Acta Biochim Biophys Sin (2008): 955-963 | © 2008 Institute of Biochemistry and Cell Biology, SIBS, CASStructure-function | All Rights Reserved relationship 1672-9145 of bifunctional scorpion toxin BmBKTx1 http://www.abbs.info; www.blackwellpublishing.com/abbs | DOI: 10.1111/j.1745-7270.2008.00479.x

Structure-function relationship of bifunctional scorpion toxin BmBKTx1

Suming Wang1,4, Lijun Huang2, Dieter Wicher3, Chengwu Chi2,4*, and Chenqi Xu4*&

1 School of Life Sciences, University of Science and Technology of China, Hefei 230027, China 2 Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai 200092, China 3 Max Planck Institute for Chemical Ecology, Department of Evolutionary Neuroethology, D-07745 Jena, Germany 4 Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

As the first identified scorpion toxin active on both big pore and use a basic residue side chain to directly clog the conductance Ca2+-activated K+ channels (BK) and small K+ flux pathway. Intermediate area block toxins are conductance Ca2+-activated K+ channels (SK), BmBKTx1 has smaller in size; they interact with small conductance Ca2+- been proposed to have two separate functional faces for two activated K+ channels (SKs) on the pore and turret areas. targets. To investigate this hypothesis, two double mutants, Charge-charge interaction is the primary interaction force K21A-Y30A and R9A-K11A, together with wild-type toxin for the SK toxin docking. The type 3 turret block toxins were expressed in Escherichia coli. The recombinant toxins interact with HERG channels. Their binding sites are were tested on cockroach BK and rat SK2 channel for located at the turret area, away from the central pore, functional assay. Mutant K21A-Y30A had a dramatic loss of and the interaction occurs with two heads of the toxin function on BK but retained its function on SK. Mutant molecule [2]. R9A-K11A did not lose function on BK or SK. These data It is quite common for one scorpion KTx to have support the two functional-face hypothesis and indicate that multiple K+ channel blocking activities. However, it has the BK face is on the C-terminal β-sheet. rarely been reported that one KTx has multiple interaction faces for different channels. In 2004, Regaya et al Keywords BmBKTx1; scorpion K+ channel toxin confirmed the existence of distinct functional faces in (KTx); K+ channel; two functional faces scorpion toxin [3]. BmTx3 is the first reported scorpion KTx to have type 1 and type 3 functional faces responsible for A-type and HERG K+ current activity, respectively Scorpion toxins are valuable pharmacological tools for [4], while BmBKTx1 is the first KTx proposed to have studies on the structure and function of potassium type 1 and type 2 faces for BK and SK function, respec- channels. The interaction modes of scorpion K+ channel tively [5]. toxins (KTxs) and K+ channels have been classified into In this work, the two functional faces of BmBKTx1 three types: (1) pore area block, (2) intermediate area block were studied by mutagenesis. Two double mutants, and (3) turret area block [1]. Pore block toxins often act K21A-Y30A and R9A-K11A, together with wild-type (WT) on the voltage-gated K+ channels (Kvs) and big conductance toxin were expressed and tested on cockroach BK channel Ca2+-activated K+ channels (BKs). They bind to the channel (pSlo) and rat SK2 (rSK2) channel. The functional data prove the existence and importance of the two functional faces, and indicate that the BK face is on the C-terminal Received: July 29, 2008 Accepted: September 27, 2008 β-sheet. This work was supported by the grants from the National Basic Research Program of China (No. 2004CB719904), the Chinese Academy of Science for Key Topics in Innovation Engineering (KSCX2-YW-R- Materials and Methods 104), and the Max Planck Society (DW) &Present address: Dana1410, Dana-Farber Cancer Institute, 44 Binney Expression and purification of recombinant toxins Street, Boston, Massachusetts 02115, USA *Corresponding authors: WT and mutant BmBKTx1 was expressed in Escherichia Chenqi Xu: Tel, 1-617-5827846; Fax, 1-617-6322662; E-mail, coli as a C-terminal fusion to a glutathione S-transferase [email protected] (GST) tag in the pGEX 4T-1 vector. The fusion protein Chengwu Chi: Tel, 86-21-54921165; Fax, 86-21-65988403; E-mail, [email protected] was induced at 37 ºC for 3 h with 0.4 mM isopropyl β-

