Biochimica et Biophysica Acta 1804 (2010) 872–883

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbapap

MeuTXKβ1, a -derived two-domain potassium channel -like peptide with cytolytic activity

Shunyi Zhu a,⁎, Bin Gao a, André Aumelas b, Maria del Carmen Rodríguez c, Humberto Lanz-Mendoza c, Steve Peigneur d, Elia Diego-Garcia d, Marie-France Martin-Eauclaire e, Jan Tytgat d, Lourival D. Possani f a Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, China b CNRS UMR5048; INSERM, U554; Université Montpellier 1 et 2; Centre de Biochimie Structurale, 29 rue de Navacelles, 34090 Montpellier, Cedex 9, France c National Institute of Public Health, Center for Infectious Diseases, Avenida Universidad, 655, Cuernavaca, Morelos 62508, Mexico d Laboratory of Toxicology, University of Leuven, O&N 2, Herestraat 49, P.O. Box 922, 3000 Leuven, Belgium e CNRS UMR 6231, CRN2M, Université de la Méditerranée et Université Paul Cézanne, 51, Bd Pierre Dramard, 13916 Marseille, France f Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Av. Universidad, 2001 Cuernavaca 62210, Mexico article info abstract

Article history: Recent studies have demonstrated that scorpion venom contains unique two-domain peptides with the Received 21 September 2009 peculiarity of possessing different functions, i.e. neurotoxic and cytolytic activities. Here we report Received in revised form 22 December 2009 systematic characterization of a new two-domain peptide (named MeuTXKβ1) belonging to the TsTXKβ Accepted 23 December 2009 molecular subfamily from the scorpion Mesobuthus eupeus by molecular cloning, biochemical purification, Available online 4 January 2010 recombinant expression, functional assays, CD and NMR studies. Its full-length bioactive form as well as 1–21 and 22–72 fragments (named N(1–21) and C(22–72), respectively) was produced in Escherichia coli by an Keywords: β fi + Defensin on-column refolding approach. Recombinant peptide (rMeuTXK 1) exhibited a low af nity for K channels Antimalarial peptide and cytolytic effects against bacteria and several eukaryotic cells. N(1–21) was found to preserve anti- Cysteine-stabilized alpha/beta motif Plasmodium activity in contrast to haemolytic activity, whereas C(22–72) retains these two activities. NMR structure Circular dichroism analysis demonstrates that rMeuTXKβ1 presents a typical scorpion toxin scaffold in water Scorpion venom and its α-helical content largely increases in a membrane-mimicking environment, consistent with the NMR structure of N(1–21) and an ab initio structure model of MeuTXKβ1 predicted using I-TASSER algorithm. Our structural and functional data clearly indicate an evolutionary link between TsTXKβ-related peptides and antiparasitic scorpines which both comprise the βSPN (β-KTxs and scorpines) family. © 2009 Elsevier B.V. All rights reserved.

1. Introduction TsTXKβ (TstβKTx) has been characterized as a blocker of Kv channel; and 2) class 2 consists of BmTXKβ, HgeβKTx, TcoKIK, TdiKIK, and Scorpion with cysteine-stabilized alpha/beta fold (CSαβ) TtrKIK. Recombinant BmTXKβ is a blocker of transient outward K+ target various ion channels of excitable membranes [1–5]. The short- current (Ito) in rabbit atrial myocytes, which is fast-inactivating and chain scorpion toxins affecting K+ channels usually are composed of associated with heteromultimeric channels with Kv4.2 and Kv4.3 23–42 amino acids with 3 or 4 disulfide bridges and are classified as α- subunits [16]. HgeβKTx exhibits strong cytolytic effects towards broad KTx molecular subfamilies [6], most of which block voltage-gated K+ targets including bacteria (Bacillus subtilis and Staphylococcus aureus), channels (Kv) by a dyad motif directly interacting with the channel human erythrocytes and frog oocytes; 3) class 3 includes some pore [7–9]. However, other interacting modes have also been proposed scorpine-related peptides (e.g. scorpine, opiscorpine1–4, HgeScplp1, to account for high-affinity binding to Kv channels for some toxins HgeScplp2, and heteroscorpine1) [17–21], a group of antimicrobial without the dyad [2,10]. defensins with sequence similarity to class 1. Whereas full-length Besides these α-KTxs, from belonging to Buthi- scorpine and HgeScplp1 are typical defensins, the K+ channel- dae, Caraboctonidae and Scorpioninae contain β-KTxs of 61–75 amino blocking effect of HgeScplp1 appears to reside in its C-terminal acids [6]. Based on sequence similarity, these molecules can be further domain. divided into three classes [11]: 1) class 1 contains TsTXKβ (TstβKTx), In this work, we report systematic characterization of a new AaTXKβ, BmTXKβ2, TdiβKTx, and TcoβKTx [12–15], of which only TsTXKβ-related peptide from the scorpion Mesobuthus eupeus venom gland by molecular cloning, biochemical purification, recombinant expression, functional assays, CD and NMR studies. rMeuTXKβ1isan ⁎ Corresponding author. Tel.: +86 010 64807112; fax: +86 010 64807099. anti-Plasmodium peptide with diverse functional features including + E-mail address: [email protected] (S. Zhu). low affinity binding to K channels and toxic effects on bacteria and

1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2009.12.017 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 873 mouse erythrocytes. Structural and functional evidence for a common 2.5. MALDI-TOF and amino-terminal sequencing ancestral origin between TsTXKβ-related peptides and the antimi- crobial scorpines has been uncovered. Molecular weights of recombinant and native peptides were determined by MALDI-TOF mass spectra on a Kratos PC Axima CFR plus 2. Materials and methods (Shimadzu Co. Ltd., Kyoto). The amino-terminal sequences of the native peptide was determined by automated Edman degradation using the 2.1. Screening of cDNA library PROCISE 492 cLC Protein Sequencer (ABI 173A, PE Applied Biosystems).

