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Originally published In Press as doi:10.1074/jbc.M202297200 on April 5, 2002

J. Biol. Chem., Vol. 277, Issue 25, 22806-22813, June 21, 2002
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Scanning Mutagenesis of a Janus-faced Atracotoxin Reveals a Bipartite Surface Patch That Is Essential for Neurotoxic Function*

Francesco MaggioDagger and Glenn F. KingDagger §

From the Departments of Dagger  Biochemistry and § Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032

Received for publication, March 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins isolated from the venom of Australian funnel web spiders. In addition to a strikingly asymmetric distribution of charged residues, from which their name is derived, these toxins contain an extremely rare vicinal disulfide bond. To shed light on the mechanism of action of these toxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for the prototypic family member, J-ACTX-Hv1c, and mapped the key functional residues using site-directed mutagenesis. An alanine scan using a panel of 24 mutants provided the first complete map of the bioactive surface of a spider toxin and revealed that the entire J-ACTX-Hv1c pharmacophore is restricted to seven residues that form a bipartite surface patch on one face of the toxin. However, the primary pharmacophore, or hot spot, is formed by just five residues (Arg8, Pro9, Tyr31, and the Cys13-Cys14 vicinal disulfide). The Arg8-Tyr31 diad in J-ACTX-Hv1c superimposes closely on the Lys-(Tyr/Phe) diad that is spatially conserved across a range of structurally dissimilar K+ channel blockers, which leads us to speculate that the J-ACTXs might target an invertebrate K+ channel.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As well as destroying an estimated 20-30% of the world's food supply (1), arthropod pests are responsible for the transmission of numerous human diseases. Mosquitoes, for example, transmit dengue fever, yellow fever, West Nile virus, filariasis, and malaria, with malaria causing more than two million deaths annually (2). These phytophagous and hematophagous insect pests have traditionally been controlled by spraying broad spectrum chemical pesticides. However, the emergence of insecticide-resistant insect populations (3, 4), as well as increasing concerns about the environmental and human health risks associated with certain chemical pesticides (5, 6), has stimulated the search for new pest control strategies.

The pioneering work of Olivera and colleagues (7, 8) has revealed that the venoms of aquatic cone snails (Conus spp.) are essentially highly evolved combinatorial peptide libraries. We have argued that spider venoms can be analogously viewed as preoptimized combinatorial libraries of insecticidal compounds (9), and therefore we decided to exploit these venoms in the search for insect-specific peptide toxins. By screening the venom of the lethal funnel web spider Hadronyche versuta (10), we discovered three families of insect-specific toxins (9, 11, 12), including an unusual family of excitatory neurotoxins that we named the Janus-faced atracotoxins (J-ACTXs; Fig. 1).1 In addition to being excellent biopesticide candidates, these toxins have allowed validation of new insecticide targets, and consequently they could be invaluable leads for the development of novel chemical insecticides (13).


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  		Fig. 1.   Primary structures of the Janus-faced atracotoxins. Comparison of the primary structures of the J-ACTXs isolated from the funnel web spider H. versuta. Identities are shaded gray, and the four disulfide bridges are indicated by solid lines below the sequences. The sequence numbering at the top of the figure refers to J-ACTX-Hv1c. The key functional residues identified in this study are indicated by the arrowheads.

In addition to a strikingly asymmetric distribution of charged residues, from which the toxin name is derived, the J-ACTXs contain an extremely rare vicinal disulfide bridge (i.e. a disulfide bond between adjacent cysteine residues) (12). The only other examples of proteins with vicinal disulfide bridges are methanol dehydrogenase (14) and the alpha  subunit of the acetylcholine receptor (15). In all three proteins, the vicinal disulfide bridge plays a key functional, rather than architectural, role; in the case of the J-ACTXs, mutation of the disulfide bridge almost completely abrogates insecticidal activity (12). This led us to speculate that the largely hydrophobic face of the toxin that encompasses the vicinal disulfide probably represents its bioactive surface (12).

