©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Genetic Engineering of Snake Toxins
THE FUNCTIONAL SITE OF ERABUTOXIN A, AS DELINEATED BY SITE-DIRECTED MUTAGENESIS, INCLUDES VARIANT RESIDUES (*)

Odile Trémeau , Caroline Lemaire , Pascal Drevet , Suzanne Pinkasfeld , Frédéric Ducancel , Jean-Claude Boulain , André Ménez (§)

From the (1) Département d'Ingénierie et d'Etudes des Protéines, DSV, CEA, Saclay, 91191 Gif-sur-Yvette, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, S18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition, new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the ``functional'' site of Ea covers a homogeneous surface of at least 680 A, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.


INTRODUCTION

Toxic proteins from animals are known to perturb physiological processes by binding to key elements, most often a receptor, an ion channel, or an enzyme. Understanding, at the molecular level, of the mode of action of a toxin requires, at least in a first step, an identification of the site by which it recognizes its target. In this respect, the functional site of snake curaremimetic toxins has been the subject of extensive studies. This site is associated with the capacity of curaremimetic toxins to bind to the nicotinic acetylcholine receptor (AcChoR)() and hence to alter nerve-muscle transmission (Changeux, 1990). Thus, curaremimetic toxins provoke paralysis of skeletal muscles, including diaphragm, and induce death as a result of respiratory failure (Chang, 1979).

Snake venom curaremimetic toxins are classified as short chain toxins (60-62 residues and four disulfides) and long chain toxins (66-74 residues and five disulfides). Despite their differences in length these toxins bind to AcChoRs in a mutually exclusive manner (Endo and Tamiya, 1991) and have a similar folding (Agard and Stroud, 1982; Laplante et al., 1990; Love and Stroud, 1986; Low et al., 1976, Low, 1979; Tsernoglou and Petsko, 1976, 1977; Walkinshaw et al., 1980; Zinn-Justin et al., 1992). On the basis of various chemical derivatizations, some residues implicated in the AcChoR-binding site of several curaremimetic toxins were tentatively identified (Endo and Tamiya, 1991). More recently, we submitted erabutoxin a (Ea) to a mutational analysis, an approach which proved well suited to map the functional site of a protein (Cunningham and Wells, 1993; Kam-Morgan et al., 1993; Prasad et al., 1993). Ea is a short chain toxin from venom of the sea snake Laticauda semifasciata (Sato and Tamiya, 1971). It possesses 62 amino acids and binds with great specificity and high affinity ( K= 7. 10 M) to AcChoR from Torpedo marmorata (Ishikawa et al., 1977; Pillet et al., 1993). Its three-dimensional structure was elucidated by x-ray crystallography (Corfield et al., 1989). The cDNA encoding Ea was cloned (Tamiya et al., 1985) and expressed in Escherichia coli as a fusion protein (Ducancel et al., 1989). Cleavage by CNBr led to a recombinant toxin having biological properties (Boyot et al., 1990) and a three-dimensional structure (Arnoux et al., 1994) indistinguishable from those of the wild-type toxin. Therefore, 10 amino acids that are commonly found in most curaremimetic toxins were submitted to site-directed mutagenesis in Ea (Hervé et al., 1992; Pillet et al., 1993). This preliminary mutational analysis together with chemical modification experiments() indicated the functional role of Ser-8, Lys-27, Trp-29, Asp-31, Arg-33, Lys-47 (probed by chemical modification), and to a lesser extent of Phe-32, Gly-34, and Glu-38. These residues form an homogeneous surface in which all residues but one (Ser-8) have their side chain oriented on the same concave face of Ea -sheet. These residues were proposed to belong to the functional site of Ea.

Two lines of evidence suggest that the functional site of Ea may include additional amino acids. First, it was noted (Pillet et al., 1993) that the previously identified functional residues cover a surface that is smaller than that currently observed in protein-protein contact areas (Janin and Chothia, 1990). Second, previous experiments made with toxin variants (Harvey et al., 1984) and receptor fragments (Basus et al., 1993; Ruan et al., 1990; Fulachier et al., 1994) suggested that several residues of the first loop may contribute to the capacity of curaremimetic toxins to recognize AcChoR. Such a possibility appeared intriguing since the first loop residues can be variable within the family of curaremimetic toxins.

By mutational analysis, we now explored the role of 26 new toxin residues located at proximity of those previously recognized as being functionally important (Pillet et al., 1993). Thus, we (i) completed the analysis of loop I by modifying Phe-4, Asn-5, His-6, Gln-7, Ser-9, Gln-10, Pro-11, Gln-12, Thr-13, Thr-14, Lys-15, and Thr-16; (ii) mutated Ser-18 and Glu-21 in the large turn between loops I and II; (iii) modified 5 residues in loop II, i.e. Tyr-25, Gln-28, Ser-30, Thr-35, and Ile-36; (iv) completed the analysis of loop III by changing Pro-44, Thr-45, Val-46, Lys-47, Pro-48, Ile-50, and Ser-53. In addition, we reinvestigated the previously tested Ser-8, Asp-31, Arg-33, and Glu-38. For each mutant, we determined the secondary structure and equilibrium binding affinity toward AcChoR from T. marmorata by, respectively, monitoring its far ultraviolet circular dichroic spectrum and measuring its ability to compete for the receptor with a tritium-labeled toxin. The data presented in this paper enabled us to propose a delineation of the functional site of Ea. The identified surface is compatible with that expected for a protein-protein contact area. Unexpectedly, it includes residues that are not conserved in all curaremimetic toxins.


