On the Convergent Evolution of Animal Toxins
CONSERVATION OF A DIAD OF FUNCTIONAL RESIDUES IN POTASSIUM CHANNEL-BLOCKING TOXINS WITH UNRELATED STRUCTURES*

(Received for publication, May 7, 1996, and in revised form, November 8, 1996)

Marc Dauplais , Alain Lecoq , Jianxing Song , Joël Cotton , Nadège Jamin , Bernard Gilquin , Christian Roumestand Dagger , Claudio Vita , Cleane L. C. de Medeiros §, Edward G. Rowan §, Alan L. Harvey § and André Ménez

From the Département d'Ingénierie et d'Etudes des Protéines, CEA, Saclay, 91191 Gif-sur-Yvette Cedex, France and the § Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G1 1XW, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

BgK is a K+ channel-blocking toxin from the sea anemone Bunodosoma granulifera. It is a 37-residue protein that adopts a novel fold, as determined by NMR and modeling. An alanine-scanning-based analysis revealed the functional importance of five residues, which include a critical lysine and an aromatic residue separated by 6.6 ± 1.0 Å. The same diad is found in the three known homologous toxins from sea anemones. More strikingly, a similar functional diad is present in all K+ channel-blocking toxins from scorpions, although these toxins adopt a distinct scaffold. Moreover, the functional diads of potassium channel-blocking toxins from sea anemone and scorpions superimpose in the three-dimensional structures. Therefore, toxins that have unrelated structures but similar functions possess conserved key functional residues, organized in an identical topology, suggesting a convergent functional evolution for these small proteins.


INTRODUCTION

Functional properties of proteins are frequently associated with a small number of important residues. For example, enzyme activities depend on a few residues that are essential for catalysis. Also, protein-protein recognition processes have been predicted (1) and recently demonstrated (2) to be energetically driven by a small proportion of the residues forming the contacting areas in protein-protein complexes, as identified by x-ray studies (3, 4). Among the proteins whose major functions require protein-protein interactions are animal toxins, which bind to various molecular targets, such as receptors or ion channels, using a small number of binding residues (5-8). As has been shown for enzymes (9), toxins with different architectures are capable of exerting similar functions (10). However, in contrast to enzymes, the molecular basis associated with the conservation of the function in structurally unrelated toxins remains unknown. In this paper, we show that two families of animal toxins with different folding patterns but a comparable capacity to bind to potassium channels include similar functional diads, composed of a critical lysine and an aromatic amino acid separated from each other by 6.6 ± 1.0 Å.


MATERIALS AND METHODS

Synthesis of Toxin and Mutants

The amino acid sequence of BgK1 was proposed a few years ago (11). However, chemical synthesis attempts, based on these data, systematically failed. The proposed amino acid sequence was therefore questioned, re-examined, and ultimately corrected.2 The revised amino acid sequence of BgK from Bunodosoma granulifera is: VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC. BgK and each alanine-substituted analog were synthesized by solid phase synthesis using an Applied Biosystems model 431A peptide synthesizer, starting from 0.1 mmol of Rink-resin (4-(2',4'-dimethoxyphenylhydroxymethylphenoxy resin; 0.48 mmol/g). A 10-fold excess (1 mmol) of Fmoc (N-(9-fluorenyl)methoxycarbonyl)-protected amino acid was used and coupled in N-methylpyrrolidone in the presence of N,N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The following side chain protections were used: t-butyl ether for Ser and Thr; t-butyl ester for Glu and Asp; trityl for Cys, His, Asn, and Gln; 2,2,5,7,8-pentamethylchromane-6-sulfonyl for Arg; and t-butoxycarbonyl for Lys and Trp. The peptide was simultaneously cleaved from the resin and deprotected by treatment (500 mg of resin/50 ml) with 90% trifluoroacetic acid, 5% H2O, 5% triisopropylsilane for 1.5 h at room temperature. The resin was filtered, and the solution was precipitated with methyl-t-butylether, washed three times with ether, dissolved in 20% acetic acid, and lyophilized.

The crude peptides (0.1 mg/ml) were oxidized in 50 mM phosphate buffer, pH 7.8, containing 5 × 10-3 M reduced and 5 × 10-4 M oxidized glutathione. Oxidation was complete after 1 h at room temperature. The solution was then acidified to pH 3.0 with acetic acid and directly applied to a Vydac C18 column (25 × 1 cm) for purification. The peptides were eluted with an acetonitrile gradient containing 0.1% trifluoroacetic acid. The fractions containing the oxidized peptides were analyzed by analytical HPLC. The adequately oxidized components were identified by mass spectrometry. The yields in purified and oxidized toxins ranged from 20 to 40%.

