Département d'Ingénierie et d'Etudes des Protéines, CEA, Saclay, 91191 Gif-sur-Yvette cedex, France
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Abstract |
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Keywords: nicotinic acetylcholine receptor/solid-phase synthesis/snake toxins
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Introduction |
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Proteins with a three-fingered fold are currently synthesized by recombinant approaches (Ducancel et al., 1991) or by the long and labor intensive fragment-by-fragment chemical methodology (Nishio et al., 1993
). Despite the large possibilities offered by the genetic approach various experimental problems are encountered. They include safety regulation with toxins, unpredicted low expression of some mutants, difficulty in introducing unnatural amino acids and in performing site-directed labeling with 13C- or 15N-labeled amino acids, so useful in NMR studies. Clearly, an alternative strategy to produce three-fingered proteins is desirable.
The aim of this paper is two fold. First, we show that the solid phase synthesis (SPPS) approach can be advantageously applied to the synthesis of a protein that adopts a three-finger fold. Thus, it is shown that (i) toxin from Naja nigricollis, a short-chain curaremimetic toxin (61 residues and four disulfides) from snake is efficiently amenable to SPPS, using the Fmoc methodology, (ii) the synthetic toxin and venom toxin have identical three-dimensional structure and biological activity, including binding affinity to the nicotinic acetylcholine receptor (AchR) from T.marmorata (Endo et al., 1991). Second, we engineered a small loop in the synthetic toxin, which is uniquely found in longer curaremimetic toxins (about 6674 residues and five disulfides) that can bind with high affinity to both muscular and
7 neuronal AchRs (Servent et al., 1997
). With this additional loop, the host short-chain toxin displays unchanged high affinity for the muscular AchR but a higher affinity for the
7 neuronal nicotinic AchR. Therefore, this paper demonstrates that SPPS is appropriate to engineer three-fingered proteins and that the engineered loop is implicated in the capacity of the long-chain toxins to bind with high affinity to this receptor.
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Materials and methods |
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Protected amino acid derivatives, resins, dicyclohexylcarbodiimide and N-hydroxybenzotriazole were purchased from Novabiochem (Meudon, France). Piperidine, N-methyl pyrrolidone, dichloromethane, methanol, trifluoroacetic acid ter-butyl methyl oxide were from SDS (Peypin, France). Phenol, ethanedithiol and thioanisole were from Aldrich (Saint-Quentin-Fallavier, France). Oxidized and reduced glutathione were from Sigma (St Louis, MO). Automated chain assembly was performed on a standard Applied Biosystems 431 Peptide Synthesizer. cDNA of the chimeric 7-V201-5HT3 receptor was kindly provided by Prof. J.P.Changeux (Institut Pasteur, Paris).
-bungarotoxin and ()nicotine were purchased from Sigma and toxin
from N.nigricollis was purified from its venom in our laboratory. [125I]-Bgtx (210250 Ci/mmol) was purchased from Amersham. The acetylcholine receptor (AChR) from the electric organ tissue of T.marmorata and the monoclonal antibodies (M
1 and M
2-3) were prepared as previously described (Saitoh et al., 1980
; Boulain et al., 1982
; Trémeau et al., 1986
).
