Eur J Biochem 271, 2504–2516 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04181.x NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical b-toxin sequence but with a-like physiological activity ´ ´ ´ Federico del Rıo-Portilla1, Elizabeth Hernandez-Marın1, Genaro Pimienta2*, Fredy V Coronas2, ´ Fernando Z Zamudio2, Ricardo C Rodrıguez de la Vega2, Enzo Wanke2,3 and Lourival D Possani2 Institute of Chemistry, National Autonomous University of Mexico, Mexico City, Mexico; 2Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico; 3Dipartimento di Biotecnologie e Bioscienze, Universita´ di Milano-Bicocca, Milan, Italy Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges Its primary structure and disulfide bridges were determined Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 lg per animal Its 3D structure was determined by proton NMR using 850 distance constraints, 36 / angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds The overall structure has a two and half turn a-helix (residues 24–32), three strands of antiparallel b-sheet (residues 2–4, 37–40 and 45–48), and a type II turn (residues 41–44) The amino-acid sequence of Cn12 resembles the b scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na+ channels in F-11 cells (mouse neuroblastoma N18TG-2 · rat DRG2), which means that it behaves more like an a scorpion toxin This behaviour prompted us to analyse Na+ channel binding sites using information from 112 Na+ channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5–S6 transmembrane segments of domain I and the S3–S4 segments of domain IV, sites considered to be responsible for binding a scorpion toxins Scorpion toxins are relatively short peptides with a variable length of amino acids, showing characteristic 3D folding comprised of an a-helix and three segments of antiparallel b-sheet structure, stabilized by several disulfide bridges [1–5] Their known physiological role is to block or modify ion-channel function, causing impairment of cellular communication, which leads to the depolarization of excitable membranes and might cause death of animals stung by scorpions [6,7] There are several reasons why the molecular basis of toxin specificity and molecular mechanism of action continue to be of scientific interest: (a) to study ion channels, the target molecules of most known scorpion toxins, in order to understand their molecular structure and function, thus learning more about cellular excitability; (b) to understand the toxic effects of scorpion venoms, a pre- requisite for the development of more effective and safer antidotes and/or vaccines; (c) to find toxins specific for invertebrate organisms with a view to developing biodegradable drugs for pest control; (d) to discover other possible unknown target molecules for which peptides were evolved in the venom of scorpions The last of these is not trivial, as the huge variability of these peptides, estimated to be of the order of 100 000 in scorpion venom alone, and of which only about 0.2% have been identified, leaves a wide open field for research [4,5,8] Several recent articles and reviews have reported on the structural and functional aspects of these peptides [3,8–18] Most dealt with scorpion toxins as ion-channels blockers or modifiers of their function However, the structural variability of peptides and the different types of receptor they recognize is steadily increasing [19], exemplified by the following novel discoveries: ERG-channel-specific toxins [20], analgesic peptides [21,22], modulators of immune response [23–25], antibiotics [26–28], antimalaria agents [29], and others For the ion-channel-specific peptides, two distinct groups of toxins have been identified based on the length of the peptide chain: short-chain peptides (23–41-amino-acids long) which recognize and bind to various types and subtypes of K+ channels [13,19], ryanodine-sensitive Ca2+ channels [11], and Cl– channels [30]; long-chain peptides (59–76 amino acids), which are specific for Na+ channels [8,18] and T-type Ca2+ channels [31,32] The purpose of this paper is to describe for the first time a Na+-channel-specific scorpion toxin (Na-ScTx), isolated from the New World scorpion Centruroides Correspondence to L D Possani, Department of Molecular Medicine and Bioprocesses, Institute of Biotechnology, National Autonomous University of Mexico, Avenida Universidad 2001, Apartado Postal 510-3, Cuernavaca 62210, Mexico Fax: + 52 777 3172388, Tel.: + 52 777 3171209, E-mail: possani@ibt.unam.mx Abbreviation: Na-ScTx, scorpion toxin specific for Na+ channel; TTX, tetrodotoxin Note: Laboratories and contributed equally to this work *Present address: EMBL, Meyerhofstrasse 1, Heidelberg D-69117, Germany (Received February 2004, revised 30 March 2004, accepted 22 April 2004) Keywords: Centruroides noxius; NMR structure; patchclamp; scorpion toxin; sodium channel Ó FEBS 2004 noxius It is structurally similar to b Na-ScTxs, but has an a-like function The 3D structure of this toxin was determined by NMR The importance of the overall charge distribution on the surface of the toxin for its activity is emphasized Furthermore, considerations related to the amino-acid sequences deduced from cloned Na+-channel genes are discussed in terms of what is known about the interacting surfaces of Na-ScTxs and Na+ channels Materials and methods Venom source, purification procedures and lethality tests Venom from scorpions collected in Nayarit State (Mexico) was obtained by electrical stimulation and separated by Sephadex G-50 gel filtration, followed by ion-exchange chromatography on CM-cellulose columns, as previously described [33] For this work, fraction II-4 (v.g., fraction II from Sephadex, and subfraction from CM-cellulose) was further separated by HPLC using previously published conditions [34] Lethality tests were conducted with mice, crickets and crayfish, using the same conditions as reported [34] Determination of amino-acid sequence and MS analysis The full amino-acid sequence of the toxin was obtained by direct Edman degradation, using an automatic Beckman sequencer (LF 3000 Protein Sequencer) and samples of native and reduced toxin Additional information was generated by sequencing subpeptides obtained by HPLC separation of toxin treated with endopeptidases lysine-C (Boehringer, Mannheim, Germany) and Staphylococcus aureus V8 (Boehringer, Mannheim, Germany), as previously described [8,33,34] The last amino-acid residue was confirmed by MS analysis, performed in a LCQDUO ion-trap spectrometer from Finnigan (San Jose, CA, USA) Determination of disulfide bridges A sample containing 100 lg toxin was digested with trypsin (Promega, Madison, WI, USA) in slightly acidic conditions, followed immediately by endopeptidase V8 digestion, as previously described [8], and separated by HPLC on a C18 reverse-phase column with a linear gradient from solution A [0.12% (v/v) trifluoroacetic acid in water] to 60% solution B [0.10% (v/v) trifluoroacetic acid in acetonitrile], run for 60 Several components were fractionated (data not shown) The component eluted at 27.78 was identified as the disulfide bridge between Cys11 and Cys65 Because of incomplete digestion, the peptide eluted at 28.92 was further treated with chymotrypsin (Boehringer) and subsequently separated by HPLC The component eluted at 26.38 (data not shown) corresponded to the disulfide bridge between Cys15 and Cys40 The two other disulfide bridges were determined by NMR analysis as discussed below For the assignment of the first two disulfide bridges, the peptides hydrolyzed by enzymatic digestion were sequenced using the Beckman LF3000 Protein Sequencer described above NMR solution structure of Cn12 (Eur J Biochem 271) 2505 Electrophysiological data Cell culture Cells of the F-11 clone (mouse neuroblastoma N18TG-2 · rat DRG) [35] were routinely cultured in Dulbecco’s modified Eagle’s medium, containing 4.5 gỈL)1 glucose and 10% fetal calf serum The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 Solutions and drugs The standard extracellular solution contained (mM): NaCl 130, KCl 5, CaCl2 2, MgCl2 2, Hepes/NaOH 10, D-glucose 5, at pH 7.40 In the high-K+ external solution ([K+]o ¼ 40 mM), NaCl was replaced by an equimolar amount of KCl The standard pipette solution at [Ca2+]i ¼ 10–7 M (pCa 7) contained (mM): K+-aspartate 130, NaCl 10, MgCl2 2, CaCl2 1.3, EGTA/KOH 10, Hepes/ KOH 10, ATP (Mg2+ salt) 1, pH 7.30 Patch-clamp recordings and data analysis The currents were recorded by means of the patch-clamp amplifier MultiClamp 700A (Axon Instruments, Foster City, CA, USA) at room temperature as previously described [36] (pipette resistance 0.8–1.2 MW); cell capacitance and series resistance errors were carefully compensated for (85–90%) before each voltage clamp protocol run The extracellular solutions were delivered through a nine-hole (0.6-mm) remote-controlled linear positioner, with an average response time of 2–3 s, placed near the cell under study The Na+-current inactivation curves were obtained by plotting the normalized peak current against Vm Final traces were corrected with traces obtained in the presence of 20 nM tetrodotoxin (TTX) The activation was derived as the normalized sodium conductance relationship (ENa ¼ )65) To determine the amplitude of the toxin-induced slow inactivation, we subtracted the control traces from the traces recorded in the presence of the toxin The amplitude of these toxin-induced currents was analyzed by plotting the value ms after the onset of the depolarizing pulse The decaying inactivating portion of the control traces and the currents in the presence of toxin were fitted to one or two exponential decaying functions, respectively, to obtain the inactivation time constants pClamp (Axon Instruments) and Origin 4.1 (Microcal Inc, Studio City, CA, USA) software were routinely used during data acquisition and analysis Preparation of the NMR sample A sample of purified Cn12 containing 6.2 mg peptide was dissolved in 0.8 mL H2O/D2O (9 : 1, v/v) to a final concentration of 0.9 mM The pH measured for this solution was 3.1 After the experiments in H2O, the peptide was lyophilized and redissolved in D2O to perform additional experiments H-NMR spectroscopy The experiments were performed in a Varian Unity Plus 500 spectrometer Data were collected at 300 K Mixing times were 35 ms for TOCSY and 80 and 100 ms for NOESY in H2O and in D2O Data were processed with NMRPIPE [37] to obtain 4K · 4K spectra Spectra were analysed using the XEASY program [38] The values of the JHN-Ha were estimated from TOCSY spectra with the modified Ó FEBS 2004 2506 F del Rı´ o-Portilla et al (Eur J Biochem 271) J doubling in the frequency domain [39,40], when possible, or with the strategy proposed by Wishart et al [41] Experimental constraints and structure calculations Most of the distance constraints were obtained from NOESY spectra in H2O; additional constraints were from