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 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: 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 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 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 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 FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 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 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 0–10 100% Buffer A, 10–20 0–27% Buffer B, 20–25 27% Buffer B, 25–58 27–42.5% Buffer B, and 58–63 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 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 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 FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 1979 A D-amino acid-containing conomarphin Y Han et al Sequence-specific resonance assignments Table 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 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 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 regions (%) Disallowed regions (%) 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 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 + 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 + 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 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 FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 1981 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 at the N-terminus (+2), and positions and 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 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 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) FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 lgỈlL)1 Trypsin was added to a ratio of : 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 : (v ⁄ v) H2O ⁄ D2O with 0.01% trifluoroacetic acid, at pH (uncorrected for the isotope effect), with a final sample concentration of approximately 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 for 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 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 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 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 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] 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 References Terlau H & Olivera BM (2004) Conus venoms: a rich source of novel ion channel-targeted peptides Physiol Rev 84, 41–68 Olivera BM (2006) Conus peptides: biodiversity-based discovery and exogenomics J Biol Chem 281, 31173–31177 Shon KJ, Olivera BM, Watkins M, Jacobsen RB, Gray WR, Floresca CZ, Cruz LJ, Hillyard DR, Brink A, Terlau H et al (1998) mu-Conotoxin PIIIA, a new peptide for discriminating among tetrodotoxin-sensitive Na channel subtypes J Neurosci 18, 4473–4481 Shon KJ, Grilley M, Jacobsen R, Cartier GE, Hopkins C, Gray WR, Watkins M, Hillyard DR, Rivier J, 13 14 15 Torres J et al (1997) A noncompetitive peptide inhibitor of the nicotinic acetylcholine receptor from Conus purpurascens venom Biochemistry 36, 9581–9587 Ferber M, Sporning A, Jeserich G, DeLaCruz R, Watkins M, Olivera BM & Terlau H (2003) A novel conus peptide ligand for K+ channels J Biol Chem 278, 2177–2183 Han YH, Wang Q, Jiang H, Liu L, Xiao C, Yuan DD, Shao XX, Dai QY, Cheng JS & Chi CW (2006) Characterization of novel M-superfamily conotoxins with new disulfide linkage FEBS J 273, 4972–4982 Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR et al (2005) Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms Biochemistry 44, 8176–8186 Buczek O, Bulaj G & Olivera BM (2005) Conotoxins and the posttranslational modification of secreted gene products Cell Mol Life Sci 62, 3067–3079 Montecucchi PC, de Castiglione R, Piani S, Gozzini L & Erspamer V (1981) Amino acid composition and sequence of dermorphin, a novel opiate-like peptide from the skin of Phyllomedusa sauvagei Int J Pept Protein Res 17, 275–283 Kuwada M, Teramoto T, Kumagaye KY, Nakajima K, Watanabe T, Kawai T, Kawakami Y, Niidome T, Sawada K, Nishizawa Y et al (1994) Omega-agatoxinTK containing D-serine at position 46, but not synthetic omega-[L-Ser46]agatoxin-TK, exerts blockade of P-type calcium channels in cerebellar Purkinje neurons Mol Pharmacol 46, 587–593 Torres AM, Menz I, Alewood PF, Bansal P, Lahnstein J, Gallagher CH & Kuchel PW (2002) d-Amino acid residue in the C-type natriuretic peptide from the venom of the mammal, Ornithorhynchus