Identification of Structural Elements of a Scorpion alpha -Neurotoxin Important for Receptor Site Recognition*

(Received for publication, October 22, 1996, and in revised form, February 26, 1997)

Noam Zilberberg Dagger , Oren Froy Dagger , Erwann Loret §, Sandrine Cestele , Dorit Arad par , Dalia Gordon par and Michael Gurevitz Dagger **

From the Dagger  Department of Plant Sciences and the par  Department of Molecular Microbiology and Biotechnology, Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel, § IBSM-LIDSM-CNRS-UPR 9027, 31 Chemin Joseph Aiguier, BP 71, 13402, Marseille Cedex 20, France, and the  Laboratoire de Biochimie, CNRS URA 1455, Faculte de Medicine, Secteur Nord, 13916 Marseille Cedex 20, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

alpha -Neurotoxins from scorpion venoms constitute the most studied group of modifiers of the voltage-sensitive sodium channels, and yet, their toxic site has not been characterized. We used an efficient bacterial expression system for modifying specific amino acid residues of the highly insecticidal alpha -neurotoxin Lqhalpha IT from the scorpion Leiurus quinquestriatus hebraeus. Toxin variants modified at tight turns, the C-terminal region, and other structurally related regions were subjected to neuropharmacological and structural analyses. This approach highlighted both aromatic (Tyr10 and Phe17) and positively charged (Lys8, Arg18, Lys62, and Arg64) residues that (i) may interact directly with putative recognition points at the receptor site on the sodium channel; (ii) are important for the spatial arrangement of the toxin polypeptide; and (iii) contribute to the formation of an electrostatic potential that may be involved in biorecognition of the receptor site. The latter was supported by a suppressor mutation (E15A) that restored a detrimental effect caused by a K8D substitution. The feasibility of producing anti-insect scorpion neurotoxins with augmented toxicity was demonstrated by the substitution of the C-terminal arginine with histidine. Altogether, the present study provides for the first time an insight into the putative toxic surface of a scorpion neurotoxin affecting sodium channel gating.


INTRODUCTION

alpha -Neurotoxins, the most abundant group in Buthidae scorpion venoms, are polypeptides composed of a single chain of 63-65 amino acids, cross-linked by four disulfide bridges, and are responsible for human envenomation (1, 2). They show variability in their apparent toxicity to mammals and insects, in their primary structures, and in their binding features to neuronal membrane preparations (3-6). Among scorpion neurotoxins, the alpha -group is the most studied and is useful in functional mapping of the sodium channel structure (6; reviewed in Refs. 7-9). Despite the reported structures (10-13), chemical modifications, and immunochemical studies (14-17) of various scorpion alpha -toxins, the structural elements dictating molecular recognition of their binding site have not been characterized. It was postulated that the molecule surface bearing a cluster of aromatic and hydrophobic residues, termed the "conserved hydrophobic surface," was associated with the toxic site (10, 13, 18). However, this postulation has not gained experimental support.

Despite the differences in their primary structures and phylogenetic selectivity, scorpion neurotoxins affecting sodium channels are closely related in their spatial arrangements and form a compact globular structure that is kept rigid by four disulfide bridges (1, 19). A genetic approach based on comparative analysis of the variable regions may clarify distinct residues involved in toxin-receptor interactions. However, due to difficulties in producing sufficient amounts of recombinant toxins (20, 21), such an approach has been limited thus far.

Recently, we have established an efficient bacterial expression system of a scorpion alpha -neurotoxin, Lqhalpha IT, displaying an exceptionally high insecticidal activity (5, 6, 22). Lqhalpha IT slows the sodium current inactivation in excitable membranes in a manner characteristic of other scorpion alpha -toxins (22). Its binding to the receptor site on neuronal membranes is competitively inhibited by the sea anemone toxin ATXII and enhanced by veratridine (6, 23, 24). Lqhalpha IT binds to a single class of high affinity sites on insect neuronal membranes (Kd = 0.2-0.5 nM and 0.03-0.04 nM in locust and cockroach, respectively (6, 23)) and competes weakly with other alpha -toxins for binding to rat brain synaptosomes. AaHII, the most potent anti-mammalian scorpion alpha -neurotoxin, competes for 125I-Lqhalpha IT binding sites on insect sodium channels, suggesting that the two scorpion alpha -toxins bind to homologous, nonidentical receptor sites on insect and mammalian sodium channels (6). Lqhalpha IT is toxic to mice at relatively high concentrations as opposed to other scorpion alpha -neurotoxins such as AaHII (5, 6), yet it appears to recognize an alpha -toxin binding site on both insect and mammalian sodium channels. Using our functional expression system, we found Lqhalpha IT very useful for a genetic study (5) and for two-dimensional 1H NMR studies (25). Here we report the results of modifications introduced at several regions of Lqhalpha IT that highlight a putative molecular surface-mediating recognition of the receptor site and intoxication.