NOESY spectra in D2O NOE intensities were evaluated from the volume of the cross-peak and calibrated internally using the CALIBA program [42] to generate a set of upper limit distances Most NOE data were obtained from resolved signals NOE signals for Hb–Hb between Cys11 and Cys65 and Cys15 and Cys40 were assigned; however, no NOE data were observed for the other Cys pairs A total of 850 distance constraints were used from which 121 are sequential, 30 medium range (1 < |i–j| < 4) and 92 long range (|i–j| > 4); the remainder were intraresidues A total of 36 / angle constraints were used based on the JHN-Ha values Using the modified J doubling method, it was possible to evaluate 23 JHN-Ha values from a trace of TOCSY spectra It was only possible to measure 13 additional coupling constants from TOCSY spectra as proposed by Wishart It was found that the modified J doubling method gives smaller values than the Wishart method [41], and in the case of the a-helix (residues 24–32), where a value of 3.9 is expected [43], values are closer depending on the structure obtained Proton– deuterium exchange of the amide groups was measured on a sample lyophilized from H2O and redissolved in pure D2O as described [43], in order to determine 22 hydrogen bond constraints Four disulfide bridge constraints were added to the calculations The dynamic annealing structure calculations were performed with the CNS software suite [44] Analysis of Na+-channel sequences By searching data banks containing sequences of Na+ channels from human, fruit fly and squid, several representative sequences were chosen (five for humans, three for Drosophila, and one for squid) With these sequences, more than 200 BLAST entries were found that matched isoforms of Na+-channel sequences An alignment was performed using the program CLUSTAL X [45], from which 50 nonredundant sequences were selected Among these are sequences from several species including mammals, insects and other invertebrates (available from the authors on request) The proposed binding sites for a Na-ScTxs, including regions corresponding to the S5–S6 segment from domain I and S3–S4 from domain IV of the Na+ channels, were re-aligned independently using CLUSTAL X, and some were chosen for our figure Among these are the ones best represented and cited in the literature on scorpion toxins Results Purification and sequencing Figure 1A shows the results of separating mg fraction II-4 (for details see Materials and methods) from the venom of the scorpion C noxius For this report, about 20 HPLC runs were performed as described, in order to obtain enough peptide for this work The component indicated by the star in Fig 1A was recovered and chromatographed again (Fig 1A, inset) to give the pure peptide, called Cn12 The name comes from the abbreviation of the scorpion species C noxius followed by the number 12, which corresponds to the 12th pure peptide fully characterized from this venom, specific for Na+ channels This peptide corresponds to  1.3% of the total venom protein concentration For determination of the primary structure, it was necessary to obtain overlapping sequences of several peptides (Fig 1B) The 36 most N-terminal amino-acid residues were identified by directly sequencing the native peptide, and the identities confirmed by sequencing a sample of reduced and alkylated cysteine residues (vinyl derivatives) of Cn12 The overlapping sequence spanning Ala33 to Gly63 was determined with a peptide obtained by Lysine-C digestion, as described in Materials and methods, and the final segment overlapping from Gly53 to Arg66 was obtained by sequencing a peptide digested with endopeptidase V8 The last residue, Ser in position 67, was determined by MS The molecular mass found by MS ionization analysis was 7139.5 Da, and the theoretically expected value was 7139.7 Da, thus confirming the full sequence Disulfide bridges As mentioned in Materials and methods, a peptide obtained by enzymatic digestion of Cn12, and separated by HPLC (elution time 27.78 min) provided a heterodimeric product when directly sequenced by Edman degradation The sequences obtained were DGYPLASNGC and GTVLWGDSGTXPCR, indicating unequivocally the position of one of the disulfide bridges, which in this case was between Cys11 and Cys65, based on the known primary structure of Cn12 (Fig 1B) Another core peptide from the initial digestion (time 28.92 min) gave several amino-acid sequences, making it impossible to assign any other disulfide bridges It was therefore further digested with chymotrypsin, which produced subpeptides, and that eluted at 26.35 (data not shown) gave the sequences FGC and GYC, corresponding to the disulfide bridge Cys15 and Cys40 The peptide eluted at 17.48 gave four sequences corresponding to segments of the primary structure containing Cys25, Cys27, Cys45 and Cys47 As we knew the primary structure, we knew that the remaining disulfide bridges were between Cys25 and Cys45 and Cys29 and Cys47, or conversely between Cys25 and Cys47 and Cys29 and Cys45 The sequences of the fragments from the chymotryptic hydrolysis showed that the other putative disulfides, i.e Cys25–Cys29 and Cys45–Cys47, were not possible because the sequence would not fit the one determined experimentally Modelling the 3D structure, based on the NMR data as discussed below, showed that the only disulfide configuration that would fit the results obtained is Cys25–Cys45 and Cys29–Cys47 Furthermore, these disulfide pairs