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Originally published In Press as doi:10.1074/jbc.M309594200 on February 19, 2004

J. Biol. Chem., Vol. 279, Issue 20, 21356-21366, May 14, 2004
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NMR Solution Structures of {delta}-Conotoxin EVIA from Conus ermineus That Selectively Acts on Vertebrate Neuronal Na+ Channels*

Laurent Volpon{ddagger}§, Hung Lamthanh¶, Julien Barbier||, Nicolas Gilles¶, Jordi Molgó||, André Ménez¶, and Jean-Marc Lancelin{ddagger}**

From the {ddagger}Laboratoire de RMN Biomoléculaire Associé au CNRS-UMR 5180, Université Claude Bernard-Lyon I, Bâtiment 308, Ecole Supérieure de Chimie Physique Electronique de Lyon, F-69622 Villeurbanne Cedex, the Département d'Ingénierie et d'Etude des Protéines, CEA Centre d'Etudes de Saclay, F-91191 Gif-sur-Yvette, and the ||Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR 9040, CNRS, 1 Avenue de la Terrasse, F-91198 Gif-sur-Yvette, France

Received for publication, August 28, 2003 , and in revised form, February 17, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
{delta}-Conotoxin EVIA, from Conus ermineus, is a 32-residue polypeptide cross-linked by three disulfide bonds forming a four-loop framework. {delta}-Conotoxin EVIA is the first conotoxin known to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6), without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel (Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685). Its structure was solved by NMR and is characterized by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR time scale. The structure of both cis and trans isomers could be calculated separately. The isomerism occurs within a specific long disordered loop 2, including residues 11-19. These contribute to an important hydrophobic patch on the surface of the toxin. The rest of the structure matches the "inhibitor cystine-knot motif" of conotoxins from the "O superfamily" with a high structural order. To probe a possible functional role of the Leu12-Pro13 cis/trans isomerism, a Pro13 -> Ala {delta}-conotoxin EVIA was synthesized and shown to exist only as a trans isomer. P13A {delta}-conotoxin EVIA was estimated only two times less active than the wild-type EVIA in binding competition to rat brain synaptosomes and when injected intracerebroventricularly into mice.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The new {delta}-conotoxin EVIA ({delta}-EVIA),1 a 32-amino acid conopeptide isolated from the venom of Conus ermineus, is the first conotoxin demonstrated to inhibit sodium channel inactivation in neuronal membranes from amphibians and mammals (subtypes rNav1.2a, rNav1.3, and rNav1.6) without affecting rat skeletal muscle (subtype rNav1.4) and human cardiac muscle (subtype hNav1.5) sodium channel subtypes (1). This important recent discovery makes {delta}-EVIA a unique tool to study the modulation mechanisms of neuronal Na+ channels. As a consequence, {delta}-EVIA may also serve as a new lead molecule for the design of new drugs to treat neurological diseases characterized by defective nerve conduction, especially those causing an axonal demyelinization (2, 3). Nerve conduction could be facilitated by specific inhibition of Na+ channel inactivation. The knowledge of the detailed three-dimensional structure is therefore the first step necessary to understand the structure-activity relationships of this new lead conotoxin.

Despite a low sequence identity with the {kappa}-, {omega}-, and {delta}-conotoxins, {delta}-EVIA clearly belongs to the four-loop family of conotoxins characterized by a similar cysteine pairing giving a conserved 3-disulfide framework as shown in Fig. 1. Until now, the three-dimensional structure of 10 conotoxins belonging to this family was already determined as follows: the conotoxin {kappa}-PVIIA (20) targeting potassium channels; conotoxins {omega}-MVIIA (21), {omega}-MVIIC (22), {omega}-MVIID (23), {omega}-GVIA (24), {omega}-SVIB (25), and {omega}-CVID (26) targeting calcium channels; the conotoxin µ-GS (27) targeting skeletal muscle sodium channels; conotoxin TVIIA (28); and the most recent conotoxin {delta}-Tx-VIA (12). The latter {delta}-TxVIA belongs to a specific class of conotoxins that affect Na+ channel inactivation exclusively in mollusks but exhibits high affinity to rat brain synaptosomes (15). In rat brain synaptosomes, {delta}-EVIA competes with {delta}-Tx-VIA for the same site 6 of voltage-dependent Na+ channels (see Ref. 1 and this work).



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  		FIG. 1.
Sequence comparison of conotoxins. Identical residues are shown in boldface type. The standard one-letter code for amino acid residues is used except for the post-translationally modified 4-trans-l-hydroxyproline (O) and {gamma}-carboxy-L-glutamic acid (X). Spacers (-) are inserted to show maximal homology based on the conserved Cys framework. {pi}, hydrophobicity calculated according to Fauchère et al. (87), without the six cysteines. Asterisks indicate an amidated C terminus. a, HWTX-I, Huwentoxin-I. b, NAR, nicotinic acetylcholine receptor. c, µ-Aga-I, µ-Agatoxin-I. d, an additional disulfide bridge occurs between Cys24 and Cys30. e, {delta}-ACTX-Hv1, {delta}-Atracotoxin-Hv1 (versutoxin). f, an additional disulfide bridge occurs between Cys16 and Cys42. {dagger}, D. Zhao, J. Yao, Q. Dai, and P. Huang, GenBankTM accession number A59135 [GenBank] .

 
Despite the differences in the loop length between cysteine residues (especially for the loops 2-4), their three-dimensional solution structures reveal a common scaffold consisting of a small {beta}-hairpin structure and several types of tight turns. All conotoxins of this group exhibit a rather well defined backbone conformation, stabilized by a number of hydrogen bonds located in the different secondary structures, and by the three disulfide bridges. The scaffold forms the classical "cystine-knot" motif known for toxic and inhibitory polypeptides (29). A remarkable feature of {delta}-EVIA is the length of the loop 2 is made of nine residues instead of six for the other conotoxins. A variability in the cystine-knot scaffold, both in sequence and length, was already described for the loop 4 of conotoxin µ-GS (27) or the {delta}-atracotoxin-Hv1 (30, Fig. 1) but not for the loop 2.