Disulfide assignments were achieved by peptide mapping using the Lys-C endoprotease (1:20 by mass) in 20 mM Tricine buffer, containing 1 mM EDTA, pH 7.6, for 7 h at 35 °C. Digests were submitted to reverse phase-HPLC on a Vydac C18 column (25 × 0.46 cm), and the resulting fragments were collected, lyophilized, and analyzed by electrospray mass spectrometry. The mass of the fragments unambiguously indicated that the pairings were Cys2-Cys37, Cys11-Cys30, and Cys20-Cys34.

Amino acid analyses were performed on an Applied Biosystems model 130A automatic analyzer, equipped with an on-line model 420A derivatizer, and mass analysis on a Nermag R10-10 mass spectrometer, coupled to an Analitica of Branford electrospray source. Amino acid compositions and mass analyses agreed with the expected theoretical values.

Peptide concentrations were determined by monitoring absorbances at 280 mm, using a molar extinction coefficient of 8100, calculated on the basis of the known amino acid content (11). For analogs where Trp or Tyr were deleted, the peptide concentrations were determined on the basis of quantitative amino acid analyses.

Circular dichroism spectra were recorded using a Jobin-Yvon CD6 dichrograph, driven by an IBM PC operating with a CD6 data acquisition and manipulation program. Spectra in the range 180-250 nm were run at 20 °C in 5 mM sodium phosphate, pH 7.0, with a 0.1 cm quartz cell and a protein concentration of 1.5-2.0 × 10-5 M.

NMR Experiments

Synthetic BgK (9.8 mg) was dissolved in 440 µl of solvent, leading to a final concentration of 4.9 mM. Solvents were either a mixture of 400 µl of water and 40 µl of D2O, or 440 µl of D2O. pH was fixed at 3.7, and chemical shifts were measured relative to TSP-d4. All NMR experiments were performed at 600 MHz (AMX600 Brüker spectrometer) at 20 and 30 °C, in order to resolve assignment ambiguities. At each temperature and in each solvent, a DQF-COSY spectrum (12), a nuclear Overhauser spectroscopy spectrum (300 ms mixing time) (13), and a total correlation spectroscopy spectrum (14, 15) were recorded. Quadrature detection was performed using the States method (16) in the indirect dimension and using simultaneous mode acquisition in the directly detected dimension. The water signal was suppressed by low power irradiation at all times except during t1 and t2. The spectra were recorded with 128 (t1) × 1024 (t2) (96 × 1024 in D2O) and with a sweep width of 7812 Hz (6578 Hz in D2O). The 3JNH-Halpha (respectively 3JHalpha -Hbeta ) coupling constants were measured from the high resolution DQF-COSY in H2O (respectively D2O).

Signal Transformation and Molecular Modeling

All data were transformed using FELIX software (17). Prior to Fourier transformation, spectra were weighted with a 18° shifted sine bell (90° for the spectra to be integrated) and were zero-filled in the t1 dimension to yield 1000 × 1000 matrices after reduction (1000 × 4000 for the DQF-COSY). Each NOE was integrated with FELIX, using a 2.48 Å distance between the delta  and the epsilon  protons of Tyr26 for calibration. A range of ±25% of the distance value was used to define the upper and the lower bounds of the restraints. Structures were obtained by molecular modeling using successively DIANA (18) for preliminary structure calculation and X-PLOR for simulated annealing (19, 20). After having solved assignment ambiguities with the help of DIANA, 50 structures were calculated using this software, and the 30 structures with a target function below 10 were further refined by simulated annealing in X-PLOR 3.1 (with files parallhdg.pro and topallhdg.pro). The 15 structures with the lowest energy were kept for analysis. Within these 15 structures, all but three distance violations are no larger than 0.3 Å and all dihedral restraints violations are lower than 5°.

Competition Binding Experiments

Competition experiments were made on rat brain synaptosomes using 125I-labeled alpha -dendrotoxin, as described previously (21).


