Proc. Nati. Acad. Sci. USA Vol. 85, pp. 3329-3333, May 1988 Biochemistry Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels (ion-channel blocker/scorpion toxin/sequence homologies/snake neurotoxin) GUILLERMO GIMENEZ-GALLEGO*t, MANUEL A. NAVIAt, JOHN P. REUBEN§, GEORGE M. KATZ§, GREGORY J. KACZOROWSKI§, AND MARIA L. GARCIA§¶ Departments of *Growth Factor Research, tBiophysics, and Membrane Biochemistry, Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, NJ 07065 Communicated by Edward M. Scolnick, January 6, 1988

ABSTRACT Charybdotoxin (ChTX), a protein present in crepancies were noted in the determination of the molecular the venom of the scorpion Leiurus quinquestriatus var. he- mass of ChTX based on amino acid composition and electro- braeus, has been purified to homogeneity by a combination of phoretic mobility of the protein, which could be explained by ion-exchange and reversed-phase chromatography. Polyacryl- inhomogeneity of the preparation. amide gel electrophoresis, amino acid analysis, and complete The present study describes the purification of ChTX to amino acid sequence determination of the pure protein reveal homogeneity, the biological activity ofthe pure toxin, and the that it consists ofa single polypeptide chain of4.3 kDa. Purified chemical characterization of this peptide in terms of amino ChTX is a potent and selective inhibitor of the -220-pS acid composition and sequence. The primary structure Ca21 -activated K+ channel present in GH3 anterior pituitary uniquely identifies this molecule and reveals its similarity to cells and primary bovine aortic smooth muscle cells. The toxin other toxins of species as phylogenetically distant as snakes reversibly blocks channel activity by interacting at the external and marine worms. Based on these similarities, a tertiary pore ofthe channel protein with an apparentKd of2.1 nM. The structural model of ChTX has been generated from the primary structure of ChTX is similar to a number of neuro- published (7) x-ray coordinates of a-bungarotoxin deposited toxins ofdiverse origin, which suggests that ChTX is a member in the Brookhaven Protein Data Bank (8). At present, ChTX of a superfamily of proteins that modify ion-channel activities. is the only agent that has been identified to cause potent On the basis of this similarity, the three-dimensional structure selectiveblockofapamin-insensitiveCa2 + -activated K + chan- of ChTX has been modeled from the known crystal structure nels, and hence it should be useful as a probe for studying the of a-bungarotoxin. These studies indicate that ChTX is useful properties of these channels. A preliminary report of these as a probe of Ca2+-activated K+ -channel function and suggest findings has been made in abstract form (9). that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block. MATERIALS AND METHODS High-conductance Ca2+-activated K+ channels have been described in a variety of electrically excitable and nonexcit- Materials. Lyophilized venom of the scorpion Leiurus able cells (1). These channels provide a pathway by which quinquestriatus var. hebraeus was obtained from Latoxan cell repolarization can occur after membrane depolarization, Scorpion Farm (Rosans, France). GH3 cells were purchased and consequently they have been implicated in the regulation from the American Type Culture Collection, while primary of neuroendocrine secretion, in the control of muscle con- cultures of bovine aortic smooth muscle were obtained as tractility, and in a number of other cellular processes. described (10). + Electrophysiological Analysis. Both GH3 and aortic smooth However, to assess the physiological role of Ca2 -activated muscle cells were grown as described (11) and cultured for K+ channels and attempt their purification, potent specific 2-4 days on 25-mm glass coverslips before use in electro- inhibitors of these channels are required. physiological experiments. Single Ca2l -activated K +-chan- The venom of the scorpion Leiurus quinquestriatus var. nel currents were monitored in outside-out excised mem- hebraeus is known to inhibit a number ofdifferent K +-channel brane patches by conventional patch-clamp procedures (12). pathways (2, 3). A minor component of the crude venom, The samples to be assayed were added directly to an termed charybdotoxin (ChTX), was discovered to block re- experimental chamber in which the microelectrode contain- versibly a large-conductance (-200 pS) Ca2"-activated K+ ing the excised patch was suspended. channel from rat skeletal muscle plasma membrane vesicles Purification of ChTX. Lyophilized scorpion venom (480 that had been reconstituted into planar lipid bilayers (4). mg) was gently homogenized in 20 mM sodium borate (pH Subsequently, ChTX was also found to inhibit low-con- 9.0), clarified by centrifugation at 27,000 x g for 15 min, ductance (=35 pS) Ca2"-activated K+ channels in neurons passed twice through Millex-GV filters (pore size, 0.2 ,um) from Aplysia californica, but not block Na+, Ca2+, transient (Millipore) and loaded onto a Mono-S column (HR5/5; K+, or delayed rectifying K + channels in this preparation (5). Pharmacia) equilibrated with the same buffer, at a flow rate The properties ofChTX have been preliminarily characterized of 0.5 ml/min. When the optical absorbance at 280 nm of the (6). The toxin was reported to be a protein with an apparent eluate decreased to within 0.06 optical density units above molecular mass of 10 kDa and to inhibit Ca2+-activated that of the elution buffer, the retained material was eluted K+-channel function with a Kd of3.5 nM. The same work also with a linear gradient of NaCl (0.75 M/hr). Fractions of this reports the amino acid composition of the protein and identi- column containing ChTX activity were loaded onto an fication of the N-terminal amino acid. However, some dis- Abbreviation: ChTX, charybdotoxin. The publication costs of this article were defrayed in part by page charge tPresent address: Centro de Investigaciones Biologicas, C.S.I.C., payment. This article must therefore be hereby marked "advertisement" Velazquez 144, 28006 Madrid, Spain. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed.

