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1 Institut de Pharmacologie Moléculaire et Cellulaire (IPMC), CNRS UMR 6097, Sophia-Antipolis, 06560 Valbonne, France
2 Architecture et Fonction des Macromolécules Biologiques (AFMB), CNRS UMR 6098 and Universités d’Aix-Marseille I and II, 13402 Marseille Cedex 20, France
3 Université Pierre et Marie Curie, Paris, France
Reprint requests to: Hervé Darbon, UMR 6098, CNRS, 31 ch. Joseph Aiguier, F-13402, Marseille Cedex 20, France; e-mail: herve{at}afmb.cnrs-mrs.fr; fax: 33 (0)4-91-16-45-36; or Michel Lazdunski, IPMC-CNRS, 660 Route des Lucioles, F-06560, Valbonne, France; e-mail: ipmc{at}ipmc.cnrs.fr; fax: 33 (0)4-93-95-77-04.
(RECEIVED February 17, 2003; FINAL REVISION March 28, 2003; ACCEPTED April 1, 2003)
4 These authors made equal contributions to this work. 
The PDB coordinate files have been deposited in the Brookhaven Data Bank (PDB code 1LMM).
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0307003.
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Keywords: Spider toxin; NMR structure; ASIC; ICK motif
Abbreviations: PcTx1, psalmotoxin 1 • NOESY, nuclear Overhauser effect spectroscopy • TOCSY, total correlation spectroscopy • COSY, correlation spectroscopy • CNS, crystallography and NMR system • ICK, inhibitor cystine knot
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Introduction |
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Numerous three-dimensional structures of peptide toxins acting on ion channels have been solved (Craik et al. 2001). In invertebrates, these peptides comprise 
15 to 70 amino acids and are usually reticulated by disulfide bridges. A large number of the peptides studied to date belong to two major structural motifs. The first one is the cystine stabilized 
/ß scaffold (CSß) comprising a short -helix and a double- or triple-stranded antiparallel ß-sheet stabilized by three or four disulfide bridges. This structural organization is essentially found in scorpion venom toxins acting on voltage-sensitive K+ and Na+ channels (Possani et al. 2000). The second type is the inhibitor cystine knot (ICK) motif which comprises several loops that emerge from a double- or triple-stranded antiparallel ß-sheet structure reticulated by at least three disulfide bridges (Pallaghy et al. 1994; Norton and Pallaghy 1998). Two of the disulfide bridges together with the amino-acid backbone form a ring, which is penetrated by a third disulfide bridge. The ICK motif is widespread in animal, plant, and fungal proteins, and has mainly been associated with inhibitory activities (Craik et al. 2001). Animal toxins structurally organized around this motif have been characterized from cone snails and spider venoms (Narasimhan et al. 1994; Norton and Pallaghy 1998; Escoubas et al. 2000b). In these peptides, the ICK fold is associated with a wide pharmacological profile, as these toxins can block voltage-dependent Na+ (Adams et al. 1989; Hill et al. 1997), K+ (Swartz and MacKinnon 1995; Savarin et al. 1998; Bernard et al. 2000), and Ca2+ (Adams et al. 1990; Olivera et al. 1991) channels as well as ryanodine-sensitive calcium channels (Mosbah et al. 2000).
Proton-sensitive inward currents activated by a drop in external pH were identified in the 1980’s (Krishtal and Pidoplichko 1981), and the underlying channels [acid-sensing ion channels (ASIC)] were cloned (Waldmann et al. 1997b). The predicted membrane topology of ASICs suggests a large extracellular loop connecting two transmembrane domains with the N and C termini inside the cell. This class of proton-gated cation channels appears to be involved in brain function and nociception (Reeh and Steen 1996; Waldmann and Lazdunski 1998; Kress and Zeilhofer 1999; McCleskey and Gold 1999; Chen et al. 2002). Several ASIC subunits and splice variants have now been described: ASIC1a (Waldmann et al. 1997b), ASIC1b (Chen et al. 1998), ASIC2a (Waldmann et al. 1997b; Champigny et al. 1998), ASIC2b (Lingueglia et al. 1997), ASIC3 (Waldmann et al. 1997a), and ASIC4 (Grunder et al. 2000). The different subtypes produce channels with different kinetics, external pH sensitivities, and tissue distribution. These different subunits can form homo- and heteromultimers in both the central nervous system and in nociceptor sensory neurons (Waldmann and Lazdunski 1998).