are at exactly the same positions as those described for other NaScTxs isolated from several other scorpion species [8] Thus, the disulfide bridges of Cn12 are assumed to be: Cys11– Cys65, Cys15–Cys40, Cys25–Cys45 and Cys29–Cys47 Ó FEBS 2004 NMR solution structure of Cn12 (Eur J Biochem 271) 2507 Fig Final purification and amino-acid sequence determination of Cn12 (A) A sample of fraction II-4 from a CM-cellulose ionexchange column [33] containing mg protein was applied to an analytical C18 reverse-phase column and eluted with a linear gradient of solution A [0.12% (v/v) trifluoroacetic acid in water] to 60% solution B [0.10% (v/v) trifluoroacetic acid in acetonitrile] run for 60 The component labelled with asterisk corresponds to  33% of fraction II-4 It was further chromatographed in the same system, but eluted with a gradient from 10% to 40% solution B over 40 Only the material eluted under the main area of the peak, indicated by the horizontal line, was collected It corresponded to pure toxin, whereas small contaminants (5%) were eliminated on the ascending and descending sections of the chromatogram (B) Direct Edman degradation of native peptide and reduced and alkylated samples provided unequivocal amino-acid sequence identification from Arg1 to Asp36 Further sequencing peptides obtained from enzymatic hydrolysis with endopeptidase Lys-C and Staphylococcus aureus protease V8 allowed us to obtain the overlapping segments from A33 to G63 and from Gly53 to R66, respectively, as indicated The last residue, S67, was identified by MS Sequence comparison of Cn12 with other Na-ScTxs A databank search with the sequence of Cn12 as the query retrieved only scorpion venom-derived peptides classified as Na-ScTxs The highest identities were found with scorpion toxins belonging to the b group (identity > 30%), including several toxins isolated from New World scorpions It also shows similarities to some depressant and excitatory toxins from Old World scorpions, as well as to the recently characterized Birtoxin, a long-chain peptide with only three disulfide bridges from the scorpion Parabuthus transvaalicus [46] The similarities to Na-ScTxs of the a group are considerably lower (identity < 25%) On the basis of these results, Cn12 should be classified as a member of the b group of Na-ScTxs To allow proper discussion in the terms of structure–activity relationship, with regard to recognition and affinity for Na+ channels, Cn12 is aligned with other Na-ScTxs in Fig For this comparison, only toxins for which the 3D structures are known were included The 3D structures of three pharmacologically different classes of Na-ScTxs are known: a Na-ScTxs (AaHII, BmKM1, BmKM2, BmKM4, BmKM8, BmK-aTx16, BmK-aIT, Bs-mktx, CsE-V, LqhII, LqqIII, Lqh-aIT), b Na-ScTxs (Cn2, CsE1, CsEv1, CsEv2, CsEv3, CsEv5, Ts1/Tsc), and an insect-specific excitatory Na-ScTx Bjxtr-IT As expected, the phylogenetic tree rooted with Bjxtr-IT places Cn12 closer to toxins described as b Na-ScTxs than to the a Na- ScTxs A recent proposal by Froy & Gurevitz [47] is that toxins CsEv1 and CsEv3 belong to the group of so-called a¢ Na-ScTxs However, the lack of pharmacological data prevents proper classification of this last group of toxins [7], and thus they are here referred to as b Na-ScTx, on the basis of their primary structure Bioassays and electrophysiological effect of Cn12 Bioassays conducted with pure Cn12 in mice, crickets and sweet-water shrimps, using concentrations up to 100 lg per animal produced inconclusive results Apparently, at this concentration, Cn12 is not toxic to any of the animals tested The immediate questions posed were: why is this component, which is present at relatively high concentration in the venom (Fig 1A), not toxic in vivo? What is the biological function of this novel peptide? To answer these questions, it was decided to verify the effect of Cn12 in vitro using patch-clamp experiments The TTX-sensitive Na+ current present in the tumour cell line F-11 [35] was used to test the properties of the toxin Cn12 Figure 3A shows representative recordings of inward currents elicited according to the protocol shown below The effects of 2.8 lM Cn12 are illustrated in Fig 3B The peak currents are higher in the presence of toxin but the most interesting effect was a net increase in the inactivation time constant Ipeak vs V plots obtained in the same cell in Ó FEBS 2004 2508 F del Rı´ o-Portilla et al (Eur J Biochem 271) Fig Multiple sequence alignment of Na-ScTxs Multiple alignment of amino-acid sequences of all Na-ScTxs for which the 3D structure is known was conducted using CLUSTAL X [45] Amino-acid sequences are followed by the abbreviated name and the corresponding PDB code The third column displays the identity scores with respect to Cn12 In the right part of the figure a simplified phylogenetic tree is included, for which the amino-acid sequence of Bjxtr-IT (a highly divergent insect-specific excitatory toxin) was used as the root The tree accurately differentiates between a and b Na-ScTxs, and the corresponding branches are labelled accordingly Amino acids shown directly to be important in pharmacological activity by site-directed mutagenesis are in bold, as reported in: Lqh-aIT [18], BmK M1 [55,56] and Bjxtr-IT [63] In the upper and lower parts of the figure, the secondary-structure elements (h, helix; b, sheet) and fully (asterisks) or partially (dots) conserved residues are indicated, respectively the control