In this study, we developed an efficient recombinant expression system for J-ACTX-Hv1c, the most pernicious and best characterized of the J-ACTXs (12),2 and used alanine scanning mutagenesis to determine the bioactive surface of the molecule. We have localized the toxin pharmacophore to seven residues that form a bipartite surface patch on one face of the toxin. However, we show that the primary pharmacophore is formed by just five residues (Arg8, Pro9, Tyr31, and the Cys13-Cys14 vicinal disulfide) that we postulate form a "hot spot" for target binding. A panel of follow-up mutations based on the alanine scan enabled us to further define the chemical nature of the toxin pharmacophore, and it also provided unexpected clues about the target of the J-ACTXs, which is presently unknown.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of a Bacterial Overexpression System-- A synthetic gene encoding J-ACTX-Hv1c was designed by annealing, extension, and amplification of overlapping oligonucleotides (Fig. 2). In the first step, four oligonucleotides with codon usage optimized for maximal expression in Escherichia coli (J-Hv1c-1, J-Hv1c-2, J-Hv1c-3, and J-Hv1c-R) were annealed and extended with polymerase. The four oligonucleotides were added at a final concentration of 2 µM to 50 µl of reaction buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-Cl, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 mg ml-1 bovine serum albumin; Stratagene) containing 400 µM dNTP mix (Invitrogen). The annealing reaction was allowed to proceed for 30 min at 60 °C. The temperature was then raised to 72 °C, and the mixture was incubated for a further 30 min following addition of 2.5 units of Pfu polymerase (Stratagene). In the second step, 20 µl of the reaction mixture was used as template for a standard PCR amplification of the entire coding sequence with primers J-Hv1c-F and J-Hv1c-R. These primers encoded 5'-BamHI and 3'-EcoRI sites, respectively, for cloning purposes (Fig. 2). The amplified PCR product was digested with BamHI and EcoRI and subcloned into BamHI/EcoRI-digested pGEX-2T vector (Amersham Biosciences) using standard methods. The resulting plasmid (pFM1) encodes J-ACTX-Hv1c as an in-frame fusion to the C terminus of Schistosoma japonicum glutathione S-transferase (GST) with an intervening thrombin cleavage site.


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  		Fig. 2.   Construction of a synthetic gene for J-ACTX-Hv1c. A synthetic gene encoding J-ACTX-Hv1c, with codons optimized for maximal expression in E. coli, was constructed in two steps. In step 1, the complementary overlapping oligonucleotides J-Hv1c-1, J-Hv1c-2, J-Hv1c-3, and J-Hv1c-R were annealed and extended with Pfu polymerase. In step 2, the entire coding sequence was PCR-amplified using the primers J-Hv1c-F and J-Hv1c-R, which encoded the 5'-BamH1 and 3'-EcoRI sites, respectively, for directional cloning into BamHI/EcoRI-digested pGEX-2T. See text for details.

Single or double point mutations were introduced into the J-ACTX-Hv1c gene using PCR with pFM1 as the template. Mutagenic primers incorporating the desired mutation proximal to the N or C terminus were used with either J-Hv1c-F or J-Hv1c-R. Mutations in the central portion of the gene were incorporated using complementary mutagenic primers followed by amplification with J-Hv1c-F and J-Hv1c-R.

Overexpression and Purification of J-ACTX-Hv1c and Mutants-- E. coliBL21 cells were transformed with pFM1, either in its original form or with one or more engineered point mutations, for overproduction of GST-toxin fusion protein. The cells were grown in LB medium at 37 °C to an A600 of 0.6-0.8 before induction of the fusion protein with 300 µM isopropyl-1-thio-beta -D-galactopyranoside. The cells were harvested by centrifugation at an A600 of 1.9-2.1 and then lysed by sonication. The recombinant fusion protein was purified from the soluble cell fraction using affinity chromatography on GSH-Sepharose (Amersham Biosciences) and then cleaved on the column by the addition of bovine thrombin (Sigma) (17). The liberated toxin was eluted from the column with Tris-buffered saline (150 mM NaCl, 50 mM Tris, pH 8.0) and either (i) purified immediately using reverse-phase (rp) HPLC (see below), or (ii) dialyzed against water in 1-kDa cut-off dialysis tubing before being lyophilized. Lyophilized toxin was then resuspended in 1 ml of 10 mM HCl prior to the addition of 1 ml of glutathione redox buffer (0.2 M 4-morpholinepropanesulfonic acid, pH 7.3, 0.4 M KCl, 2 mM EDTA, 4 mM reduced GSH, and 2 mM oxidized GSH). The toxin was then allowed to "fold" for 2-4 h before final rpHPLC purification.

The correctly folded recombinant toxin was then separated from non-native disulfide bond isomers and other contaminants by rpHPLC using a Vydac C18 analytical column (4.6 × 250 mm, 5-µm pore size). The toxins were eluted from the column at a flow rate of 1 ml min-1 using a linear gradient of 15-22% acetonitrile over 15 min. Correctly folded toxin eluted as the major peak with a retention time of 8-14 min depending on the variant being purified. The toxin molecular weight was verified using electrospray mass spectrometry.

CD Spectroscopy-- CD spectra were recorded at 4 °C using a Jasco J-715 spectropolarimeter. Toxins (25 µM) were dissolved in 1 mM sodium phosphate, pH 7.0, and loaded into a 0.1-cm rectangular quartz cell for spectral analysis. The spectra were the averages of 5-16 scans obtained using a scan rate of 20 nm min-1 and a response time of 4 s. A blank spectrum consisting of buffer was run under identical conditions and subtracted from each of the toxin spectra.