Pharmacological studies will be essential to understand the exact role of these channels in brain function and nociception. Amiloride blocks the inactivating phase of all of these channels, but it does so at relatively high concentrations at which the drug also blocks other transport systems.
The first-identified potent and specific peptide blocker of ASIC1 channels is psalmotoxin 1 (PcTx1). It was isolated from the venom of the South American tarantula Psalmopoeus cambridgei (Escoubas et al. 2000a) and very specifically blocks homomultimers of ASIC1a. It has been essential in characterizing the stoichiometry and role of these homomeric assemblies of ASIC1a in different neuron types (Escoubas et al. 2000a). The primary sequence of PcTx1 was found to be related to that of other spider venom ICK peptide toxins identified as inhibitors of voltage-dependent calcium or potassium channels. This paper reports the recombinant production of PcTx1 in Drosophila melanogaster S2 cells, and the determination of its solution structure by means of 1H 2D NMR spectroscopy.
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Results and Discussion |
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ß hairpin fold of scorpion toxins, which readily refold in vitro. Thus, direct production of refolded peptides is most desirable when attainable. Examples of recombinant production of unfolded peptide toxins in E. coli followed by in vitro disulfide formation include potassium channel scorpion toxins (Park et al. 1991), huwentoxin I (Li et al. 2000), and the J-atracotoxins (Maggio and King 2002). Although production yields can be up to 10–20 mg/L (Li et al. 2000), the refolding step is usually the limiting factor in obtaining sufficient amounts of active toxin, as the refolding yield can be less than 10% of the original product. Other strategies based on fusion proteins have permitted the production of soluble, folded forms of the toxins in E. coli using a thioredoxin reductase-deficient strain (Johnson et al. 2000) or two protein A IgG-binding domains (ZZ) leading to periplasmic expression (Bouhaouala-Zahar et al. 1996; Korolkova et al. 2001). A yeast expression system has also been used successfully with yields in the 8–10 mg/L range (Wu et al. 2002). Several drawbacks of the methodology include the need for proteolytic or chemical cleavage of the fusion proteins, lowering final yields and sometimes leaving undesired residues in the toxin sequence, as well as the difficulty in obtaining posttranslational modifications, often necessary for toxin activity.
In addition, our experience with oxidative refolding of synthetic PcTx1 and other ICK spider peptide toxins has shown that obtaining the correct disulfide bridge pairing under classical redox conditions is difficult (P. Escoubas and G. Lambeau, unpubl.), yielding amounts of toxin insufficient for structural and physiological studies.
The Drosophila S2 cell production system permits direct recombinant production of correctly folded toxins that can be easily purified from the culture medium. This system also permits the production of glycosylated proteins and thus offers significant advantages over chemical synthesis for ICK peptides, in particular for mutagenesis studies. It was therefore chosen for the production of recombinant PcTx1.
Pilot purification of PcTx1 from 2L of S2 cell culture supernatant demonstrated the presence of active folded recombinant PcTx1 (PcTx1r). An optimized purification scheme (Fig. 1A
) was developed after extensive pilot studies in which either ion-exchange HPLC or RP-HPLC was used as the primary separation method. The complexity of the S2 cell supernatant and the very low relative amounts of the recombinant peptide did not permit resolution by direct HPLC fractionation. Two batch separations involving elution steps were found to be necessary to obtain a fraction containing PcTx1, which was manageable by RP-HPLC for final purification (Fig. 1B). The final ion-exchange HPLC step was done for final polishing to >99% purity. Treatment of a total of 12L of cell culture supernatant permitted the purification of 5.5 mg of PcTx1r (final yield 0.48 mg/L). Throughout the purification procedure, MALDI-TOF MS analysis of the chromatographic fractions allowed easy monitoring of the presence of PcTx1r as indicated by observation of a peptide ion at m/z 4690 in linear mode (Fig. 1B,C). MALDI-TOF MS thus proved to be a rapid, sensitive, and reliable method for the assessment of recombinant toxin production.