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D-thiogalactopyranoside (IPTG), and purified by a 1 mM CaCl2, 0.33 mM NaH2PO4, 2 mM Na-pyruvate, glutathione-agarose (GSH) column (Amersham 10 mM glucose, 10 mM HEPES. For measuring SK Biosciences, Piscataway, USA). The fusion protein was currents, the bath solution differed and contained 110 mM then digested by Factor Xa (New England Biolabs, NaCl and 30 mM KCl. The pipette solution contained Ipswich, USA) at 23 ºC for 12 h in a digestion buffer 140 mM KCl, 4 mM NaCl, 2 mM Mg-ATP, and 10 mM containing 20 mM Tris-HCl, 100 mM NaCl and 2 mM HEPES. The free Ca2+ concentration of pipette solutions

CaCl2 (pH 8.0). was measured with calcium-sensitive electrodes (KWIK The cleavage products were loaded onto a GSH column tips; WPI, Berlin, Germany), and was adjusted to 2.5 μM to separate the recombinant toxins from the GST tag and to measure SK currents and to 25 μM to measure Slo uncleaved fusion proteins. The recombinant toxins were currents. The pH of the bath solution and pipette then purified to a high homogenecity on a reverse-phase solution was adjusted to 7.4 and 7.25, respectively. high-performance liquid chromatography (HPLC) Zorbax Liquid junction potentials between the pipette and bath C18 column (Agilent, Palo Alto, USA). solutions were taken into account before establishing the seal. The holding potentials for Slo and SK current Verification of the correct folding of the recombinant measurements were −90 mV and −40 mV, and the K+ toxins equilibrium potentials amounted to −84 mV and −40 mV, The hydrophobicity of the recombinant toxins was respectively. compared with that of previously synthesized BmBKTx1 For toxin application the bath perfusion system BPS4 by passing them through the Zorbax C18 column [5]. The (ALA, Westbury, USA) was used. elution gradient: 20% to 50% buffer B (0.1% trifluoroacetic acid in acetonitrile) in 20 min. Results

Circular dichroism (CD) spectroscopy Design of BmBKTx1 mutants Samples for CD spectroscopy were dissolved in 10mM To investigate the functional faces of BmBKTx1 for BK phosphate buffer, pH 7.0. The final concentration of the and SK channels, we aligned the sequences of scorpion samples was 0.8 mg/ml for R9A-K11A, 1 mg/ml for WT BK and SK toxins with BmBKTx1 [Fig. 1(A)]. Most of and 1.2 mg/ml for K21A-Y30A. Spectra were obtained on the BK toxins are active on Kv channels as well, and their a Jasco J-715 spectrometer in 1 mm cells at 20 ºC. The Kv functional sites have been studied extensively [6−12]. results were analyzed with standard analysis software The BK (and/or Kv) toxin functional residues cluster on (Jasco, Tokyo, Japan). the C-terminal β sheet face, and a conserved functional dyad (Lys-Tyr) plays a central role in their blocking activity. Functional assay of recombinant toxins By contrast, SK toxin functional residues, all of which are For electrophysiology, pSlo and rSK2 channels were basic residues, are found on the N-terminal α-helix face transiently expressed in HEK293 cells [5]. Cells were [13−15]. Although they belong to the same superfamily, cultured at a density of ~2×104/35 mm dish and trans- BK and SK channels show low homology in the pore region, fected with 1 μg of channel DNA (Slo: pSlo/pcDNA3.1 which may provide an evolutional reason to explain the (splice variant AAAA); SK: rSK2/pcDNA3.1) and 0.5 μg development of scorpion toxins with diverse functional of pEGFP-C1 (Clontech Laboratories Inc, Palo Alto, USA) faces [Fig. 1(B)]. using Roti-Fect transfection kit (Roth, Karlsruhe, BmBKTx1 is shorter than other BK toxins, but it is the Germany). same size as SK toxins. It contains the conserved dyad Whole-cell ion currents in HEK293 cells expressing (K21-Y30) in its C-terminus, which is why it was chosen pSlo or SK were measured at room temperature using a as the mutation sites for BK function. There are no basic whole-cell patch-clamp that appropriately compensated residues in BmBKTx1 on the conventional SK sites. Two series resistance and capacitive currents. Current neighboring basic residues (R9-K11) of BmBKTx1 were measurements and data acquisition were performed with mutated to investigate the SK functional face. an EPC9 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany), which was controlled by Expression and purification of recombinant toxins PatchMaster software (HEKA Elektronik). In order to obtain high expression yield, we optimized the The bath solution for measuring Slo currents induction temperature (18−37 ºC), the induction time (2− − contained: 135 mM NaCl, 5 mM KCl, 1 mM MgCl2, 18 h) and the IPTG concentration (0.2 0.8 mM). The