anatinus, the Australian platypus FEBS Lett 524, 172–176 Ohta N, Kubota I, Takao T, Shimonishi Y, Yasuda-Kamatani Y, Minakata H, Nomoto K, Muneoka Y & Kobayashi M (1991) Fulicin, a novel neuropeptide containing a D-amino acid residue isolated from the ganglia of Achatina fulica Biochem Biophys Res Commun 178, 486–493 Jimenez EC, Olivera BM, Gray WR & Cruz LJ (1996) Contryphan is a D-tryptophan-containing Conus peptide J Biol Chem 271, 28002–28005 Jimenez EC, Watkins M, Juszczak LJ, Cruz LJ & Olivera BM (2001) Contryphans from Conus textile venom ducts Toxicon 39, 803–808 Massilia GR, Eliseo T, Grolleau F, Lapied B, Barbier J, Bournaud R, Molgo J, Cicero DO, Paci M, Schinina ME et al (2003) Contryphan-Vn: a modulator of Ca2+-dependent K+ channels Biochem Biophys Res Commun 303, 238–246 FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS 1985 A D-amino acid-containing conomarphin Y Han et al 16 Hansson K, Ma X, Eliasson L, Czerwiec E, Furie B, Furie BC, Rorsman P & Stenflo J (2004) The first gamma-carboxyglutamic acid-containing contryphan A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus J Biol Chem 279, 32453–32463 17 Jimenez EC, Craig AG, Watkins M, Hillyard DR, Gray WR, Gulyas J, Rivier JE, Cruz LJ & Olivera BM (1997) Bromocontryphan: post-translational bromination of tryptophan Biochemistry 36, 989– 994 18 Buczek O, Yoshikami D, Watkins M, Bulaj G, Jimenez EC & Olivera BM (2005) Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily FEBS J 272, 4178–4188 19 Dutertre S, Lumsden NG, Alewood PF & Lewis RJ (2006) Isolation and characterisation of conomap-Vt, a D-amino acid containing excitatory peptide from the venom of a vermivorous cone snail FEBS Lett 580, 3860–3866 20 Pisarewicz K, Mora D, Pflueger FC, Fields GB & Mari F (2005) Polypeptide chains containing D-gammahydroxyvaline J Am Chem Soc 127, 6207–6215 21 Pardi A, Billeter M & Wuthrich K (1984) Calibration of the angular dependence of the amide proton-C alpha proton coupling constants, 3JHN alpha, in a globular protein Use of 3JHN alpha for identification of helical secondary structure J Mol Biol 180, 741–751 22 Du W, Han Y, Huang F, Li J, Chi C & Fang W (2007) Solution structure of an M-1 conotoxin with a novel disulfide linkage FEBS J 274, 2596–2602 23 McDougal OM & Poulter CD (2004) Three-dimensional structure of the mini-M conotoxin mr3a Biochemistry 43, 425–429 24 Rogers JP, Luginbuhl P, Shen GS, McCabe RT, Stevens RC & Wemmer DE (1999) NMR solution structure of alpha-conotoxin ImI and comparison to other conotoxins specific for neuronal nicotinic acetylcholine receptors Biochemistry 38, 3874–3882 25 Al-Sabi A, Lennartz D, Ferber M, Gulyas J, Rivier JE, Olivera BM, Carlomagno T & Terlau H (2004) KappaM-conotoxin RIIIK, structural and functional novelty in a K+ channel antagonist Biochemistry 43, 8625–8635 26 Nielsen KJ, Adams DA, Alewood PF, Lewis RJ, Thomas L, Schroeder T & Craik DJ (1999) Effects of chirality at Tyr13 on the structure–activity relationships of omega-conotoxins from Conus magus Biochemistry 38, 6741–6751 27 Hofmann MW, Poschner BC, Hauser S & Langosch D (2007) pH-Activated fusogenic transmembrane LV-peptides Biochemistry 46, 4204–4209 28 Zhang N, Chen X, Li M, Cao C, Wang Y, Wu G, Hu G & Wu H (2004) Solution structure of BmKK4, 1986 29 30 31 32 33 34 35 36 37 38 39 40 the first member of subfamily alpha-KTx 17 of scorpion toxins Biochemistry 43, 12469–12476 Yuan DD, Han YH, Wang CG & Chi CW (2007) From the identification of gene organization of alpha conotoxins to the cloning of novel toxins Toxicon 49, 1135–1149 Fan CX, Chen XK, Zhang C, Wang LX, Duan KL, He LL, Cao Y, Liu SY, Zhong MN, Ulens C et al (2003) A novel conotoxin from Conus betulinus, kappa-BtX, unique in cysteine pattern and in function as a specific BK channel modulator J Biol Chem 278, 12624–12633 Richter K, Egger R & Kreil G (1987) D-alanine in the frog skin peptide dermorphin is derived from L-alanine in the precursor Science 238, 200–202 Jilek A, Mollay C, Tippelt C, Grassi J, Mignogna G, Mullegger J, Sander V, Fehrer C, Barra D & Kreil G (2005) Biosynthesis of a D-amino acid in peptide linkage by an enzyme from frog skin secretions Proc Natl Acad Sci USA 102, 4235–4239 Shikata Y, Watanabe T, Teramoto T, Inoue A, Kawakami Y, Nishizawa Y, Katayama K & Kuwada M (1995) Isolation and characterization of a peptide isomerase from funnel web spider venom J Biol Chem 270, 16719–16723 Torres AM, Tsampazi M, Tsampazi C, Kennett EC, Belov K, Geraghty DP, Bansal PS, Alewood