Downloaded by guest on September 26, 2021 3329 3330 Biochemistry: Gimenez-Gallego et al. Proc. Natl. Acad. Sci. USA 85 (1988)

Ultrapore RPSC reversed-phase column (Beckman) equili- 0 brated with 10 mM trifluoroacetic acid, and eluted at a flow 1. 100 rate of 0.5 ml/min with a 0-20% linear gradient of isopro- c panol/acetonitrile (2:1) over 30 min. Fractions to be assayed from this chromatography were made either 350 mM in NaCl L or 0.5% in bovine serum albumin, lyophilized, and later L m reconstituted to their original volume with 20 mM sodium 4.) .0 borate (pH 9.0). L 0 . 50 U U Polyacrylamide Gel Electrophoresis. Samples were heated of (1000C, 2 min) with or without dithiothreitol (100 mM) in 3% mM of NaDodSO4/63 Tris*HCl, pH 6.2. Gels (1.6 mm thick) z a continuous concentration (25.7% polyacrylamide; acrylam- T- ide/bisacrylamide ratio, 37:1) were prepared and run for 24 m 0 hr at 3.3 V per cm of gel as described (13). Gels were fixed U, a 0 m as described (14) except that the first two steps were for 2 hr E: 5 10 15 5 10 15 each with hourly changes of solution and the glutaraldehyde EFFLUENT VOLUME (ml) treatment was also carried out for 2 hr. Fixed gels were silver stained (15). FIG. 1. Purification of ChTX. (A) Mono-S chromatography of 80 mg oflyophilized scorpion venom treated as described. Buffer B was Amino Acid Analysis and Extinction Coefficient Determina- 375 mM NaCl in 20 mM sodium borate (pH 9) and the gradient is tion. The optical absorbance spectrum of protein samples depicted as a dashed line. ChTX activity eluted where indicated by eluted from the reversed-phase column was digitized in a the horizontal bars. (B) Reversed-phase chromatography of an Hewlett-Packard 8450A UV/VIS spectrophotometer. Ali- aliquot of fraction A of the Mono-S chromatography. Buffer B is quots (o10-3 optical absorbance units at 280 nm) were isopropanol/acetonitrile (2:1). (Inset) A silver-stained NaDodSO4/ analyzed for their amino acid composition as described (16) polyacrylamide gel of reduced ChTX. Approximately 280 ng of the and the protein content was correlated with the recorded material obtained from the reversed-phase chromatography was absorbance. treated as described. Molecular mass standards are expressed in Amino Acid Sequence Determination. Pure ChTX was kDa. alkylated, digested with Staphylococcus aureus V8 protease, and sequenced as described (17, 18). Endoproteinase Lys-C column (51 mg). Reversed-phase chromatography of pool A (Boehringer Mannheim GmbH) digestion was carried out at (Fig. LA) resulted in a single major peak, which coelutes with 370C for 24 hr in 20 mM sodium phosphate (pH 7.6) at a ratio ChTX activity (Fig. 1B). In the case of pool B, two peaks of 55 pFg of ChTX per enzyme unit. Phenylthiohydantoin- were separated (data not shown); one with an identical derivatized amino acids were detected by using an on-line elution time, specific activity, and amino acid composition as phenylthiohydantoin analyzer (Applied Biosystems, Foster the one of Fig. 1B, while the other is an unrelated protein. City, CA; model 120A). Pyroglutamate aminopeptidase Together, both active fractions represent 60o of the total (Boehringer Mannheim GmbH) digestion was carried out protein content ofpools A and B (Fig. LA). Typically, 100 mg according to described procedures (19) with a molar ratio of of lyophilized venom yields 230 ug of ChTX. enzyme to ChTX of 1:10 and adding all enzyme at the Characterization of ChTX. NaDodSO4/polyacrylamide beginning of the reaction. gels of purified ChTX with (Fig. 1B, Inset) or without (data