The cDNA library of the M. eupeus venom gland constructed by a 2.6. CD spectroscopy PCR-based approach has been described previously [22]. Clones β – containing an insert of 300–600 bp potentially encoding open reading CD spectra of rMeuTXK 1 and C(22 72) were recorded on a JASCO frames (ORFs) of polypeptide precursors were selected for DNA J-715 spectropolarimeter (Jasco, Tokyo, Japan) at a protein concen- sequencing. Primer T25V (5′-TTTTTTTTTTTTTTTTTTT TTTTTTA/G/C- tration of 0.3 mg/ml dissolved in both water and 50% TFE. Spectra 3′) was used for random sequencing of positive clones. Nucleotide were measured at 20 °C from 250 to 190 nm by using a quartz cell of sequences of MeuTXKβ1 and MeuTXKβ2 have been deposited in the 1.0 mm thickness. Data were collected at 0.2 nm intervals with a scan GenBank database (http://www.ncbi.nlm.nih.gov) under accession rate of 200 nm/min. The CD spectra measure was performed by – numbers of EF190325 and EF190324, respectively. averaging three scans; CD spectra of N(1 21) peptide were recorded at 20 °C on a Chirascan dichrograph (Applied Photophysics) in a quartz cell of 0.5 mm thickness. N(1–21) was initially dissolved in water at a 2.2. Peptide purification from the M. eupeus venom concentration of 0.2 mg/ml. Spectra were recorded both in water and in the presence of increasing amounts of TFE. Spectra are an average of The M. eupeus crude venom collected by an electrical stimulation 2 scans recorded from 180 to 260 nm with a scan speed of 1 nm/s. method was resuspended in 0.1% trifluoroacetic acid (TFA, v/v) and Data are expressed as mean residue molar ellipticity ([θ]) and directly subjected to RP-HPLC isolation. All well-defined peaks were percentages of various secondary structure elements in rMeuTXKβ1 separately collected and rerun on the same column to purify these and N(1–21) were calculated with the DICHROWEB software [24]. peptides further. Purified peptides were identified by MALDI-TOF and Edman degradation to determine their N-terminal sequences. 2.7. Expression of ion channel genes in Xenopus oocytes and electrophysiological recordings 2.3. Construction of prokaryotic expression plasmids

Methods for the expression of ion channel genes (Kv1.1−Kv1.6, To produce MeuTXKβ1inEscherichia coli, we constructed two ShakerIR, hERG, Nav1.2, Nav1.3, and Nav1.5) in Xenopus oocytes and β prokaryotic expression plasmids (pGEX-6P-1-MeuTXK 1 and pET- two-electrode voltage-clamp recordings of these channels have been β ′ 28a-MeuTXK 1) using primers SCPF (5 -ATGGATCCGATGACGATGA- described previously [9]. CAAGGGTTTTAGAGAGAAGCATT-3′)andSCPR(5′-ATGTCGACC- ′ TAAAAGCCCATGGGAATGC-3 ) according to the previously described 2.8. Competition experiments on rat brain synaptosomes method [23]. In these two cases, MeuTXKβ1 was expressed in a fusion form with a GST-tag carrier of 26 kDa or a His-tag carrier of 4.15 kDa, Iodination of kaliotoxin and the binding conditions in the in which an enterokinase (EK) cleavage site (DDDDK) was introduced competition experiments on rat brain synaptosomes were performed for the removal of these carrier proteins from fusion products. according to the literature [25]. Incubation buffer: 50 mM Tris–HCl, Constructed recombinant plasmids were transformed into E. coli BL21 50 mM NaCl, BSA 0.1%, pH 7.2. Washing buffer: 50 mM Tris–HCl, (DE3) for protein expression. 150 mM NaCl, BSA 0.1%, pH 7.2.

2.4. In vitro folding of rMeuTXKβ1 2.9. Antibacterial, antiparasitic and haemolytic assays

Degradation of rMeuTXKβ1 occurred when GST-DDDDK- Antibacterial assays were carried out according to the literature

MeuTXKβ1 was digested by EK. However, the pET system generated [26]. Lethal concentration (CL) was calculated by the Hultmark's a correct expression product, described as follows. Expression of method [27]. Microorganisms used in the inhibition zone assay fusion protein was induced with 1 mM IPTG at OD600 of 0.25. Cells include: 1) gram positive bacteria: Bacillus megaterium and Bacillus were harvested 4 h later and the pellet was suspended in resuspen- sp. DM-1; and 2) gram-negative bacteria: E. coli ATCC 25922 and sion buffer (100 mM Tris–HCl, 150 mM NaCl, pH 8.0). After sonication Stenotrophomonas sp. YC-1. Sources of these bacteria have been and subsequent centrifugation, the pellet was washed using isolation described previously [26]; antiparasitic assays against Plasmodium buffer (2 M urea and 2% Triton X-100 in the resuspension buffer). berghei Anka 2.34 were performed according to the previously Following centrifugation, pellets were resuspended in solubilization described method [17]; and haemolytic activity against fresh mouse buffer (6 M guanidinium hydrochloride, 10 mM β-mercaptoethanol blood was assayed according to the standard method [26]. and 10 mM imidazole in the resuspension buffer) for 1 h at room temperature followed by centrifugation and the supernatant was 2.10. NMR spectroscopy and calculation of structures loaded to Ni-NTA resin pre-equilibrated by solubilization buffer. Refolding was initiated by a linear urea gradient from 6 M to 0 M. The sample of N(1–21) was prepared in a 95:5 (v/v) mixture of

Refolded fusion protein was eluted by elution buffer (200 mM H2O:D2O to yield 1.5–2.0 mM solutions, pH 4.5. As previously imidazole and 3 mM β-mercaptoethanol in the resuspension buffer) described [28], all 1H-NMR experiments were recorded on a Bruker and the imidazole in the eluate was completely removed by RP-HPLC. Avance 600 spectrometer equipped with a triple resonance cryoprobe The lyophilized fusion protein was digested in PBS buffer (140 mM at temperatures ranging from 13 to 27 °C. The study in the presence of

NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH7.3) by EK at trifluoroethanol was carried out with 24.8 and 52.1% trifluoroethanol room temperature for 2 h. rMeuTXKβ1 and its truncated derivatives (v/v) at 17 °C by recording a similar data set as in water. Data were were finally collected by RP-HPLC under the same conditions as processed with XWINNMR software. Full sequential assignment was described here. achieved using the general strategy described by Wüthrich [29]. 874 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883