MATERIALS AND METHODS

Probes for Site-directed Mutagenesis

We used the following nucleotide probes for mutagenesis: F4A (5`-ATGAGGATATGTGCTAACCATCAGTCA-3`), N5V (5`-AGGATATGTTTTGTCCATCAGTCATCG-3`), H6A (5`-ATATGTTTTAACGCTCAGTCATCGCAA-3`), Q7L (5`-TGTTTTAACCATCTGTCATCGCAACCG-3`), S8T (5`-TTTAACCATCAGACCTCGCAACCGCAA-3`), S9G (5`-AACCATCAGTCAGGTCAACCGCAAACC-3`), Q10A (5`-CATCAGTCATCGGCTCCGCAAACCACT-3`), P11N (5`-CAGTCATCGCAAAACCAAACCACTAAA-3`), Q12A (5`-TCATCGCAACCGGCTACCACTAAAACT-3`), T13V (5`-TCGCAACCGCAAGTGACTAAAACTTGT-3`), T14A (5`-CAACCGCAAACCGCTAAAACTTGTTCA-3`), K15A (5`-CCGCAAACCACTGCTACTTGTTCACCT-3`), T16A (5`-CAAACCACTAAAGCTTGTTCACCTGGG-3`), EaS18 (5`-ACTAAAACTTGTCCTGGGGAGAGC-3`), E21A (5`-TGTTCACCTGGGGCTAGCTCTTGCTAT-3`), Y25F (5`-GAGAGCTCTTGCTTCAACAAGCAATGG-3`), Q28A (5`-TGCTATAACAAGGCATGGAGCGATTTC-3`), I36R (5`-TTCCGTGGAACTCGTATTGAAAGGGGA-3`), E38L (5`-GGAACTATAATTCTGAGGGGATGTGGT-3`), P44V (5`-GGATGTGGTTGCGTCACAGTGAAGCCC-3`) I50Q (5`-GTGAAGCCCGGTCAGAAACTCAGTTGT-3`).

The probes were synthesized using an Applied Biosystems 381A synthesizer and purified on denaturing polyacrylamide gel electrophoresis. Site-directed mutagenesis was performed using the Muta-Gene M13 in vitro mutagenesis kit from Bio-Rad. Mutated codons are underlined, except in the case of the deletion of Ser-18 indicated as EaS18. After digestion with EcoRI and BamHI, the 0.4-kilobase fragments encoding mutated Ea cDNAs were purified and cloned into either the expression/secretion vector pEZZ 18 (Protein A gene Fusion Vector purchased from Pharmacia) or the cytoplasmic expression vector pET3a (Studier and Moffatt, 1986).

Expression and Purification of Fused Mutants

The bacterial host used for expression of ZZ-Ea hybrid mutants was E. coli HB 101 (Boyer and Roulland-Dussoix, 1969). Bacteria were grown in a 5-liter fermentor (LSL Biolafitte) with an initial working volume of 4 liters of TSB (Tryptic Soy Broth) medium (Difco) supplemented with 5 g/liters of glucose and 200 mg/liters of ampicillin. The temperature was kept at 37 °C, the pH was maintained at 7.2 with 5 M NaOH, and oxygen concentration was adjusted to 80% saturation by adding an increasing proportion of pure oxygen to the air supply. When the initial glucose concentration fell to 0.5 g/liter, feeding was initiated by adding glucose (40%) as carbon source. The feeding rate was adjusted to give a growth rate of 0.3 h. Culture was stopped at the early stationary phase. Cells were discarded by centrifugation, the culture supernatant was filtered through a 0.2-µm filter, and concentrated by ultrafiltration on a membrane with a cut-off mass of 10 kDa (FILTRON). The solution was then applied on top of a 10-ml IgG-Sepharose column as described previously (Ducancel et al., 1989). The hybrid was lyophilized and dissolved in HCl 0.1 N in the presence of a 500-fold excess of CNBr for 24 h. The resulting recombinant toxin was separated from ZZ moiety and other side products by chromatography on a reverse phase HPLC column (Vydac, C, 5 µm 10 250 mm). The column was equilibrated in 0.1% trifluoroacetic acid, and elution was performed using a trifluoroacetic acid/CHCN/HO gradient system. The recombinant toxin was further purified by chromatography on a Mono S (Pharmacia) HPLC column equilibrated in ammonium acetate 0.01 M, pH 4.5, and eluted by a gradient of 0.01 M to 1.5 M ammonium acetate at the same pH. Purity of the hybrid mutant was checked by SDS-polyacrylamide gel electrophoresis and isoelectric focusing.