We describe here the three-dimensional NMR structure in aqueous solution of {delta}-EVIA. The specific difference with the solution conformation of the other members of the four-loop family of conotoxins is located in loop 2, which is characterized by an apparent low conformational order corroborated by a 1:1 cis/trans isomerism of the Leu12-Pro13 peptide bond in slow exchange on the NMR chemical shift time scale. Both cis Leu12-Pro13 and trans Leu12-Pro13 bonds were separately solved based on two separate sets of experimental restraints. For comparison purposes, a Pro13 -> Ala single mutant was also prepared. The NMR data for P13A mutation are consistent with a 12-13 peptide bond purely trans, whereas the global structure of the toxin is maintained. The binding of the P13A {delta}-EVIA to rat brain synaptosomes as well as its activity, measured by intracerebroventricular injection to mice, is reduced about 2-fold.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Toxin synthesis-solid phase synthesis of the linear {delta}-EVIA and P13A {delta}-EVIA peptides were done with an ABI 430A synthesizer (PerkinElmer Life Sciences), using Fmoc chemistry with 1,3-dicyclohexylcarbodiimide/HOAt as the condensation reagent (31) and 0.1 mmol of Rink amide resin. After the last Fmoc-amino acid coupling, the Fmoc group was kept bound to the N-terminal amino acid of the linear peptide in order to simplify the read out of the HPLC profile during its purification. The N-{alpha}-Fmoc linear peptide was cleaved from the resin and purified with a reverse phase preparative column (Vydac, 218TP, 250 x 25 mm). The peptide was eluted by a linear gradient of A and B solvents, 30-100% B in 70 min; A is an aqueous solution of 0.1% (v/v) trifluoroacetic acid, and B is an aqueous solution containing 60% (v/v) acetonitrile and 0.1% trifluoroacetic acid. After elution, the major component (151 mg), corresponding to the target Fmoc peptide (mass spectrum, m/z 3515.32), was deprotected by incubation (5 min, 22 °C) with a mixture composed of N,N-dimethyl formamide (5 ml) containing 20% piperidine (v/v) and 1% 1,8-diazabicyclo[5.4.0]undec-7-ene (v/v) and 5 ml of mercaptoethanol (72 mmol). After deprotection, the medium was diluted with deionized water (final volume ~500 ml) and acidified to pH 4 with trifluoroacetic acid, and the linear peptide was desalted by reverse phase preparative HPLC, as described previously. A pool of the {delta}-EVIA-containing fractions (~100 ml) was diluted with deionized water (final volume, ~500 ml) containing the redox couple cysteine/cystine, 2/0.5 mmol. The volume was adjusted to 1 liter with degassed buffer (0.1 M NH4SO4, 0.1 M ammonium acetate, 1 mM EDTA, pH 8.5), and the solution was adjusted to pH 8.5 with NH4OH and renatured by incubation (48 h at 4 °C, followed by 12 h at 22 °C). The mixture of oxidized peptides was adjusted to pH 4 with trifluoroacetic acid, loaded on reverse phase preparative column (Vydac, 218TP, 250 x 25 mm), and eluted with the acetonitrile gradient described previously. Based on HPLC co-elution experiments and electrospray ionization mass spectrometry, {delta}-EVIA was identified as a minor product under the oxidation conditions used. The peptide was further purified with a semi-preparative column (Zorbax SB C18, 250 x 9.4 mm).

Disulfide Pairing Assignment—{delta}-EVIA disulfide pairing pattern was determined after partial reduction with tris(2-chloroethyl)phosphate (40 °C, 5 min), as reported previously (32). The reverse phase chromatographic profile of the mixture is shown in Fig. 2A. The fingerprint pattern displayed a huge peak of nonreduced {delta}-EVIA and three intermediates with one or two open disulfide bonds (peaks A-C). The fully reduced peptide (SH)6 eluted earlier than the nonreduced {delta}-EVIA and the three intermediates. After the intermediates in peaks A-C were alkylated with a large excess of iodoacetamide and purified by reverse phase HPLC, the amino acid sequences of their Cys (S-carboxamidomethyl) derivatives were determined (Fig. 2B). Sequencing the Cys (S-cam) derivative in peak A indicated SS bridging of Cys3 and Cys21. Sequencing the Cys (S-cam) derivative in peak B revealed an increased signal for Cys (S-cam) at cycles 3, 10, 21, and 25. Thus, peak B has the disulfide bridge Cys10, Cys25 in addition to the disulfide bridge Cys3, Cys21. Analysis of peak C yielded a significant increase in the Cys (S-cam) signal at cycles 10 and 25, due to the open disulfide bridge Cys10, Cys25. Therefore, we deduce SS bridging between Cys20, Cys29,in addition to the detected Cys3, Cys21 and Cys10, Cys25 disulfide bonds. The cystine framework of {delta}-EVIA was summarized as Cys3, Cys21, Cys10, Cys25, and Cys20, Cys29.



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  		FIG. 2.
Disulfide bond assignment of synthetic {delta}-EVIA and the co-elution profile of synthetic and native {delta}-EVIA. A, reverse phase HPLC of partially reduced, synthetic {delta}-EVIA by tris(2-chloroethyl)phosphate (10 mM, pH 3.5 at 40 °C), showing peaks A-C (arrows) and the nonreduced {delta}-EVIA peak (SS)3. The retention time of the fully reduced {delta}-EVIA is indicated by (SH)6 (arrow). The peptides were subsequently eluted from a C18 Vydac analytical column (250 x 4.6 mm), with a linear gradient of A-B solvents (30-100% B, 70 min): solvent A is an aqueous solution of trifluoroacetic acid (0.1%, v/v), and solvent B is an aqueous solution containing 60% (v/v) acetonitrile and 0.1% trifluoroacetic acid. B, amino acid sequences of the purified Cys (S-carboxamidomethyl) derivatives of the intermediates in peaks A-C. C, reverse phase HPLC of synthetic {delta}-EVIA (top), and co-elution of synthetic and native {delta}-EVIA (bottom). The peptide was eluted from a C18 Vydac analytical column (125 x 4.6 mm) with a linear gradient of A-B solvents (30-100% B, 70 min).