Synthesis
Assembly of the different peptides was carried out using the stepwise solid-phase method with dicyclohexylcarbodiimide/hydroxybenzotriazole as coupling reagents and N-methyl pyrrolidone as a solvent. Fmoc protected amino acids were used with the following side-chain protections: t-butyl ester (Glu, Asp), t-butyl ether (Ser, Thr, Tyr), trityl (Cys, His, Asn, Gln), 2,2,5,7,8-pentamethyl-chromane-6-sulfonyl (Arg), t-butyloxycarbonyl (Trp) (Riniker et al., 1993). For toxin
from N.nigricollis and the chimeric form, the C-terminal Asn amino acid was introduced using N-
-Fmoc-L-aspartic acid-
-t-butyl ester by coupling the side chain carboxyl group to a resin bearing the Rink-amide linker. Upon treatment with trifluoroacetic acid (TFA), the peptide-resin is cleaved and generates an Asn residue. The
-Bgtx C33S-C37S derivative was assembled on a Fmoc-Gly-Wang resin (loading: 0.47 mMol/g). The different syntheses were run on a modified version of the Applied Biosystems standard 0.1 mmol small-scale program using 0.05 mmol resin (loading 0.47 mmol/g). This program carries out UV monitoring of the deprotection and extends automatically the deprotection time (20 min) and the coupling time (30 min) when the deprotection is too slow after two successive deprotections of 3 min. At the end of the synthesis the peptide resins were treated with TFA (10ml), crystalline phenol (0.75 g), 1,2-ethanedithiol (0.25ml), thioanisole (0.5ml) and 0.5 ml distilled water. The peptides were thus cleaved from the resin and the protecting groups were removed from amino acid side chains. After 2 h incubation, the mixture was filtered in cold t-butyl methyloxyde and centrifuged three times. The precipitates were dissolved in a solution of 10% acetic acid and lyophilized. The toxins were purified by reverse phase HPLC using a Vydac C18 column (250x10 mm) with a gradient of 035% of solvent B in 6 min, 3550% of B in 29 min and 50100% of B in 10 min (A, 0.1% TFA in H20; B, 60% acetonitrile and 0.1% TFA in H20). The flow rate was 5 ml/min and the detection was followed at 280 and 214 nm.
Disulfide bond formation and protein purification
The reduced synthetic toxin from N.nigricollis (1 mg) was subjected to an oxidative reaction in 0.1 M sodium phosphate buffer (pH 7.8), in the presence of reduced (GSH) and oxidized glutathione (GSSG). The following ratios of GSH/GSSG were tested: 10:1, 4:2, 2:4 and 1:10 mM. Each resulting mixture was separated on RP-HPLC and the proportion of protein eluting like the native toxin was estimated. The ratio of 4:2 gave the highest yield of oxidation. Therefore, the refolding of the chimeric toxin and the
-Bgtx analog was carried out on a larger scale on this basis. Typically, the deprotected but reduced synthetic protein (1 mg) was dissolved in 0.2 ml 0.1% TFA and immediately diluted in 10 ml oxidation buffer (0.1 M phosphate buffer, pH 7.8, 4 mM reduced glutathione and 2 mM oxidized glutathione). After 12 h at room temperature in the dark, the pH was lowered to pH 3 by the addition of 30% TFA and the mixture was loaded on a Vydac semi-preparative column (250x10 mm) equilibrated with 0.1% TFA in water. Then, the column was submitted to the gradient previously used to purify the reduced toxin forms. The concentration of the different proteins was evaluated spectrometrically (
M at 278 nm is equal to 9000 for toxin
from N.nigricollis and the chimeric toxin and 9500 for the
-Bgtx).
Mass analysis, amino acid composition and sequence determination
Mass determination was performed on a Nermag spectrometer coupled to an analytical (Brandford) electrospray source. For amino acid composition, the different peptides were hydrolysed in a sealed vial heated at 120°C in the presence of 6 N HCl for 16 h. The hydrolysates were analysed using an Applied Biosystems Model 130A Automatic Analyser equipped with an on-line 420A derivatiser for the conversion of the free amino acid into phenyl thiocarbamoyl derivatives. Amino acid sequences were determined using an Applied Biosystems 477 A Protein Sequencer.
Circular dischroism
Circular dischroism spectra were monitored at 20°C, from 186 to 260 nm using 0.1 cm pathlength quartz cells and using a Jobin Yvon CD6 spectropolarimeter. Each spectrum represents the average of four spectra.