In the case of cytoplasmic expression, the pET expression system was used as follows. The ZZ-mutant toxin hybrid protein, without the signal sequence, was cloned in the pET3a vector, and E. coli BL21(DE3) was used as a host. Culture was made in TSB medium supplemented with 5 g/liter of glucose and 200 mg/liter of ampicillin. Induction was triggered with isopropyl-1-thio-- D-galactopyranoside (0.5 m M final concentration) when the ODreached 0.5. After 3 h cells were harvested by centrifugation and resuspended in lysis buffer (30 m M Tris, 5 m M EDTA, 20% sucrose, 0.1 mg/ml lysozyme, 0.1 mg/ml DNase I, pH 8). Cells walls were disrupted by three cycles of freezing and thawing, the supernatant was clarified by centrifugation for 30 mn at 10,000 rpm, and applied to an IgG-Sepharose column. The column was washed with 0.1 M phosphate buffer (pH 8) containing 0.1% Tween 20. Washing was continued until the Areached base line. Then two volumes of renaturation buffer (0.1 M phosphate buffer, 5 m M EDTA, 2:4 m M GSH/GSSG) were passed through the column which was then incubated 24 h at room temperature. The gel was subsequently washed with 5 m M ammonium acetate (pH 5), and the hybrid protein was eluted with 0.5 M acetate (pH 3.4). The eluted fraction was then submitted to cleavage with CNBr, and the resulting recombinant toxin was chromatographed on two successive HPLC columns, as indicated above.

Preparation and Characterization of the Mutants

Each mutant was submitted to amino acid sequencing, amino acid analysis, and isoelectric focusing. Protein concentrations were determined in solution from molar absorbancies at 278 nm evaluated as described previously (Pillet et al., 1993). This value was equal to 9000 for all mutants and 7500 for Ea Y25F.

Dichroic spectra were recorded at 22 °C using a CD III or CD VI Jobin-Yvon dicrograph.

Specific binding of toxin mutants to acetylcholine receptor-rich membranes was determined from competition experiments (Faure et al., 1983) using a H-labeled toxin as a radioactive tracer (Ménez et al., 1971). Equilibrium dissociation constants were determined from competition binding experiments according to Ishikawa et al. (1977). This was done by assuming that the two toxin-binding sites present per AcChoR molecule (Changeux, 1990) are equivalent and in particular that their affinities for the toxin and each mutant are identical. The precision of the experiments is compatible with such a hypothesis, and indeed all competition binding data fit nicely with theoretical curves in which a homogeneous class of binding sites was assumed to be present per molecule of AcChoR (see ``Results'').


RESULTS AND DISCUSSION

Choice of the Mutations

Curaremimetic toxins constitute a large family of proteins that bind to AcChoR. They share several common amino acids (Endo and Tamiya, 1991). Ten of them were recently mutated in Ea from L. semifasciata, a typical short chain toxin (Pillet et al., 1993). Mutations at Ser-8, Lys-27, Trp-29, Asp-31, Arg-33, and to a lesser extent at Phe-32, Gly-34, and Glu-38 resulted in a decrease in binding affinity of Ea for AcChoR, whereas mutations of Gly-49 and Leu-52 had no effect on Ea binding affinity. We now mutated several additional residues which, as indicated in Fig. 1, are often in proximity to those previously investigated and spread on the three toxin loops, including both faces of the toxin (Corfield et al., 1989). Mutations were chosen so as to produce the maximal change in the original chemical function and the minimal structural perturbations. The newly introduced residues were therefore often chosen for their propensity to adopt the same secondary structure as that of the wild-type residues (Chou and Fasman, 1978; Sibanda et al., 1989; Wilmot and Thornthon, 1988, 1990). Preparation and Characterization of the Mutants

The method, that consists of producing a weakly active fusion toxin in bacteria, is labor intensive but is compatible with the rules of our National Control Committee for Biohazards. Previously, we fused the toxin to a large protein A fragment, with the resultant fusion protein accumulated in the periplasm of the bacteria (Ducancel et al., 1989; Pillet et al., 1993). We now replaced protein A by its smaller two IgG-binding domains (ZZ) (Löwenadler et al., 1987; Nilson et al., 1987), so that the synthesized ZZ toxin hybrids were directly secreted into the growth medium of E. coli HB101, making them easy to purify by affinity chromatography on an IgG-Sepharose column. Treatment with CNBr (Boyot et al. 1990) followed by HPLC led to highly purified recombinant toxins with production yields routinely ranging between 0.025 and 0.5 mg toxin/liter of culture. This procedure was quite appropriate, since the resulting recombinant Ea is indistinguishable from Ea purified from venom, regarding both the biological properties (Boyot et al., 1990; Hervé et al., 1992; Pillet et al., 1993) and three-dimensional structure (Arnoux et al., 1994).