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). The inhibitory activity of PcTx1r on heterologously expressed ASIC1a was the same as that of native and synthetic PcTx1 (Escoubas et al. 2000a), and a dose-response curve yielded an IC50 value of 1.2 nM (data not shown). A properly folded, active form of the PcTx1 toxin can thus be produced in the Drosophila S2 cell expression system, in amounts compatible with the requirements of 2-D NMR analysis and physiological experiments. Further improvement of the purification process (fraction recycling) could improve the final production yield. It should be noted that the toxin is produced folded, without the need for a fusion protein. This strategy avoids the supplementary steps associated with removal of the fusion protein (Bernard et al. 2000; Li et al. 2000) and thus limits losses of material. Direct production of a correctly folded form of the toxin also avoids the in vitro disulfide reoxidation step necessary for synthetic peptides and responsible for the very low final yields that have been obtained for other ICK toxins (Pennington et al. 1992; Lew et al. 1997; Sasaki et al. 1999).
The recombinant production strategy which has been used here for the first time for an ICK spider toxin holds promise for further production of short arthropod toxins.
NMR resonance assignment
Sequential assignment was obtained by the now standard method first described by Wüthrich (Wüthrich 1986) and successfully applied to various arthropod toxins such as HpTx2, Ptu1, and Maurocalcine, respectively from the spider Heteropoda venatoria, the assassin bug Peirates turpis, and the scorpion Scorpio maurus (Bernard et al. 2000, 2001; Mosbah et al. 2000). The spin systems were identified on the basis of both COSY and TOCSY spectra recorded at 300 and 283K. The use of two temperatures for recording allowed us to resolve overlapping signals in the fingerprint region, and thus intraresidue HN-H cross-peaks were unambiguously assigned. At the end of the sequential assignment procedure, almost all protons were identified and their resonance frequency determined (BMRB ID 5495). The repartition of the H/HN and HN/HN correlations showed that the toxin is essentially organized in loops, beside three extended regions characterized by strong H/HN correlations (Fig. 2).
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. In addition, 12 hydrogen bond restraints and 21 dihedral angle constraints derived respectively from proton exchange and coupling constants have been included, as well as nine distance restraints derived from the three disulfide bridges which had been deduced from visual analysis of the previously obtained solutions and which are identical to that of other toxins folded into the same ICK motif (Escoubas et al. 2000b). Altogether, the final experimental set corresponded to 10.4 constraints per residue on average. The structures were calculated with DIANA using a distance geometry protocol, and energy minimized by CNS. The best-fit superimposition of backbone atoms for the 20 best structures gives RMSD values of 2.51 +/-0.51 Å for backbone atoms and 3.58 +/-0.40 Å if all nonhydrogen atoms are included. These rather high values are explained by the existence of structurally undefined N and C termini and two flexible regions encompassing residues 10 to 17 and 26 to 30. A summary of the structural statistics and the values of RMSD calculated on the well-defined regions are given in Table 1. All of the calculated structures have good nonbonded contacts and good covalent geometry, as shown by the low values of CNS energy terms and low RMSD values for bond lengths, valence angles, and improper dihedral angles. Correlation with the experimental data shows no NOE-derived distance violation greater than 0.2 Å. The analysis of the Ramachandran plot for the ensemble of the 20 calculated structures (in PROCHECK software nomenclature) shows 92.5% of the residues in the most favored and additional regions, 7.5% in the generously allowed regions, and none in the disallowed regions (data not shown).
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, and this reveals a differential flexibility with regard to the secondary structure. N and C termini as well as loops are less resolved than the central core of the toxin. This does come from a lack of NOEs in these regions, resulting from the internal dynamics of the protein. This feature has already been mentioned in most of the solution structures of ICK motifs solved to date.
The three-dimensional structure (PDB accession code 1LMM) of PcTx1 consists of a compact disulfide-bonded core from which three loops and the N and C termini emerge. Figure 4A shows a stereopair representation of the best-fit superimposition of the C traces of the 20 best structures. The main element of secondary structure is a three-stranded antiparallel ß-sheet comprising residues 21–24 and 31–34. Two of the three strands are stabilized by NH/CO hydrogen bonds involving amide protons from residues 22, 24, 32, and 34 and are well detected by the PROCHECK-NMR software. The third peripherally extended strand composed of residues 7 to 9 is very poorly defined and is not detected by the PROCHECK-NMR software. Nevertheless, the existence of HN8-HN33, H9-H32 correlations, and the fact that the amide proton of residue 33 is in slow exchange (engaged in an hydrogen bond with the carbonyl group of residue 8) allowed us to describe this region as the third strand of the ß-sheet. Overall, the structure shows a strong geometric anisotropy and can be related to a truncated cone.
