STRUCTURE-FUNCTIONS
OF NEUROTOXINS
be recognized in the formulation of models or mechanisms of cooperative ligand binding t o hemoglobin. Acknowledgment We extend our appreciation t o Mrs. Verna R . Laman, Miss Ursula Feucht, and Mrs. Ester Gayle for their excellent technical assistance and t o Dr. Donald G. Davis for his preliminary contributions t o this study. We are grateful t o Dr. Max F. Perutz who personally transported the sample of Hb Sydney from Cambridge t o Pittsburgh for our nmr study and t o Dr. William D. Phillips for helpful and stimulating discussions on the intensity calibration of the ring-currentshifted resonances. References Abraham,R. J. (1961), Mol. Phjs. 4,145. Antonini, E. (1965), Physiol. Rer;. 45,123. Carrell, R. W., Lehmann, H., Lorkin, P. A., Raik, E., and Hunter, E. (1967), Nature (London) 215,626. Dadok, J., Sprecher, R. F., Bothner-By, A. A., and Link, T. (1970), Abstr. 11th Exp. N M R Conf., Pittsburgh, Pa. Dayhoff, M. 0. (1969), Atlas of Protein Sequence and Structure, Silver Spring, Md., National Biomedical Research Foundation. Dozy, A. M., Kleihauer, E. F., and Huisman, T. H. (1968),
J. Chromatogr. 32,723. Drabkin,D. L. (1946), J. Biol. Chem. 158,703. Geraci, G., Parkhurst, L. J., and Gibson, Q. H. (1969), J . Biol. Chem. 244,4664. Glasoe, P. D., and Long, F. A. (1960), J. Phys. Chem. 64,188. Ho, C., Davis, D. G., Mock, N. H., Lindstrom, T. R., and Charache, S. (1970), Biochem. Biophys. Res. Commun. 38,779. Johnson, C. E., and Bovey, F. A. (1958), J . Chem. Phys. 29, 1012. McDonald, C. C., and Phillips, W. D. (1967), J. Amer. Chem. SOC.89,6332. McDonald, C. C., Phillips, W. D., and Vinogradov, S. N. (1969), Biochem. Biophys. Res. Commun. 36,442. Muller, C. J., and Kingma, S. (1961), Biochim. Biophys. Acta 50,595. Perutz, M. F. (1969), Proc. Roy. SOC.,Ser. B 173, 113. Perutz, M. F. (1970), Nature (London) 228,726. Schroeder, W. A., Shelton, J. R., Shelton, J. B., Cormick, J., and Jones, R . T. (1963), Biochemistry 6,2395. Shulman, R . G., Wiithrich, K., Yamane, T., Patel, D. J., and Blumberg, W. E. (1970), J . Mol. Biol. 53,143. Sternlicht, H., and Wilson, D. (1967), Biochemistry 6,2881. Van Gelder, B. F., and Slater, E. C. (1962), Biochim. Biophys. Acta 58, 593. Zade-Oppen, A. M. M. (1963), Scand. J . Clin. Lab. Incest. 15,491.
Structure-Function Relationships of Neurotoxins Isolated from Naja haje Venom. Physicochemical Properties and Identification of the Active S t e t Robert Chicheportiche, Catherine Rochat, Francois Sampieri, and Michel Lazdunski*
Neurotoxins I and I11 of Naja haje, like other miniproteins with a high content of disulfide bridges, present an unusually high resistance to denaturing conditions a t neutral pH. Optical rotatory dispersion measurements with neurotoxin I indicate 2 conformational changes a t acidic pH. One of them is controlled by the unmasking of a carboxylate side chain having an apparent pK of 2.0 a t 13". Titration, nitration, and acetylation show that tyrosine-24 is masked in the native conformation of neurotoxin I. This residue is essential ABSTRACT:
S
nake neurotoxins are made of single peptide chains of 60-74 amino acids cross-linked internally by 4 or 5 disulfide bridges. These miniproteins are well suited for comparative
t From the Centre de Biologie Moleculaire du Centre National de la Recherche Scientifique 3 1, chemin Joseph Aiguier, Marseilles, France, and the Laboratoire de Biochimie, Facult6 de Medecine Secteur Nord, Marseilles, France. This work was supported by the Centre National de la Recherche Scientifique (R. C . P. no. 166), the Delegation Gknirale de la Recherche Scientifique et Technique and the Commissariat a I'Energie Atomique.