We prepared 30 new mutants of Ea. In experiments not reported in this paper, we determined that all these mutants (i) were encoded by genes having the expected nucleotide sequences; (ii) had the expected amino acid compositions and amino acid sequences at least up to the site of mutation. In addition, all chemical analyses (reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis) indicated that the mutants had the expected molecular weight. In most cases, we met no particular difficulty with production of the mutants. However, when Phe-4 was changed to Ala, we detected no ZZ mutant in the medium. We therefore produced this mutant using the cytoplasmic expression system pET-3a described under ``Materials and Methods.'' The hybrid was retained on an IgG column and oxidation of the disulfides was conveniently performed by applying a mixture of oxidized and reduced glutathione to the column. The hybrid was cleaved by CNBr, and 2 mg of purified mutant were obtained per liter of culture. That the EaF4A mutant could not be produced using a secretion system which was otherwise appropriate for most mutants might indicate that the Phe-4 plays a key role in the dynamic of in vivo folding of the toxin and/or its translocation process.

The far ultraviolet circular dichroic spectra of all mutants were recorded. These spectra will not be presented in this paper; however, they are available upon request. All spectra were virtually superimposable with that of the wild-type toxin, a result that indicates that the secondary structure of each mutant is similar to that of the wild-type Ea and that underlines the high permissivity of the toxin structure toward mutagenesis.

Binding affinities of all mutants for AcChoR from the electric fish T. marmorata were determined on the basis of competition experiments, using a protocol previously described in detail (Pillet et al., 1993). As an illustration of our experimental data, Fig. 2 shows the competition binding curves obtained with some mutants modified at a single residue of the first loop of Ea. It was previously reported that such binding data nicely correlate with the toxic and probably therefore with the curaremimetic function of the toxin (Ishikawa et al., 1977). Mutational Analysis

Definition of a Functional Residue

Residues implicated in contact surfaces of protein-protein complexes can be accurately identified by structural approaches, especially x-ray analyses (Janin and Chothia, 1990). On the other hand, residues that may be important for protein-protein interactions can be identified by functional assays, such as mutational studies. At present, the relationship between determinants defined by structural and functional analyses is not entirely clear. However, determinants characterizing protein-antibody (Prasad et al., 1993) and hormone-receptor (Cunningham and Wells, 1993) complexes have been identified using structural and functional approaches. These studies demonstrate that determinants identified by both types of methods do largely overlap. The structurally defined determinants are apparently somewhat larger, but this difference might be associated with the observation that, in a small number of particular cases, more than a single mutation is needed to identify the actual functionality of a residue (Kam-Morgan et al., 1993). The comparative analysis described by Prasad et al. (1993) reveals that functional residues for which mutations cause more than 10-fold affinity changes also belong to the structurally defined determinant, whereas residues for which mutations cause lower affinity changes are at the border of the structural determinants. Finally, several reports suggested that only a small subset of side chains, i.e. three to four, dominate the binding affinity of protein-protein interactions (Jin et al., 1992; Cunningham and Wells, 1993; Nuss et al., 1993; Prasad et al., 1993). Therefore, in the following, we will consider as functional, a residue whose mutation causes substantial affinity change of Ea for AcChoR. In view of the above observations, we infer that this residue has a great probability to belong to the interacting area between Ea and AcChoR; however, one should keep in mind that its actual contribution to the receptor binding remains to be determined.

Loop I

Loop I of Ea has 13 residues located between Cys-3 and Cys-17, forming a two-stranded -sheet joined by a -turn (Corfield et al., 1989). The effects of mutations of these residues on binding affinities for AcChoR can be classified in two categories (Table I). First, changing Phe-4, Asn-5, Pro-11, Gln-12, Thr-13, Thr-14, Lys-15, and Thr-16 virtually produced no effect on the affinity. Therefore, these residues are likely to be excluded from the toxin-receptor interaction. Previously, it was shown that neutralization by acylation of the positive charge of Lys-15 of Eb, an isoform of Ea (Hori and Tamiya, 1976) or of Eahas no effect on toxin activity. The present data confirm this finding and further show that replacement of Lys-15 by the smaller alanine can be also achieved without altering the toxin affinity. Therefore, most residues at the base of loop I may not be implicated in the functional site of Ea.

Second, mutations at the tip of the first loop, i.e. in the stretch His-6-Gln-7-Ser-8-Ser-9-Gln-10, resulted in affinity decreases (). We previously showed that mutation of Ser-8 to Gly induces a 176-fold affinity decrease (Pillet et al., 1993). We confirmed the critical role of this residue which occupies the i+1 position in the 7-10 -turn, since changing Ser-8 to Thr causes an even greater affinity decrease of nearly three orders of magnitude (780) . This is the largest effect observed so far for any single mutation in Ea. The dichroic spectrum of this mutant was superimposable with that of Ea (not shown), excluding the possibility that the large affinity change that is observed is associated with major modification of the toxin secondary structure. Clearly, a Ser to Thr change cannot be considered as functionally equivalent, despite the presence in both cases of a hydroxyl group. Gln-7 and Gln-10, which respectively occupy positions i and i+3 in the -turn, are also critical residues as inferred from the substantial affinity decreases caused by their respective mutations. Strikingly, neither of these 2 residues had ever been suggested to play a role in the function of a curaremimetic toxin. The neighboring Ser-9 is less important since its mutation into Gly causes only a 9-fold affinity decrease. That mutation of the adjacent Ser-8 into Gly causes a much larger affinity decrease (Pillet et al., 1993), emphasizes the fine specificity of the mutational analysis and suggests that of the 2 adjacent serine residues Ser-8 and Ser-9 at the tip of the first loop only Ser-8 is highly important for the binding function of Ea. Another residue of loop I whose mutation moderately affects Ea affinity is His-6. This residue appears inaccessible to solvent since it has a low p K around 3, a low exchange rate with DO when in solution (Inagaki et al., 1978), and a weak chemical reactivity toward iodine (Sato and Tamiya, 1970). Despite its structural involvement, His-6 can be changed into Ala without causing more than a 6-fold affinity decrease.