 
Radioiodination and Binding Assays—{delta}-Conotoxin TxVIA was radio-iodinated by using 1 nmol of toxin, 0.5 mCi of carrier-free Na125I in a potassium phosphate buffer, pH 7.25, containing H2O2 (10 µl of 1:50,000 solution) and lactoperoxidase (0.7 unit, EC 1.11.1.7 [EC] from bovine milk) for a 2-min incubation time. The monoiodotoxin was purified on a Vydac C18 column. Rat brain synaptosomes were prepared from adult Sprague-Dawley rats (300 g), according to the method described by Kanner (33). Equilibrium competition assays were performed using increasing concentrations of unlabeled toxins in the presence of a constant low concentration of the radioactive toxin. Competition binding experiments were analyzed by the program Kaleidagraph (Synergy Software) by using a non-linear Hill equation (for IC50 determination). The Ki values of {delta}-EVIA were calculated by the equation Ki = IC50/(1 + (L*/Kd)) where L* is the concentration of the hot {delta}-TxVIA, and Kd is its dissociation constant (34). Standard binding medium composition was (in mM) as follows: choline Cl 130, CaCl2 1.8, KCl 5, MgSO4 0.8, HEPES 50, glucose 10, and 2 mg/ml bovine serum albumin. Following incubation for the designated times, the reaction was terminated by dilution with 2 ml of ice-cold wash buffer of the following composition (in mM): choline Cl 140, CaCl2 1.8, KCl 5.4, MgSO4 0.8, HEPES 50, pH 7.2, 5 mg/ml bovine serum albumin. Separation of free from bound toxin was achieved by rapid filtration under vacuum using Whatman GF/C filters preincubated with 0.3% polyethyleneimine. The filter discs were then rapidly washed twice with 2 ml of buffer. Nonspecific toxin binding was determined in the presence of a high concentration of the unlabeled toxin.

Biological Activity—To quantify the biological activity of native and synthetic conotoxins, Swiss-Webster mice ({approx}15 g) were injected intracerebroventricularly with a stereotaxic system (Harvard/ASI Apparatus, UK). The ED50 value was defined as the dose that produces hyper-activity in 50% of the tested animals within 12 h postinjection.

NMR Spectroscopy—{delta}-EVIA was dissolved at 2.2 mM (4 mg/550 µl) in either 90% H2O, 10% D2O, or 100% D2O. pH was adjusted to 3.0 (direct uncorrected pH-meter reading) using microliter increments of 0.1 N HCl. The P13A analog was prepared at 0.6 mM in 90% H2O, 10% D2O, pH 3.0. NMR spectra were recorded on a Bruker Avance DRX 500 spectrometer using a 5-mm (1H, 13C, 15N) triple-resonance probe head, equipped with a supplementary self-shielded z gradient coil. Spectra were processed using Bruker XWINNMR and GIFA V.4 (35) software. The solvent signal was suppressed with the WATERGATE sequence using a 3-9-19 pulse sequence with z gradient (36, 37). The DQF-COSY experiment (38) was recorded at 283 K in H2O with a very low power presaturation of the water resonance during the recycle delay to minimize the radiation damping effect. TOCSY/HOHAHA experiments (39, 40) were collected in both H2O and D2O at 283 K with a spin-lock time of 40 and 80 ms and at 290 and 297 K with a spin-lock time of 80 ms. An MLEV pulse sequence was used for the isotropic mixing arranged as the clean-TOCSY scheme (41). NOESY spectra (42, 43) were collected at 283, 290, and 297 K with a 1.5-s recycle time and a 150-ms mixing time. Supplementary NOESY spectra were collected at 283 K in H2O and D2O with mixing time of 75 and 250 ms, respectively. In these NOESY experiments, a selective flip-back pulse was applied on water resonance, and gradients were added during t1 to minimize the radiation damping effect (44). Except for the NOESY experiment at 283 K in H2O where 96 scans per t1 increments were accumulated, spectra were registered with 512 (t1) x 1024 (t2) complex data points and 32 scans per t1. The quadrature detection in the t1 dimension was achieved using the States-TPPI method (45). Chemical shifts were quoted relative to the solvent (H2O) chemical shift at the respective temperature (4.92 ppm at 283 K). The spectral width of all experiments was 10.96 ppm (5482.5 Hz) with a carrier frequency on-resonance with the water resonance. For the P13A conotoxin {delta}-EVIA, the TOCSY spectrum was collected with a spin-lock time of 80 ms, and NOESY spectrum was recorded with a 150-ms mixing time.

Deuterium exchange of labile protons was monitored just after dissolution of the lyophilized sample from H2O at pH 3.0 in neat D2O. The residual NH signal was followed with time at 283 K by analysis of TOCSY spectra recorded at 0.5, 1, 3, 19, 36, and 54 h.

Experimental NMR Restraints—Interproton-distance constraints were classified into four categories according to the cross-peak intensity of the NOESY spectrum at 283 K and 150-ms mixing time in H2O and D2O. Upper bounds were fixed at 2.7, 3.3, 5.0, and 6.0 Å for strong, medium, weak, and very weak correlations, respectively. The lower bound for all restraints was fixed at 1.8 Å, which corresponds to the sum of the hydrogen van der Waals' radii. The calibration for the NOE intensities was achieved using the cross-peak intensity H{delta}-H{epsilon} of Tyr7. Pseudo-atom corrections (46) of the upper bounds were applied for magnetically equivalent aromatic protons (+2 Å) and unresolved diastereotopic methyl or methylene protons (+ 1 Å). 0.3 Å was added to NOEs involving amide protons. Dihedral angle restraint {chi}1 was deduced from the stereospecific assignments of diastereotopic {beta}-protons (±40° from the ideal staggered conformation) (47, 48). The {varphi} dihedral angle restraints were set to -65 ± 25° for 3JHN-Ha >8 Hz and to -120 ± 40° for 3JHN-Ha <5 Hz. After the initial calculation, 22 distance restraints were applied to restrain 11 hydrogen bonds that were unambiguously defined by both the exchange rate and the NOEs, and by the fact that they appear in ~75% of the models. Target values of 1.5-2.3 Å for NH(i)-O(j) and 2.5-3.3 Å for N(i)-O(j) were used.

Structure Calculations—Models were calculated following a protocol described previously (49) using the X-PLOR software version 3.851 (50). Structures were analyzed using aqua/procheck-nmr (51) and promotif (52) and displayed using MolMol version 2.4 (53) and Molscript (54). The structure was generated by the hybrid distance geometry dynamically simulated annealing method (55, 56). In a first stage, the substructures generated using metric matrix distance geometry algorithms were regularized and refined by a high temperature simulated annealing protocol, using the parallhdg.pro force field of X-PLOR. The non-bonded van der Waals' interactions were represented by a simple repulsive quadratic term (55, 57). The experimental distance restraints were represented as a soft asymptotic potential, and electrostatic interactions were ignored. The force constant associated with the distance restraints was kept to 50 kcal·mol-1·Å-2 throughout the protocol. One cycle of simulated annealing refinement consisted of 1,500 steps of 3 fs at 1,000 K followed by 3,000 cooling steps of 1 fs from 1,000 to 300 K. At the end, each structure was subjected to 1,500 steps of conjugate gradient energy minimization.