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). This positive patch is surrounded on its top by three aromatic residues (Trp7, Trp24, and Phe30). Another ring of four basic residues lies on top of the aromatic residues (Fig. 4C).
Comparison with related toxins
The PcTx1 fold can be classified as an inhibitor cystine knot (ICK) fold already described for numerous toxic and inhibitory animal venom peptides (Norton and Pallaghy 1998). It belongs to the class of small disulfide-bridged peptides classified as knottins in the SCOP database (http://scop.mrc-lmb.cam.ac.uk/scop/data/scop.b.h.d.html). Many ion channel effectors from marine snail, spider, and scorpion venoms share the same ICK fold, although they possess very different pharmacological profiles (Fig. 5; Olivera et al. 1991; Norton and Pallaghy 1998; Escoubas et al. 2000b; Craik et al. 2001). Among ICK spider toxins, PcTx1 is the only peptide known to act on ASIC1a channels, whereas µ-agatoxin I (Omecinsky et al. 1996), the 
-palutoxins (Corzo et al. 2000), and the -atracotoxins (Fletcher et al. 1997) are active on Na+ channels. In contrast, hanatoxins, HpTx2, and stromatoxins, respectively from the spiders Grammostola spatulata (Takahashi et al. 2000), Heteropoda venatoria (Bernard et al. 2000), and Stromatopelma calceata (Escoubas et al. 2002) act against voltage-dependent K+ channels. Other spider ICK toxins have been reported to act on Ca2+ channels, such as the 
-agatoxins IVA and IVB (Adams et al. 1993; Mintz and Bean 1993), huwentoxin I (Peng et al. 2001), SNX482 (Newcomb et al. 1998), -GsTx SIA (Lampe et al. 1993), and the -atracotoxins (Wang et al. 1999), respectively from the venoms of the spiders Agelenopsis aperta, Selenocosmia huwena, Hysterocrates gigas, Grammostola spatulata, and Hadronyche spp. More recently, the ICK fold has also been found in the spider peptide toxins GsMTx-4 and GsMTx-2, which block mechanosensitive cationic ion channels in heart cells (Oswald et al. 2002).
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PcTx1 has a unique pharmacology, which distinguishes it from all other ICK toxins. Although its overall structure fits into the canonical ICK fold of related Ca2+, Na+, and K+ channel toxins, its selectivity and mode of action are totally different and place it in a unique pharmacological class. Previous results (Escoubas et al. 2000a) demonstrated that PcTx1 has a very high selectivity for the homomultimeric ASIC1a channel subtype and does not affect other ion channels, including other subtypes or heteromultimeric assemblies of ASIC subunits. PcTx1 perfectly illustrates the often mentioned capability of the ICK scaffold reticulated by three or four disulfides to bear a variety of pharmacological activities through selective mutagenesis of the solvent-exposed loops. The evolutionary mechanism leading to the extraordinary pharmacological diversity of venom peptides has been extensively studied in cone snails (Espiritu et al. 2001), and a similar model would be suitable to describe the molecular diversity of spider venom peptides.