Insect Biological Toxicity Assays-- Toxins diluted in insect saline (18) were injected into house flies (Musca domestica) for quantitative determination of toxicity. The flies (9-20 mg, sex undetermined) were injected with 1-2 µl of toxin at concentrations of 10-106 pmol g-1. The experiments were performed in duplicate with a cohort of 10 flies for each toxin concentration. The control flies were injected with 2 µl of insect saline. Dorsal thoracic injections were dispensed with an Arnold microapplicator (Burkard Scientific Supply, Rickmansworth, UK) furnished with a 29-gauge needle. The flies were kept immobilized at 4 °C during all injections and were subsequently transferred to room temperature (24 °C). LD50 values (i.e. the dose corresponding to 50% lethality of the test population at 24 h post-injection) were calculated from the dose-response data as described previously (19).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Production of a Recombinant Expression System for J-ACTX-Hv1c-- We attempted to develop an efficient recombinant expression system for J-ACTX-Hv1c so that its key functional residues could be delineated using site-directed mutagenesis. However, despite its small size (37 residues), J-ACTX-Hv1c contains eight cysteine residues that can be paired in 105 different ways to form four disulfide bonds. Thus, expression of the toxin in a foreign host necessitates consideration of how proper disulfide oxidation might be accomplished.

The thioredoxin and glutaredoxin disulfide-reducing pathways in E. coli ensure that all cytoplasmic cysteine residues, with a few notable exceptions (20), are in the reduced state (20, 21). However, we recently showed that expression of the insect-specific calcium channel blocker omega -ACTX-Hv1a (which contains three disulfide bonds) as a GST fusion protein in E. coli enables 65-70% of the soluble toxin to be recovered with the correct disulfide framework (19). Because the yield of correctly folded toxin was not enhanced in a strain with defective thioredoxin reductase (which should facilitate the formation of cytoplasmic disulfide bonds) (22), we postulated that most, if not all, disulfide oxidation occurred after the cells had been lysed and the fusion protein was exposed to the periplasmic Dsb system, which normally catalyzes disulfide formation in E. coli (23). We also reasoned (19) that active involvement of the Dsb system, in which DsbC catalyzes disulfide isomerization, might explain why the yield of correctly folded recombinant omega -ACTX-Hv1a (65-70%) is much higher than that obtained from in vitro folding of synthetic toxin (~15%) (24).

Given our previous success with omega -ACTX-Hv1a (19), we decided to develop a similar recombinant expression system for production of J-ACTX-Hv1c. We expressed J-ACTX-Hv1c in E. coli BL21 cells as a fusion to the C terminus of GST. SDS-PAGE analysis (Fig. 3A) revealed that the GST-toxin fusion protein was efficiently expressed and predominantly soluble (Fig. 3A, compare lanes 1 and 2). Following cell lysis, the fusion protein could be quantitatively bound to a GSH-agarose affinity column (Fig. 3A, compare lanes 3 and 4). On-column thrombin cleavage (Fig. 3A, lane 5) liberated the recombinant toxin, which could then be eluted from the column with buffer.


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  		Fig. 3.   Purification and characterization of recombinant J-ACTX-Hv1c. A, SDS-polyacrylamide gel illustrating the expression and affinity purification of J-ACTX-Hv1c. Lanes 1 and 2, soluble and insoluble fractions, respectively, resulting from centrifugation of E. coli BL21/pFM1 lysate; lane 3, eluate obtained from application of soluble lysate fraction to GSH-Sepharose affinity column; lane 4, GSH-Sepharose beads after washing with buffer, showing quantitative extraction of the 30.1-kDa GST-toxin fusion protein (marked with an arrow) from the cell lysate; lane 5, GSH-Sepharose beads after incubation with thrombin and elution of the liberated toxin with buffer. The disappearance of the GST-toxin band and the appearance of a GST band (marked with an arrow) indicate that the proteolytic cleavage has gone to completion. B, rpHPLC purification of recombinant J-ACTX-Hv1c. The major peak, which corresponds to correctly folded recombinant toxin, had almost identical retention time to that of native J-ACTX-Hv1c (not shown), which was purified from venom as described previously (12). The early eluting minor peaks correspond to misfolded toxin, whereas the late-eluting minor peaks correspond to GST and GST-toxin fusion protein. The horizontal bar below the major peak denotes the toxin fraction that was used for subsequent structure-function analyses. C, dose-response curves resulting from injection of native (WT) or various mutant recombinant toxins into M. domestica. Each data point is the mean of two independent experiments. The solid line represents the fit to the data that were used to extract the LD50 values shown in Table I.