RESULTS

Structure Determination

BgK was isolated from B. granulifera (11), and its amino acid sequence (see Fig. 1) has been recently corrected.2 BgK, like a number of scorpion toxins such as charybdotoxin, (i) binds to potassium channels, (ii) contains 37 residues, and (iii) possesses 6 half-cystines. However, our attempts to align the amino acid sequences of BgK and scorpion toxins have failed, suggesting that BgK adopts a structure that differs from the well-known alpha /beta fold of scorpion toxins (22). We therefore decided to elucidate its structure by NMR and molecular modeling.


Fig. 1. NMR data used for the sequence-specific assignment and the secondary structure identification. The data are compiled from nuclear Overhauser spectroscopy and DQF-COSY spectra at 20 °C (pH 3.7). 3Jalpha N coupling constant: square , 3Jalpha N <=  6 Hz; , 6 Hz <=  3Jalpha N <=  9 Hz; black-square, 3Jalpha N >=  9 Hz. Chemical shift index (C.S.I.): according to the Wishart method (47), a chemical shift variation larger than 0.1 from the average value of the alpha -proton for the concerned residue is represented by an arrow, directed upward for a positive difference and downward for a negative difference. A shift increase is expected in beta -sheet or extended strand, while a helical conformation will yield a decrease of the chemical shift.
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Structural Assignment

Three unique amino acids having specific spin systems were used as starting points for sequential assignments. These are Trp5, His13, and Gly18. The unique tyrosine (Tyr26) was also used to assign the C-terminal part of the amino acid sequence of BgK. As no proline was present in the protein, these four starting points as well as the existence of all but one sequential NH-NH connectivities allowed us to achieve the complete assignment of the amino acid sequence of BgK (Table I).

Table I.

Proton chemical shifts of BgK

Measurements were made at 293 K and pH 3.7. Data were obtained in H2O/D2O, 10:1 (v/v), and in D2O. Chemical shifts are in ppm relative to TSP-d4. Protein concentration was 4.9 mM.
Residue Chemical shift (ppm)
NH Halpha Hbeta Hgamma Others

Val1 3.78 2.18 0.99
Cys2 9.00 4.92 3.07, 2.82 
Arg3 8.46 4.52 1.58 1.31, 0.53 delta H: 2.59, 2.49 
Asp4 8.73 4.84 3.28, 2.68 
Trp5 8.68 4.94 3.47, 3.34 epsilon H: 7.76, eta H: 7.22, zeta H: 7.49, 7.14 
Phe6 7.84 5.23 3.44, 3.34 1.33 delta H: 7.20, Cepsilon H2: 6.93 
Lys7 8.56 4.27 2.10, 1.87 1.71, 1.65 delta H: 1.79, epsilon H: 3.07 
Glu8 8.90 4.07 2.24, 2.13 2.43
Thr9 8.42 3.91 4.16 1.30
Ala10 6.92 4.38 1.67
Cys11 8.59 4.83 3.05, 2.90 
Arg12 9.22 3.89 1.98 1.64 delta H: 3.24, 3.18 
His13 8.02 4.47 3.41, 3.46 delta H: 7.39, epsilon H: 8.71 
Ala14 8.14 3.75 0.90
Lys15 8.56 4.16 2.04, 1.66 1.49, 1.37 delta H: 1.66, epsilon H: 3.07 
Ser16 8.00 4.29 4.02
Leu17 7.15 4.36 1.52 1.43 delta H: 0.76, 0.62 
Gly18 7.71 4.41, 4.01
Asn19 7.80 4.74 2.69, 2.25 
Cys20 8.69 4.35 3.12, 3.02 
Arg21 7.59 4.35 2.01, 1.95 1.68, 1.57 delta H: 3.26 
Thr22 7.38 4.43 4.41 1.20
Ser23 7.66 4.96 4.41, 4.12 
Gln24 9.31 4.00 2.12 2.49
Lys25 8.22 3.98 1.85, 1.42 1.27, 0.74 delta H: 1.62, epsilon H: 2.82 
Tyr26 7.88 4.04 2.69 delta H: 7.40, epsilon H: 6.92 
Arg27 8.42 3.77 1.76, 1.69 1.93, 1.92 delta H: 3.10, 2.83 
Ala28 7.61 4.20 1.37
Asn29 7.13 4.84 1.45, 1.29 
Cys30 8.05 5.54 3.44, 3.24 
Ala31 9.38 3.83 1.46
Lys32 7.31 4.10 1.69, 1.24 1.57 delta H: 1.64, epsilon H: 2.94 
Thr33 10.61 3.88 4.00 1.06
Cys34 8.95 4.67 3.20, 2.88 
Glu35 8.07 4.15 2.36, 2.29 
Leu36 8.86 4.52 1.74, 1.65 1.48 delta H: 0.95, 0.84 
Cys37 7.76 4.40 3.39, 2.99