Functional surface of PcTx1
To analyze the putative functional surface of peptide toxins, we used a prediction method based on the orientation of the dipole moment resulting from the electrostatic anisotropy of the toxin. The dipole is used as a guideline to predict a putative functional surface (Cui et al. 2001, 2002; Fu et al. 2002). The highly undefined N and C termini (residues 1, 2, and 38 to 40) were not included in the calculation of the dipole of PcTx1, but it was clearly the same as the dipole calculated for the averaged complete molecule. Assuming that the dipole may work as an orientation force in the electric field of the receptor (Fremont et al. 1997; Bernard et al. 2000; Mosbah et al. 2000), a proposed functional surface for PcTx1 is presented in Figure 4C. Two possible sites of action for peptide toxin blockade of ion channels have been proposed. Current inhibition may result from pore occlusion, as observed for the scorpion and cone snail toxins blocking voltage-dependent potassium channels, or via steric hindrance of ion conduction by binding at the mouth of the pore. The latter has been proposed as the binding mechanism of voltage-gating modifiers such as the hanatoxins, which might also impede the movement of ion channel transmembrane segments in response to a depolarizing voltage and therefore affect ion conduction. Recent work also supports the "hot spot" model of protein-protein interaction (Maggio and King 2002) for toxin binding to ion channels. The hot spot on the toxin surface has been proposed to involve a functional dyad comprising a critical lysine residue involved in pore blocking and a neighboring aromatic residue such as tryptophan or phenylalanine (Dauplais et al. 1997).
This toxin-channel interaction model has been validated by mutagenesis studies on scorpion (Dauplais et al. 1997) and cone snail toxins (Jacobsen et al. 2000), and the sea anemone toxins BgK (Alessandri-Haber et al. 1999) and ShK (Pennington et al. 1996a,b), and more recently on the insecticidal J-atracotoxins from spider venom (Maggio and King 2002). This structural motif appears to be found in various channel inhibitors such as snake dendrotoxins, charybdotoxin, agitoxin, noxiustoxin, and related scorpion peptides (Dauplais et al. 1997), the newly discovered 
-hefutoxins (Srinivasan et al. 2002), -conotoxin (Savarin et al. 1998), and BgK and ShK from sea anemones (Pennington et al. 1996a,b; Alessandri-Haber et al. 1999). All these toxins block ion channels through pore occlusion, and the association of a hydrophobic residue with a patch of positive residues or a single basic amino acid such as Lys or Arg has been demonstrated to be crucial for channel recognition and blockade (Pennington et al. 1996a). As novel peptides are described and characterized, evidence is mounting to support the crucial role of the functional dyad in many toxin-channel interactions.
Another interaction model has been proposed for gating-modifier toxins such as the hanatoxins (Takahashi et al. 2000). In this case, the interaction hot spot would be represented by a patch of hydrophobic residues forming a contact surface surrounded by charged residues anchoring the toxin to its target surface through formation of salt bridges. In the hot spot model, residues surrounding the toxin hot spot are proposed to serve as "gaskets," preventing interaction of water molecules with the target residues on the channel surface. Although the hydrophobic patch model appears to be relevant in other potassium channel blockers such as the stromatoxins (Escoubas et al. 2002), in which a dyad Lys-Phe can also be found, no mutagenesis data are available to validate its relevance.
Examination of the structure of PcTx1 reveals that it does not conform to the hydrophobic patch model but bears a considerable number of positively charged residues in the ß-turn linking the two ß-strands (loop 4). The stretch K25R26R27R28 in fact forms a contiguous positive surface protruding from the rest of the molecule (Fig. 4C). Additionally, three aromatic residues (Trp7, Trp24, and Phe30) are found in the vicinity of the basic residues and may possibly be involved in the formation of functional dyads equivalent to that of other ion channel toxins. Judging from interatomic distances, only the dyad Arg26/Trp24 (6.8 Å) would fit the proposed pattern in which distances between basic and aromatic residues vary between 5.8 and 7.6 Å (Srinivasan et al. 2002). Arg28 and Phe30 are located further apart (14 Å) in the PcTx1 structure. Figure 6 shows the structural similarity of basic and aromatic residue side chains in several of these toxins. Loop 4 in PcTx1 thus appears to be a potentially important structural feature, in accordance with the electrostatic anisotropy data presented above. We propose that the positively charged patch formed mostly by residues K25–R28, accompanied by aromatic side chains and other surrounding hydrophobic or negatively charged residues could form the channel recognition surface of PcTx1. It should be noted that negatively charged residues are located primarily in the N-terminal part of the peptide, and on the opposite side of the K25–R28 loop, forming a "negative crown" above the positively charged patch.
