Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel期刊界 All Journals 搜尽天下杂志传播学术成果专业期刊搜索期刊信息化学术搜索

Drosomycin, an Innate Immunity Peptide of Drosophila melanogaster, Interacts with the Fly Voltage-gated Sodium Channel

Authors:	Lior Cohen Yehu Moran Amir Sharon Daniel Segal Dalia Gordon Michael Gurevitz

Affiliation:	From the Departments of ^‡Plant Sciences and ;^§Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, 69978 Tel Aviv, Israel

Abstract:	Several peptide families, including insect antimicrobial peptides, plant protease inhibitors, and ion channel gating modifiers, as well as blockers from scorpions, bear a common CSαβ scaffold. The high structural similarity between two peptides containing this scaffold, drosomycin and a truncated scorpion β-toxin, has prompted us to examine and compare their biological effects. Drosomycin is the most expressed antimicrobial peptide in Drosophila melanogaster immune response. A truncated scorpion β-toxin is capable of binding and inducing conformational alteration of voltage-gated sodium channels. Here, we show that both peptides (i) exhibit anti-fungal activity at micromolar concentrations; (ii) enhance allosterically at nanomolar concentration the activity of LqhαIT, a scorpion alpha toxin that modulates the inactivation of the D. melanogaster voltage-gated sodium channel (DmNa_v1); and (iii) inhibit the facilitating effect of the polyether brevetoxin-2 on DmNa_v1 activation. Thus, the short CSαβ scaffold of drosomycin and the truncated scorpion toxin can maintain more than one bioactivity, and, in light of this new observation, we suggest that the biological role of peptides bearing this scaffold should be carefully examined. As for drosomycin, we discuss the intriguing possibility that it has additional functions in the fly, as implied by its tight interaction with DmNa_v1.The cysteine-stabilized αβ scaffold, CSαβ, contains an α-helix packed against a two-stranded β-sheet stabilized by three spatially conserved disulfide bonds (reviewed in Ref. 1). The CSαβ motif appears in a number of polypeptide families that can exert various biological functions such as: short chain (30–50 residues long) and long chain (60–76 residues long) scorpion toxins that affect voltage-gated ion channels, antimicrobial peptides (of insect and plants) as well as plant protease inhibitors (see Fig. 1) (2, 3).Open in a separate window FIGURE 1.Diversity of peptides containing the CSαβ motif. Representatives from each of five major groups of peptides containing a CSαβ motif are aligned according to their conserved disulfide bridging and common structural features: two β-strands packed against an α-helix. The featured molecules are from a diverse array of organisms. Scorpion α-toxins: P01484 (Aah2 of the North African scorpion Androctonus australis hector), AAB30413 (Ts4 of the Brazilian scorpion Tityus serrulatus); Scorpion β-toxins: P60266 (Css4 of the Mexican scorpion Centruroides suffusus suffusus), 1BCG_A (Bj-xtrIT of the Israeli black scorpion Hottentota judaica); Scorpion potassium channel blockers: P13487 (charybdotoxin of the Israeli yellow scorpion Leiurus quinquestriatus hebraeus), P0C194 (α-KTx 6.11 of the scorpion Opisthacanthus madagascariensis of Madagascar); Insect antimicrobial peptides: NP_523901 (drosomycin of the fruit fly Drosophila melanogaster), 1I2U_A (heliomicin of the tobacco budworm Heliothis virescens); plant γ-thionins: 1N4N (defensin of the garden petunia Petunia hybrida), AAL85480 (defensin of peach Prunus persica), AAM62652 (protease inhibitor II of the thale cress Arabidopsis thaliana).Analysis of the structure-function relationships of several representatives of a subclass of the long chain scorpion toxins family, the scorpion β-toxins (activators of voltage-gated sodium channels (Na_vs)⁵), elucidated their bioactive surfaces including those of the anti-insect excitatory and depressant toxins Bj-xtrIT and LqhIT2 (from Hottentota judaica and Leiurus quinquestriatus hebraeus, respectively (4 –6)) and the anti-mammalian β-toxin Css4 (from Centruroides suffusus suffusus (7)). These