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Molecular basis of the mammalian potency of the scorpion -like toxin, BmK M1

Li-Hui Liu^*,1, Frank Bosmans^,1, Chantal Maertens, Ron-Han Zhu^*, Da-Cheng Wang^*,2 and Jan Tytgat^,2

^* Center for Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, People’s Republic of China; and
Laboratory of Toxicology, University of Leuven, Leuven, Belgium

²Correspondence: (D.-C. W.) Centre for Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, P.R. China. E-mail: dcwang{at}ibp.ac.cn/wangdcibp@yahoo.com.cn; (J. T.) Laboratory of Toxicology, University of Leuven, E. Van Evenstraat 4, Leuven B-3000, Belgium. E-mail: jan.tytgat{at}pharm.kuleuven.ac.be

SPECIFIC AIMS

In this study, an effective yeast expression system was used to study the role of 14 N- and C-terminal residues from the -like toxin BmK M1 from the Chinese scorpion Buthus martensii Karsch. Using site-directed mutagenesis, all of these residues were individually substituted by one or more amino acids, resulting in a total of 19 mutants. These were then subjected to a bioassay on mice, an elaborate electrophysiological characterization on three cloned voltage-gated Na⁺ channels (Na_v1.2, Na_v1.5, and para), and a circular dichroism analysis. Our results reveal large mutant-dependent differences that emphasize important and specific roles for the studied residues. By mutating single amino acids we were able to redirect the -like characteristics of BmK M1 (active on mammals and insects) to much higher mammal specificity or, in a few cases, total insect specificity. This study therefore represents a thorough mapping and elucidation of three epitopes that underlie the molecular basis of the mammalian and insecticidal potency of the scorpion -like toxin BmK M1 on voltage-gated Na⁺ channels.

PRINCIPAL FINDINGS

Fourteen N- and C-terminal residues from the -like toxin BmK M1 were analyzed by site-directed mutagenesis. This NC domain, constituted by the five-residue turn (residues 8–12) and the C-terminal region (residues 56–64), is important for toxin function and specificity.

Correlating data from high impact substitutions such as glycine and differently charged residues (positive to negative and vice versa), partially based on sequence similarity with other insect and/or mammal specific scorpion toxins, our results clearly indicate that most of these residues are specifically involved in either structural stability or pharmacological function, or both. To discriminate between effects resulting from structural alterations and those reflecting a pure pharmacological interaction with the voltage-gated Na⁺ channel (VGSC) receptor site, CD spectroscopy was used.

We hypothesize that there are three epitopes that determine the toxin’s target specificity: 1) the first three N-terminal residues; 2) the five-residue turn (residues 8–12) in combination with the C-tail (residues 57–61); and 3) the loop between the ß₂ and ß₃ sheet including the adjacent glycine in the ß₃ sheet (residues 40–43).

1. Residues involved in structural stability (mutants with altered CD spectrum)
Expression of the P52S mutant in a trace amount indicates that replacement of a proline at this position by a serine is hampering its folding. A CD spectrum and a bioassay on mice could not be performed. It seems that the position of this proline contributes to the final turn and orientation of the C terminus. The efficacies of this mutant remain unaltered but the potency is reduced on para/tipE, resulting in a less potent but more mammal-specific toxin (Fig. 1 ).

View larger version (22K): [in this window] [in a new window]   Figure 1. Bar diagram representing the maximum efficacies (upper panel) of the studied mutants referenced to rBmK M1 (100) on Nav1.2/ß1 (yellow), Nav1.5/ß1 (red), and para/tipE (blue). Lower panel: potencies obtained after sigmoidal fit of the data of studied mutants on Nav1.5/ß1 and para/tipE referenced to rBmK M1. Data represent the mean ± SE of at least 3 measurements.

The general consensus among the mutants of which the CD spectrum has been critically altered is a much lower potency in the bioassay on mice and on the individual VGSCs (Table 1 ). R2E, I6G (loss of aromatic side chain that is part of the conserved hydrophobic core), and G34A (disrupted ß₂ sheet) mutants have gained mammal specificity but their potency has decreased dramatically (Table 1 , Fig. 1 ). The L51G (loss of aromatic side chain, part of the conserved hydrophobic core) and G61A mutant seem to be more insect selective although their potency has decreased. The D3A mutant, protruding from the surface of the molecule, still has a high potency in the bioassay but the effect on Na_v1.5/ß₁ in oocytes is severely diminished indicating an augmented insect specificity. However, the efficacy of this mutant, located in the first ß sheet, on Na_v1.2/ß₁ is greatly increased. This feature may explain the toxicity level in the bioassay. It can be hypothesized that substitution of the aforementioned residues causes profound structural changes and, as a consequence, the pharmacological properties of rBmK M1 are influenced.

2. Residues involved in pharmacological function
No structural perturbations were present in the following series of mutants since their CD spectra did not change with respect to rBmK M1. In general, the results of the bioassay on mice of the mutants are in concordance with the measurements on the VGSCs expressed in oocytes.