The eluted toxin was either purified immediately using rpHPLC or incubated for 2-4 h in a GSH redox buffer prior to rpHPLC purification. Correctly folded recombinant J-ACTX-Hv1c was always the major peak in the rpHPLC chromatogram as verified by electrospray mass spectrometry and biological toxicity assays (Fig. 3B). The yield of properly folded toxin in the eluate (as a percentage of total recombinant toxin observed in the rpHPLC trace) was estimated from integration of the relevant HPLC peaks to be ~85% (Fig. 3B). This is significantly higher than the yield of correctly folded omega -ACTX-Hv1a (65-70%) that we obtained previously using a similar GST fusion expression system (19). This difference in the percentage of yield was not unexpected given the in vitro folding properties of the two toxins; in vitro oxidation of chemically synthesized J-ACTX-Hv1c and omega -ACTX-Hv1a in a GSH redox buffer gives yields of ~100% (12) and ~15% (24), respectively, of the native disulfide isomer.

The yield of properly folded wild-type toxin was not increased by incubation of the lyophilized eluate for 2-4 h in a GSH redox buffer that promotes disulfide shuffling (25). However, for some of the point mutants, incubation of the eluate in this buffer did increase the yield of properly folded toxin (data not shown). Thus, coupling GST fusion protein expression with an efficient disulfide shuffling system provides us with an efficient means of producing recombinant toxin with the correct disulfide framework.

Alanine Scanning Mutagenesis of J-ACTX-Hv1c-- The efficient recombinant expression system described above allowed us to determine the functional relevance of almost every residue in J-ACTX-Hv1c using alanine scanning mutagenesis. We previously demonstrated that deletion of the N-terminal residue (Ala1) and the two C-terminal residues (Glu36 and Pro37) does not affect toxin function,2 and consequently the scanning mutagenesis was restricted to residues 2-35. Residues Cys3, Cys10, Cys16, Cys17, Cys22, and Cys33 were excluded from the mutational analysis because their side chains are involved in disulfide bonds that form the cystine knot that is critical to the structure of the toxin (12). The two cysteine residues that form the unusual vicinal disulfide (Cys13 and Cys14) were also excluded from the alanine scanning mutagenesis because it was previously shown that the vicinal disulfide is critical for toxin function (12), and hence mutation of either residue is certain to be highly deleterious. The remaining 22 non-alanine residues were mutated to alanine, whereas the four alanine residues were mutated to serine.

All of the mutant toxins were successfully expressed as soluble GST fusion proteins except for the G5A and G19A mutant proteins, which proved to be completely insoluble as determined by SDS-PAGE analysis of soluble and insoluble cell fractions (data not shown). Both Gly5 and Gly19 are located in beta -turns where they are necessary to achieve a sharp chain reversal (Fig. 4 in Ref. 12), and hence it is likely that the insolubility of the G5A and G19A mutants results from impaired folding. Mutation of these residues was not pursued any further, leaving us with an initial panel of 24 point mutants.

Alanine is generally chosen for scanning mutagenesis because it can be accommodated in most types of secondary structure (beta -sheet, alpha -helix, and beta -turn), thus minimizing the chances that the point mutation will induce major structural perturbations. Nevertheless, it is essential to check this experimentally. We did this by acquiring far UV CD spectra of the native and mutant recombinant toxins. Because the dominant structural element of J-ACTX-Hv1c is an antiparallel beta -hairpin that protrudes from the globular cystine-rich core, we anticipated that the CD spectrum would exhibit a typical beta -sheet signature (minimum at 210-216 nm) (26), as observed previously for omega -ACTX-Hv1a (19). However, the CD spectrum of recombinant J-ACTX-Hv1c displayed a maximum at 224 nm and a minimum at ~195 nm (Fig. 4A), which is not typical of any one type of secondary structure. The spectrum is very different from that of unfolded polypeptides, which have no positive ellipticity and display a deep minimum at approximately 200 nm.


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  		Fig. 4.   Structural analysis of native and mutant recombinant toxins. Comparison of the far UV CD spectrum of recombinant J-ACTX-Hv1c (WT) with spectra of A6S and Y31A mutants (A); A12E, S21A, V29A, and K34A mutants (B); and P9A and R33Amutants (C) is shown.