In the second stage of the calculation, structures with good experimental and geometric energies were further refined using the full CHARMM22 force field of X-PLOR. In this stage of the calculation, the non-bonded interactions such as electrostatic interactions and van der Waals' interactions (described by the Lennard-Jones empirical energy function) were taken into account. An approximate solvent electrostatic screening effect was introduced by using a distance-dependent dielectric constant and by reducing the electric charges of the formally charged amino acid side chain (Asp and Lys) to 20% of its nominal charges defined in the CHARMM22 force field. The force constant used for the NOE potential was reduced to 25 kcal·mol-1·Å-2. After 1,500 steps of conjugate gradient energy minimization, the dynamic was initiated at 750 K. The system was equilibrated for 0.5 ps with an integration step of 1 fs and then coupled to a heat bath at 750 K, and the molecule was allowed to evolve for 10 ps before a slow cooling to 300 K for a period of 5.4 ps and allowed to evolve again for 15 ps. Finally, the structures were energy-minimized by 1,500 steps of the conjugate gradient algorithm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
{delta}-EVIA was synthesized using Fmoc chemistry, based on its amino acid sequence. The N{alpha}-Fmoc linear/denatured and reduced peptide was obtained in high yield (151/350 mg of calculated Fmoc peptide). Its folding/renaturation was the limiting step in the synthesis of bioactive {delta}-EVIA. Typically, the folding procedure yielded only 5-10 mg of synthetic and bioactive {delta}-EVIA (3.3-6.6% yield, based on 151 mg of Fmoc linear peptide). The HPLC pattern of the preparation obtained after the folding procedure indicated the presence of a large quantity of monomer isomass peptides (data not shown), probably because of misfolding of the linear, reduced peptide. Synthetic {delta}-EVIA co-eluted with native {delta}-EVIA (Fig. 2C), and the predicted MW of synthetic {delta}-EVIA was confirmed by ESI-MS (observed MH+ 3288.1; calculated MH+ 3288.4).

Sequence Resonance Assignments of the Spin Systems—From the DQF-COSY and TOCSY spectra, we first noticed the presence of 53 amide protons (HN), instead of the 30 expected. The sequence-specific resonance assignment (46) was undertaken and revealed two distinct NOESY walking with only Asp1, Asp2, Lys5, Hyp6, Gly8, Cys21, and Leu32 common to the two systems. The d{alpha}N connectivities proceeded unambiguously for the two sets of resonances except for Cys21/Ser22. Tyr7, Phe9, Pro13, Ala24, and Ala30 spin systems were used as starting points for the sequential assignment due to their specific pattern. In one of the two systems, the Leu12-Pro13 peptide bond was unambiguously assigned to the cis peptide conformation due to the observation of a strong and a weak in NOESY spectra. For the second system, the peptide bond conformation was unambiguously assigned to the trans conformation due to the observation of a strong with no . Then the two systems clearly resulted from a Leu12-Pro13 cis/trans mixture of {delta}-EVIA in slow exchange on the time scale of the NMR chemical shift. No exchange peaks between the two conformers were detected either in the TOCSY or the NOESY experiments (Fig. 3). The chemical shift differences (where {Delta}{delta}c-t indicates difference of the chemical shifts between cis and trans conformers) observed in the NMR spectra between {delta}-EVIA 12-13cis and 12-13trans are the most important for the HN compared with the C{alpha}H protons and very small for all the side chain protons but four of the nine residues in loop 2 (Ser11, Leu12, Pro13, and Asn17) (see "Discussion"). By taking into account the experimental uncertainties and using the relative intensities of the TOCSY cross-peaks of the same proton pair in the two different conformers (the spin system of Cys10, Ser22, Gly27, and Cys29 were the best resolved), a 1:1 ratio of each population was estimated. This ratio is not temperature-dependent between 283 and 313 K.



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  		FIG. 3.
Aliphatic F2 to aliphatic F1 region of the TOCSY spectrum of {delta}-EVIA recorded in D2O solution at 500 MHz and 283 K with a 40-ms mixing time, pH 3.0. The two different spin systems for Pro13 cis or trans are indicated (see the text).

 
The assignment of the Hyp6 was straightforward with strong and no between Lys5 and Hyp6, indicating that the peptide bond is in a trans conformation. However, the observation of between Lys5 and Hyp6 could not be observed due to the superposition of the strong intra-residual NOE C{alpha}H-C{alpha}H' of Gly23. A number of cross-peaks were not found in the HN-aliphatic region of the DQF-COSY spectrum. It was the case for Lys5, Lys16, Cys20, Cys21, and Ala30. The two sequence-specific resonance assignments of {delta}-EVIA are given as Supplemental Material.

Secondary Structure and Molecular Topology—On the basis of the NMR data (Fig. 4 and Fig. 5), we identified the presence of a small {beta}-sheet composed of three short antiparallel strands, involving Gly8-Cys10 (strand 1), Gly23-Val26 (strand 2), and Val28-Asp31 (strand 3). The double-stranded anti-parallel {beta}-sheet comprising strands 2 and 3 are connected by a typical {gamma}-turn Val26-Val28 and forms a {beta}-hairpin structure. Finally, two {beta}-turns including amino acids Lys5-Gly8 and Cys20-Gly23 were identified according to the standard criteria (58).



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  		FIG. 4.
Schematic representation of the consensus hydrogen bond network for the two models of {delta}-EVIA with Leu12-Pro13 cis and trans conformation. Only the central portion of the molecule is shown. All depicted hydrogen bonds were restricted except between Ser22 (HO) and Ala30 (O) (see text). Observed interstrand NOEs are represented by arrows. Some canonical inter-strand NOEs, such as Cys25 C{alpha}H-Cys29 C{alpha}H, were not observed due to the spectral degeneracy.

 



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  		FIG. 5.
Summary of the sequential NOE connectivities, 3JHN-H{alpha} coupling constants, slowly exchanging backbone NH protons, and side chain rotamers for the Leu12-Pro13 trans conformation of {delta}-EVIA. The sequential NOEs, d{alpha}N, dNN, dNN (i, i + 2), and d{alpha}N (i, i + 2) are indicated by bars between two residues and classified from strong to very weak according to the height of the filled bars. Open and filled circles indicate backbone NH protons that did not exchange with D2O in a TOCSY spectrum recorded after 3 and 19 h, respectively; open and filled squares indicate NH protons that are still observed in a NOESY spectrum recorded after 36 h and in a TOCSY spectrum recorded after 54 h, respectively. The side chain rotamers were determined from coupling constant, and NOE data are also indicated: g+, g-, and t correspond to g2t3 (-60°), g2g3 (+60°), and t2g3 (180°), respectively.