RESULTS AND DISCUSSION 100 Protein Purification. The biological activity of ChTX has been ascertained by electrical analysis of single high- HI- 0 conductance (=220 pS) Ca2'-activated K+ channels in 75 Z isolated plasma membrane patches derived from either aortic U0 smooth muscle or GH3 pituitary cells possessing this activity. Z U Measurements have been accomplished by using patch- 50 clamp techniques with excised patches of membranes ori- Z z ented with an outside-facing-out polarity. Inhibition ofCa2"- 'a- activated K+ channels by ChTX in these preparations results I CL 25 in an increase in silent periods between bursts of channel activity, as has been described with reconstituted Ca2"- activated K+ channels from skeletal muscle (4, 6). The appearance of such characteristic behavior was used to follow ChTX activity during purification. The purification protocol that was developed is based on the procedures of - LOG CChTX (CM) Smith et al. (6). However, the first chromatofocusing step in FIG. 2. Inhibition ofCa2+-activated K+ channels in GH3 cells by SP-Sephadex has been replaced with cationic-exchange chro- ChTX. A 5- to 10-MQ microelectrode containing (in mM) 140 KCI, matography in a Mono-S column. In addition, both the 2 MgCl2, 7 EGTA, 6 CaCl2, and 10 Hepes (pH 7.3) was used to excise organic and the aqueous solvents in the reversed-phase an outside-out membrane patch from a GH3 cell. The patch was chromatography have been changed. These higher-resolution bathed in a medium consisting of(in mM) 135 NaCl, 5 KCI, 10 CaCl2, chromatographic steps have increased considerably the re- 2 MgCl2, and 10 Hepes (pH 7.3), and held at a potential of + 20 mV. and the At After recording channel activity, increasing concentrations of puri- producibility efficiency of purification procedure. fied ChTX were added to the bath and allowed to equilibrate. pH 9.0, 84% of the 280-nm absorbance of the filtered extract Inhibitory activity was monitored as the ratio of events per sec with is not retained by the column and ChTX activity eluted ChTX to events per sec in the control. Between additions of ChTX, between 300 and 340 mM NaCl in two consecutive peaks that the chamber was perfused with toxin-free medium, whereupon were collected separately (Fig. LA). The active protein control channel activity was regained, indicating that ChTX block is constitutes 0.61% of the total protein injected onto the completely reversible. Downloaded by guest on September 26, 2021 Biochemistry: Gimenez-Gallego et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3331

F (Q.-I i ______A- _fw _f- _Am _am _am _~. _f. t--t._m_M- 80 _tm _m ism JEW _. _10 _m _m _t M_ _ft A_ _~ _N _m Am _00 _m. *VS-i I, VS-3 I UELC-4 I -LC-5M 1 i- cC-1 I- ELC-3 - FIG. 3. The complete amino acid sequence of ChTX. peptides. *, N terminus-blocked peptide that was treated on the sequenator glass fiber filter with 25 Al of 11.7 M HC1. All residues identified by amino-terminal sequence determinations are denoted by single-headed arrows pointing to the right. Residues identified at the carboxyl terminus by timed digestion of 5 nmol of ChTX with 500 pmol of carboxypeptidase A (Boehringer Mannheim GmbH) (16) are denoted with single-headed arrows pointing to the left. The observed and integer amino acid compositions ofChTX, determined as described in Materials and Methods, are as follows: Asx, 2.89, 3; Glx, 2.77, 3; Ser, 4.79, 5; Gly, 1.17, 1; His, 1.09, 1; Arg, 3.08, 3; Thr, 4.05, 4; Tyr, 1.12, 1; Val, 1.98, 2; Met, 1.24, 1; Cys, 5.%, 6; Leu, 1.14, 1; Phe, 0.97, 1; Lys, 3.76, 4; Trp, not determined. not shown) disulfide reduction by dithiothreitol display a was shown to be devoid of lysine by amino acid analysis. single band at 4.35 kDa. Because the molecular mass is not Given the specificity of this enzyme, this peptide must be altered by reduction, ChTX appears to be composed of a derived from the carboxyl-terminal end of the protein. The single polypeptide chain. Since the lower limit of protein sequence of this peptide correlates with the last residues detection in these polyacrylamide gels is =1 ng, the most identified by sequencing peptide V8-2 and pyroglutaminase- abundant contaminant in this preparation must be <0.25% of treated ChTX. The carboxyl-terminal