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Fig. 1 Design of BmBKTx1 mutants (A) Sequence alignment of scorpion toxins active on BK (and/or Kv) or SK channels. The functional residues for BK (and/or Kv) and SK toxins were marked in red, and the mutations sites of BmBKTx1 were marked in blue. (B) Sequence alignment of the BK and SK channel pore regions. d, Drosophila; h, human; m, mouse; p, Periplaneta; r, rat; BK, big conductance Ca2+-activated K+ channels; Kv, voltage-gated K+ channels; SK, small conductance Ca2+-activated K+ channels. Black denotes the same amino acid residues in the primary structure of these channels; gray denotes the similar amino acid residues in the primary structure of these channels.

best condition to induce the cells was at 37 ºC for 3 h with per liter, which is a substantial improvement over previ- 0.4 mM IPTG [Fig. 2(A)]. ous studies [16,17]. The GST-BmBKTx1 fusion proteins were cleaved by In order to verify the folding of the recombinant toxins, Factor Xa, which has a high cleavage efficiency. Another the fully functional synthetic BmBKTx1 was used as a advantage of Factor Xa is that it leaves no additional reference to test whether the recombinant BmBKTx1 had residues on the target protein after cleavage. As shown in the same hydrophobicity as the synthetic one [5]. Both Fig. 2(B), more than 90% of GST-BmBKTx1 fusion protein synthetic and recombinant BmBKTx1 were eluted at the was cleaved after 12 h of digestion in Factor Xa at 23 ºC same time on the same reverse-phase HPLC C18 column [Fig. 2(B)]. [Fig. 3(A,B)]. Equal amounts of the recombinant and syn- The recombinant toxins were purified to a high thetic toxins were then premixed and loaded onto the same homogeneity by reverse-phase HPLC [Fig. 2(C)], and then C18 column again [Fig. 3(C)]. Only one elution peak was lyophilized. The molecular weights of the recombinant observed, indicating that the two samples have the same toxins determined by mass spectroscopy were consistent characteristics. The functional assay also confirmed that with their theoretical values [Fig. 2(C) and Table 1]. The the recombinant toxins had the same function as the na- final yields of the recombinant toxins were almost 6 mg tive one [5].

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Fig. 2 Expression and purification of recombinant toxins (A) Fusion protein expression in Escherichia coli. Lane 1, whole-cell lysate before induction; lane 2, whole-cell lysate after IPTG induction; lane 3, GST-BmBKTx1 fusion protein purified by GSH column. (B) Cleavage of fusion proteins. Lane 1, fusion proteins before Factor Xa cleavage; lane 2, fusion proteins after cleavage. (C) Reverse-phase high-performance liquid chromatography profile of recombinant BmBKTx1. Inset, mass spectrum of rBmBKTx1-Wild type. GSH, glutathione-agarose; GST, glutathione S-transferase; IPTG, isopropyl β-D-thiogalactopyranoside.

Table 1 Sequences and molecular weights (MW) of recombinant toxins

Sample name Sample sequence Calculated MW Observed MW

Wild type AACYSSDCRVKCVAMGFSSGKCINSKCKCYK 3336.0 3336.0 K21A-Y30A ------A---A- 3186.8 3187.0 R9A-K11A ------A-A------3193.8 3194.0 The marked “A” in K21A-Y30A and R9A-K11A denote the substitution of relative amino acid residues by alanine.

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Fig. 4 Secondary structure determinations of recombinant BmBKTx1 and its two mutants Top, R9A-K11A mutant; middle, wild-type; bottom, K21A-Y30A mutant. CD, circular dichroism.

of K21A-Y30A mutant’s BK blocking function does not result from mutation-induced conformational changes.