Structure calculation was also carried out as previously described 3. Results [28] by using the DYANA program [30]. NMR-derived constraints measured on the NOESY spectrum recorded in the presence of 52.1% 3.1. Molecular and biochemical characterization of MeuTXKβ1 TFE were converted into interproton upper distance limits of 2.5, 3.0, 4.0 and 5.0 Å, for strong, medium, weak and very weak intensities, From the cDNA library of the venom gland of M. eupeus,we respectively. Finally, we used 157 NOE-derived distances and 10 isolated and identified two clones (BeL-62 and BeL-226) coding for dihedral constraints to calculate 100 conformers of the N(1–21) two peptide precursors with only one amino acid substitution. Fig. 1A solution structure. The resulting 10 structures with minimal restraint shows the nucleotide and deduced amino acid sequences of these two violations (no violation N0.3 Å) were analyzed with INSIGHT 97 cDNAs which are 491 bp containing an ORF encoding a peptide of 91 (Molecular Simulation Inc., San Diego). Ramachandran analysis was amino acid in length. The deduced amino acid sequence starts with a performed with PROCHECK [31]. The limits of the secondary structure 19-residue signal peptide and ends with a 72-residue mature peptide. elements were determined with STRIDE [32,33]. Chemical shifts and These two peptides are named MeuTXKβ1 and MeuTXKβ2, respec- NMR-derived constraints of N(1–21) have been deposited in the tively. To confirm their presence in the venom, we isolated single Biological Magnetic Resonance Bank (BMRB) (accession number peptide components from the M. eupeus venom by RP-HPLC and 20075) (http://www.bmrb.wisc.edu/). characterized them by MALDI-TOF and amino-terminal sequencing (Fig. 1B and C). We found that the peak at 36.5 min retention time has a detected MW of 8256.81 Da, in good agreement with the theoretical 2.11. Bioinformatics MW of MeuTXKβ1 (8255.56 Da). Edman degradation confirmed that the N-terminal 4 residue of the peak is GFRE, consistent with the See Supplementary materials. predicted post-translational processing of its precursor. Such a

Fig. 1. Molecular and biochemical characterization of MeuTXKβ1 from the scorpion M. eupeus venom gland. A. Nucleotide and deduced amino acid sequences of MeuTXKβ1 and MeuTXKβ2. Signal and mature peptides are highlighted in blue and green, respectively. The codon CCC for a proline in MeuTXKβ1 is substituted by TCC for a serine in MeuTXKβ2, in which a base substitution that leads to the amino acid change is shown in red; B. RP-HPLC showing the separation of the crude venom. The Agilent Zorbax 300SB-C18 (4.6×150 mm, 5 μm) was equilibrated with 0.1% TFA in water (v/v) and peptide components were eluted from the column with a linear gradient from 0 to 60% acetonitrile in 0.1% TFA in water (v/ v) within 60 min with a flow rate of 1 ml/min. The UV absorbance trace was followed at 225 nm. Note: peaks eluted from the retention times of 17 to 25 min corresponding to some short-chain K+ channel toxins (K-Tx); peaks from 27 to 29 min corresponding to Cl−-channel toxins (Cl-Tx); peaks from 31 to 35 min corresponding to Na+-channel toxins (Na-Tx); and the peak at 39 min corresponding to an antimicrobial peptide (AMP) (these data will be published elsewhere). MeuTXKβ1 was eluted at 36.5 min (indicated by an arrow); C. MALDI-TOF of the native MeuTXKβ1. S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 875 processing pattern was only observed in Tco42.14 (here called HgeScplp2. About 29–50% sequence identity can be found between TcoβKTx, Fig. 2), a member of the TsTXKβ subfamily. For other the C-terminal region of these two peptides and AITDs. Only two members, N-terminal 8 residues (previously called propeptide) are BmTXKβ-related peptides (e.g. TtrKIK and TcoKIK, sequences not normally cut off for the release of the mature K-channel blockers from shown here) show some weak sequence identity to their C-terminal their precursors. In the M. eupeus venom, we did not detect a region. According to sequence alignment presented in Fig. 2, these truncated version derived from the precursor of MeuTXKβ1. scorpion venom-derived peptides related to AITDs are clearly divided into two distinct classes: class 1 and class 3 (see Section 1). Peptides within a group are more highly conserved, showing up to 60% 3.2. Sequence comparison between MeuTXKβ1 and other sequence identity, whereas peptides between two groups show related peptides greater divergence (approximately 40% identity).

Searching the GenBank database using the peptide precursor as a query identified hits, including scorpion venom-derived TsTXKβ- 3.3. Production of recombinant MeuTXKβ1 related and scorpine-related peptides as well as some ancient invertebrate-type defensins (AITDs) from diverse origins (scorpion, Due to the very small amount of native MeuTXKβ1 available from tick, insect, oyster, mussel, and fungus) (Fig. 2). As expected, these the venom, we expressed and purified its recombinant peptide in E. two peptides share the highest sequence identity (86–88%) to coli for structural and functional characterization (Fig. 3A and B). BmTxKβ2 and AaTXKβ, two TsTXKβ-related peptides, respectively Purifi ed fusion proteins were subjected to EK digestion and from Mesobuthus martensii (a sibling species of M. eupeus) and And- subsequent RP-HPLC isolation (Fig. 3C and D), from which we roctonus australis, and 58–61% identity to other TsTXKβ-related collected three major fractions (F1–F3), corresponding to retention peptides, such as TsTXKβ, TtrβKTx, TcoβKTx, and TdiβKTx, and times of 19.2, 22.8 and 25.8 min, respectively, for determination of moderate identity (36–43%) to scorpine-related peptides, such as molecular weights (MWs) by MALDI-TOF analysis (Fig. S1, provided scorpine, heteroscorpine1, opiscorpine1–4, HgeScplp1 and as Supplemental materials). The MWs of the former two fractions are

Fig. 2. Multiple sequence alignment of scorpion venom two-domain peptides and AITDs. Id: identity. Identical amino acids or constitutive replacements are shadowed in yellow and grey, respectively. Secondary structures of MeuTXKβ1 were defined according to STRIDE. Consensus: h: hydrophobic residue; p: polar residue; x: any amino acid. Sequence sources are provided in Supplementary materials. 876 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883

Fig. 3. Expression, purification and characterization of rMeuTXKβ1 and its truncated derivatives. A. Construction of pET-28a-MeuTXKβ1 expression vector. The coding region of MeuTXKβ1 was inserted into BamHI and SalI sites of pET-28a with an EK cleavage site at the 5′ end of the cDNA; B. SDS-PAGE showing the expression and purification process. Lane 1: total cell extract of E. coli carrying pET-28a-MeuTXKβ1 without IPTG; lane 2: IPTG-induced total cell extract; lanes 3 and 4: pellet and supernatant, respectively, from the cell lysate prepared by sonication; lane 5: the eluted fusion protein from Ni-NTA HisBind resin after refolding; and lane 6: EK-digested product of fusion protein. Released recombinant peptide is labelled by an arrow; C. RP-HPLC showing the purified fusion protein. C18 column was equilibrated with 0.1% TFA in water (v/v) and the purified proteins were eluted from the column with a linear gradient from 0 to 60% acetonitrile in 0.1% TFA within 40 min; D. RP-HPLC showing separation of EK-digested product. SDS-PAGE showing the pure rMeuTXKβ1 at 26 min of retention time (inset). Experimental MWs detected by MALDI-TOF for each component are shown here.