EXPERIMENTAL PROCEDURES

Bacterial and Animal Strains

Escherichia coli DH5a cells were used for plasmid constructions. E. coli BL21 cells lysogen with DE3 phage derivative bearing the T7 RNA polymerase gene under control of the lac promoter (26) were used for expression. A kanamycin-resistant derivative of pET-11c vector (26) was used for expression (5). Sarcophaga falculata blowfly larvae were bred in the laboratory. Albino ICR mice were purchased from the Levenstein farm (Yokneam, Israel).

Site-directed Mutagenesis, Functional Expression, and Purification of Toxins

Substitutions K8A, K8D, Y10S, Y10W, and K8D/N9D/Y10V were generated by using back-to-back primers and inverse polymerase chain reaction (27) with pBluescript bearing Lqhalpha IT cDNA (28). Engineering of the 5' and 3' termini of the Lqhalpha IT cDNA for expression was described previously (5). Substitutions E15A, F17G, R18A, G40S/K41P, and K8D/E15A were performed according to the method of Deng and Nickoloff (29). Expression of the recombinant polypeptides in a nonsoluble form, renaturation, and purification of the active toxin were performed as described previously (5). Sequences of the mutated cDNAs were verified prior to expression with Sequenase version II (U. S. Biochemical Corp.). Quantification of the purified recombinant Lqhalpha IT variants and verification of their composition was performed by amino acid analysis.

Toxicity Assays

Four-day-old blowfly larvae (S. falculata; 100 ± 20 mg body weight) were injected intersegmentally. A positive result was scored when a characteristic paralysis (immobilization and contraction) was observed 5 min after injection. Nine larvae were injected with five concentrations of each toxin in three independent experiments. ED50 values were calculated according to the sampling and estimation method of Reed and Muench (30). Toxicity to mammals was determined by subcutaneous injection to female mice (20 ± 3 g).

Competition Binding Experiments

Preparation of radioiodinated Lqhalpha IT was performed according to Gordon and Zlotkin (23). Cockroach (Periplaneta americana) synaptosomes (P2L fraction) were prepared from the central nervous system by established methods (24, 31). The binding assays were performed in the form of equilibrium competition assays using increasing concentrations of the unlabeled toxin in the presence of a constant low concentration of the labeled toxin (30-50 pM) (6). Analyses of binding assays were carried out by using the iterative computer program LIGAND (Elsevier Biosoft). Each experiment was performed at least three times.

Circular Dichroism Measurements and Molecular Modeling

The protein samples used for CD spectrum analyses were in 20 mM phosphate buffer, pH 7. Spectra were measured at 20 °C in 0.05-mm path length cuvette from 260 to 178 nm with a JOBIN-YVON (Long-Jumeau, France) UV CD spectrophotometer (Mark VI). Calibration was performed with (+)-10-camphorsulfonic acid. A ratio of 2.2 was found between the positive CD band at 290.5 nm and the negative band at 192.5 nm. Data were collected at 0.5-nm intervals with a scan rate of 1 nm/min. CD spectra are reported as Delta epsilon per amide. The protein concentration was in the range of 0.5-1 mg/ml as determined on a Beckman amino acid analyzer. The secondary structure content was determined according to the method of Manavalan and Johnson (32).

Structural models were constructed using the Quanta-Charmm program by MSI Ltd. and Insight II-Discover software from MSI Technologies, Inc. (United Kingdom) running on a Silicon Graphics VGX R4000 Crimson Workstation. Models were built by the Homology module of Quanta on the basis of 59.4% identity to AaHII neurotoxin (10). The structures were minimized in vacuo using the Charmm force field. One hundred steps of minimization were performed using the steepest descent algorithm, followed by 5000 steps using the conjugate gradient method until a root mean square deviation of 0.001 was obtained. Electrostatic potentials of the unmodified and mutant toxins were calculated using Delphi software (MSI Technologies, Inc. (UK)).


RESULTS

Selection of sites to be modified was based on comparison between two homologous alpha -toxins, AaHII and Lqhalpha IT, displaying two extremes in their phylogenetic preferences. AaHII, the strongest anti-mammalian scorpion alpha -neurotoxin (6, 33), and Lqhalpha IT, the most insecticidal toxin among the alpha -group (5, 6, 22), compete very poorly with each other for their binding sites on insect or mammalian sodium channels. This pharmacological difference can be attributed to nonhomologous residues or structural motifs located on their surfaces. Site-directed modifications were introduced to Lqhalpha IT to elucidate the molecular surface involved in recognition of the receptor site on the sodium channel. All toxin variants were purified by high performance liquid chromatography using the toxicity assay on blowfly larvae as a quick and direct measure of activity. Binding assays to a cockroach neuronal membrane preparation were used as a measure of direct activity at the receptor site. Possible structural alterations were assessed by CD spectroscopy.