Activity assay of recombinant toxins An earlier study demonstrated that BmBKTx1 effectively blocks pSlo [5]. Here we first tested whether its muta- tion––a mutation of the basic residues R9 and K11 of toxin R9A-K11A––would likewise affect toxin action (Fig. 5, left). Compared with the concentration-dependent recom- binant WT toxin, there was no significant difference in the R9A-K11A mutant’s effect [Fig. 5(B), left]. However, the mutation of K21A and Y30A (K21A-Y30A) in the C- terminal β-sheet region of BmBKTx1 caused a dramatic loss of blocking potency (Fig. 5, left). At 10 μM, the WT toxin reduced pSlo currents to 0.19±0.08 of control (n=6) and R9A-K11A to 0.24±0.05 of control (n=6). K21A-Y30A reduced the current to only 0.56±0.07 of control (n=6), Fig. 3 Verification of correct folding of recombinant BmBKTx1 which differs significantly from both other WT and R9A- (A) The recombinant BmBKTx1 and (B) synthetic BmBKTx1 have K11A recombinant toxins (P<0.01). Furthermore, the ef- the same elution time on the column. (C) Moreover, only one peak fects of the toxin develop more slowly for K21A-Y30A was observed for the elution of the mixture of recombinant BmBKTx1 mutant [Fig. 5(C), left]. The time constant describing the and synthetic BmBKTx1. time course of toxin block (1 μM) is 161 s for R9A-K11A mutant and 210 s for K21A-Y30A mutant. In contrast to pSlo, there is no difference in the action of R9A-K11A and K21A-Y30A on rSK2 (Fig. 5, right). The secondary structure of rBmBKTx1 and its two mu- There is neither a significant difference in its blocking ef- tants were also examined by CD spectroscopy (Fig. 4). ficiency nor in its time course [Fig. 5(B,C), right]. The There were no differences in the secondary structures activities of the two mutants on rSK2 are comparable to among these three peptides, which indicates that the loss the WT toxin [5].

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Fig. 5 Effect of BmBKTx1 mutants on pSlo (left) and on rat SK2 channels (right) (A) Left, sample traces of pSlo currents activated by depolarization from −90 mV to +60 mV before (control) and 7 min after application of 1 μM R9A-K11A mutant (top) and K21A-Y30A mutant (bottom). Right, recordings of rSK2 currents activated by voltage ramps from −100 mV to +100 mV before (control) and 5 min after application of 1 µM R9A-K11A mutant (top) and K21A-Y30A mutant (bottom). (B) Left, concentration-dependence of pSlo current block by recombinant toxins. Right, fraction of total rSK2 current remaining 6 min after application of 1 µM indicated mutants. (C) Development of pSlo (left) and rSK2 (right) current block by 1 μM R9A-K11A mutant (triangle) and K21A-Y30A mutant (circle). Data are presented as mean±SEM, n=6. WT, wild type.

blocking toxins with unrelated structures have a con- Discussion servative functional dyad composed of a Lys residue and an aromatic residue (tyrosine or Phe) [18]. Breaking this The interaction of BmBKTx1 and BK channels dyad will result in the loss of channel block activity. Dauplais et al proposed the idea that potassium channel- Mouhat et al modified this theory in 2005 [19]. In the

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Fig. 6 Structural aspects of BmBKTx1 functional sites (A) The structure of pSlo S5-S6 region modeled on Streptomyces lividans K+ channel (KcSA) crystal structure. D281, Y279 and Y283, the potential interaction sites between pSlo and K21, are marked in blue. Y268 and F269, the potential interaction sites between pSlo and Y30, are marked in red. (B,C) The NMR (Nuclear Magnetic Resonance) structure of BmBKTx1 with the dyad residues highlighted in red (B) or the N-terminal basic residues highlighted in red (C). The structures of SK toxins P05 (D) and LeTxI (E) with their functional residues highlighted in red. SK, small conductance Ca2+-activated K+ channels.

new hypothesis, toxins first recognize the various Kv1 [20]. Based on the Streptomyces lividans K+ channel channel subtypes through various sets of residues (KcSA) structure [21], the pore region structure of the distinct from the functional dyad; the functional dyad then pSlo channel was modeled by the Swiss-model server potentially serves as a supplementary anchoring point (http://swissmodel.expasy.org/SWISS-MODEL.html) and a first efficient physical barrier. In BmBKTx1, the [Fig. 6(A)]. Based on the other Kv blocking models [1], conserved dyads (K21 and Y30) were found in the C- we propose that the K21 side chain might directly dip terminal β-sheet face. As expected, the dyad mutation into the channel pore by interacting with pSlo D281 via substantially reduced pSlo’s blocking potency and charge-charge interaction and by forming hydrogen bonds slowed the development of the blocking effect. It is with neighboring residues such as Y279 and Y283. The interesting to note that the K21 side chain points out from residues Y268 and F269 from four pSlo subunits form a the β-sheet surface, while the Y30 side chain is in a hydrophobic ring on the pore entrance, which might parallel position to the β-sheet [Fig. 6(B)]; this suggests interact with the Y30 of BmBKTx1 via hydrophobic that they might have different interaction sites on pSlo interaction.

