondence C.. Conomarp
Trang 1acid-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 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
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
frame-work in the primary sequence and their different tar-gets [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 sig-nal 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-super-family conotoxins have the ‘-CC-C-C-CC-’ pattern,
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: chunguangwang@mail.tongji.edu.cn
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: whdu@chem.ruc.edu.cn
*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 conocono-marphin 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 310-helix at the C-terminus By con-trast, no loop in l-Phe13-conomarphin was found, which suggests that
d-Phe13 is essential for the structure of conomarphin In conclusion, cono-marphin may represent a new conotoxin family, whose biological activity remains to be identified
Trang 2but 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
amida-tion, 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
mod-ifications to take place in a defined cluster of gene
products Cone snails have probably developed a
com-plicated 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
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 peppep-tide 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 shares no sequence homology with other M-superfam-ily conotoxins Its solution structure, including the
Table 1 The identified cysteine-free conopeptide families c, c-carboxylated glutamate; *, amidated C-terminus; , glycosylation; O, hydroxy-proline; V, D -Val; F, D -Phe; NMDA, N-methyl- D -aspartate.
Family Example Sequence Target Reference
Conantokin Conantokin-G GEccLQcNQcLIRcKSN* NMDA receptor McIntosh et al [51] Contulakin Contulakin-G ZSEEGGSNAT KKPYIL Neurotensin receptor Craig et al [52] Conorfamide Conorfamide GPMGWVPVFYRF* FR amide receptor Maillo et al [53] Conomap Conomap-Vt AFVKGSAQRVAHGY* Unknown Dutertre et al [19] Conophan Conophan gld-V AOANSVWS Unknown Pisarewicz et al [20] Conomarphin Conomarphin DWEYHAHPKONSFWT Unknown This work
Table 2 D -Amino acid-containing peptides from different organisms D -Amino acid residues are underlined *, amidated C-terminus; O, hydroxyproline; c, c-carboxylated glutamate.
Organism Example Sequence Position Reference
Cone snail Conomarphin DWEYHAHPKONSFWT )3 This work
r11a GOSFCKADEKOCEYHADCCNCCLSGICAOSTNWILPGCSTSSFFKI )3 Buczek et al [35] Contryphan-R GCOWEPWC* )5 Jimenez et al [13] Glacontryphan-M NcScCPWHPWC* )5 Hansson et al [16] Conophan gld-V AOANSVWS )3 Pisarewicz et al [20] Conomap-Vn AFVKGSAQRVAHGY* +2 Dutertre et al [19] Snail Fulicin FNEFV* +2 Ohta et al [12] Frog Dermorphin YAFGYPS* +2 Montecucchi et al [9] Spider x-Agatoxin EDNCIAEDYGKCTWGGTKCCRGRPCRCSMIGTNCECTPRLIMEGLSFA )3 Kuwada et al [10] Australian
platypus
C-type natriuretic
peptide
LLHDHPNPRKYKPANKKGLSKGCFGLKLDRIGSTSGLGC +2 Torres et al [11] Defensin-like peptide IMFFEMQACWSHSGVCRDKSERNCKPMAWTYCENRNQKCCEY +2 Torres et al [39]
Trang 3effect 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
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 corre-sponded to a novel 15 amino acid peptide with the
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 in conomarphin
In order to obtain more material for structural studies, conomarphin was chemically synthesized using stan-dard Fmoc–l-amino acids However, to our great sur-prise, 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 conomar-phin 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 syn-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
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,
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 mLÆmin)1 The peak labeled with an asterisk is conomarphin.
Trang 4retention times, which indicated that one or more of
the four C-terminal amino acids of natural
conomar-phin 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-configu-ration 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
(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
a conserved M-superfamily signal peptide of 25 resi-dues, a proregion of 29 resiresi-dues, the conomarphin mature peptide, and the two additional residues, Leu and Val, at the C-terminus, which are cleaved during maturation
Fig 2 cDNA and the deduced precursor
sequence of conomarphin The signal
pep-tide is shadowed and the mature peppep-tide is
underlined The polyA signal AATAAA in the
3¢-UTR is also underlined The cDNA of
con-omarphin has been deposited in the
Gen-bank database with the accession number
EU048276.
Trang 5Sequence-specific resonance assignments
Complete proton resonances for both conomarphin
and l-Phe13-conomarphin were assigned by
well-established 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
spec-tra 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
veri-fied in a relevant DQF-COSY spectrum For
l-Phe13-conomarphin, 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-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
used from J coupling constants For
l-Phe13-conomar-phin, 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
exper-iments indicated the hydrogen bonds that might exist
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 addi-tional 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
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 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
(2–15)
0.54 ± 0.23 0.63 ± 0.30 Mean global heavy
rmsd (2–15)
1.59 ± 0.46 1.88 ± 0.44 Ramachandran statistics from PROCHECK-NMR
Most favored regions (%) 87.8 68.9 Additional allowed
regions (%)
12.2 31.1 Generously allowed
regions (%)
Disallowed regions (%) 0 0
Trang 6refined for simulated annealing and restrained energy
minimization [24,28], using the sander module of the
amber9.0 package The resulting conformers
con-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-conocono-marphin (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 310 -helix The helix was supported by Oi-HNi+ 3 hydro-gen 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 con-tained 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 cou-pling 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 resi-dues 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
conomar-phin (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.
Trang 7Cone 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
identi-fied 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
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
the recognition sequence for post-translational
modifi-cations, namely hydroxylation of Pro10 and
epimeriza-tion 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
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 neurotrans-mitters 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 pep-tide 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-ter-minus (+2), and positions 3 and 5 at the C-terN-ter-minus ()3 and )5) (Table 2) It is noteworthy that epimeriza-tion at each of these three posiepimeriza-tions 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 posiepimeriza-tion 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 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
Trang 8helix was two residues longer Superimposition of the
C-terminal helices of conomarphin and
l-Phe13-cono-marphin 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
glacontry-phan-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
ter-minal 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
dra-matically 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 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)
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
cono-marphin is shown in stick mode, and the
side chain of L -Phe13 in L
-Phe13-conomar-phin is shown as a gray stick.
Trang 9The 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
chemis-try 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 lgÆlL)1 Trypsin was added to a ratio of
1 : 20 The digestion was carried out at 25C 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 CaCl2buffer with the same
peptide concentration and enzyme ratio The reactions were
kept at 25C 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
resulting PCR products were inserted directly into the pGEM-T Easy vector for sequencing
NMR experiments
Samples of conomarphin and l-Phe13-conomarphin for NMR studies were prepared in either 99.99% D2O (Cam-bridge 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 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 D2O 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-fication and volume integration of the NOESY crosspeaks were performed with the interactive program sparky (v 1.113) Non-stereospecifically assigned atoms were trea-ted 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
JNHacoupling constants derived by analysis of a high-res-olution one-dimensional proton spectrum of the conotoxin conomarphin For peaks that did not show a splitting pattern, the 3
JNHa 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 3
JNHa values > 8.0 Hz, it was constrained in the range )120 ± 40 [21,45] Backbone
Trang 10dihedral 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–Ojbonds and 2.8–3.2 A˚ for Ni–Ojbonds, 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
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
cono-marphin was done using molmol [49] software, and the
geometric qualities of the obtained structures were assessed
with procheck-nmr software [50]
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-YW-R-104) Chunguang Wang is supported by the
Pro-gram for young excellent talents in Tongji University
(2006KJ063) and Dawn Program of Shanghai
Educa-tional Development Foundation (06SG26)
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