The CD spectrum of J-ACTX-Hv1c most closely resembles the beta -turn spectrum derived from combined analysis of Fourier transform infrared and CD spectra (27), which is consistent with the high beta -turn content of J-ACTX-Hv1c (43%). However, the single tyrosine residue in J-ACTX-Hv1c (Tyr31) most likely contributes to the maximum at 224 nm, because a detailed analysis using model peptides revealed that tyrosine side chains have a significant positive CD band with a maximum at approximately 225-230 nm (28). Consistent with this hypothesis, we see a small loss of intensity of the positive CD band over the region 224-238 nm in a Y31A mutant (Fig. 4A). We conclude that the small spectral changes observed for the Y31A mutant are due to loss of the Tyr31 side chain chromophore and are not the result of a minor structural perturbation.

The CD spectra of 20 of the other 23 mutant toxins were essentially superimposable on the CD spectrum of the native recombinant toxin (see examples in Fig. 4B), indicating that none of these mutations significantly perturb the structure of the toxin (see Table I for a summary of the CD results). Two mutants, P9A and R33A, caused minor changes in the CD spectra that were suggestive of small structural perturbations (Fig. 4C). In the case of R33A, the maximum was reduced in intensity and slightly blue-shifted from 224 to 220 nm. The P9A mutant caused a small reduction in intensity of the maximum without inducing a peak shift. The significance of these spectral changes is discussed in more detail below.

                              
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Table I
Insecticidal activity of recombinant J-ACTX-Hv1c and various point mutants

In striking contrast to the other 23 mutants, the A6S mutation caused a major structural perturbation with the CD spectrum (Fig. 4A), indicating that the mutant protein is largely unfolded. Although it was not anticipated that this point mutation would have such a dramatic effect on folding, similar results have been observed during mutagenesis of other cystine knot toxins. For example, during alanine scanning mutagenesis of the N-type calcium channel blocker omega -conotoxin GVIA, it was shown that less than 3% of G5A, N20A, and T23A mutants were correctly folded (29). The A6S mutant was discarded from further analysis, leaving us with a panel of 23 first round mutants for biological assays.

Activity of Alanine Scan Mutants-- The insecticidal potency of each of the mutants was examined by comparison of their LD50 values in house flies with that of unmutated recombinant toxin. As shown in Table I, 16 of the 23 mutants caused a 5-fold or lower increase in the LD50, and one of the mutants (P18A) caused a marginal enhancement of insecticidal potency. We conclude that none of these 17 residues are critical to toxin function. The I2A and V29A mutants caused 7- and 13-fold decreases in activity, respectively, and we conclude that these residues are important but not absolutely critical for toxin function. The R33A mutant caused an 11-fold decrease in insecticidal potency, but the slightly perturbed CD spectrum of this mutant precluded a definitive conclusion about its functional significance without further mutational analysis (see below). Three mutants (R8A, P9A, and Y31A) caused very significant reductions in LD50 (98-270-fold), and we conclude that these residues are critical for the insect-specific neurotoxic activity of J-ACTX-Hv1c.

Second Round Mutagenesis-- The initial screen indicated that residues Arg8, Pro9, and Tyr31 are likely to be key residues for the insecticidal activity of J-ACTX-Hv1c, with Ile2, Val29, and Arg33 being somewhat less important. To further probe the functional relevance of these residues and to investigate the role of individual chemical moieties in toxin activity, we designed a panel of additional mutants.

We first addressed the functional role of Arg33, because the R33A mutant toxin had a slightly perturbed CD spectrum (Fig. 4C). We were concerned that the 11-fold decrease in insecticidal potency of the R33A mutant might be due primarily to the structural perturbation induced by the mutation rather than a consequence of the change in chemical nature of the side chain. We therefore constructed and analyzed R33L and R33H mutations. Both mutants had wild-type CD spectra and caused less than 2-fold increase in the LD50 (Table I). Because at least one of these mutations (R33L) involves a radical change in the side chain, we conclude that Arg33 is not a functionally important residue and that the loss in activity of the R33A mutant is a consequence of an induced structural perturbation.

We next probed the features of Tyr31 that make this residue critical for toxin function. Mutation of this residue to Ala caused a 162-fold decrease in insecticidal potency. The CD spectrum of a Y31F mutant indicated that it was properly folded, and it displayed wild-type activity (Table I). We conclude that the hydroxyl group is unimportant and that the aromatic ring is the key functional moiety of Tyr31.

Finally, we further probed the role of Arg8, the only functionally important charged residue, with an R8E mutation. If Arg8 makes an ionic interaction with a negatively charged group on the target, then we might expect an R8E mutant to be even less potent than the R8A mutant because it will introduce repulsive electrostatic interactions. The R8E mutant was properly folded as judged from its CD spectrum, but its activity was reduced a further 3-fold compared with the R8A mutant (Table I). Although a larger decrease in potency might be expected from introduction of a repulsive electrostatic interaction with the toxin target, it should be noted that we are measuring the effect of the mutation on LD50 rather than target affinity. Previous mutagenesis studies with the insecticidal neurotoxin Lqhalpha IT showed that this tends to attenuate the effects of the mutation; the decrease in target binding was typically 10-50-fold greater than the decrease in ED50 for key functional residues (30). Our own studies with omega -ACTX-Hv1a have shown that, compared with binding studies, the effect of the mutation can be attenuated up to 40-fold in LD50 assays.3 Given this caveat, we tentatively conclude that Arg8 makes an electrostatic interaction with a negatively charged moiety on the toxin target.