sequence was con- the total amount of ChTX present. firmed by digestion of the whole protein with carboxypepti- The amino acid composition ofChTX is given in the legend dase A (Fig. 3). The molecular mass of ChTX, calculated of Fig. 3. This amino acid composition resembles that of from this sequence, is 4353 Da. Although two potential Smith et al. (6) except in proline content, ifit is assumed that asparagine glycosylation sequences (Asn-Xaa-Ser/Thr) exist the alanine residue in their determination is a contamination at positions 4 and 22 of ChTX, it is unlikely that this protein and if the scaling is consequently done on the basis of either is glycosylated since the yield ofasparagine at these positions leucine, phenylalanine, or tyrosine. The extinction coeffi- during sequencing is not less than expected, and there is good cients of ChTX calculated from the amino acid analysis as agreement in the molecular mass of ChTX as calculated from described in Materials and Methods are 1.52 (mg/ml)-l electrophoretic mobility and amino acid composition. cm 1 at 280 nm and 15.05 (mg/ml)-1'cm-' at 215 nm. Amino Acid Sequence Similarity. In a search ofthe National Single-channel analyses of the action of pure ChTX reveal Biomedical Research Foundation protein sequence data that the toxin reversibly blocks the =220-pS Ca2 '-activated bank,11 ChTX appears to be unique. ChTX was also com- K+ channel of GH3 cells by binding only at the external face pared with other known peptide inhibitors of K+ channels. A of the channel, as suggested by Miller et al. (4), without high level of similarity was detected between ChTX and affecting mean channel conductance or significantly modify- noxiustoxin, a toxin from the venom of the scorpion Centu- ing individual channel open times. According to previous roides noxius that blocks the delayed rectifier K+ channel of work (6), ChTX appears to block channel activity by a simple the squid giant axon (20). The best alignment between ChTX bimolecular inhibition process. Therefore, determination of and noxiustoxin (Fig. 4, I) is of 8.17 SD from random. the concentration of ChTX that results in a 50% probability Interestingly, ChTX displays little similarity with apamin, an of channel block should yield an apparent dissociation inhibitor of a low conductance Ca2+-activated K+ channel constant (Kd) for ChTX under defined experimental condi- (22), or with dendrotoxin and mast-cell degranulating pep- tions. As shown by the data of Fig. 2, pure ChTX blocks the tide, both blockers of rapidly inactivating K+ channels Ca2 + -activated K+ channel ofGH3 cells with a Kd of2.1 nM. (A-type current; ref. 23). In addition, purified ChTX (100 nM) has no effect on either The ChTX sequence has a remarkably high concentration rapidly inactivating or slowly inactivating Ca2+ currents or of basic residues between positions 21 and 28, making this on rapidly inactivating K+ currents in GH3 cells, indicating region a potential site of interaction with the Ca2 +-activated specificity of action of the toxin. Titrations of ChTX activity K+ channel. Therefore, a search of the National Biomedical performed with isolated membrane patches obtained from Research Foundation data bank was repeated to detect bovine aortic cells give a nearly identical Kd value. possible homologies with this region. Thirteen neurotoxins Amino Acid Sequence. The complete amino acid sequence belonging to species as distant as scorpions, marine worms, of ChTX is given in Fig. 3. The 37-residue sequence is well and snakes were listed (Fig. 4, legend). Of these, the best determined, with all regions confirmed by overlapping pep- alignment is between ChTX and neurotoxin P2 of the scor- tides. Attempts to sequence ChTX from its amino-terminal pion Androctonus mauretanicus var. mauretanicus (Fig. 4, end were unsuccessful, indicating a blocked N terminus. No I), whose mechanism of action is unknown (24). This align- phenylthiohydantoin-derivatized amino acids were detected ment is 4.1 SD from random, which corresponds to a 1 in after three Edman degradation cycles of peptide V8-1, 32,000 chance that the alignment is nonsignificant. Alignment indicating that this peptide was derived from the amino- of the 21-28 segment of ChTX with the equivalent region of terminal end of the protein. The peptide was unblocked by the marine worm neurotoxins (25) indicated the cysteine treating the sequenator glass fiber sample filter with HCO for residues to be in register between amino acids 10 and 45. This 30 min (Fig. 3, legend). The resulting sequence agrees with alignment vs. one of the homologous worm sequences at 2.5 the amino acid composition of this peptide except for two SD above random is shown (Fig. 4, II). The probability is missing amino acids, phenylalanine, and glutamic acid or <0.62% that this value reflects zero correlation between the glutamine, suggesting that the blocked amino-terminal resi- compared sequences. It is noteworthy that the marine worm due is pyroglutamine. Equivalent results were obtained with neurotoxins act by prolonging the repolarization phase ofthe peptide ELC-4, although in this case the HCO treatment was action potential in crustacean axon (25), a mechanism con- extended to 60 min. A sequence of37 residues, accounting for sistent with K+-channel block. The list of homologous all residues of the amino acid composition of ChTX, except sequences (Fig. 4, legend) also includes 10 well-characterized one glutamic acid or glutamine, was obtained when the protein was treated with pyroglutaminase. A peptide was IProtein Identification Resource (1986) Protein Sequence Database generated by digestion with endoproteinase Lys-C, which (Natl. Biomed. Res. Found., Washington, DC), Release 8.0. Downloaded by guest on September 26, 2021 3332 Biochemistry: Gimenez-Gallego et al. Proc. Nad. Acad Sci. USA 85 (1988)

ChTX EI *V S C-T T E C i-S-V C Q-R [ H-N-T S-R-G- -K-C-H-N K F-C R C-Y s

1 (39) T-I-I N --V K C-T S- K Q C S-K-P C K-E L Y-G-S S ilGilK-C-N-N- G I-C K C-Y N-N j... . .I . .. . U' '--SU''-L.*' *'- 2 (35) -C: T -D-P-YT E C A-T-C C - -- - -G-G R-G--K-C V-G-P-Q CN-R-I

II ChTX E-F-T-N-V-S C T-T-S- -K-E C W-S-V C Q-R-L-H-N-T-SR C N-NR RC - --- -R VY-S

3 (55) P-A-C-E-N-N C R--Q-Y-D-D C -I-K C Q-G-K-W-A-G-K [-G-i-C A-A-H- C A-V-Q-T-I-S C N--

III ChTX E-F N ]S T-T-S-K-E- W-S- -V- Q-R-L-H-N -S-R-G-K C-N-N-K-K -R y[~ 56 4 (73) -P-S-S-A1VT P-P-G-E-N-L Y-R-K-N-i D-A-F-C- -S S-R-G-K V-V-E-L-G A-A-T P Sl 8 LU 4t 5 (71) -D-V [ S-Q-I A-D-G- -N-V y-T-K-TiW D-N-F-C- -A S-R-G-K R-V-D-L-G A-A-T P-T- 81 47 6 (71) -fD-I T S-K-D P-N-G- -R-V Y-T-K-T-i D-G-F-C- -S S-R-G-K R-V-D-L-G A-A-T P-T- 81 48 7 (73) -D-A - S-Q-T P-D-G-Q-D-I Y-T-K-T-i D-G-F-C- -S S-R-G-K R-I-D-L-G A-A-T P-K- 48 FIG. 5. Modeled three-dimensional structure of ChTX. The 8)73) -D-V-K-S-E-I P-A-G-Q-D-I Y-T-E-T-W D-A-W-C- I-S-R-G-K R-V-D-L-G A-A-T P-I- a-carbon backbone of ChTX (green) is shown superimposed on that ofa-bungarotoxin (red). The overlap between the structures is shown in yellow, where red and green mix. The conserved disulfide bridges FIG. 4. The following scorpion (I), marine worm (II), and snake appear in white, while those exclusive to either a-bungarotoxin or toxins (III) are compared with ChTX: 1, noxiustoxin, Centruroides ChTX appear in orange and blue, respectively. Both structures are noxius; 2, neurotoxin P2, Androctonus mauretanicus var. maureta- assigned the equivalent a-bungarotoxin residue numbers. The ami- nicus; 3, neurotoxin B-II, Cerebratulus lacteus; 4, a-bungarotoxin, no-terminal residues ofa-bungarotoxin and ChTX are labeled N1 and Bungarus multicinctus; 5, long neurotoxin 2, Naja melanoleuca; 6, N10, whereas the corresponding carboxyl termini are labeled C74 long neurotoxin 1, Naja naja; 7 and 8, long neurotoxin 2, and 1, and C50, respectively. Positively charged