2597.12 Da and 5676.69 Da, respectively, and well match the theoretical forming activity or cytolysis as induced by rMeuTXKβ1 (i.e. induction values of N(1–21) and C(22–72) of rMeuTXKβ1 (2595.99 Da and of ‘leaky cells’). In the absence of the peptide, the conductance over 5677.59 Da, respectively), suggesting that a non-specific cleavage the membrane of the oocytes remains minimal over a large voltage occurred between 21Rand22T of rMeuTXKβ1. For the third fraction range, which is indicative for a healthy cell (Fig. 4A). As the cytolytic collected, MALDI-TOF detected a major MW of 7896.94 Da and a small damage of the oocytes at high concentrations makes it impossible to component with MW of 8256.73 Da. These two components respec- evaluate K+ channel-blocking activity of rMeuTXKβ1 in this way, we tively correspond to C(4–72) of MeuTXKβ1 and the full-length used the competition binding experiment to assess its capacity of MeuTXKβ1 (their theoretical values being 7895.14 and 8255.56, replacing 125I-kaliotoxin bound on rat brain synaptosomes. Kaliotoxin respectively). Clearly, C(4–72) is a product of an additional non-specific is a well-characterized scorpion toxin and exhibits no activity on cleavage between R3 and E4. rMeuTXKβ1 contains three Arg residues Kv1.2, little specificity for Kv1.1 and high-affinity blockade of Kv1.3 (R3,R9 and R21) and only Arg-polar or -charged residue is the susceptible [25]. Our results demonstrate that rMeuTXKβ1 exhibits a low affinity bond of EK non-specific cleavage. Here, we called the third fraction (F3) binding for the kaliotoxin receptor on rat brain synaptosomes and the rMeuTXKβ1 because it contains the full-length molecule and a 125I-kaliotoxin is partially removed from its site by rMeuTXKβ1, with truncated version with only three N-terminal residues removed. the IC50 of 0.1 μM(Fig. 4B), about 10 times lower than ChTx under similar conditions [25]. From this result, it can be concluded that our 3.4. Biological activity of rMeuTXKβ1 and its two truncated derivatives recombinant peptide is a weak Kv channel interacting toxin. Next, we assayed activities of rMeuTXKβ1 against bacteria, the As a new member of class 1 of the β-KTxs, we firstly evaluated the parasite P. berghei, and mouse blood cells (Fig. 5). At a high ability of rMeuTXKβ1 in blocking Kv channels expressed in Xenopus concentration, it displayed weak antibacterial activity against Steno- oocytes. We found that rMeuTXKβ1 exhibits no effect on Kv channels trophomonas sp. YC-1 with a lethal concentration (CL)of21μM. + (Kv1.1−Kv1.6, ShakerIR and hERG) and Na channels (Nav1.2, Nav1.3 However, rMeuTXKβ1 strongly inhibited the development of the and Nav1.5) at 1 μM concentration. At 2.5 μM concentration, an P. berghei ookinetes in the concentration range from 10 to 20 μM outwardly rectifying toxin-dependent conductance was observed in (inhibition percentage: 89.0±2.0 to 98.8±1.0). For scorpine, only non-injected oocytes, which can be interpreted as evidence for pore- 2 μM is needed for 90% development inhibition to the P. berghei S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 877

Fig. 4. Pore-forming and K-channel binding effects of rMeuTXKβ1. A. A non-injected oocyte becomes “leaky” in the presence of 2.5 μM of rMeuTXKβ1; B. effect of rMeuTXKβ1 on the 125I-kaliotoxin binding to rat brain synaptosomal membranes. Rat brain synaptosomal membranes (30 μg/assay) were incubated with 40 pM 125I-kaliotoxin and increasing concentrations of kaliotoxin (black dots) and rMeuTXKβ1 (open dots). Competition experiments were done twice in triplicate. ookinetes [17]. Although lower activity against P. berghei than 3.5. NMR structure of N(1–21) scorpine, rMeuTXKβ1 is more potent than two well-characterized antiparasitic peptides, e.g. Shiva-3, a cecropin-like synthetic peptide Circular dichroism (CD) spectra of N(1–21) were recorded in [34], and gambicin, a cysteine-rich antimicrobial peptide (AMPs) of water or TFE, a membrane-mimicking environment (Fig. 6A). In 8 kDa from Anopheles gambiae [35]. At the above two concentrations, water, an essential random coil spectrum was obtained (≈8% α- rMeuTXKβ1 displayed slightly hemolytic effect on mouse blood cells helix). In the presence of various percentages of TFE, spectra were (10.3% and 14.8% haemolysis, respectively). progressively modified with characteristic bands at 193, 207 and For comparison, we parallelly evaluated cytolytic effects of the two 222 nm. The mean residue ellipticity changes at 222 nm are consistent truncated derivatives under the same conditions: N(1–21) led to with an increase of the α-helical content. 12.0, 34.0, 52.0, 51.0, and 62.1%±1.0% and 77.7%±0.3% inhibition to development of the P. 51.0% α-helices were observed at 17.4, 25.0, 31.8, 38.0, and 43.6% TFE, berghei ookinetes at 10 and 20 μM, respectively. For C(22–72), the respectively. Obviously, CD spectra remained similar in ≥38% TFE, corresponding values are 36.3%±3.3% and 71.5%±1.9%, respectively. indicating that the conformation of N(1–21) remained unchanged in For haemolysis, C(22–72) displayed almost the same efficiency with these high percentages of TFE. the full-length molecule, 6.7% and 17.1% at 10 and 20 μM, respectively. In the 1H-NMR spectrum of N(1–21) recorded in water at 27 °C, all On the contrary, N(1–21) lacked this effect even in 100 μM, suggesting amide signals gathered in the 8.5 to 7.8 ppm range, suggesting that that the functional region of rMeuTXKβ1 for haemolysis resides in its the peptide is mainly unstructured (data not shown). Sequential NOEs C-terminus. These two truncated peptides lack anti-Stenotrophomonas of strong intensity facilitated the assignment of all the spin systems. activity at the concentration effective for the full-length rMeuTXKβ1. Four NN NOES of weak intensity (R9−F10,F10−V11, and V11−K12) Finally, toxicity assays demonstrated that MeuTXKβ1 was non- were also observed, suggesting that residues 9–12 tend to adopt a toxic on mice at 20 μg peptide for a 25 g-mouse (n=3) and on the poorly stable α-helical structure in water. Overall, the absence of insect Tenebrio molitor at 30 pmol for 100 mg body weight. medium- and long-range NOEs indicates that N(1–21) is essentially 878 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883

character mainly in its helical part (Fig. 6E and F). The hydrophobic cluster is composed of F7,F10,V11,A14,V15 and P16, whereas the hydrophilic cluster is bigger and consists of R3,E4,K5,H6,Q8,R9,K12 and Y13. The unique proline residue (P16) located at the C-terminus of the helix probably acts as a helix breaker to prevent its extension.