NMR
For NMR measurements, about 8 mg of native and synthetic toxin from N.nigricollis were dissolved in 0.4 ml of solvent to a final concentration of 3 mM. The solvent used was a mixture of 95% (v/v) H2O and 5% (v/v) D2O at pH 3.5, as adjusted by addition of diluted HCl. A proton 2D TOCSY experiment (Braunschweiler and Ernst, 1983
) was carried out at 308 K on a Bruker AMX500 spectrometer. A WALTZ 16 composite pulse was used for the 80 ms isotropic mixing period. The spectrum was recorded according to the time-proportional phase incrementation method (Marion and Wütrich, 1983) in the t1 dimension and in simultaneous acquisition mode in the t2 dimension. The water signal was suppressed by low-power irradiation of the solvant resonance at all times except during t1 and t2. The experiment was acquired with 512(t1)x2048(t2) data points and with a spectral width of 7812.5 Hz. It was zero-filled in the t1 dimension in order to achieve the same resolution in each dimension. It was multiplied by a phase-shifted sine bell function in both dimensions prior to Fourier transformation. Data processing was carried out with the FELIX program on a Silicon Graphics station.
7 expression in HEK cells
Chimeric cDNA ( 7-5HT3), kindly provided by Prof. J.P.Changeux (Institut Pasteur, Paris) was transfected into human embryonal kidney cells (HEK 293, ATCC CRL 1573) by calcium precipitation with careful control of pH (6.95), CO2 level (3%) and cDNA amount (15 µg) as previously described (Chen and Okayama, 1987
). Two days after transfection, the cells were harvested in phosphate-buffered saline (PBS) and 5 mM EDTA, rinsed twice in PBS and finally resuspended in this buffer (3 ml by plate). This high-efficiency transfection protocol allowed direct binding experiments on intact cells for 2 days.
Binding assays
The affinity of [125I]--Bgtx for the
7 receptor was tested by incubating the labeled toxin (5 nM final concentration) with 350 µl cells suspended in PBS. The non-specific binding was determined in the presence of 1 mM ()-nicotine. After 40 min of incubation, the sample was diluted with 3 ml cold PBS, filtered through a GF/C filter (Whatman) previously dipped in 1% non-fat dried milk and rinsed with 3 ml cold PBS. The radioactivity of the filter was determined on a
-counter. For competition experiments with the
7 receptor, we examined the effect of the short-chain, long-chain or chimeric toxin on the initial rate of [125I]-
-Bgtx binding. The competitors were preincubated, at different concentrations, for at least 45 min with the cell suspension and filtrated 6 min after the addition of 5 nM [125I]-
Bgtx. The protection constant (Kp) calculated by fitting the competition data by the Hill equation was shown to correspond to the Kd value (Weber and Changeux, 1974
). Competition experiments made with AChR from T.marmorata were performed at equilibrium, using [3H]-labeled toxin
from N.nigricollis as a radioactive tracer. Varying amounts of natural or chimeric toxins were incubated with 2 nM AChR and [3H]-toxin
from N.nigricollis (27 Ci/mmol, 10 nM) for 18 h at 20°C and the mixture was filtered through Millipore filters (HAWP) which had been soaked in ringer buffer. The filters were washed with 10 ml ringer buffer, dried and after addition of 10 ml scintillation solution (Lipoluma) counted on a Rackbeta counter (LKB). Equilibrium dissociation constants were determined from competition experiments as previously described (Ishikawa et al., 1977
). Specific binding of different toxins to the monoclonal antibodies (M
1 and M
2-3) were performed by radioimmunoassays by precipitation of AgAc complexes with PEG 6000 (12.5%) after incubation with the [3H]-toxin
from N.nigricollis (12,13).
The equation used to calculate dissociation constants from competition binding data is
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Toxicity
Toxicity of synthetic toxin from N.nigricollis was determined by intraperitoneal injections of various doses (from 4 to 0.8 mg) of toxin in 0.9% NaCl in 20 g female Swiss mice (three mice per dose) and the number of dead mice was recorded 24 h later.
