Solution structure of an M-1 conotoxin with a novel disulfide linkage Wei-Hong Du 1,2, *, Yu-Hong Han 3,4, *, Fei-juan Huang 1, *, Juan Li 2 , Cheng-Wu Chi 3,4 and Wei-Hai Fang 2 1 Department of Chemistry, Renmin University of China, Beijing, China 2 Department of Chemistry, Beijing Normal University, China 3 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai, China 4 Institute of Protein Research, Tongji University, Shanghai, China Over their 50 million years of evolution, cone snails have developed a series of small disulfide-rich peptides (conotoxins) in their venoms. Each peptide can selec- tively target a specific isoform of ion channel or mem- brane receptor [1,2]. Although it is estimated that each species of cone snail possesses 50–200 conotoxins in its arsenal, and there are more than 50 000 known cono- toxins, the majority belong to several gene super- families and only several structural motifs are widely shared. M-superfamily conotoxins form a group with a typ- ical cysteine arrangement of (-CC-C-C-CC-) and par- ticularly highly conserved signal peptide sequences. Depending on the number of residues located in the last cysteine loop, the M-superfamily has been provi- sionally divided into four branches, M-1, M-2, M-3 and M-4 [3]. The M-superfamily conotoxins l-, w- and jM-conotoxins (22–24 amino acids) have all been iden- tified from fish-hunting cone snails and belong to M-4 branch [4–6]. Although they have diverse molecular targets (Na + channel, nicotinic acetylcholine receptor and K + channel, respectively), they share a disulfide connectivity (C 1 –C 4 ,C 2 –C 5 ,C 3 –C 6 ) and common backbone scaffold [7]. In contrast to the M-4 branch, Keywords disulfide linkage; M-conotoxin; mr3e; NMR; solution structure Correspondence W H. Fang, Department of Chemistry, Beijing Normal University, 19 Xin Jie Kou Wai St., Beijing 100875, China Fax: +86 10 5880 2075 Tel: +86 10 5880 5382 E-mail: fangwh@bnu.edu.cn C W. Chi, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China Fax: +86 21 5492 1011 Tel: +86 21 5492 1165 E-mail: chi@sunm.shcnc.ac.cn *These authors contributed equally to this study (Received 10 December 2006, revised 7 March 2007, accepted 16 March 2007) doi:10.1111/j.1742-4658.2007.05795.x The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C- CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divi- ded into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D 1 H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six u dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 ± 0.19 A ˚ . Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a ‘flying bird’. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold. 2596 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS the other three branches of the M-superfamily are rel- atively small (12–19 amino acids) and are found mostly in mollusk- and worm-hunting cone snails. Disulfide linkage analyses of two M-2 branch conotoxins, mr3a and tx3c, have shown that they possess a distinctive disulfide bond arrangement of C 1 –C 6 ,C 2 –C 4 and C 3 –C 5 [3]. In addition, BtIIIB, another M-2 branch conotoxin from the venom of a vermivorous cone snail Conus betulinus, has been proven to have the same disulfide linkage as mr3a and tx3c [8]. Recently, a third disulfide bond arrangement within M-superfamily conotoxins has been characterized. Two M-1 branch conotoxins, mr3e (Fig. 1) and tx3a were found to have a new disulfide linkage (C 1 –C 5 ,C 2 –C 4 , C 3 –C 6 ), which differs from those seen in M-2 and M-4 branch conotoxins [9]. Here we report the 3D structure of mr3e, a novel M-1 branch conotoxin with the above new disulfide connectivity. Results Sequence-specific resonance assignments 2D NMR spectroscopy was used to investigate the 3D structure of conotoxin mr3e in aqueous solution at pH 3. Proton resonances for conotoxin mr3e were assigned using established methods [10]. Fourteen of the 16 spin systems were found in the ‘fingerprint’ region of a 120 ms TOCSY spectrum. TOCSY assign- ments for Val1, Pro4, Phe5, His9, Leu11, Tyr13 and Asp16 