and during the application of 0.7 and 2.8 lM Cn12 are shown in Fig 3C In the voltage range )20 mV to +20 mV, the decaying inactivation time course was fitted with one single exponential time constant in the control, but, in the presence of the toxin, we were forced to add another slower time constant while maintaining unaltered the fast time constant used in the control The ratio of the fast to slow amplitudes was  0.1 ± 0.02 (n ¼ 4) The complete results of four experiments are shown in Fig 3D where it can be seen that the control data (open squares) and the Cn12-induced slow inactivation component data (open circles) differ by about one order of magnitude Normalized voltage-dependent activation is illustrated in Fig 3E both in the control (open squares) and during the action of Cn12 (open triangles); the data show no significant difference Classical double-pulse inactivation protocols were used to investigate the voltage-dependent steady-state inactivation process In this case also (Fig 3E), data did not differ in the control (open squares) and in the presence of 2.8 lM Cn12 (open triangles) As the toxin induced the development of a novel slowly inactivating component (Fig 3D), we subtracted the control traces from the toxin traces and plotted the steady state inactivation of this toxin-induced component (closed squares) This resulted in a right shift by about 12 mV Overall, these data suggest that Cn12 behaves like a weak a Na-ScTx because it induces supplementary slow inactivation of the Na+ channel In other words, Cn12 interferes with cellular communication at the level of the Na+ channels diagram of sequential and medium range data, the chemical shift index, and coupling constants used for the structure calculation Figure 4B shows a ribbon diagram of Cn12, in which the most obvious 3D elements are shown This was possible because of the use of 850 distance constraints, 36 / angles derived from 23 coupling constants measured from TOCSY experiments using the modified J-doubling method and 13 additional coupling constants using the Wishart method [41], plus 22 hydrogen bonds added after the first calculations, and the four disulfide constrains (see Materials and methods) Over 250 structures were calculated, from which 19 with the smallest total energy and no NOE ˚ violations greater than 0.2 A and no angle constraints violations greater than ° were used to draw Fig 4C Table shows the rmsd values from different regions in the peptide and energy mean values of calculated structures All three prolines were determined in trans position because of the presence of NOE data between the previous Ha with the proline HDX protons [Ha(i–1) Hc(i)] For each proline, at least one NOE was assigned Concerning the disulfide bridges mentioned above, if a distinct disulfide pairing constraint is used to calculate the plausible 3D structures, the model obtained does not fit the NMR results well, because several NOE violations are produced and structures with very high total energy are obtained These data led to the conclusion that the two disulfide bridges not yet directly determined are indeed between Cys25 and Cys45 and Cys29 and Cys47, as mentioned above Additional details of NMR experimental results of Cn12 can be found in the databanks (PDB entry 1PE4 and BRMB number 5913) Discussion NMR solution structure of Cn12 Figures 4, and summarize the most important data obtained from the NMR analysis of pure Cn12 It was possible to analyze the NMR data because of well-dispersed signals obtained at 11.75 T Figure 4A shows the NOE Purification, sequence and function of known Na-ScTxs The purification procedure and sequence determination of Cn12 are clearly described in the Results section and require no further discussion However, as this paper reports the Ó FEBS 2004 NMR solution structure of Cn12 (Eur J Biochem 271) 2509 Fig Biophysical modifications produced by Cn12 on the TTX-sensitive sodium currents of the neuroblastoma cell line F-11 (A-B) Na+ current elicited from a holding potential of )100 mV to the test potentials from )30 to +20 according to the protocol shown below A, in the control and in the presence of 2.8 lM toxin in B (C) The I–V plot of one experiment in which Cn12 was used at concentrations of 0.7 and 2.8 lM in the same cell (D) Plot of the inactivation time constant (sh) as a function of test potential in the control (h) and from the traces showing the slow toxin-induced component (s) (Materials and methods) Insets: left, control inward current at mV (CON) and in the presence of 2.8 lM Cn12; right, the slowly inactivating toxin-induced current obtained by subtracting the control current from the trace in the presence of toxin (E) Plot of the voltagedependent activation and steady-state inactivation (n ¼ 4; Materials and methods) The Boltzmann relationships (dashed lines) were fitted with the following parameters (mV): activation (control) V½–10, slope 7.1; inactivation (control) V½ 41.7, slope 5.2; inactivation (slow component) V½ 54, slope 6.2 The amplitude of these slowly inactivating traces ms after the onset was plotted (j) as a function of the conditioning potential The left insets show the currents elicited at 10 mV from )100, )60 and )20 mV in the control (upper) and in the presence of the toxin (lower) The central inset shows the recordings obtained after subtraction of the control traces from the 2.8 lM traces TTX-sensitive current data are plotted structure and function of the