Modification of Tight Turns

Comparison between the three-dimensional structures and amino acid sequences of AaHII (10) and Lqhalpha IT (5, 25) revealed major differences in the five-residue turn (residues 8-12) and in the C-terminal region: (i) residues Asp8-Asp9-Val10 in AaHII are located on an external region of the toxin surface and are conserved in a large group of alpha -toxins, whereas these positions are occupied by Lys8-Asn9-Tyr10 in Lqhalpha IT (22, 28) (Fig. 1); (ii) the disposition of the C termini relative to the five-residue turns shows a clear difference between Lqhalpha IT and AaHII (10, 25).


Fig. 1. Alignment of the amino acid sequences of several scorpion alpha -neurotoxins. Residues homologous to those in Lqhalpha IT are designated by dashes. Dots indicate gaps in the aligned sequences. Modified sites are designated by asterisks (modifications at positions 49, 50, and 54 were described previously (5)), and secondary structure motifs (25) are indicated. AaH2, Androctonus australis Hector toxin 2 (AaHII); Lqq4 and Lqq5, Leiurus quinquestriatus quinquestriatus toxins 4 and 5, respectively; Amm5, A. mauretanicus mauretanicus toxin 5; Bot3, Buthus occitanus tunetanus toxin 3 (3).
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We initiated the mutagenesis program by substituting residues Lys-Asn-Tyr (positions 8-10) into Asp-Asp-Val (as appear at these positions in AaHII) and produced the recombinant Lqhalpha IT variant using our expression-reconstitution system (5). This modification affected the activity of the toxin dramatically: the apparent affinity for the insect receptor site decreased 21,000-fold, and the toxicity was practically lost (Fig. 2A, Table I). To determine whether this detrimental effect was due to a conformational change of the overall structure, the mutant toxin was analyzed by CD spectroscopy (Fig. 3A). Despite some changes in the spectrum, the calculated secondary structure content (32) was similar to that of the unmodified toxin. This result suggested that the mutation might have generated a local effect on the active molecular surface.


Fig. 2. Competitive inhibition of the binding of unmodified 125I-labeled Lqhalpha IT by various recombinant Lqhalpha IT toxins. The unmodified toxin is compared with mutants modified in turn structures (A), mutants modified at residues presumably interacting with the receptor site (B), and mutants modified at the C-terminal region (C). Cockroach neuronal membranes (25 µg of membrane protein) were incubated for 60 min at 22°C in the presence of 0.03 nM 125I-labeled Lqhalpha IT and increasing concentrations of unmodified or mutant toxins. Nonspecific binding of 125I-labeled Lqhalpha IT, determined in the presence of 1 µM Lqhalpha IT (corresponding to 15-25% of total binding), was subtracted. The binding was determined as described (see "Experimental Procedures" and Ref. 6) and analyzed by the LIGAND computer program.
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Table I. Biological activity of the recombinant toxins


Toxin ED50a Toxicity IC50b Increase in IC50

ng/100 mg body weight % nM -fold
Unmodified 12.8 100 0.035  ± 0.01 1
K8D/N9D/Y10V 3500 0.4 745.6  ± 130.5 21,303
K8D 2100 0.6 56.4  ± 11.0 1,611
K8A 50 26 1.38  ± 0.41 39.4
Y10W 30 43 0.13  ± 0.04 3.7
Y10S 129 10 4.0  ± 0.6 114.3
K8D/E15A 238 5 2.5  ± 0.5 71.4
E15A 55 23 0.021  ± 0.013 0.6
F17G 92 14 4.6  ± 0.9 160
R18A 59 22 8.1  ± 1.9 231.4
D19N 11 115 0.043  ± 0.01 1.2
D19H 52 25 0.21  ± 0.1 6
R18K/D19N 31 41 0.016  ± 0.008 0.46
G40S/K41P 20 64 0.9  ± 0.3 25.7
K62L 69 19 7.7  ± 3.1 220
R64N 12 106 2.05  ± 0.7 58.6
R64D 258 5 83.8  ± 26.0 2,394
R64A 24 53 1.9  ± 0.8 54.3
R64H 4 320 0.012  ± 0.009 0.33
R64H/65R 28 46 0.06  ± 0.03 1.7

a ED50 was determined on blowfly larvae (100 ± 20 mg).
b IC50 was determined on cockroach neuronal membrane preparations.