were verified in the fingerprint region of a DQF-COSY spectrum. The sequential assignments of amino acids in the primary sequence started with the unique residues Leu11 and His9. A NOESY ‘walk’ toward the N-ter- minus identified the residues from Leu11 to His9 and one toward the C-terminus identified the residues from Cys12 to Asp16. Two of the glycine spin systems iden- tified in the TOCSY spectrum were encountered in a NOESY walk at positions 6 and 7. Residues from Cys2 to Cys8 were then assigned along the ‘walk’. The phenylalanine at position 5 was confirmed during the process. Pro4 was assigned by its TOCSY spin system and the NOEs from its a-proton to the amide proton of Phe5, and from its d-proton to the a-proton of Gly7. The valine residue at position 1 was finally assigned on the basis of NOEs from the a- and b-pro- tons of Val1 to the amide proton of Cys2. The amide proton of Val1 disappeared in the H 2 O spectrum, possibly because of its special position at the N-terminus and fast exchange in water. NOESY data acquired at 294 K for conotoxin mr3e showed a large number of NOEs which suggested that the structure of the peptide was sufficiently constrained for distance- geometry calculations. Figure 2 shows the sequential d aN(i,i+1) connectivities on the CaH-NH fingerprint region of the NOESY spectrum with a mixing time of 200 ms. All chemical shifts are listed in Table 1. Structure calculation and evaluation NMR experiments provided enough distance and angle constraints to calculate the structure of mr3e. The con- straints for structure elucidation were determined from a survey of NMR data using the traditional visual ana- lysis method developed by Wuthrich [10]. In total, 169 distance constraints were obtained from the 200-ms NOESY spectrum. Six u angle constraints and three disulfide bonds from Cys2 to Cys14, Cys3 to Cys12, and Cys8 to Cys15 were added to the distance constraints for primary structure determination. A set of 20 structures was generated with a mean global rmsd of 1.88 A ˚ using the dyana (v. 1.5) [11] software package. The lowest energy structure was then dis- played, and ambiguous NOESY signals were evaluated Fig. 1. Conotoxin mr3e sequence and its disulfide linkage. 3.5 E10 V1 G6 F5 Y13 L11 C3 D16 C8 C2 C15 G7 C14 C12 H9 4.0 4.5 5.0 9.5 9.0 8.5 8.0 7.5 D1 (p.p.m.) D2 (p.p.m.) Fig. 2. Sequential d aN(i,i+1) connectivities in the CaH-NH fingerprint region of the NOESY spectrum. The mixing time for the NOESY spectrum is 200 ms. Sequential d aN connectivities are shown for residues 1–3 and 5–16. Residue 4 is proline. W H. Du et al. Solution structure of an M-1 conotoxin FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2597 compared with those of the partially minimized struc- ture. Twenty-one distance constraints were added on the basis of this analysis, and the minimization process was repeated to generate a set of 15 structures with a mean global rmsd of 0.69 A ˚ . dyana was used to provide hydrogen-bond informa- tion during the minimization. Deuterium-exchange studies indicated that hydrogen bonds might form exCysist between the amide protons of Gly7, Cys8 and Cys12 and nearby oxygen or nitrogen atoms. The reso- nances of amide protons in these residues were not diminished after 3 h in D 2 O at 294 K in a 1D proton time course experiment. dyana provided hydrogen bond acceptor oxygen and nitrogen atoms for each of the amide protons from these four residues. The hydrogen bonds for Gly7, Cys8 and Cys12 were used as constraints. Thus, six upper and six lower distance constraints were added for the hydrogen-bond interac- tions, and another round of minimization was per- formed. The result was a final set of 20 structures with a mean global backbone rmsd of 0.56 ± 0.16 A ˚ and a mean global heavy atom rmsd of 1.30 ± 0.28 A ˚ . Finally, refinement of the structure was carried out using amber 5 [12] for energy minimization. An ensemble of 20 structures with lower energy and better Ramachandran plots was chosen to represent the 3D solution fold of conotoxin mr3e and the mean struc- ture was