first a-toxin specific for Na+ channels, isolated from C noxius, it is important to review briefly the field, considering the data and current ideas on the interacting surfaces of scorpion toxins and Na+ channels The best studied scorpion toxins with respect to interacting surfaces are the K+ channel-specific toxins [9,13,15,16,19] Less information is available for those specific for Na+ channels The Na-ScTxs were initially classified as a and b toxins [48,49] The a Na-ScTxs bind to site of the receptor from vertebrates in a voltagedependent manner, whereas the b Na-ScTxs bind to site 4, producing a shift to more negative potential and the binding is independent of the membrane potential [18,50,51] On the basis of electrophysiological recordings and binding and displacement experiments performed with different animal models (mammals, insects and crustaceans), several subgroups of a, a-like, and b Na-ScTxs have been proposed [7,8,47] Still more recently, another novel toxin, Cn11 from C noxius was described, the first example of a Na+ channel blocker peptide in crustacean preparations, in contrast with all other known Na-ScTxs that are modifiers of channel function [34] However, in reality a general classification of the different types and subtypes of Na-ScTxs is not available, which is due in part to the lack of knowledge about their structural and functional relationships In addition, none of the different types and subtypes of Na+ channel have had their 3D structure determined, unlike the K+ channels [52] Previous studies conducted with both a and b Na-ScTxs showed that small differences such as single amino-acid modifications or deletion of short stretches of sequence were Ó FEBS 2004 2510 F del Rı´ o-Portilla et al (Eur J Biochem 271) Fig Structure of Cn12 (A) The most representative data for the structure determination of Cn12 (sequential and medium range NOEs, exchangeable amide proton, chemical shift index, and coupling constants) are shown A strong NOE is represented by a bigger rectangle Exchange behaviour of amide protons is indicated by black rectangles; the bigger the rectangle the slower the exchange Arrows indicate b-strands and zig-zag lines indicate the a-helix (B) Ribbon diagram of Cn12 showing an a-helix at residues 24–32, three strands of antiparallel b-sheet comprising residues 2–4, 37–40 and 45–48, and a type II turn at residues 41–44 (C) The models of 19 out of 250 NMR structures calculated for Cn12 were superimposed, showing well-defined secondary structures for segments of amino-acid residues in positions: 3–4, 24–32, 37–48, except for the N-terminal and C-terminal regions enough to cause a dramatic change in toxicity to mice [53,54] In recent years, many publications have contributed novel data and identified possible residues directly involved in the recognition and binding to Na+ channels [4,5,18,33,55–65] However, a structural and functional characteristic unique to all ScTXs and Na+ channels has not been found Rather, each novel toxin needs to be analysed in the specific context of the structural determinants of its functions and putative receptor or binding sites For these reasons, the report of a novel function or structural feature of an undescribed Na-ScTx should be taken as an important contribution to the field We have described here a novel peptide isolated from C noxius which displays a-like activity We show that the charge distribution on the surface of the peptide is probably one of the significant structural features that govern its function Furthermore, if we comparatively rotate the 3D structure of the known Na-ScTxs, it is evident that there are several equivalent spatial orientations for which a clear difference in charge distribution exists among these toxins Here, one such orientation, called face C, was arbitrarily chosen to illustrate the point 3D Structural elements of Cn12 There are three secondary-structural elements in Cn12, a two and a half turn a-helix (residues 24–32), three strands of antiparallel b-sheet (residues 2–4, 37–40 and 45–48), and a type II turn (residues 41–44) (Fig 4B) These elements are common to all Na-ScTxs No significant changes were found in the secondary and tertiary structures It can be seen in Fig 4C that the segment from residue to 11 and that from residue 53 to 67 is not well defined because of the lack of long-range NOEs, probably because of the high mobility of these regions The a-helix is linked to the b-sheet by two disulfide bridges, which are conserved in all long-chain toxins [8], the CS-a/b motif [2] Alignment of Fig 2, using the CLUSTAL X program, shows a clear cut separation of all the b Na-ScTxs from all the a Na-ScTxs The a Na-ScTxs have identities of the order of 50% among themselves, the same being true for all the b Na-ScTxs (data not shown) However, when the a and b Na-ScTxs are compared, the identities fall below 30% In addition to the eight highly conserved cysteines, Tyr4 and Gly38 are strictly conserved in all toxins, but further Ó FEBS 2004 NMR solution structure of Cn12 (Eur J Biochem 271) 2511 Fig Electrostatic surface potentials of selected Na-ScTxs (A) Ribbon diagram of Cn12 (blue) superimposed with Bjxtr-IT (red), Lqh-aIT (green) and BmK M1 (orange), taken from PDB: 1PE4, 1BCG, 1LQH and 1SN1, respectively This figure was generated after overlapping the solved 3D structures according to the positions occupied by similar secondary-structure elements, as indicated in Fig The orientation