3.6. Structural features of rMeuTXKβ1

The CD spectra of rMeuTXKβ1 recorded in H2O was similar to those of other scorpion toxins (e.g. KAaH1 and KAaH1) [36] and drosomycin [23] between 190 and 250 nm. A positive band at 190 nm and a negative band at 206 nm represent a typical curve of CSαβ peptides. However, in the presence of 50% TFE, the CD spectra of rMeuTXKβ1 changed dramatically (Fig. 7), suggesting its structural flexibility. A negative minimum with an ellipticity of −35.9307 degree.cm2/dmol at 208.8 nm and a shoulder at 220 nm are indicative of an α-helix. Based on the CD data, we estimated the content of secondary structure elements in rMeuTXKβ1 by DICHROWEB, which showed that the recombinant peptide in water contains 17% α-helix and 21% β-sheet, compatible with the scorpion toxins and drosomycin [23,36]. In 50% of TFE, the α-helical content largely increases (from 17% in water to 55%) whereas the β-sheet content only changes slightly (from 21% in water to 17%). The 38% increase of the helical content in 50% of TFE could be attributed to its N-terminal region (Fig. 6). This is further supported by

the observation of the CD spectra of C(22–72) in H2O and 50% of TFE (Fig. 7). The rMeuTXKβ1 and C(22–72) spectra are overall similar in

H2O, consistent with the essential random coil of N(1–21) in H2O, but significantly different in 50% TFE. In this membrane-mimicking environment, C(22–72) only increased 10% of helical content due to the deletion of the N-terminal 21 residues.

3.7. Model structure of MeuTXKβ1

Fig. 5. Biological activities of rMeuTXKβ1 and its truncated derivatives. A. rMeuTXKβ1 inhibited growth of Stenotrophomonas sp. YC-1. 0.1% TFA, the solvent of the peptide, To obtain a reliable full-length structure model with compatible showed no antibacterial activity; B. antiparasitic activity. Percentages of inhibition of secondary structure content with the CD results, we applied the I- ookinete development were recorded at 24 h after adding the peptide; C. haemolytic TASSER algorithm to ab initio model the rMeuTXKβ1 structure. TM- activities against mouse erythrocytes. Experiments in B and C were repeated three score of the first model is 0.51±0.15, implying that it represents a times. Meucin-18 [26] and AdDLP [40] were used as positive and negative controls. correct topology. The model built in this way exhibits a two-domain architecture containing two independent structural domains respec- tively composed of N-terminal residues 1–34 and C-terminal residues unstructured in water. The addition of 24.8% TFE generated a significant 35–72. Based on the overall structural feature of MeuTXKβ1, we effect on the overall 1H-NMR spectrum. Amide signals were more named these two domains N-terminal α-helical domain (NHD) and C- spread, from 8.85 to 7.75 ppm, whereas the chemical shifts of all methyl terminal CSαβ domain (CCD) (Fig. 8A). The model shows a reasonable resonances were altered. The main chemical shift alterations were main chain geometry, in which some hydrophobic residues (such as observed for F2,R3,K12 and Y13 amide resonances and for V11 and T19 F7,F10,V11,A14,V15,P16,L20,V23,L24,V27,V28, and V31 from NHD, and methyl signals. Although all sequential NOEs were still of high intensity, F36,I52,C64,C66,I68, and M70 from CCD) form a stable molecular core to more NN NOEs than in water were observed, suggesting the presence of facilitate the interaction of the two domains, whereas the molecular asignificant percentage of α-helical structure, in equilibrium with surface of MeuTXKβ1 displays a strong hydrophilic feature with the random coil (data not shown). With 52.1% of TFE, these alterations were exception of five fully exposed hydrophobic residues (F2,A40,F58,F62 slightly enhanced (amide signals in the 8.95 and 7.60 ppm range). In and F72) which tightly contact the hydrophobic core (Fig. 8B). addition, a weak line broadening was observed for the whole spectrum. Remarkably, such a structural core appears to be also present in The NOESY spectrum displayed several successive NN NOEs of strong some MeuTXKβ1-related peptides listed in Fig. 2 since the majority of intensity (Fig. 6B), indicating the formation of a more stable helical these hydrophobic residues are conserved across the alignment. These structure, consistent with the CD data. residues include F7(V/I/A), V11(I), V15(I/L/M), L20(V/I), V23(I/M/A), We used a set of 167 NMR-derived constraints (157 distance L24(I/M/A), V27(I/L/A), V28 and V31(I/M/A/I) from NHD and F36,C64 constraints and 10 angle constraints) to calculate the structure of N and C66 from CCD (numbered according to MeuTXKβ1). When the (1–21) in the presence of TFE. A family of 10 conformers is displayed model was compared with the structure-associated experimental in Fig. 6C and D. The Ramachandran plot indicated that 94.4% of data, its rationality is further strengthened in two aspects: 1) residues were in the most favored and allowed regions and 5.6% were secondary structure element contents of the model are 54% α-helix in the generously allowed region. The main part of the structure and 14% β-sheet, highly consistent with those calculated from the CD consists of a well-defined α-helical structure spanning residues R3/ data of rMeuTXKβ1 (55% α-helix and 17% β-sheet in 50% TFE); and 2) E4–A14. In contrast, residues from V15 to R21 appeared less con- the model of N(1–21) in MeuTXKβ1 shows structural similarity to its strained. The superimposition of all these conformers showed a good NMR structure with an rmsd of 1.03 Å for 15 Cα atoms, in which six overlay for backbone atoms of residues 4–16 with an average rmsd of hydrophobic residues involved in the interaction of two domains 0.20±0.07 Å. The N(1–21) structure displays a marked amphipathic possess similar conformation (Fig. 8C). S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 879

Fig. 6. The NMR structures of N(1–21). A. CD spectra obtained in water, and with various percentages of TFE; B. Part of the NOESY experiment showing the dNN NOEs (17 °C, 52.1% of TFE and 200 ms mixing time); C and D. Superposition of 10 conformers of N(1–21) determined in 52.1% TFE with different display styles (Cα wire and solid ribbon, respectively) in identical orientation. E and F. Two views of N(1–21). The helical structure spans residues 4–14, in which an amphiphilic structure can be identified. 880 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883

Fig. 7. CD analysis of rMeuTXKβ1. A. The CD spectra of rMeuTXKβ1 and C(22–72) in water and 50% TFE. Both were recorded from 190 to 250 nm. Spectra were taken at a peptide concentration of 0.3 mg/ml. B. Comparison of estimated secondary structure element contents from the CD data between rMeuTXKβ1 and C(22–72) is also shown here.