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Recombinant toxin purification and characterization
Drosophila S2 cell culture medium was centrifuged at 5000 rpm for 30 min. One liter of the supernatant was then diluted twofold with equilibration buffer (H2O/10% acetonitrile/1% acetic acid) and loaded overnight in batch mode on 300 mL of SP Sephadex C-25 cation-exchange resin (Amersham Pharmacia Biotech) prewashed and equilibrated in H2O/10% acetonitrile/1% acetic acid. Two washing steps were performed with (1) equilibration buffer and (2) equilibration buffer containing 0.4M ammonium acetate/10% acetonitrile. Recombinant PcTx1 was then eluted with 1.5M ammonium acetate/10% acetonitrile. After a twofold dilution with water and acidification with TFA to pH 3.0, the PcTx1 fraction was loaded in batch mode on 100 mL of C18 reversed-phase silica (20–43 µm; Merck) pre-equilibrated in 0.1% TFA. Three consecutive washing steps (500 mL each) were performed with (1) water/0.1% TFA; (2) 5% aqueous acetonitrile/0.1% TFA; (3) 10% aqueous acetonitrile/0.1% TFA. PcTx1 was eluted with 500 mL of 50% aqueous acetonitrile in 0.1% TFA, and the elution volume was diluted threefold with water and freeze-dried.
Throughout the batch purification scheme, the presence of recombinant PcTx1 was monitored in all fractions by analysis of a small aliquot by MALDI-TOF MS in linear mode. For diluted fractions, a solid-phase extraction with C18 ZipTip microconcentrating devices (Millipore) was performed prior to mass spectrometry analysis.
Further purification was performed by cation-exchange HPLC on a preparative SP5PW column (150 x 21 mm, Tosoh-Haas) equilibrated with 10% aqueous acetonitrile/1% acetic acid, using a linear gradient of 10% to 90% of 2M ammonium acetate/10% acetonitrile (1%/min), at 6 mL/min. PcTx1 fractions from several HPLC runs were pooled and then freeze-dried, and a final purification step was done by reversed-phase HPLC on a C18 semipreparative column (250 x 10 mm, 5µm, 100 Å, Nacalai Tesque), using a linear gradient of acetonitrile/0.1% TFA in water/0.1%TFA (5% to 35% acetonitrile in 60 min, 2 mL/min). Final quantification of the toxin was done by optical density measurement at 280 nm, using a calculated 
280 of 11740.
Full characterization of recombinant PcTx1 was achieved by automated N-terminal Edman sequencing on an Applied Biosystems Procise gas-phase sequencer, monoisotopic molecular weight analysis by MALDI-TOF MS, and HPLC co-elution experiments with the native toxin. Co-elution of recombinant and native PcTx1 was performed by cation-exchange on an analytical SP5PW column (75 x 7.5 mm, Tosoh-Haas) using a linear gradient of 20mM to 1M ammonium acetate in 50 min (0.5 mL/min) and by reversed-phase on a Merck Purospher STAR 55–4 column (55 x 4 mm, 3 µm), using a linear gradient of acetonitrile in water (constant 0.1% TFA) at 1 mL/min and 0% to 35% acetonitrile/min over 35 min.
MALDI-TOF mass spectrometry
Peptides or HPLC fractions mixed with -cyano-4-hydroxycinnamic acid (-CHCA, Aldrich) matrix (10 mg/mL) were analyzed on an Applied Biosystems Voyager DE-Pro system in positive reflector (pure peptides) or linear (fractions) mode. Mass spectra (200–300 scans) were calibrated with external or internal standards and analyzed with Data Explorer software.
Electrophysiological characterization
Activity of recombinant PcTx1 was tested against cloned ASIC1a channels heterologously expressed in COS cells as described (Escoubas et al. 2000b). Recombinant PcTx1 was applied in a range of concentrations to establish its dose-response curve (N=5 per dose).
Sample preparation for NMR
Recombinant PcTx1 (4 mg) was solubilized in 450 µL of an H2O/D2O mixture (9:1 v/v), to give a protein concentration of 2.9 mM at pH 3.0. This rather low pH is used to reduce as much as possible the amide proton exchange rate. The exchange rate was determined after lyophilization of this sample and dissolution in 100% D2O to check that the conformation (on the sight of NH resonance frequencies) was the same at pH 3 than the one at pH 5, which is used to test the pharmacological activity of the toxin.