generated using molmol [13]. The program procheck was used to analyze the family of 20 struc- tures [14]. Structural statistics are shown in Table 2. The 20 structures converged to a common fold; the rmsd values of 20 structures are low. The coordinates for the family of 20 structures and NMR constraints file have been deposited in the Brookhaven Protein Data Bank (PDB) with accession number 2EFZ. 3D structure of mr3e Figure 3 shows an overlay of the backbone atoms for the 20 structures of mr3e. The overall rmsd reported for the final 20 structures (0.43 ± 0.19 A ˚ ) is influenced by disorder in the C-terminal residue Asp16. When Asp16 is eliminated and the molecule is minimized by considering the first 15 residues only, the mean global backbone rmsd decreases from 0.43 to 0.24 A ˚ . Unlike the N-terminal portion, the C-terminal portion of the molecule is poorly resolved. The refined structure of conotoxin mr3e contains two turns defined by residues Phe5 to Cys8 and His9 to Cys12 (Fig. 4). The residues from Phe5 to Cys8 are characteristic of a type I b-turn with a glycine residue (Gly7) at position i+2. The glycine residue is required to accommodate the necessary angle constraints of the turn. The second turn in the region between His9 and Cys12 is apparently stabilized by a hydrogen bond between the carbonyl oxygen of His9 and the amide proton of Cys12. The interaction is characteristic of a type II b-turn. Table 1. Proton resonance assignments (p.p.m.) for mr3e. Residue HN abOther Val1 3.88 2.25 c: 1.06 ± 1.03 Cys2 8.79 4.92 2.72, 2.51 Cys3 8.49 4.49 3.72, 3.47 Pro4 4.6 2.30, 2.03 c: 2.11, 1.81 d: 3.77, 3.61 Phe5 8.75 4.2 3.05, 2.98 d: 7.26 e: 7.31 f: 7.54 Gly6 8.65 4.03, 3.53 Gly7 8.18 4.58, 3.31 Cys8 8.42 4.7 3.06 His9 7.3 4.89 3.53, 3.26 d: 7.29 e: 8.67 Glu10 8.92 4.15 2.13 c: 2.55 Leu11 8.45 4.08 2.01, 1.72 c: 1.61 d: 0.94 ± 0.89 Cys12 7.57 4.46 3.22, 3.17 Tyr13 9.08 4.34 3.23, 3.04 d: 7.17 e: 6.88 Cys14 7.96 4.64 3.79, 3.16 Cys15 9.44 5.07 3.42, 3.03 Asp16 8.76 4.49 2.69, 2.52 Table 2. Structural statistics for the family of 20 structures of cono- toxin mr3e. Experimental constraints Number Intraresidual 122 Sequential (|I ) j | ¼ 1) 48 Medium range 7 Long range 13 AMBER energies, kcalÆmol )1 Bond 2.822 ± 0.136 Angle 34.311 ± 1.454 Dihedral 36.114 ± 2.807 VDW )55.829 ± 1.478 EEL )442.969 ± 13.886 H-bond )4.178 ± 1.079 Constraints 2.551 ± 0.438 Total )98.210 ± 8.976 rmsd to mean coordinates Backbone atoms 0.43 ± 0.19 A ˚ Nonhydrogen heavy atoms: 1.26 ± 0.30 A ˚ Rachandran statistics from PROCHECK-NMR Most favored regions, % 78.6 Additional allowed regions, % 14.1 Generously allowed regions, % 7.3 Disallowed regions, % 0 Solution structure of an M-1 conotoxin W H. Du et al. 2598 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS The 3D structure of mr3e is well defined. Figure 5 shows the backbone structure along with front, side and back views of the surface of the peptide. The double- turn conformation in conotoxin mr3e produces an over- all shape of a ‘flying bird’ when viewed from the front. Discussion M-superfamily conotoxins, one of the major groups of disulfide-rich peptides, are widely distributed in the venoms of all three feeding types of cone snails. Depending on the number of residues located in the last Cys loop, M-superfamily conotoxins have been provisionally divided into four branches, namely M-1, -2, -3, -4. Interestingly, to the best of our knowledge, three different disulfide linkages can be found in M-1 (1–5, 2–4, 3–6), M-2 (1–6, 2–4, 3–5) and M-4 (1–4, 2–5, 3–6) branch conotoxins, respectively. mr3e is an M-1 branch conotoxin purified from the venom of a mollusk-hunting cone snail, C. marmoreus; it has 16 amino acids in its mature peptide. Previously, we have shown that mr3e is characterized by its dis- tinctive disulfide connectivity (C 1 –C 5 ,C 2 –C 4 ,C 3 –C 6 ) [9], which is completely different from those of well- studied l-, w- and jM-conotoxins (M-4 branch) and the recently reported, comparatively small, excitory M-superfamily conotoxins mr3a, mr3b and tx3c (M-2 branch). In