Functional Significance of Pro9-- The LD50 of a P9A mutant was found to be increased 270-fold compared with the unmutated recombinant toxin (Table I). However, the CD spectrum of this mutant was slightly perturbed compared with the wild-type toxin (Fig. 4C), raising the possibility that the observed reduction in insecticidal potency is due to an induced structural perturbation rather than loss of the proline side chain. For example, it is plausible that mutation of Pro9 alters the conformation of the spatially proximal Arg8 and Tyr31 residues, which we have demonstrated are crucial for toxin function. However, the perturbation of the CD spectrum induced by the P9A mutation is relatively minor in that we observe only a slight reduction in intensity of the maximum at 224 nm. This is inconsistent with a major conformational rearrangement, and the minor changes in the positive CD band may be due largely to an increase in the solvent accessibility of the nearby Tyr31 residue.

Although the reduction in insecticidal potency caused by an R33A mutation does appear to be due to an induced structural perturbation, the loss of potency is only 11-fold. In contrast, the P9A mutation caused a 25-fold greater loss of potency without perturbing the CD spectrum as much as the R33A mutant (Fig. 4C). This argues that much of the reduction in insecticidal potency in the P9A mutant is due to loss of the proline side chain rather than an induced structural perturbation. We therefore conclude that Pro9 is a critical residue for toxin function.

The Vicinal Disulfide-- We previously showed that deletion of the vicinal disulfide by isosteric substitution of both Cys13 and Cys14 with Ser led to serious impairment of insecticidal activity (12), but the level of impairment was not quantitated. To rank the functional importance of key toxin residues, we examined the potency of a synthetically produced C13S,C14S mutant in M. domestica and showed that the LD50 of this mutant was reduced 425-fold compared with the unmutated recombinant toxin. We previously showed using NMR spectroscopy that this mutant assumes the native fold (12).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Insecticide Design-- The Janus-faced atracotoxins are a recently discovered family of insect-specific excitatory neurotoxins (12). The presence of a functionally significant vicinal disulfide encompassed within a conserved hydrophobic patch distinguishes these toxins from all other known members of the inhibitory cystine knot motif family (31). These toxins are lethal to both phytophagous insects and various dipterans (12),2 but they are inactive in vertebrates (12), making them valuable leads for the design of novel insecticides.

To elucidate the molecular mechanism of action of the Janus-faced atracotoxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for J-ACTX-Hv1c and mapped the key functional residues using site-directed mutagenesis. We constructed and analyzed a total of 30 mutants (Table I), which enabled us to explore the functional relevance of all residues except Gly5, Ala6, and Gly19. This analysis revealed that Arg8, Pro9, and Tyr31, as well as the vicinal disulfide, are key functional residues, whereas Ile2 and Val29 are somewhat less important. These residues are conserved in all members of the J-ACTX family (Fig. 1).

From an insecticide design viewpoint, there are two important conclusions that emerge from the mutagenesis results: (i) The insecticidal potency of the Janus-faced atracotoxins appears to be conferred by a relatively small number of residues. If the marginally important Ile2 and Val29 are excluded, only five of the 37 residues of J-ACTX-Hv1c appear to be critical for toxin function. Similar results have been obtained from scanning mutagenesis of other cystine-rich toxins. For example, alanine scanning mutagenesis of the K+ channel blocker BgK revealed five key functional residues, of which three were deemed critical for channel binding (32), and a similar number of functionally important residues were elucidated from mutagenesis of the N-type calcium channel blockers omega -conotoxin GVIA (29) and omega -conotoxin MVIIA (33). (ii) The functionally important residues identified from the mutagenesis studies are located on a single face of the toxin, where they form two closely apposed patches on the surface of the protein (Fig. 5, A and B). This distinguishes J-ACTX-Hv1c from other cystine-rich toxins such as omega -conotoxin GVIA where functionally important residues are found on multiple faces of the protein (29). Restriction of the pharmacophore to a single surface of the toxin should increase the probability of designing functional small molecule mimics of the toxin.