chosen corresponds to the original face B described in [66] (B) Electrostatic surface potential of the same toxins calculated with the MOLMOL program [71], using fully ˚ charged residues, shown at 1.4 A van der Waals radius Visible charged residues for each toxin are indicated by the one-letter code, in which red means negatively charged, blue positively charged Neutral or hydrophobic residues are in white, but not individually marked Axes in the middle of the figures represent the selected orientation similarities are present in positions: R1, D2, G3, G10, Y46 and V50 (numbered according to the sequence of Cn12) Location of charge distribution and the binding affinities for Na+ channels Data from lethality tests conducted in vivo usually correlate well with electrophysiological data obtained in vitro A high toxicity of Na-ScTx usually means high affinity for ion channels This seems to be the case for Cn12 It is not toxic in vivo at concentrations at which other toxins from the same scorpion, such as Cn2 (a mammalian-specific toxin), Cn5 (a crustacean-specific toxin) and Cn10 (an insectspecific toxin) are very effective (doses of 0.4–40 lg per individual [57,58]) As shown in Fig the affinity of Cn12 for the Na+ channel model chosen for this study (F-11 clone, see Materials and methods) indicates that the affinity is low (high nanomolar or even  1.0 lM) Thus, it seems to fit the rule: low toxicity in vivo, low affinity in vitro However, this simple observation could be misleading There are too many variables in the toxin–channel interactions The different tissues of the experimental animals are differently susceptible to different scorpion toxins, as mentioned above Peptides not toxic when intraperitoneally injected can be highly toxic when intracranially injected [18,22] The types and subtypes of ion channels and other possible receptor targets for the Na-ScTx, and their distribution in cell membranes, are extremely variable and may explain the differences It is quite clear that scorpions have evolved huge variability in peptides to capture their prey or defend themselves from predators A plausible explanation for this is the presence of a coevolutionary 2512 F del Rı´ o-Portilla et al (Eur J Biochem 271) Ó FEBS 2004 Fig Electrostatic surface potentials for face C of selected Na-ScTxs Same electrostatic surfaces for toxins as in Fig 5B, rotated from face B by 87.5 ° in the z-axis direction, 62.5 ° in the y-axis direction, and 10.0 ° in the x-axis direction, following exactly this order of rotation This is one of the orientations in which a pronounced difference was found Toxin structures are labelled as in Fig Table Experimental constraints and structural statistics (A) Distance constraints Intraresidue 607 Sequential 121 Medium-range 30 Long-range 92 Total 850 (B) Angle constraints 36 (u) ˚ (C) Cartesian coordinate rmsd (A) in backbone atoms All 1.994 Backbone All 1.097 Residues 11–52 0.968 Helix(24–32) 0.259 b-strand(2–4) 0.421 b-strand(37–40) 0.261 b-strand(45–48) 0.173 b-sheet 0.532 b-strand and helix 0.612 (D) Energy (kcalỈmol)1) calculated from CNS (19 structures) Total 97.9 (+15.6) Bonds 5.0 (+1.1) Angles 28.7 (+4.3) van der Waals 35.8 (+5.5) NOE 21.4 (+5.8) Dihedral 1.1 (+0.5) Impropers 6.0 (+1.5) process Whenever the channel changes, the toxin also changes in order to most efficiently fit its binding site [64,65] However, as shown in Fig 5A, the Cn12 scaffold is similar to the others, and yet it is a weak modifier of Na+ channel function Figure 5B compares the distribution of the charge of Cn12 with three toxins in which site-directed mutations were performed, and the corresponding function studied [18,56,57,63] It is clear that the a-like and a anti-insect toxins (BmK M1 and Lqh-aIT, respectively) show a quite similar overall charge distribution, when analyzed in the orientation originally described as face B [66] Although Cn12 shows an a scorpion toxin effect, it does not have the same charge distribution, suggesting that other faces of the 3D structure of the toxins contribute to this effect Similarly, the insect-excitatory toxin Bjxtr-IT, defined as b scorpion toxin, has a different charge distribution, as would be expected The results of site-directed mutagenesis show that face A does not seem to be important for channel recognition Rather, it probably has a role in maintaining the correct 3D folding of the molecules [55] For example, the residues shown to be important for the function of the a toxins Lqh-aIT [18] and BmK M1 [55,56] are: K8, Y10, F17, R18, W38, N44, R58, V59 and K8, W38, Y42, K62, H64, respectively For these two toxins, the residues in question are mostly situated in face B, as shown in Fig 5B However, this seems not to be case for the b toxin Bjxtr-IT Ó FEBS 2004 [63], in which the important residues, although clustered in two patches (E15, V19, N20, I22, A23, P24, H25, Y26, E30, V34, V71, Q72, I73 and I74), are situated in different orientations of the molecule On further analysis of the structures available, another face was found, defined here as face C, which is quite distinct with respect to charge distribution of the toxins under analysis Face C was obtained by rotating the four superimposed models through 87.5 ° in the z-axis direction, 62.5 ° in the y-axis direction, and 10.0 ° in the x-axis direction, as shown in Fig Other toxins for which the 3D structures are known were similarly analyzed: BmK M2, BmK M4, BmK M8, AaH II, CsE-V (a toxins), and