In conclusion, the loop I possesses functionally important residues, especially Gln-7, Ser-8, and Gln-10, which all belong to the tip the loop.

The Stretch between Loop I and Loop II

The stretch between Cys-17 and Cys-24 forms a large turn. In Ea, it comprises 6 residues whereas in other toxins it sometimes includes one or two deletions (Endo and Tamiya, 1991). We therefore deleted Ser-18, a residue which is located at position i of the -turn 18-21 in Ea (Corfield et al., 1989). A recent structural and immunological analysis suggested that this deletion is likely to provoke a shift of the type II -turn from residues 18-21 to residues 17-20 (Zinn-Justin et al., 1994). Notwithstanding this substantial structural change, the deletion has no consequence on Ea binding affinity (), indicating that Ser-18 and its vicinal region are not implicated in the functional site of Ea. Accordingly, mutation of Glu-21, a residue located at position i+3 of the -turn, also has no effect on toxin affinity (). We suggest, therefore, that the whole stretch 17-24 is not implicated in the functional site of Ea.

Loop II

Lys-27, Trp-29, Asp-31, Arg-33 and to a much lesser extent Phe-32, Gly-34, and Glu-38 were previously shown to be functionally important residues (Pillet et al., 1993). Most of them have their side chains on the toxin concave face. We now probed the two remaining side chains of this face of loop II, i.e. Tyr-25 and Ile-36. Tyr-25 is highly conserved among toxins that adopt a three-finger structure (Endo and Tamiya, 1991; Dufton and Hider, 1991). Its high p Ksuggests that its hydroxyl is hydrogen bonded, probably to Glu-38 as indicated from x-ray data of Ea (Corfield et al., 1989) or the isotoxin Eb (Bourne et al., 1985). Since this bond was previously anticipated to be important for maintaining the biologically active conformation of some toxins (Endo and Tamiya, 1991), we replaced Tyr-25 by Phe. Not only was the mutant found to have a circular dichroic spectrum similar to that of the native toxin (not shown), but also, as shown in Table II, the mutation has little effect on the toxin binding affinity. The hydroxyl group of Tyr-25 and its hydrogen bond with Glu-38 do not constitute critical requirements for Ea to fold into a functional conformation. Why then do so many toxins possess a tyrosine at position 25? We are currently addressing this question with new mutants.

That Ile-36 might play some role in the function of curaremimetic toxins has been previously anticipated (Ménez et al., 1982, 1984). We mutated Ile-36 into Arg, a side chain frequently found at this position in other toxins, and strikingly we observed a substantial increase in toxin affinity. Although competition experiments do not allow an accurate evaluation of such increments, we estimate it to be close to 7-fold. This is the first time that an affinity increase is seen upon individual mutation of an Ea residue. Recently, Mori and Tu (1991) demonstrated that Arg-36 of Lapemis neurotoxin becomes inaccessible to an arginine-specific reagent upon complexation of the toxin with AcChoR. This observation together with our own findings suggest that position 36 is functionally important, at least for some curaremimetic toxins. That the I36R mutation provokes a substantial affinity enhancement indicates that the interface between the wild-type toxin and AcChoR does not correspond to an optimized binding.

Previously, we showed that mutation of Asp-31 to His causes an affinity decrease of Ea for AcChoR by a factor of 46 (Pillet et al., 1993). In contrast, Rosenthal et al. (1994) found that mutation of Asp-31 to Ala does not affect the affinity of -bungarotoxin for AcChoR. In agreement with this result, we found that substitution of Asp-31 to Asn does not affect Ea affinity. At least two explanations can account for the observation that Ea affinity is uniquely affected by introduction of His at position 31. First, Asp-31 may play no functional role but the presence of the imidazole ring could induce some local conformational change. Since it is not detected by CD analysis, we infer that the putative change would be small and that Asp-31 would be at proximity of the functional site. Second, Asp-31 may be functionally important, but only mutation to His is capable of highlighting it. Interestingly, such a situation has been previously encountered by Kam-Morgan et al. (1993). These authors observed that mutation in lysozyme (HEL) of Asp-101to Gly had virtually no effect on the affinity of HEL for a HEL-specific antibody, whereas mutations of Asp-101to bulkier residues such as Arg or Phe, provoked affinity decreases by factors of 36 and 39, respectively. Notwithstanding this selective mutational sensitivity, the side chain and backbone atoms of Asp-101formed clear contacts with the antibody, as judged from the crystal structure of the HEL-antibody complex (Kam-Morgan et al., 1993). Although no definite explanation accounts for their observations, these authors demonstrated that a single mutation does not necessarily inform about the actual role of an amino acid. Similarly, mutation of the highly conserved Glu-38 of Ea into Gln and Lys was previously shown to cause no and moderate effect on Ea affinity, respectively (Pillet et al., 1993), whereas introduction of a Leu residue, whose side chain cannot establish hydrogen bonds, produces a 25-fold affinity decrease (Table II). As in the case of Asp-31 it is clear that more than one mutation is required at position 38 to observe a substantial decrease in affinity. In view of the high degree of conservation of Asp-31 and Glu-38, especially in short chain toxins (Endo and Tamiya, 1991), we wish to suggest that the affinity decreases that are selectively observed upon mutation to His and Leu, somehow reflect the functional character of both residues, although the actual nature of these functionalities remains unknown. We can only conclude in both cases that the negative charge is unlikely to be a critical functional parameter.