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  		Fig. 5.   The bioactive surface of J-ACTX-Hv1c. A, molecular surface of J-ACTX-Hv1c (Protein Data Bank code 1DL0) illustrating the key functional residues identified using site-directed mutagenesis. These residues form two closely apposed patches (orange and red) on one face of the toxin. B, side view of the J-ACTX-Hv1c pharmacophore illustrating the alignment of key functional residues along a single face of the toxin. Note the location of Ile2 and Val29 at the periphery of the bioactive surface. C, superposition of the side chains of the functionally critical Arg8 and Tyr31 residues of J-ACTX-Hv1c (green) on the Lys-Tyr/Phe diad of the K+ channel blockers agitoxin 2 (blue; Protein Data Bank code 1AGT) and BgK (red; Protein Data Bank code 1BGK). The three-dimensional folds of these toxins are very different, and thus, for the sake of clarity, only the backbone of J-ACTX-Hv1c is shown (thin green tube, except for the arrows representing the two beta -strands).

Hot Spot versus "Gasket" Residues-- The key functional residues revealed by the mutagenesis studies (apart from the vicinal disulfide) were Arg8, Pro9, and Tyr31. Arg and Tyr are two of the three most highly enriched residues at the hot spots of protein-protein interfaces (only Trp is more common) (34), which leads us to speculate that these residues, along with the vicinal disulfide, form the primary binding site, or hot spot, for interaction of J-ACTX-Hv1c with its target.

A recent survey of protein-protein binding interfaces (34) revealed that most interfaces consist of a central hot spot surrounded by residues whose primary role is to occlude water from the critical interacting residues; in other words, these peripheral residues act like a gasket to seal off the hot spot from bulk solvent (34). Remarkably, the exact chemical nature of the gasket residues appears to be relatively unimportant; alanine is usually sufficient to provide a water-resistant seal (34). This might explain why mutation of Ile2 to Ala caused only a 7-fold drop in activity (Table I), whereas in a previous study2 deletion of Ile2 led to a substantial 70-fold drop in insecticidal potency. If Ile2 is a gasket residue, then these results would be expected, because mutation of the residue to Ala would maintain the water-resistant seal, whereas complete removal of this residue would expose the hot spot to bulk solvent and therefore significantly reduce target binding and hence insecticidal potency. Given its location at the opposite end of the hot spot (Fig. 5, A and B), Val29 might also be a gasket residue that helps to prevent bulk solvent penetrating into the binding site.

What Is the Target of the Janus-faced Atracotoxins?-- The three-dimensional fold of cystine knot toxins generally provides little insight into their mode of action; the cystine knot simply provides the structural framework onto which diverse functional motifs can be grafted (13, 31, 35). However, for cystine knot and other disulfide-rich ion channel toxins, the topological disposition of key functional residues, regardless of three-dimensional scaffold, is often informative of function. The classical example is the diad of Lys and Tyr/Phe residues that is topologically conserved across a wide range of otherwise structurally dissimilar potassium channel blockers (32). Thus, we wondered whether the topological arrangement of the J-ACTX-Hv1c pharmacophore might provide some insight into the mode of action of the toxin.

Peptide toxins bind to voltage-gated sodium channels at a number of pharmacologically and apparently topologically distinct sites; µ-conotoxins (CTXs) and delta -CTXs bind to sites 1 and 6, respectively, whereas scorpion alpha -toxins and various sea anemone toxins bind to site 3 (36). Mutagenesis of µ-CTXs has revealed that the key residue for blockage of vertebrate voltage-gated sodium channels is an arginine (Arg13 in µ-CTX GIIIA) that projects into the pore of the channel (37, 38). However, in striking contrast to J-ACTX-Hv1c, the critical arginine is surrounded by an array of positively charged residues (Arg1, Lys11, Lys16, and Arg19 in µ-CTX GIIIA) that also interact with the channel according to mutant cycle analyses (39). Although scorpion alpha -toxins bind to a different locus on sodium channels than µ-CTXs, their bioactive surface also comprises a critical positive residue (Lys8 in the insecticidal scorpion alpha -toxin Lqhalpha IT) surrounded by several other important positively charged residues (30). The bioactive surface of the sea anemone toxins appears to be more chemically diversified, with both positively and negatively charged residues as well as hydrophobes being important for channel binding (40, 41). Thus, if J-ACTX-Hv1c interacts with sodium channels, it must do so in an entirely different manner from that of the µ-CTXs, sea anemone toxins, and scorpion alpha -toxins.