Experimental Restraints, r.m.s.d., and Energetical Parameters

A total of 767 distance restraints deriving from NOEs (316 intra-residual correlations, 172 sequential, 155 short range (|i - j<=  4), 124 long range (|i - j| > 4) and 12 dihedral restraints (7 from phi  angles, 5 from chi 1 angles) deriving from coupling constants was used. This large set of constraints (21 restraints by residue) yielded a well defined structure, as reflected by the mean r.m.s.d. value of its backbone, which was as low as 0.8 ± 0.2 Å between two structures. The calculated structures were consistent with both experimental data and the standard covalent geometry. The structures had no distance violations larger than 0.32 Å, and only three distance violations were larger than 0.3 Å. All dihedral restraint violations were lower than 5°. The covalent geometry was respected, as revealed by the low < r.m.s.d.> values of the bond length (0.003 Å) and the valence angles (1.52°).

Backbone Structure Description

The existence of two helices, running from residues 9 to 16 and residues 24 to 31, was clearly indicated by the presence of numerous short-range NOEs between halpha (i) and hn(i + 3) protons and between halpha (i) and hbeta (i + 3) protons (Fig. 1). No other regular secondary structure emerged from these data. Fig. 2a shows the 15 best structures of BgK, which result from transformation of NMR data and molecular modeling. The two helical stretches are well defined with r.m.s.d. values of 0.4 ± 0.1 Å and 0.5 ± 0.2 Å for the position 9-16 and 24-31 stretches, respectively. Though to a lesser extent, the other parts of the toxin structures were also relatively well defined. Panels b and c of Fig. 2 show, respectively, the overall fold of BgK and the spatial organization of its secondary elements. The N- and C-terminal regions are maintained in spatial proximity by the disulfide 2-37, whereas the third disulfide 20-34 brings the loops 16-24 and 31-37 inside the center of the molecule, providing the toxin with a globular shape. The 11-30 disulfide, which links the two helices, adopts a unique conformation, which is almost left-handed (23). It is centrally located in the structure, consistently with a tight organization of a local hydrophobic core. The other disulfide bonds adopt either two conformations (disulfide 20-34) or no prevailing conformation (disulfide 2-33).


Fig. 2. a, stereoview (48) of the 15 best structures of BgK. Disulfides are in yellow. b, a ribbon (49, 50) structure of the lowest energy structure of BgK. The secondary elements are indicated in blue and the disulfides in yellow. c, schematic representation of the fold of BgK.
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Mutational Analysis

To identify functional residues of BgK, we submitted the toxin to an alanine-scanning experiment, producing all single point variants by chemical synthesis, as described under "Materials and Methods." Competition experiments were performed between the variants and radiolabeled alpha -dendrotoxin on membrane from rat brain synaptosomes. Twenty-five variants have been synthesized and investigated regarding their effects on dendrotoxin binding. Fig. 3 shows five inhibition curves obtained with native BgK and four variants, which display a substantially lower competition activity, as compared with native BgK. Except for the variant T33A, all inhibition curves are parallel to that observed with the native toxin. Similar parallel inhibition curves were obtained for other variants, except K25A, for which no inhibition was observed even in the presence of 3 × 10-7 M protein. In the presence of the variant F6A, a 10% increase in binding of labeled dendrotoxin was observed. Although we are not able to explain this observation, as yet, one should recall that different subtypes of potassium channels are present in brain and are composed of heterooligomeric mixtures of different protein subunits. Experiments with subtype-specific antibodies reveal that most of the channels in the brain contain Kv1.2 (~80%) or Kv1.1 (~50%) subunits (24). alpha -Dendrotoxin blocks Kv1.1 and Kv1.2 channels with almost equal affinity (IC50 values ~ 20 nM), and has greater than 10 times less affinity for other cloned channels (25). Therefore, in view of such complexity, the present competition data have to be considered with caution. From curves similar to those shown in Fig. 3, we deduced IC50 values for all variants. These values are compiled in Table II. The correlation coefficient for fitting the data points was calculated from the Hill equation, y = Rmax/[1 + (X/IC50)P]. As can be seen from Table II, a value close to 1 was obtained in all cases. Moreover, preliminary competition experiments performed with cloned Kv1.2 channels and a number of the variants exhibiting a lower inhibitory capacity, as compared to native BgK, nicely paralleled those reported in Table II.3 Therefore, we anticipate that data obtained with brain synaptosomes mostly reflect the functional importance of residues implicated in the capacity of BgK to bind to Kv1.2 potassium channels. Nevertheless, in order not to overinterpret our data, we considered only the relative inhibitory capacity of the variants, without any attempt to deduce their binding affinities.