Two remarkable observations can be made as to this model toxin-like peptide MeuTXKβ1. As a new anti-Plasmodium peptide, structure: 1) the MeuTXKβ1 NHD is disrupted by a hinge region rMeuTXKβ1 exhibited diverse cytolytic activities. Apart from the whose location significantly differs from cecropin, an antimicrobial inhibition of the parasite P. berghei, it can bind K+ channels on rat and antiparasitic peptide previously thought to be the structural brain synaptosomes, and lyse the bacterium and eukaryotic cells homologue of the scorpine NHD. The hinge region includes residues (oocytes and mouse erythrocytes). Remarkably, two truncated V15PES18 in MeuTXKβ1andG23P24 in cecropin [37],reflecting derivatives (N(1–21) and C(22–72)) also displayed clear anti-Plas- differences between them. A short insert (residues MIGVPVV) was modium activity. The lack of haemolysis together with 3D structural found to be located at the hinge region of HgeScplp2; 2) VAST search availability makes N(1–21) a candidate for the construction of detected a total of 55 structural homologues, including ancient transgenic malaria-resistant mosquitoes and drug design. invertebrate-type defensins, classical insect-type defensins, plant/ insect-type defensins, and scorpion (data not shown). 4.1. Structural insights into cytolytic effect of rMeuTXKβ1 Among these peptides the closest relatives to MeuTXKβ1 are two ancient invertebrate-type defensins from the oyster C. gigas (CgDEF, Initial functional analysis suggests that rMeuTXKβ1 only partly pdb entry 2B68) (Fig. 8C) and the mussel M. galloprovincialis (Mgd-1, replaced 125I-kaliotoxin bound on rat brain synaptosomes. The weak pdb entry 1FJN). In addition, VAST search characterized a putative tail affinity for K-channels may be explained by the extension of several lysozyme from the bacterium Geobacter sulfurreducens (pdb entry residues in the N-terminus of rMeuTXKβ1 when compared with 2IA7) as the structural analogue of MeuTXKβ1 with 1.86 Å of rmsd TsTXKβ. An N-terminal extension has been found to affect activity of between their 39 Cα atoms (Fig. S2, provided as Supplemental two K-channel toxins: 1) an amino terminus-extended analogue of materials). kaliotoxin 2 with three additional residues has 1000-fold less affinity for the 125I-kaliotoxin binding on rat brain synaptosomes than 4. Discussion kaliotoxin 2 [38]; and 2) although the HgeScplp1-derived C-terminal fragment (C-HgeScplpDig, residues 33 to 76) obtained by chemical The present study reports molecular cloning, biochemical purifi- digestion can efficiently inhibit ion currents in Kv1.1, Kv1.2 and Kv1.3, cation, recombinant expression, chemical refolding and structural/ a naturally-occurring HgeScplp1-derived peptide (Hge36) lacks functional characterization of a new scorpion long-chain K+ channel blocking effect on Kv1.2 and Kv1.3 channels and has two-fold lower S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 881

Fig. 8. Structural model of MeuTXKβ1. A. Overall folding of rMeuTXKβ1 predicted using the ab initio modeling method (I-TASSER). NHD: N-terminal helical domain (residues 1–34). CCD: C-terminal CSαβ domain (residues 35–72). A sphere model is also displayed as two distinct domains in different colors; B. hydrophobic interaction between two domains of MeuTXKβ1. Hydrophobic residues of MeuTXKβ1, as shown in a surface model, of which some are found to be located in the interface of two domains, as marked by a dotted cycle; C. Structural superimposition. Left: the N(1–21) NMR structure and the corresponding region of the ab initio model, in which six hydrophobic residues with similar conformation are displayed as stick model. Right: structural evidence for the existence of the CSαβ fold in the C-terminal domain of MeuTXKβ1.The ancient invertebrate-type defensin from C. gigas (CgDEF, pdb entry 2B68) (blue) can well fitted with the CCD (red).