studies highlighted a conserved pharmacophore positioned on the CSαβ protein core (7). The C-tail, loops, turns, and unstructured stretches that connect to the CSαβ protein core in long chain scorpion toxins constitute a large portion of their exteriors and bear residues that participate in bioactivity (reviewed in Ref. 8). We have recently reported that truncated scorpion β-toxins, lacking the N- and C-terminal regions of the parental peptides but maintaining the CSαβ motif (ΔΔβ-toxins), are able to interact at high affinity with Na_vs (9). Although by themselves, the ΔΔβ-toxins (ΔΔCss4 and ΔΔBj-xtrIT) were nontoxic and did not bind at the receptor sites of the parental toxins, they exhibited an unexpected ability to allosterically facilitate the activity of a scorpion α-toxin (inhibition of Na_v fast inactivation), which binds at receptor site-3 on insect Na_vs (10), and the effect of the marine polyether toxin brevetoxin-2 (PbTx-2, facilitator of Na_v activation), which binds to receptor site-5 (11). However, a short chain potassium channel blocker (charybdotoxin) with a CSαβ structural fold did not exert any of these effects (9). These results indicated that it is not only the CSαβ motif but that specific amino acids at key sites on the protein exterior that can interact with ion channels and either block voltage-gated potassium channels or induce conformational alteration of voltage-gated sodium channels. From a structural viewpoint, the ability of ΔΔBj-xtrIT and ΔΔCss4 to bind to the Na_v, as manifested in modulation of the interaction of receptor site-3 and -5 ligands, suggests that by truncation of the two β-toxins, a masked functional surface was exposed. Because the CSαβ motif appears in several protein families including antimicrobial peptides, potassium channel blockers, and sodium channel gating modifiers (Fig. 1) (2, 3), we explored the possibility that a well characterized CSαβ peptide may exert an additional function known for other peptides bearing this scaffold.For this aim, we tested the ability of a well characterized Drosophila melanogaster anti-fungal peptide drosomycin (DRS) to interact with voltage-gated sodium channels. The solution structure of DRS indicates that this 44-amino acid peptide is cross-linked by four disulfide bonds, of which three render a CSαβ structural fold (Fig. 2) (12). Sequence comparison of the truncated scorpion β-toxin ΔΔCss4 with DRS indicates moderate identity (34%) and similarity (50%), including conservation of six cysteine residues that stabilize the CSαβ motif, which is manifested by a remarkable structural similarity (Fig. 2). Moreover, Lys-3, Asp-11, Asn-12, Glu-13, Gln-21, and Gln-22 of ΔΔCss4, which are involved in the interaction with insect Na_vs, are spatially conserved in DRS (Fig. 2) (9) but not in potassium channel blockers (Fig. 1). In light of the resemblance between the truncated scorpion β-toxin and DRS, we tested whether DRS is able to interact with the D. melanogaster voltage-gated sodium channel DmNa_v1.Open in a separate window FIGURE 2.Sequence alignment and three-dimensional structures of ΔΔCss4 and DRS. A, schematic diagrams of the Cα model structures of ΔΔCss4 and DRS covered by semitransparent molecular surfaces. The structure of DRS (right panel) is derived from the Protein Data Bank code 1MYN. The ΔΔCss4 model (left panel) is based on the NMR structure of Cn2 (Protein Data Bank code 1Cn2) and is spatially aligned with that of DRS. A was prepared using PyMOL. B, sequences were aligned according to the conserved cysteine residues, and the disulfide bonds formed between cysteine pairs are marked in solid lines. Dashes indicate gaps. Amino acid residues that were identified as part of the interacting surface of ΔΔCss4 with insect Na_vs (9) are shown in sticks according to their chemical nature (blue, positive charge; red, negative charge; green, nonpolar) and are also highlighted in the sequence alignment. Corresponding residues in DRS according to sequence and structural alignments are also shown in sticks.

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