Fig. 3. Inhibition of 125I-labeled dendrotoxin to rat brain synaptosomes by various concentrations of the wild type BgK and four variants in which an alanine was introduced in place of Phe6, Ser23, Tyr26, and Thr33. Nonspecific binding was approximately 10% of the total binding.
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Table II.

Effect of introduction of an alanine residue at 25 individual positions of BgK on the ability of the toxin to inhibit binding of 125I-labeled alpha -dendrotoxin to membranes from rat brain synaptosomes

All residues except half-cystines, alanines, and the unique glycine have been mutated. Mutation at Asp4 yielded no refolded compound. The S.E. values for IC50 values are derived from independent experiments (n = 5 for the wild type toxin and three for each variant). The displacement curves were fitted with a sigmoid curve for a single binding site. The correlation coefficients for fitting the data points were calculated from the Hill equation, y = Rmax/[1 + (X/IC50)P]. Due to various reasons (see text) the decrease in inhibitory activity observed upon mutation at Thr33 uniquely was not considered to reflect the functional importance of the threonine residue. WT, wild type.
Affinity variations of refolded alanine mutants
IC50 Correlation coefficient Mutant/WT

nM
WT 3.6  (±0.6) 0.99 1
V1A 1.6  (±0.1) 0.99 0.4
R3A 7.7  (±0.9) 0.99 2.1
W5A 2.6  (±0.4) 0.99 0.7
F6A 167  (±48) 0.90 46
K7A 5.7  (±0.6) 0.99 1.6
E8A 1.5  (±0.1) 0.99 0.4
T9A 1.6  (±0.3) 0.99 0.4
R12A 3.6  (±0.3) 0.99 0.9
H13A 20  (±3) 0.99 5.6
K15A 5.5  (±1.4) 0.99 1.5
S16A 2.8  (±0.3) 0.99 0.8
L17A 2.3  (±0.2) 0.99 0.6
N19A 6.3  (±1) 0.99 1.8
R21A 4.6  (±1) 0.99 1.3
T22A 2.5  (±0.5) 0.99 0.7
S23A 30  (±6.9) 0.96 8.3
Q24A 1.5  (±0.3) 0.99 0.4
K25A >300 >80
Y26A 100  (±33) 0.95 28
R27A 5.1  (±1.8) 0.98 1.4
N29A 4.2  (±0.8) 0.99 1.2
K32A 5.5  (±0.5) 0.99 1.5
T33A 70  (±9.2) 0.99 19
E35A 3.0  (±0.3) 0.99 0.8
L36A 6.4  (±0.8) 0.99 1.8

Competition experiments, shown in Table II, revealed that mutation of lysine 25 into alanine is associated with the largest affinity decrease. This mutation, however, caused no significant change in the secondary structure of the toxin, as inferred from the circular dichroism spectra of the variant which is quite similar to the spectrum of the native toxin (Fig. 4). We conclude, therefore, that the low competition ability of this mutant is not due to distortions in the toxin structure but to the absence of the lysine side chain and possibly to the loss of its positive charge. Upon mutations of three other residues, Phe6, Tyr26, and Thr33, BgK was a less potent inhibitor, since its competition capacity decreased by factors of 46, 38, and 19, respectively. However, while the circular dichroism spectra of the first two variants were indistinguishable from that of the native toxin, the spectrum of the T33A variant showed some distortions (data not shown), which could not be readily interpreted but which might reflect some structural perturbations. Additionally, the inhibition curve with the T33A variant (see Fig. 3) was not quite parallel to those observed with the native BgK or other variants. In addition, synthesis of this variant was particularly difficult to achieve, leading to a relatively low yield of recovery. Therefore, all these observations suggest that introduction of an alanine at position 33 may be associated with structural perturbations, which might account for the substantial decrease in inhibitory activity of BgK. As a result, one cannot safely propose that the side chain of residue 33 is implicated in the binding of the toxin to the BgK target. Therefore, in the absence of further data, only the side chains of Lys25, Phe6, and Tyr26 are concluded to be involved in the functional site of BgK. Mutations at two positions, Ser23 and His13, also induced approximately 8- and 6-fold decrease in competition capacity. Although relatively low, these values might also reflect involvement of these residues in the binding site of BgK. Mutations at the other positions either had no effect on the affinity of BgK or caused changes in inhibitory capacity that are lower than 3-fold. These side chains are not considered, therefore, as actors in the recognition capacity of BgK for dendrotoxin-sensitive binding sites. In summary, alanine-scanning-based experiments indicated that three, and perhaps five, residues of BgK belong to the surface by which the toxin interacts with the channels.