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relevance to not only scorpion toxins but also other animal The interaction of BmBKTx1 and SK channels toxins. It has been reported that three basic residues (R6, R7 and R13) located at the α-helix face of the toxins are crucial References for SK blocking [13]. In BmBKTx1, there are no basic residues on these sites, and Arg9 and Lys11 are the only 1 Rodriguez de la Vega RC, Merino E, Becerril B, Possani LD. Novel basic residues on the α-helix face. The mutation of R9 interactions between K+ channels and scorpion toxins. Trends − and K11 to alanine does not affect the SK function, Pharmacol Sci 2003, 24: 222 227 2 Xu CQ, Zhu SY, Chi CW, Tytgat J. Turret and pore block of K+ suggesting that BmBKTx1 might have an unconventional channels: what is the difference? Trends Pharmacol Sci 2003, 24: form of interaction with SK channels. 446−448 We compared the structure of BmBKTx1 with that of 3 Regaya I, Beeton C, Ferrat G, Andreotti N, Darbon H, De Waard SK toxins P05 and LeTXI [Fig. 6(C–E)]. The functional M, Sabatier JM. Evidence for domain-specific recognition of SK amino acid residues in P05 (R6, R7 and R13) and in and Kv channels by MTX and HsTx1 scorpion toxins. J Biol Chem 2004, 279: 55690−55696 LeTxI (R6 and R13) are fully exposed on the surface, 4 Huys I, Xu CQ, Wang CZ, Vacher H, Martin-Eauclaire MF, Chi point in the same direction and form a positive charge CW, Tytgat J. BmTx3, a scorpion toxin with two putative func- pincer [22,23]. Although R9 and K11 in BmBKTx1 are tional faces separately active on A-type K+ and HERG currents. also exposed on the surface, they point in opposite Biochem J 2004, 378: 745−752 directions and thus do not form a positive charge pincer. 5 Xu CQ, Brône B, Wicher D, Bozkurt O, Lu WY, Huys I, Han YH et al. BmBKTx1, a novel Ca2+-activated K+ channel blocker puri- Furthermore, compared with other SK toxins, the two fied from the Asian scorpion Buthus martensi Karsch. J Biol Chem basic residues of BmBKTx1 (R9 and K11) are too close 2004, 279: 34562−34569 to each other, so they might not be capable of contacting 6 Aiyar J, Rizzi JP, Gutman GA, Chandy KG. The signature se- the corresponding binding sites of the SK channel in quence of voltage-gated potassium channels projects into the ex- − conformation. This difference may explain why R9 and ternal vestibule. J Biol Chem 1996, 271: 31013 31016 7 Stampe P, Kolmakova-Partensky L, Miller C. Intimations of K+ K11 are not functional sites for SK, and it also further channel structure from a complete functional map of the molecu- supports the notion that the scorpion toxin function is lar surface of charybdotoxin. Biochemistry 1994, 33: 443−450 precisely regulated by its residue type and side chain 8 Blanc E, Romi-Lebrun R, Bornet O, Nakajima T, Darbon H. Solu- orientation. tion structure of two new toxins from the venom of the Chinese scorpion Buthus martensi Karsch blockers of potassium channels. Biochemistry 1998, 37: 12412−12418 Two separate functional faces for BK and SK 9 Srinivasan KN, Sivaraja V, Huys I, Sasaki T, Cheng B, Kumar TK, Although the SK functional face has not been determined, Sato K et al. Kappa-Hefutoxin1, a novel toxin from the scorpion the hypothesis that BmBKTx1 has two separate functional Heterometrus fulvipes with unique structure and function. Impor- faces has been substantially supported by this study. The tance of the functional diad in potassium channel selectivity. J − fact that the mutation of dyad residue K21-Y30 Biol Chem 2002, 277: 30040 30047 10 Huys I, Tytgat J. Evidence for a function-specific mutation in the dramatically affects the BK function but not the SK neurotoxin, parabutoxin 3. Eur J Neurosci 2003, 17: 1786−1792 function indicates that BK and SK functional faces are 11 Gilquin B, Racapé J, Wrisch A, Visan V, Lecoq A, Grissmer S, fully separated. BmBKTx1’s BK functional face is located Ménez A et al. Structure of the BgK-Kv1.1 complex based on on the C-terminal β-sheet; we predict that the SK face distance restraints identified by double mutant cycles. Molecular might be on the N-terminal α-helix, on which other basis for convergent evolution of Kv1 channel blockers. J Biol Chem 2002, 277: 37406−37413 residues (besides the basic residues R9 and K11) are the 12 Huys I, Olamendi-Portugal T, Garcia-Gómez BI, Vandenberghe I, primary interaction sites with SK channels. Future research Van Beeumen J, Dyason K, Clynen E et al. A subfamily of acidic will be necessary to find the functional residues for SK alpha-K+ toxins. J Biol Chem 2004, 279: 2781−2789 blocking activity. 13 Shakkottai VG, Regaya I, Wulff H, Fajloun Z, Tomita H, Fathallah M, Cahalan MD et al. Design and characterization of a highly selective peptide inhibitor of the small conductance calcium-acti- Concluding remarks vated K+ channel, SkCa2. J Biol Chem 2001, 276: 43145−43151 In this study, we established a high-yield system for 14 Cui M, Shen J, Briggs JM, Fu W, Wu J, Zhang Y, Luo X et al. expression and purification of rBmBKTx1 and its mutants. Brownian dynamics simulations of the recognition of the scor- Our data validated the existence and importance of the pion toxin P05 with the small-conductance calcium-activated po- − conservative dyad of functional residues and proved the tassium channels. J Mol Biol 2002, 318: 417 428 15 Pedarzani P, D’hoedt D, Doorty KB, Wadsworth JD, Joseph JS, hypothesis of separated functional faces in one toxin. The Jeyaseelan K, Kini RM et al. Tamapin, a venom peptide from the multi-functional face phenomenon may have a general Indian red scorpion (Mesobuthus tamulus) that targets small con-