N- and P/Q-type voltage-gated calcium channels are usually distinguished by their susceptibility to blockage by omega -CTXs and omega -agatoxins, respectively. At this stage, the residues responsible for interaction of omega -agatoxins with P/Q-type channels have not been identified. However, mutagenesis studies have revealed that N-type calcium blockers such as omega -CTX GVIA (29), omega -CTX MVIIA (33), and Ptu1 (42) contain a functionally critical Lys-Tyr diad. The lysine, but not the tyrosine, residue is conserved in the recently discovered L-type calcium channel blocker omega -CTX TxVII (43). The Lys and Tyr residues in the N blocker diad are separated by a considerable distance and are located on opposite faces of the toxin (see Fig. 8 in Ref. 29); this diad bears no resemblance to the J-ACTX-Hv1c pharmacophore. In any case, blockage of calcium channels is inconsistent with the excitatory phenotype induced in insects by J-ACTX-Hv1c (12).

Intriguingly, the functionally critical Arg8 and Tyr31 residues in J-ACTX-Hv1c align extremely well with the Lys-Phe/Tyr diad that is conserved across structurally dissimilar potassium blockers such as BgK and agitoxin 2 (Fig. 5C). Similar overlays (not shown) can be made with the functionally critical diad residues in numerous other K+ channel blockers such as charybdotoxin (44) and kappa -conotoxin PVIIA (45). Thus, although the three-dimensional fold of J-ACTX-Hv1c is homologous to the sodium channel modulators conotoxin GS, µ-agatoxin-I, and delta -ACTX-Hv1a (12), the hot spot identified by site-directed mutagenesis provides circumstantial evidence that the J-ACTXs might target invertebrate K+ channels. The excitatory phenotype induced by the J-ACTXs in Drosophila melanogaster4 and other insects (12) is consistent with a potassium channel blocking activity. Indeed, prior to death, the fruit flies exhibit a leg shaking phenotype that is reminiscent of that induced by conditional mutants of Drosophila K+ channels such as EAG and Shaker (46).

Thus, the topological arrangement of key functional residues, rather than the three-dimensional structure per se, allows us to speculate that the J-ACTXs target invertebrate K+ channels. We plan to test this hypothesis by employing a variety of biochemical, electrophysiological, and genetic screens to identify the exact molecular target of these toxins.

Mode of Target Recognition-- Regardless of the molecular nature of the target of the J-ACTXs, the mutagenesis studies allowed us to make some concrete conclusions about the types of interactions that must occur between the toxin and its target (Fig. 5B). These data, which help to chemically define the toxin pharmacophore, are likely to be useful for insecticide design even in the absence of specific information about the target.

The near wild-type activity of a Y31F mutant (Table I) indicates that Tyr31 interacts with the target primarily via its aromatic ring and not the hydroxyl group. The much reduced activity of the R8E mutant compared with an R8A mutant suggests that Arg8 makes an electrostatic interaction with a complementary negatively charged group on the target. If Arg8 inserts into the pore of a KcsA-like K+ channel (16), the complementary charge could be provided by acidic residues lining the external entrance to the pore and/or the polarized oxygen atoms of backbone carbonyl groups that line the selectivity filter and are oriented to coordinate and stabilize cations (16). The way in which the vicinal disulfide interacts with the toxin target remains enigmatic. However, along with Ile2, it may simply form an apolar patch (colored orange in Fig. 5A) that interacts with hydrophobic residues on the target. Consistent with this hypothesis, disruption of the hydrophobic patch by mutation of the spatially proximal Ala12 to Glu caused an 11-fold drop in insecticidal potency (Table I). Additional mutations of the vicinal disulfide residues might help to better define the role of this structural feature in toxin binding and activity.

In summary, we have developed a recombinant expression system for one of the Janus-faced atracotoxins and undertaken a comprehensive site-directed mutagenesis study to elucidate the key functional residues. This has provided us with the first complete view of the bioactive surface of a spider toxin. We have shown that the J-ACTX pharmacophore is relatively small and localized to a single face of the toxin. The data provide a solid framework for the rational design of novel insecticides based on the Janus-faced atracotoxins, as well as clues to the likely target of these toxins.

    ACKNOWLEDGEMENTS

We thank Dr. Nicolas Gilles and Dr. Graham Nicholson for critical appraisal of the manuscript and Dr. Zheng-yu Peng for use of the CD spectropolarimeter.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB9983242 (to G. F. K.) and a University of Connecticut Health Center Postgraduate Student Fellowship (to F. M.).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 Biochemistry, MC3305, Univ. of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032. Tel.: 860-679-8364; Fax: 860-679-1652; E-mail: glenn@psel.uchc.edu.

Published, JBC Papers in Press, April 5, 2002, DOI 10.1074/jbc.M202297200

2 F. Maggio and G. F. King, submitted for publication.

3 H. W. Tedford, N. Gilles, and G. F. King, unpublished data.

4 F. Maggio, R. A. Reenan, and G. F. King, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: J-ACTX, Janus-faced atracotoxin; GST, glutathione S-transferase; rp, reverse-phase; HPLC, high pressure liquid chromatography; CTX, conotoxin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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