affinity for Kv1.1 channels due to the extension of four amino acids at sequence similarity [15]. A phylogenetic analysis showed that the N-terminus compared with C-HgeScplpDig [15]. TsTXKβ-related β-KTxs constitute a sister clade relative to scorpine- However, the anti-Plasmodium activity of rMeuTXKβ1 appears to related peptides with about 40% sequence identity between them, require the N-terminal sequence because C(22–72) alone showed supporting the existence of a closely related peptide family. For lower potency than the full-length molecule. This is not surprising in convenient description, we named it the βSPN (β-KTxs and scorpines) that the extended region in rMeuTXKβ1 is also present in scorpine. family. Besides their CCDs resembling the ancient invertebrate-type Residues 3–8 of the N-terminal extension constitute the half of the N defensins (AITDs), the βSPN family NHDs are also structurally similar, (1–21) helix and provide both hydrophobic and hydrophilic side- as identified by a hinge-forming region linking two α-helical chains to form an amphipathic architecture, thus representing a fragments with a consensus sequence motif: xxhpEKphQpxhxxxh in functionally related region. Lower potency against P. berghei observed helix 1 and xxxhxxhhpxhVHKhxKp in helix 2 (Fig. 2). On the contrary, in N(1–21) and C(22–72) than in rMeuTXKβ1 suggests the functional BmTXKβ-related peptides exhibit no sequence similarity in their importance of the interactions between the two domains. Such extended NHDs to that of the βSPN family. Moreover, their CCDs interactions could also be needed for rMeuTXKβ1 to exhibit lethal resemble more the α-KTxs and thus they are often clustered together effect to Stenotrophomonas sp. YC-1 in that two separate domains lose in a phylogenetic tree when all scorpion toxins are taken into antibacterial activity. However, the haemolytic activity of rMeuTXKβ1 consideration. In particular, a conserved functional motif (Gly-Lys- seems to reside in its C-domain in that removal of N(1–21) does not Cys) in α-KTxs with its crucial residue Lys directly inserting into the significantly change its effect and N(1–21) lacks such activity. channel pore is also present in the BmTXKβ -related peptides (class 2), suggesting that α-KTxs and the BmTXKβ-related β-KTxs might adopt 4.2. Recognition of the βSPN family a similar manner to block K+ channels [15] and could have an evolutionary link. A structural analysis provides additional evidence As mentioned previously, scorpion venom-derived two-domain for exclusion of the BmTXKβ-related β-KTxs in the βSPN family. molecules can be categorized into three different classes based on Although adopting an overall similar fold, the extended NHDs 882 S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 between them appear to be structurally unrelated. In the βSPN family [5] M.F. Martin-Eauclaire, B. Ceard, F. Bosmans, J.P. Rosso, J. Tytgat, P.E. Bougis, New 16 17 “Birtoxin analogs” from Androctonus australis venom, Biochem. Biophys. Res. members, the residue P or G in an equivalent position (Fig. 2), as a Commun. 333 (2005) 524–530. helical breaker, disrupts NHD and forms a hinge region itself to link [6] J. Tytgat, K.G. Chandy, M.L. Garcia, G.A. Gutman, M.F. Martin-Eauclaire, J.J. van der two helical fragments, whereas the β-KTxs represented by BmTXKβ Walt, L.D. Possani, A unified nomenclature for short-chain peptides isolated from scorpion venoms: α-KTx molecular subfamilies, Trends Pharmacol. 20 (1999) lack this key structural residue and likely fold into a complete helix. 444–447. Both scorpion neurotoxins targeting animal ion channels and [7] M. Dauplais, A. Lecoq, J. Song, J. Cotton, N. Jamin, B. Gilquin, C. Roumestand, C. Vita, invertebrate defensins acting on bacterial membranes adopt the CSαβ C.L. de Medeiros, E.G. Rowan, A.L. Harvey, A. Ménez, On the convergent evolution – scaffold and thus are classified into the same structural superfamily. of animal toxins, J. Biol. Chem. 272 (1997) 4302 4309. [8] P. Savarin, M. Guenneugues, B. Gilquin, H. Lamthanh, S. Gasparini, S. Zinn-Justin, A. + Recent discovery of bi-functions (K channel-blocking and antimi- Ménez, Three-dimensional structure of kappa- PVIIA, a novel potassium crobial activities) of two members (e.g. ChTx and HgeScplp1) in this channel-blocking toxin from cone snails, Biochemistry 37 (1998) 5407–5416. superfamily [15,39] raises an intriguing question of whether these [9] K.N. Srinivasan, V. Sivaraja, I. Huys, T. Sasaki, B.S. Cheng, T.K. Kumar, K. Sato, J. β Tytgat, C. Yu, B.C. San, S. Ranganathan, J.H. Bowie, R.M. Kini, P. Gopalakrishnakone, peptides originated from a common ancestor. The SPN family κ-Hefutoxin1, a novel toxin from the scorpion Heterometrus fulvipes with unique carrying these two functions described above provides a new example structure and function: importance of the functional diad in potassium channel to support an evolutionary link between neurotoxins and defensins: selectivity, J. Biol. Chem. 277 (2002) 30040–30047. [10] K.M. Giangiacomo, Y. Ceralde, T.J. Mullmann, Molecular basis of alpha-KTx 1) at the sequence and structural levels, the CCD of this family is specificity, Toxicon 43 (2004) 877–886. closely related to the AITDs; and 2) both rMeuTXKβ1 and some AITDs [11] E. Diego-García, E.F. Schwartz, G. D'Suze, S.A. González, C.V. Batista, B.I. García, R.C. exhibit antibacterial and antiparasitic activities. The TsTXKβ-related de la Vega, L.D. Possani, Wide phylogenetic distribution of Scorpine and long- chain beta-KTx-like peptides in scorpion venoms: identification of “orphan” peptides (class 1), all derived from Buthidae, is separated from the components, Peptides 28 (2007) 31–37. scorpines(class3)fromCaraboctonidae and Scorpioninae in a [12] R.S. Rogowski, B.K. Krueger, J.H. Collins, M.P. Blaustein, R.S. Rogowski, B.K. Krueger, phylogenetic tree (Fig. S3, provided as Supplemental materials), J.H. Collins, M.P. Blaustein, Tityustoxin K alpha blocks voltage-gated noninactivat- ing K+ channels and unblocks inactivating K+ channels blocked by alpha- suggesting that divergence of these two subfamilies occurred after in synaptosomes, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 1475–1479. speciation. This tree defines an early duplication event of an AITD gene [13] C. Legros, B. Céard, P.E. Bougis, M.F. Martin-Eauclaire, Evidence for a new class of in the scorpion lineage due to the existence of AaTXKβ and AaDEF in scorpion toxins active against K+ channels, FEBS Lett. 431 (1998) 375–380. A. australis, by which a duplicated copy could finally develop into the [14] S. Zhu, W. Li, X. Zeng, D. Jiang, X. Mao, H. Liu, Molecular cloning and sequencing of two ‘short chain’ and two ‘long chain’ K+ channel-blocking peptides from the βSPN family through the assembly of the NHD in the ancestral AITD Chinese scorpion Buthus martensii Karsch, FEBS Lett. 457 (1999) 509–514. scaffold. [15] E. Diego-García, Y. Abdel-Mottaleb, E.F. Schwartz, R.C. de la Vega, J. Tytgat, L.D. Possani, Cytolytic and K+ channel blocking activities of beta-KTx and scorpine-like peptides purified from scorpion venoms, Cell. Mol. Life Sci. 65 (2008) 187–200. 5. Conclusion [16] Z. Cao, F. Xiao, F. Peng, D. Jiang, X. Mao, H. Liu, W. Li, D. Hu, T. Wang, Expression, purification and functional characterization of a recombinant scorpion venom peptide BmTXKbeta, Peptides 24 (2003) 187–192. In this work, we extended the functional spectrum of the two- [17] R. Conde, F.Z. Zamudio, M.H. Rodríguez, L.D. Possani, Scorpine, an anti-malaria and domain TsTXKβ molecular subfamily and further clarified its anti-bacterial agent purified from scorpion venom, FEBS Lett. 471 (2000) 165–168. structural features. rMeuTXKβ1 is the first TsTXKβ-related peptide [18] S. Zhu, J. Tytgat, The scorpine family of defensins: gene structure, alternative – characterized to have cytolytic effect against bacteria, oocytes, mouse polyadenylation and fold recognition, Cell. Mol. Life Sci. 61 (2004) 1751 1763. [19] N. Uawonggul, S. Thammasirirak, A. Chaveerach, T. Arkaravichien, W. Bunya- erythrocytes and parasites, which supports close relationship be- tratchata, W. Ruangjirachuporn, P. Jearranaiprepame, T. Nakamura, M. Matsuda, tween scorpion venom-derived TsTXKβ-related peptides and antimi- M. Kobayashi, S. Hattori, S. Daduang, Purification and characterization of crobial scorpine-related peptides. Successful expression of Heteroscorpine-1 (HS-1) toxin from Heterometrus laoticus scorpion venom, Toxicon 49 (2007) 19–29. β rMeuTXK 1inE. coli lays the basis for further investigation of the [20] R. Carballar-Lejarazú, M.H. Rodríguez, F. de la Cruz Hernández-Hernández, J. structure–function relationship of venom two-domain peptides with Ramos-Castañeda, L.D. Possani, M. Zurita-Ortega, E. Reynaud-Garza, R. Hernán- cytolytic effects. dez-Rivas, T. Loukeris, G. Lycett, H. Lanz-Mendoza, Recombinant scorpine: a multifunctional antimicrobial peptide with activity against different pathogens, Cell. Mol. Life Sci. 65 (2008) 3081–3092. Acknowledgements [21] E.F. Schwartz, T.S. Camargos, F.Z. Zamudio, L.P. Silva, C. Bloch Jr., F. Caixeta, C.A. Schwartz, L.D. Possani, Mass spectrometry analysis, amino acid sequence and biological activity of venom components from the Brazilian scorpion Opistha- This work was supported by the following grants: 1) the National canthus cayaporum, Toxicon 51 (2008) 1499–1508. Natural Science Foundation of China (30730015 and 30621003); 2) the [22] S. Zhu, B. Gao, Molecular characterization of a new scorpion venom lipolysis fi 973 Program from the Ministry of Science and Technology of China activating peptide: evidence for disul de bridge-mediated functional switch of peptides, FEBS Lett. 580 (2006) 6825–6836. (2010CB945304); 3) Bilateral Cooperation for the 16th Session of the [23] Y. Yuan, B. Gao, S. Zhu, Functional expression of a Drosophila antifungal peptide in Sino-Belgian S&T Mixed Commission to S. Z; and 4) OT/05/64, FWO- Escherichia coli, Protein Expres. Purif. 52 (2007) 457–462. Vlaanderen (G.0330.06 and G.0257.08), BIL 07/10 (China) to J.T. [24] L. Whitmore, B.A. Wallace, DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data, Nucl. Acids Res. 32 (2004) W668–W673. Appendix A. Supplementary data [25] R. Romi, M. Crest, M. Gola, F. Sampieri, G. Jacquet, H. Zerrouk, P. Mansuelle, O. Sorokine, A. Van Dorsselaer, H. Rochat, M.F. Martin-Eauclaire, J. Van Rietschoten, Synthesis and characterization of kaliotoxin, J. Biol. Chem. 268 (1993) Supplementary data associated with this article can be found, in 26302–26309. the online version, at doi:10.1016/j.bbapap.2009.12.017. [26] B. Gao, P. Sherman, L. Luo, J. Bowie, S. Zhu, Structural and functional characterization of two genetically related meucin peptides highlights evolution- ary divergence and convergence in antimicrobial peptides, FASEB J. 23 (2009) References 1230–1245. [27] D. Hultmark, in: Wiesner, et al., (Eds.), Techniques in Insect Immunology, 1998, [1] L.D. Possani, B. Becerril, M. Delepierre, J. Tytgat, Scorpion toxins specific for Na+- pp. 103–107. channels, Eur. J. Biochem. 264 (1999) 287–300. [28] S. Campagna, N. Saint, G. Molle, A. Aumelas, Structure and mechanism of action of [2] R.C. Rodríguez de la Vega, E. Merino, B. Becerril, L.D. Possani, Novel interactions the antimicrobial peptide piscidin, Biochemistry 46 (2007) 1771–1778. between K+ channels and scorpion toxins, Trends Pharmacol. Sci. 24 (2003) [29] K. Wüthrich, NMR of Proteins and Nucleic Acids, John Wiley and Sons, New York, 1986. 222–227. [30] P. Guntert, C. Mumenthaler, K. Wuthrich, Torsion angle dynamics for NMR [3] E. Carbone, E. Wanke, G. Prestipino, L.D. Possani, A. Maelicke, Selective blockage of structure calculation with the new program DYANA, J. Mol. Biol. 273 (1997) voltage-dependent K+ channels by a novel scorpion toxin, Nature 296 (1982) 283–298. 90–91. [31] R.A. Laskowski, J.A. Rullmannn, M.W. MacArthur, R. Kaptein, J.M. Thornton, AQUA [4] B. Inceoglu, J. Lango, J. Wu, P. Hawkins, J. Southern, B.D. Hammock, Isolation and and PROCHECK-NMR: programs for checking the quality of protein structures characterization of a novel type of neurotoxic peptide from the venom of the South solved by NMR, J. Biomol. NMR 8 (1996) 477–486. African scorpion Parabuthus transvaalicus (Buthidae), Eur. J. Biochem. 268 (2001) [32] D. Frishman, P. Argos, Knowledge-based protein secondary structure assignment, 5407–5413. Proteins 23 (1995) 566–579. S. Zhu et al. / Biochimica et Biophysica Acta 1804 (2010) 872–883 883