 
Structure Calculations—The input data for the structural calculation consisted of 206 distance and 24 dihedral restraints. The 206 distance restraints include 56 intraresidual, 68 sequential (i, i + 1), 27 medium range (i, i + 2 to i, i + 4), and 55 long range (>i, i + 4) restraints (Fig. 5A) for the trans conformer. The number of distance restraints is a little different for the cis conformer with 134 and 56 distance restraints for the sequential and medium range restraints, respectively. Sixteen slowly exchanging amide protons were assigned from the D2O exchange experiment, and 11 of them were used to define 22 hydrogen-bond restraints (see under "Experimental Procedures"). These 11 with the 5 remaining slowly exchanging amide protons will be discussed below. Dihedral angle restraints included 15 {chi}1 angles and 9 {varphi} angles.

Both cis and trans Pro13 were modeled separately with the appropriate {omega} angle restraints in the simulated annealing calculations using parallhdg.pro force field. Thirty initial structures were calculated, leading to 16 and 18 final structures for the trans and the cis conformer, respectively. None of these models have an NOE violation exceeding 0.15 Å or a dihedral angle violation exceeding 5°. These structures were refined with the CHARMM22 force field and show a few distance violations lower than 0.25 Å and no dihedral angle violations exceeding 5°. The geometric and energetic statistics of these final structures, given in Table I, satisfy both the force fields and the experimental restraints. Side-by-side views of the final structures (Fig. 6A) show that residues 3-10 and 20-30 (corresponding to loops 1, 3, and 4) have well defined backbone dihedral angles (Fig. 7, B, C, and E) and low r.m.s.d. for the backbone atoms (Fig. 7D), whereas loop 2 (residues 11-19) appears completely disordered. The two residues of the N- and C-terminal parts also exhibit higher disorder. This correlates with the small number of medium and long range NOEs for these parts of the molecule (Fig. 7A).


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  		TABLE I
Structural statistics for the 16 final structures of {delta}-conotoxin EVIA

Data are given for the trans conformer of the Leu12-Pro13 peptide bond determined from NMR data at pH 3.0 and 10 °C.

 



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  		FIG. 6.
Stereoview of the NMR structures of conotoxin {delta}-EVIA. A, stereoview 16-structure superimposed for a minimum r.m.s.d. to the (C, C{alpha}, and N) atoms from residues Cys3 to Cys10 and Cys20 to Asp30; B, Molscript (54) scheme of the backbone polypeptide folding of the closest solution structure to the geometric average. The structural topology of the triple-stranded antiparallel {beta}-sheet is shown with the three disulfide bridges and the Pro13 residue. N-term and C-term indicate N-terminal and C-terminal positions.

 



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  		FIG. 7.
Structural data of the 16 NMR models of {delta}-EVIA as function of the protein sequence. A, distribution of the distance restraints classified from black to light gray into intraresidual, sequential, medium range, and long range restraints. B and C, values of the {varphi} (B) and {psi} (C) backbone dihedral angles describing {delta}-EVIA. D, r.m.s.d. from the average structure for the backbone atoms. E, order parameter S of the {varphi} (shaded bars) and {psi} (solid bars) angles; 0 = randomly distributed, and 1 = perfectly defined.

 
Analysis of the Hydrogen Bond Network—The hydrogen-exchange rates in D2O identified 16 slowly exchanging amide protons in the structure. It concerned the Ile4, Lys5, Tyr7, Gly8, Phe9, Cys10, Ile14, Leu15, Cys21, Ser22, Gly23, Val26, Val28, Cys29, and Asp30 amide protons. Eleven of them were used as restraints in the structure calculation from which 7 were involved in the triple-stranded antiparallel {beta}-sheet (Fig. 4). Two amide protons, Gly8 and Gly23, were involved in the formation of {beta}-turns. The last two exchanging amide protons, Ile4 and Cys21, restrained as hydrogen-bonded with Leu19 (O) and Ile4 (O), respectively, characterize an additional {beta}-bridge between residues Ile4 and Cys20, providing an additional stabilization to the N terminus. Such hydrogen bonds were also found for the {omega}-GVIA (24), µ-GS (27), {kappa}-PVIIA (20), and {delta}-TxVIA (12) conotoxins. Furthermore, three other hydrogen bonds, which were not experimentally imposed, participate in the stability of these two regions together with the C-terminal part in the models. These hydrogen bonds involve the {delta}-hydroxyl proton (HO) of Hyp6 with the hydroxyl oxygen (OH) of Ser22, the Ser22 (HO) with Asp30 (O), and the Ser22 (HN) (corresponding to a slowly exchanging amide proton) with Hyp6 (OH). Each of them was found in 12, 16, and 7 of the 16 refined structures, respectively.

The four remaining slow exchanging amide protons (Lys5, Phe9, Ile14, and Leu15) are more rapidly exchanged than the preceding ones, except for Lys5 as shown in Fig. 5. The two first amide protons corresponding to the second and the last residues of the first loop are also slowly exchanged in {omega}-MVIIA and {omega}-SVIB conotoxins (25), but a posteriori no justification was given. The slow exchange of Ile14 and Leu15 amide protons, located in the disordered loop, could be due to the high hydrophobicity in this region (see "Discussion" below).

Description of the Three-dimensional Structure—{delta}-EVIA is characterized by a well defined antiparallel triple-stranded {beta}-sheet of classification +2x, -1 (59). Each of the strands, comprising residues Gly8-Cys10 (strand 1), Gly23-Val26 (strand 2), and Val28-Asp31 (strand 3) (Fig. 6B), interacted with its neighbor with 3 hydrogen bonds for strand 1 - strand 3 and 4 hydrogen bonds for strand 3 - strand 2 (Fig. 4). The strands 2 and 3 are hydrogen-bonded from Ala24 (HN) to Val26 (O) and from Val28 (HN) to Ala30 (O), respectively (i.e. practically 3 residues in the two cases), whereas strands 3 and 1 were hydrogen-bonded from Gly27 (O) to Cys29 (O) (i.e. 2 residues) and from Tyr7 (HN) to Cys10 (HN) (i.e. 3 residues), respectively. This is consistent with the large 3JHN-H{alpha} coupling constants measured for the residues of strands 2 and 3, the small 3JHN-H{alpha} measured for the residues of the strand 1, and the peripheral distorted strand 1 observed in the models that therefore do not constitute a complete {beta}-strand. It is noteworthy that the same feature was detected for {omega}-GVIA (24), {kappa}-PVIIA (20), {omega}-MVIIA, and {omega}-SVIB (25) conotoxins.