Fig. 4. Circular dichroism spectra of synthetic BgK (- - -) and K25A mutant (---). Spectra were recorded at 20 °C with a 15-20 nM concentration.
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DISCUSSION

Potassium channels constitute a major target for various toxins produced by venomous animals from distinct phyla, including cnidaria (26), arthropods (10), and reptiles (27). Thus, a number of toxins, found in venoms of (i) sea anemones such as BgK (11) or ShK (28), (ii) scorpions like charybdotoxin (29) or noxiustoxin (30), and (iii) snakes like dendrotoxin (27), bind to dendrotoxin-sensitive K+ channels (31, 32). However, it is unclear as to whether these proteins share any structural or chemical elements that could account for their common capacity to recognize similar channels.

In this paper, we first provided evidence that BgK from the sea anemone B. granulifera is structurally unrelated to scorpion toxins, although these two groups of toxins share a similar number of residues, the same number of disulfides, and comparable functional properties. The present structural analysis revealed that the fold adopted by BgK contains two nearly perpendicular stretches of helices, with no additional canonical secondary structures (Fig. 2). The globular architecture of the toxin is stabilized by the three disulfides, one of them linking the two helices. This structure is similar to that of ShK, as indicated by a paper that was published during the review of our manuscript (33). ShK, from the Stichodactyla helianthus sea anemone, is a 35-residue toxin that also binds to potassium channels (28). As in BgK, the structure of ShK includes two helices running from residues 14-19 and 21-24. However, these two stretches are not located at identical corresponding positions in both toxins. In fact, the absence of four consecutive residues in the amino acid sequence of ShK, as compared to BgK (residues 12-15 using BgK numbering), leads to a different organization of the N-terminal part of the toxin. More precisely, the N-terminal part of BgK is successively composed of an extended strand (1-8), an helix 1 (9-16), and a loop (17-23), whereas in ShK, it is composed of an extended strand (1-8), a loop (9-13), and helix 1 (14-19). The structures of the C-terminal parts of the two toxins are more similar in the two toxins. Therefore, BgK and ShK adopt the same type of fold with, however, a number of marked structural differences located mostly in the N-terminal regions. Quite distinctly, charybdotoxin (ChTX), a typical scorpion toxin that blocks potassium channels (29), adopts a different fold with a short helix linked by two disulfides to a three-stranded beta -sheet (22). No beta -sheet structure was found in BgK.

Although BgK and ChTX have different structures, they both inhibit the binding of labeled dendrotoxin to potassium channels (11, 31), suggesting that they bind to regions of the channels that are recognized by dendrotoxin. In agreement with this view, previous observations reported that the sites recognized by ChTX (34, 35) and dendrotoxin (36, 37) commonly include the very conserved P-region of the pore of the channels. The channel region that is recognized by BgK is unknown; however, the toxin binds to several subtypes of Kv1 channels (i.e. Kv1.1, Kv1.2, and Kv1.3) with almost equal affinity,2 suggesting that the target of BgK also includes a highly conserved region of the channels, possibly the P-region. In the absence of further data on channel regions that are recognized by these toxins, one way to understand the molecular basis associated with their common capacity to inhibit the binding of dendrotoxin consists in comparing the sites by which these toxins recognize their targets.