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Results |
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The chemical synthesis of toxin from N.nigricollis was performed with a modified version of the Fmoc/small-scale (0.1 mmol) program developed by Applied Biosystem (see Materials and methods). The synthesis proceeded smoothly and no failure in the deprotection monitoring was detected. Introduction of the C-terminal Asn was achieved by coupling a N-
-Fmoc-L-aspartic acid-
-t-butyl ester by its side chain to a resin bearing the amide linker (Albericio et al., 1990
; Breipohl et al., 1990
). Thus, upon treatment with a TFA cleavage mixture (King et al., 1990
), the linkage generated an Asn that, interestingly, was free of racemization and of dipeptide formation. As shown in Figure 1
, the final TFA cleavage led to a crude mixture in which one component predominated. This component corresponded to approximately 55% of the total reaction mixture. It was the reduced form of the toxin. Optimal conditions for disulfide formation required the presence of reduced and oxidized glutathione (4:2 mM, see Materials and methods) to improve the formation of correct species and the final oxidation yield. As shown in Figure 1
, the synthetic toxin folded as a major component and eluted in RP-HPLC like native toxin
from N.nigricollis (not shown). The toxin was obtained in 40% overall yield (after cleavage of the blocked peptide from resin, refolding and purification). Amino acid composition, sequence analysis (not shown) and electrospray mass analysis confirmed the purity and identity of the synthetic toxin. The mass of the protein was 6786.1 Da (theoretical 6786.5 Da).
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Design of the chimeric toxin
Short-chain (6062 residues and four disulfides) and long-chain curaremimetic toxins (6674 residues and five disulfides) adopt similar architectures, five strands of antiparallel ß-sheets organized in three adjacent loops (Figure 4). Both types of toxin bind with high affinity to muscular acetylcholine receptors (AchR), but only the long-chain toxins bind also with high affinity to neuronal
7 AchR (Servent et al., 1997
). It was proposed that the additional loop cyclized by the fifth disulfide and which is uniquely present in the central finger of long-chain toxins may be associated with their unique capacity to bind to neuronal receptors with high affinity. We therefore decided to transfer this loop from the donor long-chain
-bungarotoxin to the host short-chain toxin
and to investigate the consequence in terms of recognition of the neuronal receptor.
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Efficiency of synthesis was lower with the chimera as compared with that observed for the native toxin. In particular, low deprotection yields were observed and extended coupling times were needed for each residue of the sequence YKKVWCDAFC. However, the RP-HPLC profile of the crude compound revealed a major fraction with the expected sequence, with a few additional peptides present at low levels (Figure 1). Folding was achieved using the experimental conditions sucessfully applied for toxin
from N.nigricollis and again a rapid and efficient folding process was observed. The different analyses unambiguously proved the identity and purity of the chimeric protein. In particular, electrospray mass analysis gave a mass of 7102.9 Da (theoretical: 7103.1 Da). Also, no free cysteine was detected, indicating that the two additional cysteines introduced at positions 33 and 37 were involved in a disulfide bond.
Secondary structure of the chimeric toxin
Introduction of a cyclized loop at the tip of the central finger did not alter much the secondary structure of the toxin. As shown in Figure 2, the CD spectrum of the chimeric toxin displayed a less intense positive band at 197 nm, a more intense negative trough at 214 nm and a weak positive band at 230 nm. In contrast, abolition of the disulfide bond in the long-chain toxin had much more profound effects, as observed with the derivative
-Bgtx C33SC37S analog whose half-cystines of the fifth disulfide were replaced by two serine residues (Figure 2
). Its CD spectrum displayed a much less intense negative band at 210 nm accompanied with a shift to 215 nm. The significance of differences is unclear; however, it suggests that the fifth disulfide bond may control some local secondary structure.
Probing tertiary structures with topographical antibodies
To get some information on the tertiary organization of the chimeric toxin we used two monoclonal antibodies, M1 (Boulain et al., 1982
) and M
2-3 (Trémeau et al., 1986
). These antibodies recognize topographical epitopes whose organization is strictly dependent on the native-like architecture of the toxin. The side chains of those residues identified as involved in the epitope recognized by M
1 are Glu2, Asn8, Thr16, Thr17, Lys18, Thr19, Pro21 and Lys68 (using numbering shown in Table I
) and the N-terminus (Zinn-Justin et al., 1993
). Those involved in the epitope recognized by M
2-3 are Gln10, Lys30, Trp32, Asp34, His36, Arg40, Ile43, Glu45, Lys56 and Ile59 (Ducancel et al., 1996
.). They were raised against toxin
from N.nigricollis and do not cross-react with long-chain toxins. M
1 recognizes the upper part (Figure 4
) of the toxin structure, whereas M
2-3 binds to the concave side of the toxin at a site that largely overlaps the receptor-binding site (Boulain et al., 1982
; Pillet et al., 1993
). As expected for two proteins that share the same architecture, the synthetic and native toxins bind the two monoclonal antibodies M
1 and M
2-3 with identical affinities, equal to 0.65 and 9 nM, respectively (Figure 5
). As expected also (Trémeau et al., 1986
), the long-chain
-Bgtx was unable to bind to any of them at concentrations up to 10 µM (Table II
).