In addition, one {gamma}- and two {beta}-turns were characterized in the models. Based on the analysis of the {varphi} and {psi} angles (60), the models describe a type II {beta} 5-8 and a type I {beta} 20-23 turn (Fig. 7, B and C). Also, the hydrogen bond between Val28 (HN) and Val26 (O) defined a classic {gamma} 26-28 turn ({gamma}c-turn).

The buried side chains correspond to residues Ile4, Hyp6, Ser11, Ser22, Ala24, Val26, Val28, and Ala30 and the six cystines. When calculated on the heavy atoms of the Cys3-Cys10 plus Cys20-Asp30 segments, the r.m.s.d. with respect to the mean coordinate positions is only equal to 0.59 Å because of the restriction of 14 {chi}1 angles (Fig. 5). The {chi}1 angles of all the Cys residues were restricted and thus contribute to stabilize the core of the molecule. As observed for the majority of the conotoxins, the Cys10-Cys25 disulfide bridge is a right-handed spiral motif, whereas the Cys20-Cys29 disulfide bridge is a left-handed spiral motif (52). The third disulfide bridge Cys3-Cys21 is less well defined due to the proximity of the undefined N-terminal part. It appears to be represented by two conformations, with 7 structures displaying a right-handed hook conformation and 9 structures having a non-standard conformation. The ordering of the three disulfide bridges with the three-stranded antiparallel {beta}-sheet form the so-called "inhibitor cystine-knot motif" described by Pallaghy et al. (29).

NMR Analysis of the Pro13 -> Ala {delta}-EVIA—The P13A mutation restores a unique NMR spin system (given as Supplemental Material), and the sequential NOEs are characteristic of a trans peptide bond between Leu12 and Ala13. The chemical shifts are very close in frequency to the trans wild-type toxin; this observation indicates a conserved global solution structure. The chemical shift similitude between the Leu12-Ala13 peptide bond in P13A mutant and the Leu12-Pro13 trans conformer in wild-type conotoxin is particularly noteworthy for the HN proton of Ile14, which has the highest difference with the cis isomer (({Delta}{delta}c-t = 0.73 ppm). Most of the observed chemical shift differences with the wild-type conotoxin are essentially located in the region of the Leu12-Ala13 dipeptide (given as Supplemental Material).

Biological Activity Pro13 -> Ala {delta}-EVIA—Competition curves for 125I-labeled {delta}-TxVIA binding inhibition by {delta}-EVIA and its P13A variant on rat brain synaptosomes revealed that both conopeptides, in a dose-dependent manner, fully displaced the radiolabeled conotoxin from its receptor site (Fig. 8). The calculated Ki values were about two times higher for the P13A variant (1,100 ± 140 nM, n = 3) than for {delta}-EVIA (475 ± 75 nM, n = 3). These results indicate that the P13A variant binds to receptor site 6 of voltage-dependent Na+ channels, like {delta}-EVIA, but with lower affinity. Consistent with this finding, a 2-fold reduction in the ED50 for the excitotoxic activity of the P13A variant was observed (following intracerebroventricular injection to mice), when compared with natural and synthetic {delta}-EVIA (Table II). Furthermore, experiments performed on isolated frog neuromuscular preparations revealed that 5 µM P13A analog selectively increased nerve terminal excitability, without affecting directly elicited muscle action potentials and excitability in muscle fibers (data not shown). These results indicate that the mutant toxin keeps it selectivity for neuronal Na+ channels but is less active than reported for {delta}-EVIA (1).



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  		FIG. 8.
Competition curves for 125I-labeled {delta}-TxVIA binding inhibition by {delta}-conotoxin EVIA and its P13A variant. Synaptosomes (65.5 µg/ml) were incubated for 60 min at 22 °C with 180 pM 125I-labeled {delta}-TxVIA and increasing concentration of {delta}-EVIA toxins. Nonspecific binding, determined in the presence of 1 µM of {delta}-TxVIA, was subtracted. The amount of 125I-labeled {delta}-TxVIA bound is expressed as the percentage of the maximal specific binding without additional toxin. Competition curves are fitted by the non-linear Hill equation (with a Hill coefficient of 1) to determine the IC50 values (see "Experimental Procedures"). The Ki values are (in nM, average of three experiments) as follows: 475 ± 75 nM (open circle) for {delta}-EVIA and 1,100 ± 140 nM (black circle) for P13A {delta}-EVIA.

 


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  		TABLE II
Biological activity of C. ermineus crude venom, native and synthetic {delta}-conotoxin EVIA after intracerebroventricular injection to mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Chemical Synthesis and Folding—As shown in Fig. 2A, the retention time (Rt) for natively folded and biologically active {delta}-EVIA was significantly longer than that of the linear, fully reduced peptide, in contrast to the observed Rt for the natively folded hydrophilic {omega}- or {kappa}-conotoxin (20, 61). This observation suggests (i) that the folding process increases the {delta}-EVIA-accessible hydrophobic area and (ii) the hydrophobic character of {delta}-EVIA likely contributes to the low yield folding process. The detergent-assisted oxidative folding process recently described by De la Cruz et al. (63) could therefore improve the {delta}-EVIA folding yield. In this respect, we have observed a higher amount of folded P13A {delta}-EVIA variant compared with that of the wild-type {delta}-EVIA, suggesting a folding effect of Pro13.

Comparison to Other Toxins—The three-dimensional structure of {delta}-EVIA consists of a {beta}-hairpin, involving residues Gly23 to Asp31 (Fig. 6B), and several turns. Such a conformation, stabilized by a number of disulfide bridges and hydrogen bonds, is adopted by several other small proteins (Fig. 1) coming from phylogenetically divergent species including spiders and cone shells but also fungi or plants (29). The r.m.s.d. of the three disulfide bridges with or without loops 1 and 3 was calculated for these different peptides relative to the {delta}-EVIA (Table III). Despite the relatively low amino acid sequence homology (Fig. 1), it reveals a strong correlation between this four-loop family and the scaffold of the peptidic backbone. In particular, the loop 1, stabilized by a {beta}-turn, and the disulfide bridges appear very close. The loop 3 is constituted of a type I ({kappa}-conotoxin PVIIA, huwentoxin-I, and {delta}-EVIA), type VIa ({delta}-atracotoxin-Hv1), or type VIII ({omega}-conotoxin GVIA, {omega}-conotoxin MVIIA, and µ-agatoxin) {beta}-turn.