Cn2, CsE-v5 and Ts1 (b toxins) Using the orientation of face C for these toxins, a distinct charge distribution is observed The toxins are like a Christmas tree, decorated in different forms to interact most efficiently with their specific targets Comparative analysis of charge distribution in Na+ channels The receptor site of Na+ channels is surmised to be formed mainly of extracytoplasmic loops of the S3–S4 segment of domain IV and, to a lesser extent, of the segment S5–S6 of domain I [14] Contact with a Na-ScTx is mediated by electrostatic and hydrophobic interactions Nevertheless, as shown in Fig 7, Na+-channel isoforms constitute a highly homogeneous family in terms of primary structure, particularly in the S3–S4 segment of domain IV The variations found among the different isoforms are reduced NMR solution structure of Cn12 (Eur J Biochem 271) 2513 to point mutations, making it difficult to explain their distinct susceptibilities to various a Na-ScTxs For example, it is well documented that some a Na-ScTxs display remarkable species specificity (e.g Na-ScTx Lqh-II binds to rat brain synaptosomes with a 100-fold higher affinity than to cockroach preparations; conversely Lqh-aIT binds to neuronal preparations from cockroach with a 10 000fold higher affinity than to rat brain synaptosomes [51]) In addition, some toxins are capable of discriminating between Na+-channel isoforms of the same organism (e.g the rat brain isoform rNaV1.1 is 10-fold more sensitive to the action of the a Na-ScTx Lqq5 than the cardiac isoform rNaV1.5 [67]) However, when the overall charge of both the toxins and the channels are considered together, a plausible explanation comes from analysis of the other region involved in receptor site 3, segment S5–S6 of domain I In general, there is greater variability in this segment Highly sensitive Na+ channels have more acidic residues than insensitive isoforms (compare rNaV1.4 and rNaV1.5 in Fig 7), which would also support the observed preferred interaction of the former with anti-mammal-specific basic a Na-ScTxs [68–70] In striking contrast, insect-specific a Na-ScTxs usually present an overall neutral or, often, a negative net charge at physiological pH It has been suggested that the presence of sialic acid in the S5–S6 segment from domain I of mammalian channels may influence the binding of nonpositively charged Na-ScTxs, disfavouring their binding because of the absence of primary electrostatic attraction [70] Cn12 has a slightly negative charge at physiological pH, and the neuroblastoma cells Fig Multiple alignment of amino-acid sequences corresponding to the receptor site of Na+ channels A total of 50 nonredundant sequences of Na+ channels available in databases were aligned with CLUSTAL X [45] The segments S5–S6 of domain I and S3–S4 of domain IV from selected sequences are shown (NaV1.1, 1.4 and 1.5 from rat; NaV1.7 from human; Para from fruit fly; NachB1 from squid) Conserved residues are indicated by asterisks, and conservative replacements by dots Acidic amino acids in the extracytoplasmic loops are shown in bold Critical binding residues determined by mutagenesis are highlighted by empty circles [67,70] 2514 F del Rı´ o-Portilla et al (Eur J Biochem 271) used in Fig (mammalian tissue) have a high content of sialic acid These two facts may explain the lower affinity found for this toxin In contrast with the charge-interacting residues, the putative hydrophobic interactions are much more difficult to estimate as there is less difference in the relative abundance and no periodicity of distribution of the hydrophobic residues The small number of site-directed mutants prepared with scorpion toxins [7,18,55,56,63,68] and the study with the Na+ channels are insufficient to confirm or refute the current views This analysis indicates that different amino acids in distinct positions of Na-ScTxs are capable of defining their function Similarly, the sites on the Na+ channels known to be responsible for the binding of Na-ScTxs are different: a vs b effect (for those that modify the gating mechanism) and Cn11 (a blocking toxin) We expect that, when more data become available, the concepts on the interaction of Na-ScTxs and Na+ channels will be better understood or even modified In this communication, we show the 3D structure of a novel scorpion toxin We conclude that the actual interacting surfaces depend on the whole toxin molecule, with an important role for charge distribution As important must be the type or subtype of Na+ channel with which the toxin interacts More work is necessary Acknowledgements This work was partially supported by grants from the Mexican Council of Science and Technology (CONACyT) Z-005 and 40251-Q to L.D.P and grant number 32000N and 38616E to F.R.P Grant IN206003 from Direccion General de Asuntos 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[8], the CS -a/ b motif [2] Alignment of Fig 2, using the CLUSTAL X program, shows a clear cut separation of all the b Na-ScTxs from all the a Na-ScTxs The a Na-ScTxs have identities of the order of. .. Discussion NMR solution structure of Cn12 Figures 4, and summarize the most important data obtained from the NMR analysis of pure Cn12 It was possible to analyze the NMR data because of well-dispersed... constraints were added to the calculations The dynamic annealing structure calculations were performed with the CNS software suite [44] Analysis of Na+-channel sequences By searching data banks