residues (i.e., lysine, Ophiophagus hannah. Numbers in parentheses indicate the number histidine, and arginine; K, H, and R, respectively) are in blue. of residues of each protein. When the comparison does not include a-Bungarotoxin coordinates were obtained from the Brookhaven the full length of the protein, the residue number at the amino and Protein Data Bank (7, 8) and were displayed and manipulated with carboxyl terminus of the considered fragment appears above the the graphics program FRODO (30, 31) operating on an Evans & sequence. Similarities were evaluated with the ALIGN program (21), Sutherland PS330 system. Amino acid substitutions, deletions, and using a 112-point-accepted mutation (PAM) matrix (I), 192-PAM additions were made according to Fig. 4, maintaining as closely as matrix (II), and 160-PAM matrix (III). The BIAS and GAP param- possible the similarity between the structures. eters were iteratively optimized. Toxins included in II and III and neurotoxin P2 of Androctonus mauretanicus (I) were detected in a zero and a SD from the mean of 1.0; ref. 28).** The search of the ChTX21-28 region in the National Biomedical Research correlation coefficient (rH) between ChTX and a-bungaro- Foundation sequence data bank, by using the IFIND program toxin is 0.71. The probability that this value reflects zero (Intelligenetics, Mountain View, CA). Only residues identical with x ChTX are framed. Additional identified toxins were as follows: correlation is 9.9 10-8. This similarity in the solvent neurotoxin B-IV, Cerebratulus lacteus; long neurotoxin 1, Naja exposure indicates a high similarity between the tertiary melanoleuca; long neurotoxin 1, Laticauda semifasciata; long neu- structure of ChTX and the corresponding region of a- rotoxin 1, Naja naja var. oxiana; long neurotoxin 1, Notechis bungarotoxin. The three-dimensional structural similarity is scutatus; long neurotoxin 1, Acantophis antarticus. The single-letter also supported by analysis ofthe minimum mutation distance amino acid code is used. between these two sequences (29), which is 1.03 mutations per amino acid, -6 SD from random. snake neurotoxins (26), which are all members of the long Given this striking similarity between ChTX and region neurotoxin family. The alignments between these neuro- 10-48 of a-bungarotoxin, a model was constructed for the toxins and ChTX that score >3 SD from random are shown three-dimensional structure of ChTX based on the solved (Fig. 4, III). The highest score (3.4 SD from random) crystal structure of a-bungarotoxin (in the following discus- corresponds to the alignment between ChTX and a- sion ofthe model, both structures are assigned the equivalent bungarotoxin. The probabilities that the obtained scores a-bungarotoxin residue number according to Fig. 4; there- reflect zero correlation between ChTX and these snake fore, the amino- and carboxyl-terminal residues of ChTX neurotoxins (Fig. 4, III) are between 1.3 x 10-3 and 4 x become Glu-10 and Ser-50, respectively). Fig. 5 illustrates an 10-4. Interestingly, the region ofgreatest similarity between a-carbon plot of the ChTX model superimposed on the ChTX and a-bungarotoxin (residues 23-27 of ChTX) corre- structure of a-bungarotoxin. A large degree of overlap sponds to a highly conserved region of the long neurotoxins (shown in yellow, where red and green mix) is evident that is critical for the interaction at their receptor (26). between these two structures. The region that shares identity The similarity of ChTX with toxins of species as phyloge- with ChTX includes the whole central loop and some residues netically distant as those listed in Fig. 4 is somewhat sur- of the first and third loops of a-bungarotoxin (26). Energy prising. Perhaps all these toxins are versions of normal minimization (32) studies indicate that the proposed model is proteins with the nontoxic physiological function of modu- a stable