Pillet et al. (1993) showed that mutations of Arg-33 to Lys or Glu caused respectively 25- and 318-fold affinity decreases, indicating the functional importance of this residue and in particular of its positive charge. This is confirmed by the new mutation of Arg-33 to the neutral Gln which causes a nearly 200-fold affinity decrease.

Finally, we mutated a number of residues which, in contrast to those that have been so far investigated, have their side chains on the toxin convex face (Fig. 1). Thus, we mutated Gln-28, Ser-30, and Thr-35 to Ala and found no alteration of Ea affinity. This result agrees with the previous observation that Eb, a natural variant differing from Ea by a single mutation at position 26 (Asn in Ea is replaced by His in Eb (Sato and Tamiya, 1971)) has virtually the same affinity as Ea for AcChoR (Ishikawa et al., 1977). Together, all these results indicate that residues on the convex side of loop II are not functionally important.


Figure 1: Three-dimensional structure of the polypeptide chain of Ea from Laticaudata semifasciata (Corfield et al., 1989; Arnoux et al., 1994). The four disulfides are indicated by gray lines. Residues that have been mutated in our previous study (Pillet et al., 1993) or chemically modified (Lys-47 and Lys-51)are indicated by black circles. New positions that have been submitted to mutagenesis are indicated by gray circles. The side chains of residues oriented toward the reader are indicated by an arrow pointing to the upper part of the figure. * indicates that Asn-26 is naturally changed to His in the isotoxin Eb (Sato and Tamiya, 1971; Saludjian et al., 1992).



Loop III

The third loop of Ea includes 10 residues located between the half-cystines 43 and 54. Previous chemical modifications of individual lysine residues revealed that the Lys-47 derivatized Ea has lower affinity for AcChoR as compared to unmodified Ea. This result agrees with both similar experiments made with other curaremimetic toxins (Ishikawa et al., 1977; Faure et al., 1983; Endo and Tamiya, 1991) and the observation that mutation of Lys-47 to Ala markedly decreases the binding affinity of Ea (). Chemical modification of Lys-51 does not affect the binding affinity of Ea.Neither the natural mutation of Lys-51 to Asn, as it occurs in the natural variant Ec, affects the toxin affinity. This result further confirms that the residue 51, whose side chain is on the convex face, is not functionally important. Pillet et al. (1993) have observed that mutations of Gly-49 and Leu-52 have no effect on Ea binding function. We now observe that mutations of Pro-44, Thr-45, Val-46, Pro-48, Ile-50, and Ile-53 have no effect on the binding affinity of Ea for AcChoR (). Therefore, the present data suggest that of all the residues that constitute the loop III of Ea only the concave face-orientated Lys-47 is functionally important. On the Functional Site of Ea

General Description

Two complementary pieces of evidence allowed us to delineate the ``functional site'' of Ea. First, we identified a large set of probably excluded residues, as inferred from the mutants displaying a similar affinity as compared to the wild toxin (Tables I and II). These residues are Phe-4, Asn-5, Pro-11, Gln-12, Thr-13, Thr-14, Lys-15, Thr-16, Ser-18, Glu-21, Gln-28, Ser-30, Thr-35, Pro-44, Thr-45, Val-46, Pro-48, Gly-49, Ile-50, Leu-52, and Ser-53. This is also the case of the N-terminal group and Lys-51 which were chemically modifiedand of Asn-26 which is naturally mutated in His in the nearly equipotent Eb (Ishikawa et al., 1977). Therefore, of the 62 residues of Ea, at least 24 seem not to belong to the toxin functional site. Interestingly enough, a large number of them, i.e. the N-terminal group Phe-4, Gln-12, Thr-14, Thr-16, Asn-26, Gln-28, Ser-30, Thr-35, Val-46, Lys-51, and Ser-53 are located on the convex side of the toxin and widely spread on loops I, II, or III. This observation strongly suggests that the convex face of the -sheet formed by the three loops of Ea is not implicated in the functional site. Moreover, a substantial proportion of the concave face also seems to be excluded from the site. It concerns the residues that are localized on the -sheet strands of loop I, i.e. Asn-5, Pro-11, Thr-13, Lys-15, between loops I and II, i.e. Ser-18 and Glu-21 and in loop III, i.e. Thr-45, Gly-49, Ile-50, and Leu-52.