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Binding to the muscular and 7 neuronal acetylcholine receptors
The synthetic and natural toxin from N.nigricollis displayed the same high binding affinities (22 ± 5 pM) for the muscular-type acetylcholine receptors. Also, they had identical low affinity for the
7 neuronal receptor, their equilibrium dissociation constants being equal to 2.0 ± 0.4 µM, only. These data definitively confirmed the identity of the natural and synthetic toxins.
As previously reported, the long-chain -Bgtx has high affinity for both muscular and neuronal receptors (Servent et al., 1997
), its affinity constants being equal to 56 ± 18 pM and 1.1 ± 0.15 nM, respectively (Figure 6A and B
). The high affinity of
-Bgtx for the neuronal receptor was proposed to be due, at least in part, to residues located within the small additional loop cyclized by the fifth disulfide bond and located at the tip of the long-chain toxin central finger (Servent et al., 1997
). This proposal was confirmed by the observation that replacement in
-Bgtx of the two half-cystines by serine caused a 35-fold affinity decrease for the neuronal receptor (Figure 6B
, Table II
). In contrast, this double replacement caused only a 5-fold affinity decrease for the muscular receptor (Figure 6A
, Table II
). These data agree therefore with the view that the fifth disulfide bond and presumably some of the residues located in its vicinity participate in the specific recognition of the long-chain toxin to the neuronal receptor.
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Discussion |
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We then engineered a new function in the short-chain toxin from N.nigricollis, with the aim of providing it with a high affinity for
7 neuronal AchR. Previously, it was shown that long-chain curaremimetic toxins have a high affinity (Kd in the nanomolar range) for this neuronal receptor whereas the short-chain toxins recognized them with 103104-fold lower affinities (Servent et al., 1997
). It was proposed that at least part of this differential affinity was due to the unique presence in the long-chain toxins of an extra loop cyclized by a disulfide bond and located at the tip of the central loop. This suggestion was supported by the selective reduction and chemical modification of the fifth disulfide with 2,2'-dithiopyridine (Servent et al., 1997
). In this study, we confirmed the implication of the fifth disulfide in the neuronal activity but we showed that the effect of the replacement of the two half-cystines of the fifth disulfide by isosteric serine residues was not as great in the case of
-bungarotoxin interaction with the
7 neuronal receptor. Therefore, we suggest that the extra loop may be sensitive to local conformational changes which are less favored upon substitutions of the cystines by isosteric residues. Furthermore, the 35-fold decrease in affinity found for the
-bungarotoxin derivative is consistent with the 50-fold decrease in affinity observed for
-neurotoxin on the neuronal
3ß2 receptor after the removal of the equivalent fifth disulfide (Grant et al., 1998
).
To definitively demonstrate the specific contribution of this region to the neuronal activity, the additional cyclized loop of -Bgtx was tentatively transplanted at an homologous position in the short-chain toxin. However, a foreign loop is not necessarily well accommodated by a protein, especially if the insertion position is imposed at a predetermined location. For the inserted loop not to affect the general structural organization of the host toxin, we carefully respected the original elements of secondary structure of the host toxin. For that, we superimposed the ß-sheet structures of the host toxin and
-Bgtx, using Trp32 as a common point and observed that Thr42 of the ß4-strand of the short-chain toxin superimposed with Val43 in the long-chain toxin. We inserted in the host toxin all the sequence located between these two homologous positions, substituting Ile43 in the short-chain toxin by the corresponding Val residue in the long-chain toxin. In this operation, the four functionally critical residues in both short- and long-chain toxins (Asp34, Phe36, Arg40 and Gly41) continued to occupy the same positions in the chimeric toxin (Table I
). However, six new amino acids (Cys33, Ala35, Cys37, Ser38, Ser39 and Lys42) were introduced in the host toxin, the two cysteines Cys33 and Cys37 forming a disulfide bond. Despite the importance of the introduced modifications, the modified toxin still bound to two toxin-specific monoclonal antibodies with high affinities and displayed comparable circular dichroic spectra, suggesting that the three-dimensional structures of the chimeric and parent toxins are similar.