molecular structure (R. Blevins, personal commu- lating ion-channel activities. It may be that all of these nication). Only one disulfide bridge (residue 16 to 44; Fig. 5, channel modulating proteins evolved from a common ances- and a- tral gene encoding a singular ion-channel modulator whose white notation) can be conserved between ChTX mode of action became diverse as ion channels evolved, bungarotoxin. A second disulfide bridge (residue 23 to 48; acquiring different functional properties. Three-Dimensional Structure of ChTX. Upon examination **By using the normalized exposure indexes, the correlation coef- ficient (rH) between the two homologous domains of rhodanase, of Fig. 4, a similarity between the hydropathic patterns of cytochrome c2 of Rhodospirillum rubrum and cytochrome c551 of ChTX and the long neurotoxins becomes evident. This can be Pseudomonas aeruginosa, and cytochrome c2 of Rhodospirillum evaluated by the method of Sweet and Eisenberg (27), with rubrum and cytochrome c of tuna are 0.43, 0.44, and 0.67, the solvent exposure index ("1-f," normalized to a mean of respectively. Downloaded by guest on September 26, 2021 Biochemistry: Gimenez-Gallego et al. Proc. Natl. Acad. Sci. USA 85 (1988) 3333 Fig. 5, blue notation) was readily formed in the ChTX model 1. Peterson, 0. H. & Maruyama, Y. (1984) Nature (London) 307, by a small reorientation of the C terminus. The remaining 693-6%. bridge (residue 17 to 27; Fig. 5, blue notation) did require a 2. Abia, A., Lobaton, C. D., Moreno, A. & Garcia-Sancho, J. (1986) Biochim. Biophys. Acta 856, 403-407. reconstruction vs. the solved a-bungarotoxin structure to 3. Castle, N. A. & Strong, P. N. (1986) FEBS Lett. 209, 117-121. make the sulfhydrils point toward each other. This results in 4. Miller, C., Moczydlowski, E., Latorre, R. & Philips, M. (1985) a more conventional disulfide bond that relieves the strong Nature (London) 313, 316-318. distortion of the structure by disulfide bond 29-33 in this 5. Hermann, A. & Erxleben, C. (1987) J. Gen. Physiol. 90, 27-47. region of a-bungarotoxin. The most 6. Smith, C., Philips, M. & Miller, C. (1986) J. Biol. Chem. 261, striking differences 14607-14613. between both structures are the large C-terminal deletion of 7. Agard, D. A. & Stroud, R. M. (1982) Acta Crystallogr. Sect. A 24 residues and a further N-terminal deletion of 9 residues. 38, 186-194. These deletions have the effect ofuncovering the central loop 8. Bernstein, F. C., Koetzle, I. F., Williams, G. J. B., Meyer, structure of ChTX. E. F., Jr., Brice, M. C., Rodgers, J. R., Kennard, O., Shima- Circular dichroism spectra of the long neurotoxins reveal nouchi, T. & Tasumi, M. (1977) J. Mol. Biol. 112, 535-542. 9. Garcia, M. L., Gimenez-Gallego, G., Navia, M., Katz, G., that these proteins contain a high proportion of (3-structure Reuben, J. P. & Kaczorowski, G. J. (1988) Biophys. J. 53, 151a (26). In the solved a-bungarotoxin structure, there are three (abstr.) strands of the polypeptide chain that adopt a triple-stranded 10. Ross, R. (1971) J. Cell Biol. 50, 172-186. antiparallel /3-pleated sheet conformation. Two of these 11. King, V. F., Garcia, M. L., Himmel, D., Reuben, J. P., Lam, strands are in the second loop, the one entirely conserved in Y. K., Pan, J. X., Han, G. Q. & Kaczorowski, G. J. (1988) J. Biol. Chem., 263, 2238-2244. ChTX. Furthermore, the disappearance in ChTX of the 12. Hamil, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth, disulfide bridge equivalent to the one between residues 29 F. J. (1981) Pflugers Arch. 391, 85-100. and 33 of a-bungarotoxin should enlarge this P-region, as is 13. Fling, S. P. & Gregerson, D. S. (1986) Anal. Biochem. 155, the case with the short neurotoxins. Hence, the relative 83-88. amount of B-structure should increase substantially in ChTX. 14. Morrisey, J. H. (1981) Anal. Biochem. 117, 307-310. 