Fig. 3. Circular dichroism spectra of unmodified and mutant Lqhalpha IT toxins. The unmodified toxin is compared with mutants modified at the five-residue turn and a suppressor mutant (A), mutants with minor structural changes (B), and mutants modified at the 40-43 beta -turn and at the C-terminal region (C). Spectra of some mutants that resembled that of the unmodified toxin, i.e. Y10W, D19N, D19H, R18A/D19N, R64N, and R64HR, were omitted.
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To identify the specific residue responsible for the detrimental effect, the following separate substitutions were conducted: (i) K8A and K8D to determine the significance of the positive charge at this position; and (ii) Y10S and Y10W to clarify whether a hydroxyl or an aromatic ring is crucial at this site. Neutralization of the positive charge at position 8 (K8A mutant) resulted in a 39.4-fold decrease in the apparent affinity for the receptor site on cockroach sodium channels and a 4-fold decreased toxicity to blowfly larvae (Fig. 2A, Table I) without a significant change in the CD spectrum (Fig. 3B). However, a severe effect was obtained when the charge at position 8 was inverted by replacing lysine with aspartate (K8D). The apparent affinity for the receptor site decreased 1611-fold, and less than 1% of the residual toxicity was determined (Fig. 2A, Table I). CD spectrum analysis of the K8D variant revealed a complete loss of the 190-nm band (Fig. 3A), suggesting changes in its secondary structure (see "Discussion").

In an attempt to visualize the impact of the charge inversion, we compared the calculated electrostatic potentials of the unmodified, the K8D, and K8D/N9D/Y10V mutant toxins (Fig. 4). The unmodified toxin was found to be highly polar. One pole, containing the N terminus, was negatively charged, and the other pole, containing Lys8, Arg18, Arg58, Lys62, and the C-terminal Arg64, was positively charged (Fig. 4, top left). The "bullet-shaped" electrostatic potential was severely disrupted in mutant K8D (Fig. 4, top right) and only mildly disrupted in mutant K8A (data not shown). We investigated whether this disruption was due to the charge inversion at position 8, causing a conformational change (Fig. 3A), or to alteration of the overall charge distribution of the molecule by neutralizing the negative charge of the adjacent Glu15 (E15A mutant). This substitution was based on results obtained by Delphi calculations with K8D and K8D/E15A computer models suggesting a detrimental effect on the electrostatic potential caused by two adjacent negative charges, i.e. Asp8 and Glu15 (Fig. 4, top right). The E15A mutation alone revealed no significant change in the binding affinity, a 4-fold decrease in toxicity (Table I, Fig. 2A), no change in the CD spectrum (Fig. 3A), and a nonsignificant change in the electrostatic potential (data not shown). However, an E15A mutation in addition to the K8D mutation (K8D/E15A double mutant) had no effect on the CD spectrum, remaining similar to that of mutant K8D (Fig. 3A), but restored the electrostatic charge distribution to a great extent (Fig. 4, top left). This restoration was reflected by substantial increases in the apparent affinity for the receptor site (22-fold) and toxicity (8.3-fold) of the double mutant compared with the K8D mutant (Fig. 2A, Table I). These results suggest that the electrostatic potential may be important for the biological activity of Lqhalpha IT.


Fig. 4. The electrostatic potentials of Lqhalpha IT and modified toxins. The positive (+1 kcal/mol) surface of the unmodified toxin is shown as a white net (top left), and the positive surfaces of the mutants are shown by blue nets. The negative (-1 kcal/mol) surfaces are indicated by the red nets or by the purple net in the double mutant K8D/E15A. The structures and electrostatic potentials of the unmodified and K8D/E15A double mutant are superimposed. N and C stand for the N and C termini, respectively. The orange ribbons, oriented similarly in all variants, indicate the carbon backbones.
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The possible role of the aromatic Tyr10, belonging to the five-residue turn, was investigated by two substitutions. A Y10W substitution had a minute effect on toxicity (Fig. 2B, Table I). However, replacement by serine caused a 114-fold decrease in the apparent affinity for the receptor site (Fig. 2B, Table I), and only 10% of the toxicity was detected (Table I). In light of the unchanged CD spectra of mutants Y10S (Fig. 3B) and Y10W (not shown), these results suggest that the aromatic side chain at position 10 may interact with the receptor site.

The 40-43 beta -turn is unique to alpha -scorpion neurotoxins and thus could pertain to their characteristic pharmacology (19). In other alpha -toxins, residues 40 and 41 are serine and proline, respectively, whereas glycine and lysine occupy these positions in Lqhalpha IT. Still, the G40S/K41P mutation had a mild effect on toxicity. Although the 190-nm band decreased substantially in the CD spectrum of this mutant (Fig. 3C), the apparent affinity for the receptor site decreased only 25.7-fold, and 64% of the toxicity was preserved (Table I). These results suggest that this tight turn does not play a critical role in the insecticidal activity of Lqhalpha IT.