for the stabilization of an active structure. Its nitration abolishes toxicity. Difference spectra and fluorescence data indicate a masking of the tryptophan-28 side chain. Formylation does not abolish toxicity. Acetylation and maleylation indicate that lysine residues are essential elements of the active site. Dansylation affects selectively 2 superreactive residues, lysines-26 and -46. The kinetics of the loss of toxicity closely parallels the covalent modification of these two lysine residues.
sequence studies, analyses of conformational properties and determinations of active sites. Due t o the increasing use of neurotoxins t o study neurotransmitters-receptors interactions (Changeux er al., 1970; Miledi et al., 1971) it is of prime importance to understand the structure-function properties of these proteins. Neurotoxins I and 111 obtained from Naja haje venom have been selected for this work. The sequence of neurotoxin I which consists of 61 amino acid residues and 4 disulfide bridges has been recently elucidated (Botes and Strydom, BIOCHEMISTRY,
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1969). The sequence of neurotoxin JII which contains 71 amino acids and 5 disulfide bridges is not yet known. Parallel studies on scorpion neurotoxins are also carried out in our laboratories (Rochat et a/., 1970a,b; Chicheportiche and Lazdunski, 1970).
e t ui.
Nitrcition. Nitration was carried otit according to the genera1 method of Sokolovsky er n / . (1966) in a 0.1 M Na phosphate buffer at p H 8.0, 20' during 4 h r . The reaction w \ stopped by filtration on Bio-Gel P2 eyuilibratcd ;it pH 6.9 with 0.1 1 1 ammonium acetate. The nitrotyrosine content was measured by amino acid analysis i n a Brckman 120 C analyser. Materials and Methods Cliet,iical Modijcarion of Curboxj~lutes.Carboxylic fiincNeurotoxins and Chemicals. Neurotoxins I and 111 were tions were transformed into amides with glycine ethyl ester under thc influence of l-ethyl-3-(3-dimethylaniinopropyl)purified from the venom of Naja liaje as previously described by Miranda et a/. (1970a). The proteins were shown to be carbodiimide by the method of Hoare and Koshland (lY67). The reaction was carried out at pH 4.7, 25". with I 11 [ I T I homogeneous by equilibrium chromatograghy on Amberlite CG-50, starch gel electrophoresis, and amino acid composiglycine ethyl ester and 0.065 \ I I-ethyl-3-i3-dirnethylamlnotion. propy1)carbodiimide. The nenrotoyin concentration ~ i i 0.33 s Toxicity was determined according to Miranda er d. mg'nil. The reaction was \topped after 3 hr by chromatography through a Sephadev G-25 ~ ~ o l t i m( n3 X 30 c m ) cqiiiii(1970b) by subcutaneous injection of the neurotoxins into mice. Neurotoxins I and I11 have an LDjo of 1.12 and 1.35 brated with 0.001 \ I tiC1. The protein peak W L I S pooled. conpg, respectively, per 20 g of body weight. centrated by partial lyophylization, and reacted with hydroxyliimine (0.5 \ I ) a t pH 7, 25". to reverse a possible cu\xlent 1-Ethyl-3-( 3-dimethy1aminopropyI)carbodiimide was obtained from the Ott Chemical Company. Dansyl chloride modification of the tyrosine residue (C'irraway iirid K o h came from Sigma and dithioerythritol from Pierce. TPCKIland. 1965). Thc: number of [ I Clglycine ethyl ester.; incortreated trypsin was a Worthington product. ['CIAcetic anporated was counted in a Pachard scintillation spectromctcr after rechroniatography on the same Sephadex coliinin. hydride, [ 14C]rnaleicanhydride, and [ ldC]glycine ethyl ester were obtained from the Commissariat 2 I'Energie Atomique Forriz.~~/ork~n C I ~Tr? prophcirn. Formylation of to\in I of (France). Najci h i e was conducted in anhydrous formic acid sattirated Oprical Rorarorj. Dispersion, Spectroplioturrietiic Tirrciricin, with gaseoits HCI ;is described by Prebiero r r iil (1967). Lleand Fluorescence Measurernenrs. Optical rotatory dispersion formylation easily occurs :at pH higher than 8 . 