[33] M. Heinig, D. Frishman, STRIDE: a web server for secondary structure assignment [37] F.M. Marassi, S.J. Opella, P. Juvvadi, R.B. Merrifield, Orientation of cecropin A from known atomic coordinates of proteins, Nucleic Acids Res. 32 (2004) helices in phospholipid bilayers determined by solid-state NMR spectroscopy, W500–W502. Biophys. J. 77 (1999) 3152–3155. [34] M.C. Rodríguez, F. Zamudio, J.A. Torres, L. González-Cerón, L.D. Posanni, M.H. [38] C. Legros, E. Feyfant, F. Sampieri, H. Rochat, P.E. Bougis, M.F. Martin-Eauclaire, + Rodríguez, Effect of a cecropin-like synthetic peptide (Shiva-3) on the sporogonic Influence of a NH2-terminal extension on the activity of KTX2,aK channel development of Plasmodium berghei, Exp. Parasitol. 80 (1995) 596–604. blocker purified from Androctonus australis scorpion venom, FEBS Lett. 417 (1997) [35] J. Vizioli, P. Bulet, J.A. Hoffmann, F.C. Kafatos, H.M. Müller, G. Dimopoulos, 123–129. Gambicin: a novel immune responsive antimicrobial peptide from the malaria [39] N.Y. Yount, M.R. Yeaman, Multidimensional signatures in antimicrobial peptides, vector Anopheles gambiae, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 12630–12635. Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 7363–7368. [36] N. Srairi-Abid, J.I. Guijarro, R. Benkhalifa, M. Mantegazza, A. Cheikh, M. Ben Aissa, [40] B. Gao, M. Rodriguez, H. Lanz-Mendoza, S. Zhu, AdDLP, a bacterial defensin-like P.Y. Haumont, M. Delepierre, M. El Ayeb, A new type of scorpion Na+-channel- peptide exhibits anti-Plasmodium activity, Biochem. Biophys. Res. Commun. 387 toxin-like polypeptide active on K+ channels, Biochem. J. 388 (2005) 455–464. (2009) 393–398.