Modification of the Loop Preceding the alpha -Helix

From the solution structure of Lqhalpha IT (25) it was apparent that residues Phe17 and Arg18 in the loop preceding the alpha -helix (residues 19-28) belong most likely to the molecular surface common to the aforementioned turns. Phe17 is unique to Lqhalpha IT, and Arg18 is found at this position in most alpha -neurotoxins with high activity against mammals (Fig. 1) (6, 34). Phe17 was replaced by glycine, found in most alpha -toxins, and Arg18 was substituted by alanine to examine the role of its charge. The IC50 of mutant F17G was affected markedly (160-fold increase), and 14% of the toxicity was determined (Fig. 2B, Table I). The apparent affinity of mutant R18A for the receptor site on the cockroach sodium channels decreased 231-fold, and 22% of the toxicity was determined (Fig. 2B, Table I). However, only minor changes in calculated electrostatic potentials (data not shown) and CD spectra (Fig. 3B) were observed.

The residue adjacent to Arg18 is the first amino acid in the alpha -helix. In Lqhalpha IT, this position is occupied by aspartate (Asp19), whereas asparagine is found at this position in most alpha -neurotoxins (Fig. 1). Mutant D19N revealed no change in the apparent affinity for the receptor site but some increase in toxicity to blowfly larvae (Table I). Mutant D19H revealed a slight decrease in activity. A double mutant, R18K/D19N, exerted some improved binding affinity but a slight decrease in toxicity (Table I). Apparently, the negative charge at position 19 has no important role in the toxicity of Lqhalpha IT.

Modifications of the C Terminus

The region connecting Cys12 (five-residue turn) with Cys63 (C-terminal region) by a disulfide bridge was shown to participate in an immunoreactive epitope whose blocking by specific antibodies inhibited the binding of AaHII to rat brain synaptosomes. Furthermore, Val10, Lys58, His54, and/or His64 were proposed to play a role in the interaction with a monoclonal antibody, which precluded binding of AaHII to the receptor site (35).

The possible involvement of the C terminus in the toxic site of Lqhalpha IT was investigated by modifying the terminal Arg64 and the adjacent Lys62. The positive charge of Arg64 was either neutralized (R64A, R64N) or inverted (R64D). Furthermore, the terminal Arg64 was substituted by histidine, found at the C terminus of several other alpha -toxins, and by a histidine-arginine pair, as found in the cDNA sequence of Lqhalpha IT (28). Lys62 was converted to leucine to assess the significance of its positive charge. As shown in Fig. 2C and Table I, the apparent affinity for the receptor site of mutants R64A and R64N decreased, whereas their toxicity (Fig. 2C, Table I) and CD spectra remained practically unchanged (not shown). Orientation of their electrostatic potentials did not change, albeit there was a reduction in the overall positive charge (data not shown). Mutant R64D, however, revealed a marked decrease in the apparent affinity for the receptor site and 20-fold reduced toxicity (Table I, Fig. 2C). These effects were in concert with a marked disruption of the electrostatic potential (Fig. 4) and a change in CD spectrum (Fig. 3C). Another severe effect occurred with mutant K62L (220-fold decrease in apparent binding affinity and 5-fold reduced toxicity) (Table I, Fig. 2C). This effect was accompanied by an increase in the 190-nm band of the CD spectrum (Fig. 3C) and a marked decrease in the positive component of the electrostatic potential (not shown).

In contrast to the mutants showing reduced activity, mutant R64H revealed a 3.2-fold increase in toxicity and a 3-fold improved binding affinity for the receptor site (Fig. 2C, Table I). The CD spectrum of this variant remained similar to that of the unmodified toxin (Fig. 3B).

All toxin variants were injected to mice in parallel with the biological tests on insects. Interestingly, the changes in the insecticidal activity of each of the mutant toxins correlated with the changes in the anti-mammalian toxicity (not shown).


DISCUSSION

The genetic approach in the present study has become possible due to an efficient expression-reconstitution system established recently for the highly insecticidal scorpion alpha -neurotoxin Lqhalpha IT and its genetic variants (5). Interestingly, the yields of functional polypeptide variants were similar to that obtained for the unmodified toxin, varying between 2 and 5 mg of protein per liter of E. coli culture, and no correlation between the yield, bioactivity, and/or CD spectrum was observed. Point mutagenesis highlighted amino acid residues having a role in receptor site recognition, structural arrangement, and formation of a polar electrostatic surface.