this report, we show that there are two classic b-turns involved in the tertiary structure of mr3e (Fig. 6A). The backbone conformation of mr3e is different from that of the M-2 branch conotoxin mr3a, which possesses a distinctive triple-turn back- bone structure motif (Fig. 6B) [15]. Such a triple-turn motif makes the mr3a molecule fold into a tight and globular structure (Fig. 6D). By contrast, the double- turn motif of mr3e, which apparently results from its differing disulfide bond arrangement, gives the mr3e a more irregular overall molecular shape, with the side Fig. 4. Backbone peptide folding of mr3e. Turn 1 between Phe5 and Cys8 and turn 2 between His9 and Cys12 are shown in green. Fig. 3. Overlay of the backbone atoms for the 20 converged struc- tures of conotoxin mr3e. The C-terminal Asp is seen to be in a poorly resolved region of the molecule. Backbone Front Back Side Fig. 5. 3D structure of mr3e. The backbone structure is shown along with front, back, and side views of the surface of M-1 branch conotoxin mr3e. Blue regions are hydrophobic, and red regions are hydrophilic. W H. Du et al. Solution structure of an M-1 conotoxin FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2599 chains of several amino acids protruding outside the molecule (Fig. 6C). In contrast to the typical excitory symptoms, such as circular movements, barrel rolling and convulsions, elicited by cranial injection of mr3a [3], mr3e has no obvious effect on mice [9]. Therefore, it is most likely that these two conotoxins have different physiological functions, and this is not surprising considering that they have completely different backbone scaffolds. Although M-1 and M-2 branch conotoxins are similar in size and cysteine framework, and are all abundant in mollusk- and worm-hunting cone snails, more evi- dence has emerged that they are phylogenetically diver- gent groups. These two groups of M-superfamily conotoxins differ with respect to signal peptide sequence, disulfide linkage, backbone scaffold and most likely molecular target. It seems to be a favored strategy of cone snails to generate different backbone scaffolds within conotox- ins by introducing different disulfide linkages into conotoxins that share the same cysteine framework. For instance, a-conotoxin and v-conotoxin share the same ‘-CC-C-C-’ cysteine framework, but differ greatly in disulfide linkage, backbone scaffold and conse- quently molecular target [16–18]. Such a strategy, which yields more structural and functional diversity in the conotoxins, will help cone snails to survive severe environmental pressures. Experimental procedures Peptide synthesis and refolding mr3e was chemically synthesized as described previously [9]. Linear peptide was oxidized by air in 50 mm NH 4 HCO 3 buffer and purified on a semi-preparative C 18 reverse-phase column. The final product was coapplied with native mr3e to an analytical C 18 reverse-phase column to verify its identity. NMR experiments Samples for NMR experiments were prepared at a concentra- tion of  2.0 mm in either 99.99% D 2 O (Cambridge Isotopes, Andover, MA, USA) or 9 : 1(v ⁄ v) H 2 O ⁄ D 2 O with 0.01% trifluoroacetic acid, at pH 3.0. NMR measurements were performed using standard pulse sequences and phase cycling on a Bruker Avance 500 NMR spectrometer at 294 K. Proton DQF-COSY, NOESY and TOCSY spectra in 99.99% D 2 O and in 9 : 1 H 2 O ⁄ D 2 O(v⁄ v) were acquired with the transmitter set at 4.80 p.p.m. and a spectral win- dow of 6000 Hz. All 2D NMR spectra were acquired in a phase-sensitive mode using time-proportional phase incre- mentation for quadrature detection in the t 1 dimension. Presaturation during the relaxation delay period was used to solvent resonance. A series of NOESY spectra was acquired with mixing times of 400, 200, 150 100 and 50 ms. TOCSY spectra under both solvent conditions were acquired with a mixing time of 120 ms. Spectra were processed using xwinnmr or topspin soft- ware. Phase-shifted sine-squared window functions were applied before Fourier transformation, with shifts of 60 or 90 ° in both dimensions. Final matrix sizes were usually 2048 · 2048 real points. To identify the slow exchange of backbone amide protons, the sample lyophilized from a H 2 O solution was redissolved in D 2 O. 1D 1 H spectra were measured after 5 min, and subsequently every 0.5 h up to 20 h. Chemical shifts were referenced to the methyl reson- ance of 4,4-dimethyl-4-silapentane-1-sulfonic acid used as an internal standard. Distance restraints and structure calculations An initial survey of distance constraints was performed on a series of NOESY spectra acquired at mixing times of 400, 200, 150, 100 and 50 ms. Build-up curves were produced which showed a leveling of the intensity of the NOE at mix- ing times > 200 ms. Quantitative determination of the cross-peak intensities was based on counting the contour levels. Off-diagonal resonances were classified as strong, medium or weak on the basis of their relative intensities and set to distance constraints of 1.8–2.5, 1.8–3.5, and 1.8–5.5 A ˚ , respectively. A set of 96 intra- and interproton distance restraints, representing unambiguously assigned dipolar AB CD Fig. 6. Comparison of 3D structure of mr3e (M-1 branch conotoxin) and mr3a (M-2 branch conotoxin). (A,B) Backbone conformation of mr3e and mr3a. (C,D) Surface representation of mr3e and mr3a. Blue regions are hydrophobic, and red regions are hydrophilic. Solution structure of an M-1 conotoxin W H. Du et al. 2600 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS couplings, was generated from the data and used as input for dyana (V.1.5). Six u dihedral angles were determined on the basis of the 3 J NHa coupling constants derived by analysis of a high resolution 1D proton spectrum of conotoxin mr3e. The u angle constraints were set to )120 ± 40° for 3 J NHa > 8.0 Hz (Gly7, Glu10) and to )65±25° for 3 J NHa < 5.5 Hz (Cys3, Phe5, Leu11, Cys12). Backbone dihedral constraints were not applied for 3 J NHa values between 5.5 and 8.0 Hz. After the initial calculation, hydrogen-bonds constraints were added as target values of 2.2 A ˚ for NH(i)– O(j) and 3.2 A ˚ for N(i)–O(j), respectively. One thousand random structures were generated by dyana (v. 1.5) that fit the primary sequence and covalent and spatial requirements of mr3e. A total of 190 distance constraints, six u angle restraints and three hydrogen bonds constraints were input for the molecular modeling protocol for the dyana algorithm. The outcome was a set of 20 structures with a mean global rmsd of 0.56 ± 0.16 A ˚ and a mean global heavy atom rmsd of 1.30 ± 0.28 A ˚ . Structural refinement was carried out using amber 5 and structure quality was analyzed using molmol and procheck-nmr. Acknowledgements This work was supported by the National Basic Research Program of China (2004CB719900) and the National Natural Science Foundation of China (20473013). 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Solution structure of an M-1 conotoxin FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS 2601 zation of a novel conus peptide with apparent antinoci- ceptive activity. J Biol Chem 275, 32391–32397. 18 Sharpe IA, Gehrmann J, Loughnan ML, Thomas L, Adams DA, Atkins A, Palant E, Craik DJ, Adams DJ, Alewood PF et al. (2001) Two new classes of conopep- tides inhibit the alpha1-adrenoceptor and noradrenaline transporter. Nat Neurosci 4, 902–907. Solution structure of an M-1 conotoxin W H. Du et al. 2602 FEBS Journal 274 (2007) 2596–2602 ª 2007 The Authors Journal compilation ª 2007 FEBS . the dyana algorithm. The outcome was a set of 20 structures with a mean global rmsd of 0.56 ± 0.16 A ˚ and a mean global heavy atom rmsd of 1.30 ± 0.28 A ˚ . Structural refinement was carried. Solution structure of an M-1 conotoxin with a novel disulfide linkage Wei-Hong Du 1,2, *, Yu-Hong Han 3,4, *, Fei-juan Huang 1, *, Juan Li 2 , Cheng-Wu Chi 3,4 and Wei-Hai Fang 2 1 Department. unambiguously assigned dipolar AB CD Fig. 6. Comparison of 3D structure of mr3e (M-1 branch conotoxin) and mr 3a (M-2 branch conotoxin) . (A, B) Backbone conformation of mr3e and mr 3a. (C,D) Surface