PF & Kuchel PW (2006) Mammalian l-to-d-amino-acid-residue isomerase from platypus venom FEBS Lett 580, 1587–1591 Buczek O, Yoshikami D, Bulaj G, Jimenez EC & Olivera BM (2005) Post-translational amino acid isomerization: a functionally important D-amino acid in an excitatory peptide J Biol Chem 280, 4247–4253 Grant MA, Hansson K, Furie BC, Furie B, Stenflo J & Rigby AC (2004) The metal-free and calcium-bound structures of a gamma-carboxyglutamic acid-containing contryphan from Conus marmoreus, glacontryphan-M J Biol Chem 279, 32464–32473 Pallaghy PK, Melnikova AP, Jimenez EC, Olivera BM & Norton RS (1999) Solution structure of contryphanR, a naturally occurring disulfide-bridged octapeptide containing D-tryptophan: comparison with protein loops Biochemistry 38, 11553–11559 Eliseo T, Cicero DO, Romeo C, Schinina ME, Massilia GR, Polticelli F, Ascenzi P & Paci M (2004) Solution structure of the cyclic peptide contryphan-Vn, a Ca2+-dependent K+ channel modulator Biopolymers 74, 189–198 Torres AM, Tsampazi C, Geraghty DP, Bansal PS, Alewood PF & Kuchel PW (2005) D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties Biochem J 391, 215–220 Buczek O, Wei D, Babon JJ, Yang X, Fiedler B, Chen P, Yoshikami D, Olivera BM, Bulaj G & Norton RS FEBS Journal 275 (2008) 1976–1987 ª 2008 The Authors Journal compilation ª 2008 FEBS Y Han et al 41 42 43 44 45 46 47 48 49 50 (2007) Structure and sodium channel activity of an excitatory I(1)-superfamily Conotoxin Biochemistry 46, 9929–9940 Pi C, Liu J, Peng C, Liu Y, Jiang X, Zhao Y, Tang S, Wang L, Dong M, Chen S et al (2006) Diversity and evolution of conotoxins based on gene expression profiling of Conus litteratus Genomics 88, 809–819 Rance M, Sorensen OW, Bodenhausen G, Wagner G, Ernst RR & Wuthrich K (1983) Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering Biochem Biophys Res Commun 117, 479–485 Jeener J, Meier BH, Bachmann P & Ernst RR (1979) Investigation of exchange processes by two-dimension NMR spectroscopy J Chem Phys 71, 4546–4553 Braunschweiler L & Ernst RR (1983) Coherence transfer by isotropic mixing: application to proton correlation spectroscopy J Magn Reson 53, 521–528 Kline AD, Braun W & Wuthrich K (1988) Determination of the complete three-dimensional structure of the alpha-amylase inhibitor tendamistat in aqueous solution by nuclear magnetic resonance and distance geometry J Mol Biol 204, 675–724 Fletcher JI, Smith R, O’Donoghue SI, Nilges M, Connor M, Howden ME, Christie MJ & King GF (1997) The structure of a novel insecticidal neurotoxin, omega-atracotoxin-HV1, from the venom of an Australian funnel web spider Nat Struct Biol 4, 559– 566 Fletcher JI, Chapman BE, Mackay JP, Howden ME & King GF (1997) The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site neurotoxins to the voltage-gated sodium channel Structure 5, 1525–1535 Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW & Kollman PA (1995) A second generation force field for the simulation of proteins, nucleic acids, and organic molecules J Am Chem Soc 117, 5179–5197 Koradi R, Billeter M & Wuthrich K (1996) MOLMOL: a program for display and analysis of macromolecular structures J Mol Graph 14, 51–55, 29–32 Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R & Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR J Biomol NMR 8, 477–486 A D-amino acid-containing conomarphin 51 McIntosh JM, Olivera BM, Cruz LJ & Gray WR (1984) Gamma-carboxyglutamate in a neuroactive toxin J Biol Chem 259, 14343–14346 52 Craig AG, Norberg T, Griffin D, Hoeger C, Akhtar M, Schmidt K, Low W, Dykert J, Richelson E, Navarro V et al (1999) Contulakin-G, an O-glycosylated invertebrate neurotensin J Biol Chem 274, 13752–13759 53 Maillo M, Aguilar MB, Lopez-Vera E, Craig AG, Bulaj G, Olivera BM & Heimer de la Cotera EP (2002) Conorfamide, a Conus venom peptide belonging to the RFamide family of neuropeptides Toxicon 40, 401–407 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 http://www.blackwell-synergy.com 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 ... Kumagaye KY, Nakajima K, Watanabe T, Kawai T, Kawakami Y, Niidome T, Sawada K, Nishizawa Y et al (1994) Omega-agatoxinTK containing D-serine at position 46, but not synthetic omega-[L-Ser46]agatoxin-TK,... Inoue A, Kawakami Y, Nishizawa Y, Katayama K & Kuwada M (1995) Isolation and characterization of a peptide isomerase from funnel web spider venom J Biol Chem 270, 16719–16723 Torres AM, Tsampazi... 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