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  		TABLE III
r.m.s.d. (Å) between {delta}-conotoxin EVIA and other four-loop peptides

The closest structure to the geometric average was used for the calculation.

 
The major structural differences between these molecules result from the different sizes of the loops 2 and 4 (Fig. 1). The resulting secondary structure in loop 2, except for peptides with nine amino acids, is a type I {beta}-turn in the majority of the cases. {delta}-EVIA, which is the first structurally resolved conotoxin with a large second loop, displays a disordered loop from Ser11 to Leu19 (Figs. 6A and 9). This loop is devoid of medium range and long range interactions with the rest of the molecule and therefore protrudes from the core of the molecule without a well defined orientation. Since the other conotoxins with such a large second loop (conotoxins MrVIA, MrVIB, and {delta}-PuIA) also contain a Pro residue in the third position (Fig. 1), it can be supposed that the same cis/trans isomerism could occur for this residue. The earlier structural studies of the four-loop peptides all concern peptides with at least four amino acids in the loop 4. This loop is characterized by a type I ({omega}-conotoxin GVIA and {kappa}-conotoxin PVIIA), type I' ({omega}-conotoxin MVIIA and {omega}-conotoxin MVIIC), or type VI (µ-conotoxin GS and µ-agatoxin) {beta}-turn and is involved in a {beta}-hairpin structure. Despite the presence of only three amino acids in the loop 4 of {delta}-EVIA, the peptide backbone adopts the same topology between the strands 2 and 3, as shown in Fig. 9, with a similar hydrogen bond network, constraining in this case the hairpin turn to form a {gamma}-turn.



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  		FIG. 9.
Solution structures of some four-loop conotoxins. A, GVIA; B, MVIIA; C, GS; D, {delta}-EVIA. The structures are superimposed to the backbone (C, C{alpha}, and N) atoms of the well defined regions encompassing residues 1-8 and 15-26 for GVIA, 1-8 and 15-25 for MVIIA, 2-20 and 25-31 for GS, and 3-10 and 20-29 for {delta}-EVIA. The structures are shown with the same orientation. Nt and Ct indicate N-terminal and C-terminal positions.

 
The cis/trans Isomerism of Pro13 in {delta}-EVIA—Considering the NMR ensemble of {delta}-EVIA, the great disorder of loop 2 could correlate with the Leu12-Pro13 peptide bond cis/trans isomerism. The absence of exchange peaks between the two conformers indicates, however, a very slow exchange rate relative to the NMR time scales. For instance, such a feature was already observed for the Lqh-8/6, a toxin from a scorpion venom (64), or for a cyclic peptide (65). Several statistical studies (66, 67) tentatively suggested possible correlations between the steric and electronic properties of the residues surrounding the proline and the stability of the cis isomer, by examination of peptide bonds from the Protein Data Bank (68). According to these studies, a cis peptide bond in a sequence Leu12-Pro13-Ile14 of {delta}-EVIA would not be frequent due to the presence of branched aliphatic residues preceding and following the proline (67).

In {delta}-EVIA, amide protons of Ile14 and Leu15 slowly exchanged with D2O. Unfortunately, the HN of Ile14 is degenerate in the trans conformer with the intra-residual C{alpha}H/HN cross-peak of Gly23, so we cannot conclude about a similar exchange for this conformer (Fig. 4). Two additional weak NOEs, Leu12 (C{alpha}H)-Ile14 (HN), and Leu12 (C{alpha}H)-Ile14 (C{alpha}H), are found for the cis isomer relative to the trans isomer. Calculation of structures for the cis conformer induced the formation of a non-hydrogen-bonded type VIb-3 {beta}-turn involving Ser11 to Ile14 (68, 67), and a significant decrease of the disorder for the {varphi} and {psi} angles of Leu12 (-122 ± 9 and 168 ± 8°) and Pro13 (-65 ± 8 and 157 ± 11°) (see Fig. 7, B and C, for comparison with the trans conformer). However, the presence of this {beta}-turn is not sufficient to render ordered the entire loop 2. The slow exchange observed for the HN of Ile14 and Leu15 would then originate in the high local hydrophobicity (Leu12-Pro13-Ile14-Leu15) rather than the formation of hydrogen bonds.

As a consequence of the cis/trans isomerism of Pro13, the {Delta}{delta}c-t is not uniformly distributed along the sequence (data given as Supplemental Material). Ile14 HN is the most affected as well as HN of Asn17, Gly18, and Leu19 located in front of Pro13 in the loop 2. In addition to the backbone protons, the {Delta}{delta}c-t of the side chain protons of Cys10, Ser11, Leu12, Pro13, and especially Asn17 residues are relatively high (0.10 to 0.30 ppm). The comparison of the structure of the two conformers in this region show more hydrogen bonds for the cis conformer involving the side chain of Asn17. The {delta}NH2 protons are hydrogen-bonded in the models with Ser11 (O{gamma}), Ser11 (O), and Pro13 (O) in 6, 6, and 10 of the 18 final structures, and they are practically absent for the trans conformer. Moreover, half of the final structures for the cis conformer have a Leu15 (HN)-Asn17 (O{gamma}) hydrogen bond, although it does not appear for the trans conformer. The expected {Delta}G of the cis-to-trans interconversion for an isolated Xaa-Pro bond is in the order of 13 kcal/mol, and the cis/trans ratio was calculated to be about 1:3 (71-73). The additional stabilization in the cis conformer could explain the 1:1 cis/trans ratio observed. It is also likely that the additional hydrogen bonds in the cis conformer could increase the interconversion energy barrier leading to a very slow exchange rate.

The fact that the Pro13 -> Ala mutant {delta}-EVIA gives a better yield of the native structure upon the air-oxidation step of the free cysteines could be interpreted as a propensity of Pro13 -> Ala {delta}-EVIA to favor low potential energy intermediates, productive for the native structure. A prolyl residue at the position 13 would slow a folding step as observed recently in the case of the systematic study of the Pro -> Ala mutations of the three prolines on the folding kinetics of the cellular retinoic acid-binding protein I (74).