A Putative Dual Role for Lys8

Since variations in animal group specificity among toxins belonging to the same neuropharmacological class are most likely related to structural differences at their putative toxic sites, we directed the initial modifications to such a variable motif, namely the five-residue turn of Lqhalpha IT. This turn (residues 8-12) in Lqhalpha IT (KNYNC), the most insecticidal scorpion alpha -toxin (6, 22), differs greatly from its corresponding turn in AaHII (DDVNC), the strongest anti-mammalian scorpion alpha -neurotoxin (6, 33). The profound decrease in toxicity and binding affinity to the receptor site caused by the K8D/N9D/Y10V mutation (Table I) suggested a major role for residues 8-10. At this point it was important to clarify whether the K8D/N9D/Y10V mutation affected the overall structure, which could consequently lead to loss of activity, or whether it was a local effect on residues exposed to the solvent and, thereby, disrupted direct interactions with the receptor binding site. CD spectrum (Fig. 3A) and singular value decomposition-derived data analyses of the mutant toxin revealed a secondary structure content similar to that of the unmodified toxin, suggesting preservation of the overall spatial arrangement of the mutant toxin. The modifications performed with either Lys8 or Tyr10 were made to determine the specific residue responsible for the detrimental effect obtained with the triple mutation. The marked decrease in the apparent binding affinity and toxicity obtained with the K8D mutation (Table I) implied a major role for this residue. This detrimental effect could be due to either direct disruption of a structural motif or indirect alteration generated by repulsion between Asp8 and Glu15, elimination of a direct interaction with a recognition point at the receptor site, and/or alteration of the polarity of the molecule. Analysis of mutants K8A, E15A, and K8D/E15A provided data enabling discrimination among these possibilities. The importance of a positive charge at this position was shown by the decrease in toxicity of the K8A mutant (Table I). Since the electrostatic potential and CD spectrum of the K8A mutant changed only slightly, the decrease in activity did not result from disruption in secondary structure or from elimination of the putative interaction with the adjacent Glu15. It is more likely that Lys8 interacts electrostatically with the receptor site, and since this interaction is part of a multipoint interacting surface (31, 36), its effect is rather slim.

However, inversion of the charge at position 8 (K8D mutant) caused a severe effect on biological activity (Table I) in addition to a complete disappearance of the 190-nm band in the CD spectrum (Fig. 3A). This band was found to correlate with either alpha -helix and/or turn structures (37). We speculate that the changes observed in the 190-nm band in the CD spectra of mutants K8D and G40S/K41P are associated with structural alterations of tight turns alone. That is because the modifications were introduced to turn structures distant from the single alpha -helix of this molecule. On the basis of the structural difference between AaHII (10) and Lqhalpha IT (25), we speculate that this structural change might cause Asp8 to form hydrogen bonds, as is found in AaHII (10), leading consequently to a buried position in the mutant K8D. Furthermore, the electrostatic potential of this variant was disrupted as well (Fig. 4). Restoration of activity obtained by the E15A suppressor mutation while the CD spectrum remained similar to that of the K8D mutant (Fig. 3A) suggested that neither the change in secondary structure nor the negative charge at position 8 were fully responsible for the detrimental effect caused by mutation K8D. The unchanged CD spectrum of mutant K8D/E15A compared with that of mutant K8D also contradicted the possibility that a repulsion between Asp8 and Glu15 caused the alteration in secondary structure (Fig. 3A). It is possible that introduction of two negative charges (Asp8 and Glu15) at the positive pole disrupts the electrostatic potential of the molecule, whereas one negative charge (Glu15 in the unmodified toxin, provided that no interaction with Lys8 exists, or Glu15 in the K8A mutant, or Asp8 in the K8D/E15A mutant) is not detrimental. This conclusion is supported by the results obtained with the K8D/N9D/Y10V triple mutant. Three negative charges (Asp8, Asp9, and Glu15) changed the electrostatic potential of the toxin (Fig. 4); indeed, the apparent affinity for the receptor site decreased 21,000-fold, and only 0.4% of the residual toxicity was measured (Table I). These results may suggest an important role for the electrostatic potential in the biological activity of the toxin.

Comparison between the structures of the five-residue turns in AaHII (10) and Lqhalpha IT (25) provides further insights regarding the critical effect obtained by the K8D substitution. In AaHII, hydrogen bonds exist between Asp8 and each of the residues Cys12, Val10, and Asn11 (10). This network stabilizes the five-residue turn and may influence the C-terminal stretch through the Cys12-Cys63 disulfide bridge. In Lqhalpha IT, however, Lys8 is exposed to the solvent, in contrast to the buried Asp8 in AaHII. Thus, stabilization of the five-residue turn in Lqhalpha IT is achieved differently. From the solution structure of Lqhalpha IT (25), it is apparent that Tyr10 interacts most likely with the terminal Arg64, and Lys8 does not seem to interact with Glu15 but rather with Tyr14. As mentioned above, the lack of interaction between Lys8 and Glu15 reflects the negligible change in the CD spectrum obtained with mutant E15A (Fig. 3A). Thus, Lys8, whose substitution by alanine had a moderate effect on toxicity, may play a dual role by interacting electrostatically with the receptor site and contributing to the polar electrostatic surface of Lqhalpha IT.

Aromatic Residues in the Putative Toxic Surface

The importance of the aromatic side chain of Tyr10 for toxicity was apparent from the unchanged toxicity of mutant Y10W compared with mutant Y10S, which revealed a 110-fold decrease in the apparent binding affinity for the receptor site (Table I, Fig. 2B). The possible interactions of Tyr10 with the C-terminal arginine, inferred from the solution structure of Lqhalpha IT (25), may suggest that the precise position of Tyr10 is important and needs special stabilizing interactions.