NMR experiments
All 1H NMR spectra were recorded on a BRUKER DRX500 spectrometer equipped with an HCN probe, and self-shielded triple-axis gradients were used. The experiments were performed at two different temperatures in order to solve assignment ambiguities (283 K and 300 K). Two-dimensional spectra were acquired using the states-TPPI method to achieve F1 quadrature detection. Water suppression was achieved using presaturation during the relaxation delay (1.5 sec), and during the mixing time in the case of NOESY experiments (Kumar et al. 1980), or using a watergate 3–9–19 pulse train using a gradient at the magic angle obtained by applying simultaneous x-, y-, and z-gradients prior to detection. NOESY spectra were acquired using mixing times of 100 msec and 120 msec. TOCSY was performed with a spin locking field strength of 8 kHz and spin lock time of 80 msec. The amide proton exchange experiments were recorded immediately after dissolution of the peptides in D2O. Four series of NOESY spectra with a mixing time of 80 msec were recorded at 283K, the first series for 1 h, the subsequent three for 4 h each.
The identification of amino acid spin systems and the sequential assignment were done using the standard strategy described by Wüthrich (1986) and regularly used by our group (Bernard et al. 2000, 2001; Mosbah et al. 2000), with the graphic software XEASY (Bartels et al. 1995). The comparative analysis of TOCSY spectra recorded in water gave the spin system signatures of the protein. The spin systems were then sequentially connected using the NOESY spectra.
The integration of NOE data was done by measuring the peak volumes using a specific routine of the XEASY package. These volumes were then translated into upper limit distances by the CALIBA routine of the DIANA software (Güntert and Wüthrich 1991). The lower limit was systematically set at 0.18 nm. The 
torsion angle constraints resulted from the 3JHN-H coupling constant measurements. They were estimated with the INFIT program (Szyperski et al. 1992). For a given residue, separated NOESY cross-peaks with the backbone amide proton in the 2 dimension were used. Several cross-sections through these cross-peaks that exhibited a good signal-to-noise ratio were selected and then summed, and only those data points of the peak region that were above the noise level were retained. The left and right ends of the peak region were then brought to zero intensity by linear baseline correction. After extending the baseline-corrected peak region with zeros on both sides, which is equivalent to oversampling in the time domain, an inverse Fourier transformation was performed. The value of the 3JHN-H coupling constant was obtained from the first local minimum. 3JHN-H coupling constants were translated into angle restraints using HABAS from the DIANA package.
We checked that increasing the pH to 5, closer to the actual activation pH of the channel, does not alter the conformation of the protein. This was checked by comparing NH resonance frequencies at both pHs. A NOESY recorded at pH 5 does not show any significant modification in NOEs collected for the structure determination.
Structure calculation
Distance geometry calculations were performed with the variable target function program DIANA 2.8. A preliminary set of 1000 structures was initiated including only intraresidual and sequential upper limit distances. From these, the 500 best as shown by the value of the target function were kept for a second round, including medium-range distances. The resulting 250 best solutions were kept for a third round, using the whole set of upper restraints. The 100 best solutions were then refined by adding the restraints coming out from the disulfide pairing. This pairing was assessed from the visual analysis of the structures obtained solely from the NOE constraints, and found to be identical to that of other ICK-folded animal toxins. Starting from the 100 best structures, one REDAC cycle (Güntert and Wüthrich 1991) was used in a last step in order to include the dihedral constraints together with the additional distance restraints coming from hydrogen bonds.
To remove residual bad Van der Waals contacts, the 30 best structures were refined by restrained molecular dynamics annealing, slow cooling, and energy minimization (parameter file: protein-allhdg in CNS). Visual analysis was done using TURBO software (Roussel and Cambillau 1989), and the geometric quality of the obtained structures was assessed by PROCHECK 3.3 (Laskowski et al. 1993) and PROCHECK-NMR software (Laskowski et al. 1996).
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References |
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Adams, M.E., Bindokas, V.P., Hasegawa, L., and Venema, V.J. 1990. -agatoxins: Novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom. J. Biol. Chem. 265: 861–867.
Adams, M.E., Mintz, I.M., Reily, M.D., Thanabal, V., and Bean, B.P. 1993. Structure and properties of -agatoxin IVB, a new antagonist of P-type calcium channels. Mol. Pharmacol. 44: 681–688.[Abstract]
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