Interaction with the Na+ Channel—As for µO- and {delta}-conotoxins targeting sodium channels, {delta}-EVIA displays a high degree of hydrophobicity and a small number of charges (Figs. 1 and 10). This fact was recently discussed for the NMR structure of {delta}-TxVIA (12), a 27-mer peptide that competes with {delta}-EVIA for the same receptor site 6 on the Na+ channels (1) (Fig. 8). In particular, a characteristic of {delta}-TxVIA is to bind strongly to the receptor site 6 of Na+ channels of mammals without modifying the spontaneous inactivation period, while it delays significantly this period in mollusks (75-77). A hydrophobic cluster was revealed on the surface {delta}-TxVIA including, in particular, Met8, Leu11, Leu12, Tyr20, Val23, Leu24, and Val25. Among these seven amino acids, five are homologously conserved in {delta}-EVIA (Phe9, Pro13, Ile14, Val26, and Val28, Fig. 1). The hydrophobic patch revealed on the surface {delta}-TxVIA is even extended by Ile4, Tyr7, and Leu12 as shown in Fig. 10. The positively charged amino acids are located at the beginning of the loop 1 (Lys5) and in the loop 2 (Lys16), whereas the negatively charged amino acids are located at the opposite N and C termini (Asp1, Asp2, and Asp31), making {delta}-EVIA quite amphipathic.



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  		FIG. 10.
Hydrophobic patch of the surface of conotoxins {delta}-EVIA (this work) and {delta}-TxVIA (Protein Data Bank entry 1FU3 [PDB] (12)). A, superposition of {delta}-EVIA (the red trace) to {delta}-TxVIA (the blue trace) as indicated in Table III. B, surface-associated electrostatic potentials of conotoxin {delta}-EVIA represented as a MolMol contact surface (53) from electronegative to electropositive by a red to blue continuous color range, respectively. The molecule is shown in the same orientation as in Figs. 7 and 8. Specific amino acids associated with loop 2 and/or the hydrophobic patch are indicated as well as charged amino acids.

 
The apparent disorder observed in the loop 2 of {delta}-EVIA, which includes Leu12, Pro13, and Ile14 participating in the hydrophobic patch, could be important for receptor binding as it was suggested for the {alpha}A-conotoxin PIVA (78), which has a flexible 3-11 loop and a rigid 14-23 loop. Such a flexible region has already been proposed to be stabilized upon interaction with the receptor (79-81). In particular, a cis/trans proline isomerization was supposed to occur in the free form of the human acidic fibroblast growth factor (80), but when complexed to the sucrose octasulfate, the slow conformational motion corresponding to this isomerization is thwarted by this binding leading to a more rigid conformation. Also, conformational changes of small peptidic ligands upon binding have been observed in other systems (82-85). It can be presumed that the loop 2, which is quite flexible in solution, might allow structural rearrangements to accommodate its binding, whereas the rigid core of {delta}-EVIA serves as a delivery scaffold, with possible direct additional interactions with the receptor.

Due to the peculiar apparent low structural order and the hydrophobic character of the loop 2 from Leu12 to Leu15, and to the specific cis/trans isomerism of the Leu12-Pro13 peptide bond, another question arose about a possible biological and functional interest of such a structural singularity. The fact is that a trans, or a cis, amino-acyl proline peptide bond does not offer at all the same local binding abilities. In the trans conformation, the peptide carbonyl (a potent hydrogen bond acceptor) of the preceding residue, is trans to the hydrophobic cyclic side chain of the proline amino acid (i.e. on the two opposite sides of the main peptide chain). In the cis conformer, the carbonyl of the peptide bond is cis to the hydrophobic proline side chain (i.e. on the same side of the main peptide chain). In terms of molecular interactions between the neurotoxin and the receptor, it could make strong and significant differences. Such differences have been demonstrated, for instance, in a study related to an opioid peptide (86) in which increasing the proportion of the cis conformation of a prolyl peptide bond affects its affinity for the receptor. If we hypothesize the requirement of a cis amino-acyl proline peptide motif for a tight molecular contact within the neurotoxin-receptor complex, the small but significant change in activity of the P13A mutant {delta}-EVIA could be interpreted as a deficiency in presenting on the same side of the main peptide chain both a hydrophobic cyclic proline side chain and a peptide bond carbonyl, a potent acceptor of a hydrogen bond. Further explorations and probing of this hypothesis are currently in progress using peptido-mimics simulating either cis or trans amino-acyl proline as well as using other single-point mutations in the regions expected to be important for the toxin-receptor interactions.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1G1P [PDB] and 1G1Z [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* Part of this work was presented at the 26th European Peptide Symposium, 10-15 September 2000, Montpellier, France. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

The on-line version of this article (available at http://www.jbc.org) contains one table of the 1H NMR chemical shifts of cis and trans conformers of {delta}-EVIA and of P13A {delta}-EVIA; one figure representing the HN-aliphatic region of the TOCSY spectra of {delta}-EVIA and P13A {delta}-EVIA; one figure representing the chemical differences between cis and trans conformers of {delta}-EVIA. Back

§ Recipient of a Ph.D. fellowship 1998-2001 from the French Ministère de l'Education Nationale de la Recherche et de la Technologie. Present address: Dept. of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada. Back

** To whom correspondence should be addressed: Laboratoire de RMN Biomoléculaire, CNRS UMR 5180 Sciences Analytiques, Université Claude Bernard-Lyon 1, Domaine Scientifique de La Doua, ESCPE-Lyon, F-69622 Villeurbanne Cedex, France. Tel./Fax: 33-4-72-43-13-95; E-mail: lancelin{at}hikari.cpe.fr.

1 The abbreviations used are: {delta}-EVIA, {delta}-conotoxin EVIA from Conus ermineus; DQF-COSY, double quantum-filtered correlation spectroscopy; Hyp, hydroxyproline; r.m.s.d., root mean square atomic deviation; HPLC, high pressure liquid chromatography; Fmoc, N-(9-fluorenyl)methoxycarbonyl; S-cam, S-carboxamidomethyl; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barbier, J., Lamthanh, H., Le Gall, F., Favreau, P., Benoit, E., Chen, H., Gilles, N., Ilan, N., Heinemann, S. F., Gordon, D., Ménez, A., and Molgó, J. (2004) J. Biol. Chem. 279, 4680-4685[Abstract/Free