Mutants with no change in selectivity: P9N/H10Y, V59G, and P60G did not alter the selectivity of rBmK M1 toward mammals or insects. The EC₅₀ values of P9N/H10Y on Na_v1.5/ß₁ and para/tipE are decreased proportionally so selectivity is not affected. In the bioassay this mutant seems more potent than rBmK M1. A plausible explanation for this can be the highly improved efficacy on Na_v1.2/ß₁(Fig. 1) . The V59G and P60G mutants revealed a slight proportional decrease of EC₅₀ values on oocytes, in concordance with the bioassay. The P60G mutant is slightly more potent than rBmK M1 on mice, which can be explained by the gain in efficacy on Na_v1.5/ß₁ of this mutant (Table 1) . P9N/H10Y, V59G, and P60G are located in the NC domain but only (slightly) affect the potency and efficacy of rBmK M1 on all VGSCs tested; there was no change in selectivity.

Mutants promoting mammal selectivity: Only one C-terminal mutant (P60A) causes rBmK M1 to become more mammal specific, although the efficacy on Na_v1.2/ß₁ has disappeared (Fig. 1) . It has been reported that the specific orientation of the C-terminal segment mediated by the five-residue turn is relevant to the preference of various -toxin subgroups for phylogenetically distinct VGSCs receptor sites, leading to the conclusion that it is mainly the NC domain that confers selectivity for a certain VGSC.

Mutants R2A, D3N, D3R, P9N, and H10Y have gained mammal specificity without changing the overall structure of rBmK M1. Especially P9N (increased efficacy on Na_v1.2/ß₁, slightly increased potency on Na_v1.5/ß₁) and H10Y (substantial increase in efficacy on Na_v1.2/ß₁, 7-fold increase of potency on Na_v1.5/ß₁) have become highly specific for mammals. Their elevated toxicity toward mammals is confirmed in the bioassay (Table 1) . It is possible that the tyrosine at position 10 (in the NC domain) can form a more tightly packed NC domain by forming more hydrogen bonds and sustaining more hydrophobic interactions with the aliphatic residues from the C-tail (e.g., ring-ring interaction with histidine at position 64). This mutation therefore results in a more hydrophobic character of the NC domain that reaches out for interaction with mammalian VGSCs. Since these mutations do not alter the general structure of rBmK M1, it does not support an earlier study describing that mammal-specific scorpion toxins lack the protruding shape of the NC domain. H10 and P60 were indicated in one of our previous studies to be evolutionarily important. This study confirms their pharmacological relevance.

Mutants promoting insect selectivity: When measured on VGSCs, the deletionV1 mutant seems to have gained insect selectivity. However, this mutant is still toxic for mice. This may be caused by its highly increased efficacy on Na_v1.2/ß₁ and Na_v1.5/ß₁. The valine at this position reaches out to the aspartic acid at position 53. Disturbing these neighbors obviously affects the potency of the toxin on Na_v1.5/ß₁. When the highly conserved glycine at position 43 is mutated to the aliphatic residue alanine (G43A), it is remarkable to see that rBmK M1 becomes totally insect specific (no activity on mice) although its potency is less than rBmK M1 itself. This residue is located next to Y42 in the loop between the ß₂ and ß₃ sheet, which is quite different in sequence and structure between - and ß-toxins. Apparently this loop contributes to the toxin’s target specificity. As seen in different crystal structures of scorpion -toxins, this loop adopts various conformational states, indicating its inherent flexibility. Recent site-directed mutagenesis of BmK M1 identified the Y42 residue as important for the pharmacological function of this toxin. Considering that G43 is adjacent to this residue, it is plausible to infer that the highly conserved G43 provides a degree of flexibility required in the functional performance of the Y42 residue. Therefore, this glycine residue should be considered as part of this functional site.

CONCLUSIONS AND SIGNIFICANCE

Based on the mutagenesis results in this study, it seems plausible that the charge distributions or hydrophobicity of the protruding NC domain determines the target specificity of scorpion -like toxins. One of the most surprising findings of our work is the fact that one point mutation (e.g., H10Y) may completely alter the target specificity of -like scorpion toxins (Fig. 2 ). A conclusion from a previous extensive study was that scorpion -toxins active on insects still possess the protruding NC domain whereas the scorpion -toxins active on mammals do not have this shape. It was stated that this phenomenon could be responsible for VGSC subtype selectivity. However, from our data it seems that it is not the presence of the protruding shape of the NC domain itself that causes the toxin to be specific for mammals or insects. In several of the mutants that have gained mammal specificity, the general structure of the toxin did not change significantly. The presence of several functionally important epitopes is more likely to be the key in this issue. An important part of these toxin regions was identified in the same study.

View larger version (51K): [in this window] [in a new window]   Figure 2. Overview of all studied mutants indicated on the ribbon structure of BmK M1. Changes in selectivity toward mammals or insects are indicated in grey.

As a result from our experiments, we propose a fine-tuning of this previous hypothesis stating that two domains, the NC and the Core domain, determine the target specificity of a scorpion -toxin. We hypothesize there are three epitopes (not two) that determine the toxin’s target specificity: 1) the first three N-terminal residues; 2) the five-residue turn (residues 8–12) in combination with the C-tail (residues 57-61); and 3) the loop between the ß₂ and ß₃ sheet, including the adjacent glycine in the ß₃ sheet (residues 40–43).

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2485fje;

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