15. Wray, W., Boulikas, T., Wray, V. P. & Hancock, R. (1981) The circular dichroism spectrum of ChTX agrees with this Anal. Biochem. 118, 197-203. prediction. Its maximum (195 nm), its minimum (217 nm), and 16. Gimenez-Gallego, G. & Thomas, K. A. (1987) J. Chromatogr. the ratio [601/[60215 (==2) are quite close to the theoretical 409, 299-304. values of poly-L-lysine in /3-conformation (33). The percent- 17. Gimenez-Gallego, G., Rodkey, J., Bennett, C., Rios Candelore, age of /3-structure content calculated from this spectrum (34) M., DiSalvo, J. & Thomas, K. (1985) Science 230, 1385-1388. is -70%, a value that is considerably higher than the one 18. Gimenez-Gallego, G., Conn, G., Hatcher, V. B. & Thomas, K. A. (1986) Biochem. Biophys. Res. Commun. 138, 611-617. calculated for the long neurotoxins (26). 19. Podell, N. N. & Abraham, G. N. (1978) Biochem. Biophys. Res. In the proposed model of the tertiary structure of ChTX Commun. 81, 176-185. (Fig. 5), there are two clusters ofpositively charged residues. 20. Carbone, E., Prestipino, G., Spadavecchia, L., Franciolini, F. & The first is at the bottom of the central loop of the molecule. Possani, L. D. (1987) Pflugers Arch. 408, 423-431. This includes three basic residues, Arg-30A, Arg-36, and 21. Dayhoff, M. O., ed. (1978) Atlas of Protein Sequence and Lys-38, and possibly a charged His-32, all of whose side Structure (Natl. Biomed. Res. Found., Washington, DC), Vol.5, chains are located on the same side of the molecule. The Suppl. 3, pp. 345-358. 22. Romey, G., Hughes, M., Schmid-Antomarchi, H. & Lazdunski, second is found at the top of the molecule and includes M. (1984) J. Physiol. (Paris) 79, 259-264. Lys-20, Lys-42, Lys-43, and Arg-45. There is, however, a 23. Bidard, J. N., Mourre, C. & Lazdunski, M. (1987) Biochem. possibility that Lys-42 forms a salt bridge with Glu-21. This Biophys. Res. Commun. 143, 383-389. hypothesis is consistent with the fact that in noxiustoxin both 24. Rochat, H., Bernard, P. & Couraud, F. (1979) Adv. Cytophar- residues are simultaneously replaced by two noncharged macol. 3, 325-334. amino acids. Interestingly, all residues of both clusters, 25. Blumenthal, K. M., Kein, P. S., Heinrikson, R. L. & Kem, except Lys-20, are on the same side of the molecule, giving W. R. (1981) J Biol. Chem. 256, 9063-9067. a 26. Dufton, M. J. & Hider, R. C. (1982) CRC Crit. Rev. Biochem. ChTX distinct charge polarity on one face vs. the other. The 14, 113-172. positively charged face might be involved in the interaction 27. Sweet, R. M. & Eisenberg, D. (1983) J. Mol. Biol. 171, 479-488. by which, as it has been proposed, ChTX blocks Ca2"- 28. Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H. & activated K+ channels (4, 35). Obviously, the proposed Zehfus, M. H. (1985) Science 229, 834-838. model is a preliminary approach to the three-dimensional 29. Dickerson, R. E. (1971) J. Mol. Biol. 57, 1-15. structure of ChTX. However, the findings presented in this 30. Jones, T. A. (1982) in Computational Crystallography, ed. study form a basis for developing the structure-activity Sayer, D. (Clarendon, Oxford), pp. 303-318. relationship of the ChTX molecule and indicate that ChTX 31. Bush, B. L. (1984) Comput. Chem. 8, 1-11. 32. Singh, V. C., Weiner, P. K., Caldwell, J. W. & Kollman, P. A. will be a useful probe for elucidating the function of Ca2+ - (1986) AMBER (Department ofPharmaceutical Chemistry, Uni- activated K+ channels. versity of California, San Francisco), Version 3.0. 33. Greenfield, N. & Fasman, G. D. (1969) Biochemistry 8, We thank William Randall for his assistance in obtaining circular 4108-4116. dichroism spectra of ChTX, John Jacobs and Chris Dunwiddie for 34. Yang, J. T., Wu, C. S. C. & Martinez, H. M. (1986) Methods useful discussions, and Kenneth A. Thomas for discussions and Enzymol. 130, 208-269. critical reading of the manuscript. 35. MacKinnon, R. & Miller, C. (1987) Biophys. J. 51, 53a (abstr.). Downloaded by guest on September 26, 2021