Another aromatic residue showing importance for toxicity is Phe17. This is inferred from the substantial decrease in biological activity (Table I) without a significant change in CD spectrum (Fig. 3B) upon its substitution. Its location within the putative molecular surface common to the five-residue turn and the C terminus suggests that Phe17 may interact directly with the receptor site.

Examination of the spatial arrangement of Lqhalpha IT suggests that the side chains of additional aromatic amino acids, such as Tyr14, Tyr21, or Trp38, are located in the vicinity of the putative toxic surface and may be important for toxicity. In accordance with this notion is the result obtained by sulfenylation of Trp38 in AaHII, which decreased the toxicity and apparent binding affinity for mammalian sodium channels by more than 1 order of magnitude, whereas the CD spectrum of the modified toxin remained unchanged (36).

Conversely, modifications of two aromatic residues that belong to the conserved hydrophobic surface, i.e. sulfenylation of Trp45 in AaHII (the equivalent of Trp47 in Lqhalpha IT) and substitution of Tyr49 by isoleucine in Lqhalpha IT (5), had a negligible effect on toxicity to mammals and insects, respectively. In both instances, the CD spectra of the modified toxins changed dramatically (Ref. 36 and data not shown). Thus, it is likely that the conserved hydrophobic surface may not be involved in the toxic surface.

Charged Residues in the Putative Toxic Surface

Our results pinpoint several charged residues, in addition to Lys8 (see above), that show significance for activity. One such residue is Arg18, as shown by the R18A mutation. The CD spectrum of this mutant (Fig. 3B) was similar to that of the unmodified toxin, and its electrostatic potential was only moderately affected (data not shown). Therefore, the decrease in activity of mutant R18A (Table I) may suggest a direct electrostatic interaction of Arg18 with the receptor site. Interestingly, substitution of the adjacent Asp19 by asparagine had no effect on toxicity, and a D19H mutation affected the activity only moderately. The double mutant R18K/D19N also had a minor effect (Table I). These results imply that residue 19, the first residue of the alpha -helix stretch (residues 19-28), may have no role in the interaction with the receptor site. This result is in concert with the finding that antibodies raised against the alpha -helix region of AaHII and the turn comprising residues 27-30 were capable of binding to the toxin when associated with the receptor site (14).

Superposition of the structures of AaHII and Lqhalpha IT reveals also that the C termini of both toxins are positioned between the five-residue turn and the beta -turn formed by residues 40-43. However, their dispositions relative to the five-residue turns differ in both toxins. Based on these observations, we modified Lys62 and Arg64 to investigate their contribution to the putative toxic surface. Neutralization of the positive charge of the C-terminal residue (R64A or R64N) decreased the apparent affinity for the receptor site more than 50-fold, although the toxicity remained practically unchanged (Table I). The discrepancy between the effects obtained in vivo, using blowfly larvae, and in vitro, using cockroach neuronal membranes, may be related to the inherent differences between fly and cockroach sodium channel structures. Differences in sodium channel proteins of various insect central nervous system membranes have been shown at the biochemical level (38, 39). Moreover, the ability of scorpion neurotoxins to distinguish among sodium channel preparations of various insects has also been reported (6, 24, 39). The similarity between the CD spectra of mutant R64N (not shown) and that of the unmodified toxin minimized the possibility that mutation R64N caused a structural change affecting activity. More likely is the suggestion that Arg64 interacts electrostatically with the receptor site.

Inversion of the charge at the C terminus, mutation R64D, had a dramatic effect: the apparent binding affinity decreased 2394-fold, and the remaining toxicity was 5% (Table I). Such a detrimental effect can result from a conformational alteration, as inferred from the altered CD spectrum (Fig. 3C), the change of the electrostatic potential of the toxin (Fig. 4), or the perturbation of the electrostatic contact with the receptor site. Except for the disruption of the contacts between Arg64 and Tyr10, Asp64 may generate a new intramolecular contact with a nearby residue, leading to some structural change, as was suggested by the altered CD spectrum of this mutant (Fig. 3C). Yet, this putative alteration, reflected by the highly sensitive measurement of the CD spectrum, had only a minute effect on the energy-minimized alpha -carbon model of the mutant toxin (Fig. 4).

Another positive charge related to the C-terminal domain is Lys62. Its putative interaction with the receptor site is shown by the 220-fold decrease in the apparent binding affinity to the cockroach sodium channel when substituted by leucine (Table I). The moderate change observed in the CD spectrum of mutant K62L (Fig. 3C) may suggest that this residue is involved in an intramolecular interaction as well.