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ductance Ca2+-activated K+ channels and after hyperpolarization J Pept Sci 2005, 11: 65−68 currents in central neurons. J Biol Chem 2002, 277: 46101−46109 20 Cai Z, Xu C, Xu Y, Lu W, Chi CW, Shi Y, Wu J. Solution structure 16 Song LZ, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, Jacobs A of BmBKTx1⎯a new BKCa channel blocker from the Chinese et al. Efficacious recombinant influenza vaccines produced by Scorpion Buthus martensi Karsch. Biochemistry 2004, 43: 3764− high yield bacterial expression: a solution to global pandemic and 3771 seasonal needs. PLoS ONE 2008, 21: e2257 21 Doyle DA, Morais Cabral J, Pfuetzner RA, Kuo A, Gulbis JM, 17 Yang R, Peng F, Liu H, Cao ZJ, Li WX, Mao X, Jiang DH. Func- Cohen SL, Chait BT et al. The structure of the potassium channel: tional analysis of a gene encoding a chlorotoxin-like peptide de- Molecular basis of K+ conduction and selectivity. Science 1998, rived from scorpion toxin. Chinese J Biochem Mol Biol 2005, 21: 280: 69−77 19−23 22 Martins JC, Van de Ven FJ, Borremans FA. Determination of the 18 Dauplais M, Lecoq A, Song J, Cotton J, Jamin N, Gilquin B, three-dimensional solution structure of scyllatoxin by 1H nuclear Roumestand C et al. On the convergent evolution of animal toxins. magnetic resonance. J Mol Biol 1995, 253: 590−603 Conservation of a diad of functional residues in potassium chan- 23 Meunier S, Bernassau JM, Sabatier JM, Martin-Eauclaire MF, Van nel-blocking toxins with unrelated structures. J Biol Chem 1997, Rietschoten J, Cambillau C, Darbon H. Solution structure of P05- − 272: 4302 4309 NH2, a scorpion toxin analog with high affinity for the apamin- 19 Mouhat S, De Waard M, Sabatier JM. Contribution of the functional sensitive potassium channel. Biochemistry 1993, 32: 11969− dyad of animal toxins acting on voltage-gated Kv1-type channels. 11976

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