The high affinity of the chimera for the monoclonal antibody M2-3 was surprising and deserves some comment. Its epitope involves 10 residues of the short-chain toxin among which Asp34, His36, Arg40 and Ile43 (Ducancel et al., 1996
) are located in the region that is concerned by the insertion. That the affinity of M
2-3 for the chimera was only 16-fold lower than for the native toxin
from N.nigricollis suggests that the insertion had not much effect on the structural organization of these critical side chains. The slight affinity decrease can even be explained by the mutation of the critical Ile43 into a Val residue (Ducancel et al., 1996
). Since the same residues are critical for the binding of the short-chain toxin to the Torpedo AChR (Ducancel et al., 1996
), we anticipated that the insertion would not much affect this binding. And indeed, we observed that the chimeric toxin and the native toxin
from N.nigricollis bind to T.marmorata AChR with identical affinity, their Kd being close to 20 pM. Therefore, though being localized in proximity of the functional region, the insertion of the additional and cyclized loop at the central finger has not altered the structural organization of the functional residues of the host toxin.
More strikingly, perhaps, the chimera displayed a 20-fold affinity increase for the 7 neuronal receptor as compared with the host toxin. This result clearly demonstrates that the inserted fragment is involved in the recognition of the
7 receptor. Which of the inserted residues effectively contributes to this affinity increment is not yet known. The disulfide bond between Cys33 and Cys37, together with the Ala35 residue inserted in the loop, most likely contribute to it. However, we cannot exclude the possibility that the other additional residues Ser38, Ser339 and Lys42 also participate in the binding to the
7 receptor.
Recent data (Servent et al., 1998; Maslennikov et al., 1999
) suggests a common structural fold between the tip of the central loop of
-Cbtx and the conotoxin ImI from Conus imperialis, a toxin that binds to the
7 neuronal receptor with a Kd in the micromolar range (Quiram and Sine, 1998
). We now demonstrate that the tip of the central loop of long-chain toxins and the cone snail toxin express similar binding function toward the
7 neuronal receptor. Evidently, the chimera still remains 20100-fold less potent than long-chain toxins at binding to the
7 neuronal receptor (Servent et al., 1997
). One or more of the regions that are structurally different between the chimera and the long-chain toxins are probably responsible for this differential behavior, but they have not been identified, as yet. It was observed that Phe65 of the long-chain
-cobratoxin is involved in the binding to the
7 receptor (D.Servent, personal communication). Possibly, the homologous residue in
-bungarotoxin (His72, using the numbering shown in Table I
) is also involved in this binding. Such a residue is missing in the chimeric toxin and it would be worth introducing it in the future.
In conclusion, we have shown that a protein adopting a `three finger fold' and containing four or five disulfide bonds is amenable to stepwise solid-phase synthesis in a rather straightforward manner. We have also shown that the chemical synthetic approach is appropriate to graft a loop that provides the toxin with a high affinity for the 7 neuronal receptor. Another group has shown that the same region in K-neurotoxins is implicated in their recognition of the neuronal
3ß2 receptor (Grant et al., 1998
). Presumably, this region is central for the recognition of three-fingered toxins for neuronal receptors. It would now be interesting to investigate whether a synthetic version of the tip of the central finger displays, by itself, any binding to the neuronal receptor. Also, it may be of interest to examine whether introduction of a combinatorial library of residues within the additional loop of a toxin would allow us to generate new specificities for other neuronal receptors.
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Acknowledgments |
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Notes |
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References |
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Received August 2, 1999; revised December 12, 1999; accepted January 6, 2000.