In a previous study, biotinylation of Lys58 of AaHII resulted in the most devastating effect obtained with chemical agents applied on scorpion toxins affecting sodium channels (16). Only 1% of the original toxicity toward mammals was determined, and no detectable displaceability of the native toxin from the mammalian receptor site could be monitored with the modified toxin. From the known structure of AaHII (10), Lys58 was suggested to interact with Asn11 and Gly61 by hydrogen bonding. This network may be important for stabilizing the C terminus relative to the five-residue turn. By analogy to Lqhalpha IT and its putative toxic surface, we speculate that the prominent effect obtained in the biotinylated AaHII could be due to a combination of structural disruption, elimination of a putative electrostatic interaction with the receptor site, and/or alteration of the electrostatic potential of the molecule.

Conclusion

Our study pinpoints a putative molecular surface involved in recognition of the receptor site that differs from the conserved hydrophobic surface located on a different side of the molecule (Fig. 5). The plausible suggestion of a multipoint interaction of scorpion alpha -neurotoxins with their binding sites (31, 36) is in concert with our results, in which each substitution of a residue participating in binding has a partial effect on activity, whereas a structurally devastating mutation may cause a more severe effect.


Fig. 5. The putative toxic surface of Lqhalpha IT. The model was constructed according to the solution structure of the toxin (25). Genetically modified residues affecting the toxic surface are designated in red. Residues whose counterparts in similar toxins were affected by chemical modifications are shown in orange. Residues whose substitution had no effect are indicated in green. The conserved hydrophobic surface, located on a different side of the molecule, appears in blue. Orientation of the carbon backbone is to a similar direction as in Fig. 4 with some variations for better visualization.
[View Larger Version of this Image (90K GIF file)]

Binding to the receptor site may involve electrostatic as well as hydrophobic interactions with opposing constituents at the receptor site. This was demonstrated in other toxin receptor site interactions, e.g. K+ channels and short scorpion neurotoxins such as charybdotoxin, leiurotoxin, and PO5 (40-43), or the nicotinic acetylcholine receptor and a sea snake toxin, erabutoxin a (44). The most recent finding, showing that inversion of a negative glutamate into a positive residue in the extracellular linker between sodium channel transmembrane segments S3 and S4 in domain IV disrupts the binding of an alpha  scorpion neurotoxin to rat brain sodium channels (45), is in concert with our suggestion. The significance of the electrostatic potential for the binding of a short scorpion toxin, Lq2, to its receptor site was inferred from the inversion of a negatively charged residue in a potassium channel, which affected association rather than the dissociation rate of the toxin (46). From the correlation between the changes in biological activities and alterations of the putative electrostatic potentials of the various modified toxins produced in the present study, the electrostatic potential seems to play a role in the biological activity of the long scorpion neurotoxin Lqhalpha IT. Whether this polarity is related to association or dissociation kinetics of the toxin is still unknown and deserves further study.

An alternative approach to the elucidation of the toxic site of neurotoxins affecting the cholinergic binding site was demonstrated by a combination of site-directed mutagenesis of isolated fragments of the receptor (47, 48) and NMR analyses of bound ligands (49).

Three types of animals, i.e. fly larvae, cockroaches, and mice, were used in the bioassays. Injection to fly larvae provided a useful and quick measure of the effects caused by the modifications and throughout the purification process of each of the toxin variants. However, the effects of the mutations on the apparent binding affinity to the cockroach central nervous system sodium channels were in most instances more intense than the effects on toxicity to fly larvae. This phenomenon may result from (i) differences in sensitivity of various insect sodium channels from different tissues or animals to Lqhalpha IT and to its modified toxin (6) or (ii) variations in susceptibility of the toxin variants to proteolysis or availability to various tissues in the whole animal that result from the chemical features of the toxin, i.e. hydrophobicity or change in charge. Hence, binding-competition studies using purified neuronal membranes are a direct measure for toxin receptor site interactions on sodium channels and thus provide a more sensitive assay than toxicity assays on whole animals.

The similar changes in toxicity toward fly larvae and mice obtained with the various mutants suggest that their corresponding receptor sites are similar. Further modifications of residues that belong to the putative recognition site may be useful for final determination of the toxic site and for clarifying the molecular basis of animal group specificity of scorpion alpha -toxins.


FOOTNOTES

*   This research was supported by Grant IS-2486-94C from BARD, The United States-Israel Binational Agricultural Research and Development Fund, and Grant 891-0112-95 from the Israeli Ministry of Agriculture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
**   To whom correspondence should be addressed: Dept. of Plant Sciences, Tel-Aviv University, Ramat-Aviv 69978, Tel-Aviv, Israel. Tel.: 972-3-6409844; Fax: 972-3-6406100; E-mail: mamgur{at}ccsg.tau.ac.il.

ACKNOWLEDGEMENTS

We thank Dr. J. C. Fontecilla-Camps for providing the coordinates for the structure of AaHII; R. Oughideni and N. Zylber (CNRS Marseille) for the amino acid analyses; and Dr. B. Musafia for help with the modeling.


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