Second, we identified a number of residues forming a homogeneous surface and whose mutations induced substantial affinity changes. These are Gln-7, Ser-8, Gln-10, Lys-27, Trp-29, Asp-31, Arg-33, Ile-36, Glu-38, and Lys-47, among which Ser-8, Gln-10, Lys-27, and Arg-33 appear to constitute a subset of more crucial residues for binding affinity. Nicely surrounding the important functional side chains are residues whose mutations caused moderate affinity decreases. These are His-6, Ser-9, Tyr-25, Phe-32, and Gly-34 which belong to the first and second loops.

Based on all available data, Figs. 3 and 4 show the functional site of Ea. It is spread on the three toxin loops, mostly on its concave face, where it forms a homogeneous surface of approximately 680 Å, a value which is compatible with that expected for a protein-protein contact area (Janin and Chothia, 1990). As emphasized in Fig. 3, the site comprises three polar clusters, Lys-27-Glu-38 and Arg-33-Asp-31 in loop II, and Gln-7-Gln-10 in loop I, which all interact with the central hydrophobic cluster Trp-29-Ile-36 in loop II. Ser-8 establishes the only main chain-main chain contact between the loops I and II in Ea, with its hydroxyl group forming an hydrogen bond with the NH group of Ile-37 (Corfield et al., 1989; Bourne et al., 1985; Low and Corfield, 1986) and Lys-47 looks surprisingly isolated on loop III.


Figure 3: Representation of the residues whose mutations causes a change in the affinity of Ea for AcChoR. In yellow are those side chains whose mutations cause a 3-9-fold affinity decrease. The side chains colored orange are residues for which at least one mutation causes an affinity decrease by a factor equal to or greater than 10. In blue is indicated the residue, Ile-36, whose mutation causes an increase in affinity. To further classify the differential effects of the mutations, we indicated by bold lines the side chains whose mutations caused more than 100-fold affinity decrease. These are Ser-8, Gln-10, Lys-27, and Arg-33. Note that Asp-31 and Glu-38 have been indicated as functional residues on the basis of the observation that at least one, although not all tested mutations causes more than a 10-fold affinity decrease.



The functional site of Ea may not be completely delineated, however. As indicated by our mutational analysis of Asp-31 and Glu-38 and by other studies (Kam-Morgan et al., 1993), more than a single mutation may be required to establish the functional importance of a residue. Since most residues that are considered to be excluded from the functional determinant have undergone a single mutation, they need to be submitted to further mutagenesis. In particular, it is intriguing that only Lys-47 plays a functional role in loop III. This is all the more surprising as it was recently suggested that a fragment of AcChoR specifically recognizes loop III (Fulachier et al., 1994), and it is unlikely that such a recognition could be specifically exerted with a single positively charged residue. Neither does our analysis inform about the interactions that the backbone atoms might establish with the receptor. Finally, the actual individual contributions of the functional residues to the establishment of EaAcChoR complex remain to be determined. In this respect, it would be of interest not only to estimate the on and off rate constants of the mutant-receptor complexes but also to determine the effects of the mutations on the thermodynamic parameters, i.e. H and S, involved in EaAcChoR interactions.

The Functional Site of Curaremimetic Toxins Has Both Invariant and Variant Residues

Curaremimetic toxins are classified as short chain toxins which, like Ea, contain 60-62 residues and four disulfides and as long chain toxins with 66-74 residues and five disulfides (Endo and Tamiya, 1991). Despite their length differences, curaremimetic toxins share a similar overall folding, as revealed by both NMR and x-ray crystallography (Agard and Stroud, 1982; Laplante et al., 1990; Love and Stroud, 1986; Low et al., 1976; Low, 1979; Tsernoglou and Petsko, 1976, 1977; Walkinshaw et al., 1980; Zinn-Justin et al., 1992). Using the common presence of eight half-cystines, the amino acid sequences of curaremimetic toxins can be aligned, revealing the existence of a number of residues that are invariant or type invariant in short chain toxins, in long chain toxins, or in both categories of toxins, as proposed by Endo and Tamiya (1991). Among the residues recognized to be highly important for the function of Ea, 4 are highly conserved in both toxin categories. These are Lys-27, Trp-29, Arg-33, and Lys-47. These invariant residues might constitute a ``common functional core'' through which curaremimetic toxins establish conservative contacts with AcChoRs. In addition, it is possible that the invariant Asp-31 and Glu-38, whose roles remain however to be clarified, may also contribute to the conserved functionality of curaremimetic toxins.

The most remarkable feature emerging from our result is that the functional site unexpectedly overlaps the first loop, in contrast to previous proposals (Low, 1979; Ménez et al., 1982, 1984). Among the residues that were not predicted to be functionally important are Gln-7 and Gln-10 which are not present in long chain toxins and which were not even considered as being conserved within the family of short chain toxins (Endo and Tamiya (1991). These residues are sometimes replaced in short chain toxins by Pro or Met and Thr or Glu, respectively. Ile-36 is another functional variable residue which is nearly totally absent in long chain toxins where it is frequently replaced by an arginine and sometimes by a valine. Therefore, the site by which the short chain toxin Ea binds to AcChoR from T. marmorata includes variant residues.