5 (['ret iero rr d.? 1967): i t uas followed at pH 9.? i n ii 0,s \ I w?diiiiii (ORD) measurements were carried out in a Fica Spectropol carbonate bulfcr containing 7 \ I urea. I spectropolarimeter. The cell could be thermostated between The extent of tryptophan reversibly transfornicd i n t o N 5 and 8 5 " -C 0.2". Optical rotatory dispersion data are given as mean residue rotation [m], in degrees X cm' X decimole- 1. forniyltryptophan was estimated spectrophotomctrically at d., 1967) in a Cary 14 298 nni ( e s r 4.8s X lo", Previero X MRW spectrophotometer. Toxicity of the modified toxin was mea[/VIx = sured after dilution of aliqtiots into a bovine serum albumin l00cl solution at 10 mg ml in 0.14 11NaCl at pH 7.0. Amino acid The mean residue weight ( M R W ) was 114 for neurotoxin analyses were carried otit with ii Technicon autoanalyser I and 113 for neurotoxin 111. after acidic hydrolysi\ in 6 \ HCI during 20 h r a t 110'. Citrate (0.01 M), acetate (0.01 xi), and phosphate (0.01 hi) It w a s verified that neuroto\in I retained a full activity were the buffers used between pH 2 and 8 in thermal denaafter staying in anhydrous formic acid, or in I N NC'I during turations studies. They all contained 0.1 M NaCI. Dissolved 100 rnin. gases were carefully removed from the buffers so that bubbles .Icer),/cirii~nt i / ? d Mci/ej~/arion.Acetylation of neuruto\ins would not form on heating. Prior t o use the solutions were I and I I I was carried otit at pH S (0.1 11Tris) a n d 3 ' by acidfiltered through Millipore filters (0.45 p). Protein concening every 10 min 1O-pl aiiqtiots of [lC]acetic anhydride to trations were estimated in a Zeiss PMQ I1 spectrophothe protein solution (1- 2 m g nil). The pH was maintained tometer. At 280 nm et'& = 13.5 for neurotoxin I and 11.5 constant in a Radiometer pH-Stat. The reaction was stopped for neurotoxin 111(Miranda et ul., 1970a). after 1 hr. The mixture was then passed through a Sephadc\ Spectrophotometric titration of the tyrosine residue of neuroG-25 column (2 X 30 cm): equilibrated in 0.1 ht ammonititn toxin I was performed with a Cary 14 spectrophotometer acetate, to separate radioactive acetate from the ;icetylated using a 0.12 mglml protein solution in a 0.01 hi phosphate protein. This protein sample was then treated with 1 11 hybuffer containing 0.1 M NaCI. The temperature was maindroxylamine pH 7 to reverse a possible acetylation of t l r o tained at 20" 0.1" in both compartments of the spectrosine. After 1 hr the excess of hydrozylamine was cliniinatcd photometer. The p H of the solution in the reference cel! was by filtration on Sephadex G-25 as described abovc. The numkept at 7.0 while the pH of the solution in the other cell was her of acetyl groups incorporated in the neurotoxin ita\ constantly varied by addition of minute amounts of NaOH estimated from radioactivity measurements in ;I I'ach~iril 1 N or 10 N . The new pH value was measured after each NaOH Tricarb scintillation spectrometer model 3375. The hinctics addition and the spectrum was recorded between 250 and of tyrosine deacylation under the influence of hydroxylamine 320 nm. were carried out at 278 nm in a Cary 1 5 spectrophotolneter. Fluorescence measurements (corrected excitation and Maleylation of neurotoxin I (1 mg;ml) was carried out a t emission spectra) were taken with a Fica 55 differential specpH 9 according to Butler er t i l . (1969) using ["C]maleIc a n trofluorimeter. All fluorescence measurements were perhydride. After I hr. the reaction mixture was p s s e d through formed at 30". The quantum yield was evaluated as described a Sephadev (3-25 column ( 2 x 20 cm) equilibrated with :I by Teale (1961). 0.1 \I phosphate bufer, pH 7.5. The number of maleyl group\ incorporated into the neurotoxin was estimatcd Ily radioiiieasurements. Demaleylation was obtained aftcr I Abbreviation used i s : TPCI