With a view toward identifying the functionally important residues of BgK, we submitted the toxin to an alanine-scanning experiment and compared the ability of all the synthetic variants to inhibit the binding of dendrotoxin membranes from rat brain synaptosomes. Twenty-five positions out of 37 have been modified. Therefore, if one excepts the six half-cystines, which are likely to play a structural role, nearly 80% of the positions of BgK have been individually explored regarding their possible involvement in the binding of the toxin to dendrotoxin-sensitive sites in rat brain synaptosomes. Our data showed that introduction of an alanine at five positions, i.e. Lys25, Phe6, and Tyr26, and, to a lesser extent, at His13 and Ser23, caused a decrease in the ability of the toxin to compete with dendrotoxin for its specific binding sites, without changing the secondary structure of the toxin. Moreover, preliminary and unpublished data performed with cloned Kv1.2 channels and a number of BgK variants parallel those obtained with rat brain synaptosomes, strongly supporting the view that the residues Lys25, Phe6, Tyr26, and perhaps His13 and Ser23, are involved in the functional site of BgK. Of these residues, however, mutation at Lys25 most dramatically affected the capacity of BgK to inhibit dendrotoxin binding, suggesting that this particular lysine is the major binding actor of BgK. This finding agrees with recent observations made with ShK, a homologous K+ channel-blocking toxin from the sea anemone S. helianthus whose lysine 22, which corresponds to Lys25 in BgK, plays a critical binding role toward the same channels (38). Previously, ChTX was submitted to site-directed mutagenesis, and the residues by which the toxin binds to the voltage-sensitive Shaker K+ channel were identified (8, 39). Clearly, Lys27 was the most critical residue, with four neighboring amino acids (Tyr36, Met29, Asn30, and Arg34) whose mutation also affected the affinity of ChTX to the channel. Thus, the functional residues of ChTX form a homogeneous area located on the flat beta -sheet face that is exposed to solvent (8, 39) and that covers approximately 200 Å2 (18 × 12 Å). Although BgK has no beta -sheet structure, it nevertheless possesses a flat surface of similar size (21 × 9 Å), which is formed by the edge of the 9-16 helix and its two flanking loops. Strikingly, this surface harbors the functional residues of BgK (see Fig. 5A). Therefore, BgK and ChTX possess comparable flat surfaces, which include a similar small number of energetically important residues, among which a lysine is the major binding actor (Fig. 5B).


Fig. 5. A, stereoview of a structure of BgK and its functional residues. Lys25, whose mutation caused the larger decrease in inhibitory capacity, is shown in red. Tyr25 and Phe6, whose mutations caused a decrease in inhibitory activity by factors higher than 10, are shown in orange. His13 and Ser23, whose mutations caused the lowest effect, are in yellow. Note that these residues occupy a flat surface defined by an edge of the longest helix and the two sandwiching loops. The homogeneous surface that is covered by these residues is also shown in B, where the toxin atoms are represented in CPK. The color code of the functional residues is the same as in A.
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If one assumes that these lysines play a similar binding role in BgK and ChTX, their superimposition can be readily associated with superimposition of Tyr26 in BgK with Tyr36 in ChTX, two aromatic residues that play an important binding function in the toxins (Fig. 6, top). The distances that separate the Calpha of lysines from the center of the benzene rings of the tyrosines are 6.6 ± 1.0 Å. The common capacity of the two toxins to recognize potassium channels is therefore associated with the conservation of a similar diad of functional residues, a lysine and a close aromatic residue. The other functional residues are different in the two toxins and do not form any evident superimposable pattern. Remarkably, however, if one rotates ChTX 90° around a central axis localized near the crucial lysine, Phe6 of BgK superimposes with Tyr36 in ChTX (Fig. 6, bottom). Thus, by virtue of the four-fold symmetrical organization of the channel (40), Phe6 in BgK is likely to interact with the same aromatic binding site as Tyr26, but on another monomer of the channel.


Fig. 6. A, superimposition of the functional diad Lys27/Tyr36 (in pink) of the scorpion charybdotoxin whose backbone is in red, with either the functional diad Lys25 (violet)/Tyr26 (yellow) (top) or Lys25 (violet)/Phe6 (orange) (bottom) of BgK whose backbone is in cyan. Note that for both superimpositions to be achieved, the structure of the scorpion toxin has to be rotated by 90° around an axis centered around the common lysine. ChTX coordinates come from the Protein Data Bank.
[View Larger Version of this Image (86K GIF file)]