It is well documented that both the short and long chain toxins bind with high affinity to AcChoR isolated from electric organ of Torpedo fish (Endo and Tamiya, 1991). This binding can be first rationalized by our finding that approximately 60% of the residues that have been identified as functionally important for Ea are also present in nearly all curaremimetic toxins. These residues may provide all these toxins with a basic moderate affinity for AcChoR from Torpedo. In Ea, the additional functional residues mostly belong to loop I. Strikingly, they are not present in long chain toxins whose loop I greatly differs from that of short chain toxins in terms of size, conformation, and amino acid composition (Endo and Tamiya, 1991). To exert their high affinity for Torpedo AcChoR, the long chain toxins are therefore anticipated to possess other functional residues, presumably also variable in nature. This suggestion is supported by a recent NMR study depicting the three-dimensional structure of the complex formed between the long chain toxin -bungarotoxin and the fragment 185-196 from the -subunit of Torpedo californica (Basus et al., 1993). This study shows that several interacting residues of -bungarotoxin are mutated or even do not exist in Ea. This is the case, for example, of Thr-5, Thr-6, Ala-7, and Ile-11 which belong to the loop I of -bungarotoxin and which all differ in Ea. This is also the case for His-68 which is uniquely located at the C-terminal tail of -bungarotoxin. Clearly, therefore, variant residues especially localized in loop I substantially contribute to the high binding affinity of curaremimetic toxins. This finding implies that there is not a unique ``functional site'' for all toxins but rather a sort of ``variation on a theme'' around a common functional core. Interestingly enough, implication of variable residues to AcChoR binding offers an explanation as to why short chain toxins are usually more toxic in rodents and birds whereas the long chain toxins tend to be more effective against receptors from humans and from some reptiles and amphibians (Lee and Chen, 1976; Chang, 1979; Burden et al., 1975; Ishikawa et al. 1985). Finally, we noted that the 3 important residues Gln-7/Ser-8/Gln-10 of Ea, a sea snake toxin, are also present in more than 80% of the other short chain toxins from sea snakes (Endo and Tamiya, 1991). This observation suggests that these residues are evolutionarily conserved to provide the short chain toxins with a high binding affinity toward AcChoR from fish, the well-known prey of sea snakes (Heatwole, 1987).


CONCLUSION

At present, 36 individual positions of Ea have been submitted to site-directed mutagenesis, two others (positions 1 and 51) have been mono-modified by chemical means, and one (position 26) has been substituted in a natural mutant. If one excludes the four disulfides whose structural role is highly probable (Rydén et al., 1973; Ménez et al., 1982, 1984), nearly 72% of the Ea residues underwent mutational analysis. Considering that (i) the functionally identified residues are located within a continuous surface surrounded by a large number of excluded residues; (ii) the number of functional residues and the surface (680 Å) covered by them is close to that currently observed for a protein determinant, we believe that most of the ``curaremimetic'' site of Ea is now identified. However. it remains to study the individual contributions of the identified residues in the formation of the toxinAcChoR complex and to identify the receptor residues that recognize the toxin functional site. Nevertheless, the present knowledge can already be exploited in various practical directions, including the preparation of controlled toxoids and the design of new curare compounds for clinical use.

  
Table: Indication of the apparent Kvalues of the wild-type and mutants of Ea

Mutations made in the first loop and in the stretch that joins loops I and II. For symbol notations see legend of Table II.


  
Table: Mutations made in loops I and II

The values were determined from competition data similar to those shown in Fig. 2 according to Ishikawa et al. (1977). The second column shows the ratio of the affinity constant of each mutant to that of the wild-type Ea. The residues for which mutations induced important affinity changes are emphasized by bold letters. The notation (a) indicates that the mutation occurs naturally in the variant Eb which differs from Ea by this single substitution at position 26. The notation (b) indicates that the mutation occurs naturally in the variant called Ec which differs from Ea by two mutations, one at position 26 where Asn is replaced by His and the other at position 51 where Lys is replaced by Asn. Note that the chemical derivative of Ea selectively monoacetylated at position 51 has a similar affinity as compared to Ec,further confirming the nonimportance of Lys-51 in Ea function. Asterisks indicate data from Pillet et al. (1993).



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: AcChoR, nicotinic acetylcholine receptor; Ea, Erabutoxin a; HPLC, high performance liquid chromatography.

O. Trémeau, C. Lemaire, P. Drevet, S. Pinkasfeld, F. Ducancel, J.-C. Boulain, and A. Ménez, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Sophie Zinn-Justin, Sylvaine Gasparini, Denis Servent, and Bernard Gilquin for their kind help and their constant interest. We are also indebted to Evelyne Lajeunesse for her technical assistance. Biohazards associated with the experiments described in this publication have been examined previously by the French National Control Committee.


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