Similar diads are present in toxins that are homologous to BgK and ChTX. Thus, the diad Lys25-Tyr26 is conserved in the three known K+ channel-blocking toxins from sea anemones (Fig. 7). The situation is slightly more complex with K+ channel-blocking scorpion toxins. Thirteen K+ channel blocking scorpion toxins are presently known. They have been previously divided into four subfamilies (41). Three of these subfamilies possess the same Lys27-Tyr36 diad as in charybdotoxin. In contrast, all toxins forming the fourth subfamily, i.e. the two kaliotoxins and the three agitoxins (AgTX1-3), have a threonine at position 36. Strikingly, all these toxins uniquely possess a phenylalanine at position 24 or 25, located on the exposed face of the beta -sheet. Mutational studies of a member of this family (AgTX2) showed that this phenylalanine is involved in the binding to the Shaker potassium channel (42). Interestingly, the diad Phe25-Lys27 in AgTX2 can be superimposed on the diad Lys27-Tyr36 in ChTX provided the beta -sheets of the two toxins form a 90° angle. We suggest, therefore, that Tyr36 in toxins of three subgroups of scorpion toxins and Phe25 in the remaining toxins occupy the same binding site on the four-fold symmetrical channel, but on different monomers. Thus, the common capacity of sea anemone and scorpion toxins to recognize K+ channels is associated with the conservation of a functional diad, composed of an essential lysine assisted by a 6.6 ± 1.0 Å distant aromatic residue whose precise nature (Tyr or Phe) and location may differ from one toxin to another.


Fig. 7. Amino acid sequences of the three known homologous toxins from sea anemone that recognize potassium channels. BgK and ShK were, respectively, isolated from B. granulifera and S. helianthus (11, 28). In both names, K stands for potassium, the targeted ion channels, and the first two letters correspond to the initials of the producing species. AsKS, also called kaliseptine, has been found in Anemonia sulcata extracts (51).
[View Larger Version of this Image (8K GIF file)]


Why is such a diad conserved among two families of functionally similar but structurally unrelated proteins? In scorpion toxins, the positively ammonium group of the lysine of the diad may mimic K+ ions entering the pore, occluding the ion pathway (39, 43, 44). A similar role may be associated with the functional lysine of the toxins from sea anemones. The role played by the aromatic residue of the diad is less obvious. Recent bimutational analyses (42) showed that Phe25 in AgTX2 mainly interacts in the Shaker with the hydrophobic Met448, a position that is conserved or conservatively mutated (Ile, Val) in other K+ channels (45). Then, the conserved aromatic residues in the diads provide the toxin with two unique features. First, they offer the possibility for local hydrophobic interactions to take place between toxins and channels. Second, since their rings are somewhat parallel to the plane defined by the flat surface of the toxin, they allow the crucial lysine to protrude outside the toxin surface, thus being in an appropriate position to plug into the pore. Evidently, residues other than those of the diads also play some functional role in the toxins. These additional residues clearly differ from one toxin to another. Therefore, we suggest that the diads constitute a conserved functional core around which less conserved residues provide the different toxins with a proper binding specificity, which varies from one toxin to another. A similar situation that has also been found with snake curaremimetic toxins (5, 46).

In conclusion, two families of toxins with distinct folding patterns display a conserved functional diad. Each diad contains a critical lysine which should possess environmental and/or structural features that other lysines do not share and which allow it to exert a predominating recognition role toward potassium channels. Its location on a flat surface, its protruding position and the constant close proximity of an aromatic ring probably contribute to give a unique role to the functional lysine. Whether similar criteria are shared by other structurally unrelated but functionally similar toxins remains to be investigated.


FOOTNOTES

*   This work was supported by the Atomic Energy Commission, Direction de Recherches et Etudes Techniques, and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    Present address: CBS, CNRS-UMR 995, Université de Montpellier, 34060 Montpellier Cedex, France.
   To whom correspondence should be addressed.
1    The abbreviations used are: BgK, B. granulifera K+ channel-blocking toxin; HPLC, high performance liquid chromatography; r.m.s.d., root mean square deviation; ShK, S. helianthus K+ channel-blocking toxin; ChTX, charybdotoxin; AgTX, agitoxin; Tricine, N-tris(hydroxymethyl)methylglycine; NOE, nuclear Overhauser effect; DQF-COSY, double quantum filtered correlated spectroscopy.
2    J. Cotton, M. Crest, F. Bouet, N. Alessandri, M. Gola, M. Forest, E. Karlsson, O. Castaneda, A. L. Harvey, C. Vita, and A. Ménez, submitted for publication.
3    A. Lecoq, J. Cotton, M. Dauplais, C. L. C. de Medeiros, E. G. Rowan, A. L. Harvey, and A. Ménez, unpublished data.

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