Eur. J. Biochem. 229, 61 -69 (1995) 0 FEBS 1995

Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin Vincenzo DE FILIPPIS I , Luca VANGELISTA’, Giampietro SCHIAVO’, Fiorella TONELLO’ and Cesare MONTECUCCO*

’ CRIB1 Biotechnology Centre, Universiti di Padova, Italy * Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Universiti di Padova, Italy

(Received 28 November 1994/16 January 1995) - EJB 94 1820/3

Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin. TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases. They consist of three domains and the proteolytic activ- ity is displayed by the 50-kDa light chain (L chain). The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy. The secondary structure content (27 % a-helix and 43 % P-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25 % a-helix and 49 % P-sheet). Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in a-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion. Both near-ultravio- let CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft.

The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluores- cence, second derivative absorption spectroscopy) as well as proteolytic probes. The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule. Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration). These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermopro- teolyticus, where both conformational and stability properties are unchanged upon zinc removal.

Keywords. Tetanus neurotoxin ; metal binding ; circular dichroism; fluorescence ; limited proteolysis.

Tetanus neurotoxin (TeNT) is the cause of all clinical symp- toms of tetanus. The toxin binds the presynaptic membrane of the neuromuscular junction, is internalized inside vesicles that move backwards inside the motor neuron axon and reaches the spinal cord. Here, TeNT penetrates the inhibitory interneurons and blocks neurotransmitter release, thus causing a spastic paral- ysis. The disease is completely prevented by vaccination with formaldehyde-treated TeNT (Simpson, 1989).

TeNT is produced by toxigenic strains of Clostridium tetani as a single 150-kDa polypeptide chain, readily cleaved by vari- ous proteases at an exposed loop to generate two disulfide- linked polypeptide chains. The heavy chain (H chain, 100 kDa) is responsible for the neurospecific binding and cell penetration, while the light chain (L chain, 50 kDa) blocks neuroexocytosis (Schiavo et al., 1993, 1994, 1995).

Recently, the L chain of TeNT was shown to be a zinc-de- pendent endopeptidase which cleaves specifically VAMPlsynap- tobrevin at a single Gln-Phe peptide bond and this cleavage of

Correspondence to V. De Filippis, Centro di Ricerca Interdiparti- mentale per le Biotecnologie Innovative, Via Trieste 75,I-35121 Padova, Italy

Abbreviations. TeNT, tetanus neurotoxin from Closfridium tetani ; L chain, fragment 1-449 of tetanus neurotoxin from C. tefani; SSVs, small sinaptic vescicles ; VAMP, vescicle-associated membrane protein.

Enzymes. Trypsin from bovine pancreas (EC 3.4.21.4) ; thermolysin from Bacillus thermoproteolyticus (EC 3.4.24.4) ; Glu-C protease from Staphylococcus aureus (EC 3.4.21.19) ; chymotrypsin from bovine pan- creas (EC 3.4.21.1); papain from Carica papaya (EC 3.4.22.2).

VAMPIsynaptobrevin is correlated to the blockade of neuroexo- cytosis (Schiavo et al., 1992a, b). Its structure, unique sequence around the zinc binding motif, site and specificity of action, both in terms of target and peptide bond cleaved, identifies TeNT and the related neurotoxins responsible for botulism as a new group of zinc-endopeptidases (Montecucco and Schiavo, 1993, 1994).

Very limited structural data are available on the TeNT L chain (Robinson et al., 1982) and they were obtained with a protein isolated after separation of the L and H chains with di- thiothreitol and urea, agents known to alter the native structure of proteins and the environment of active-site metal atoms (Creighton, 1993). Here, we describe an alternative procedure for the isolation of the L chain from the bacterial culture which avoids protein denaturation. Given the importance of TeNT both as a vaccine and as the most intensively studied clostridial neu- rotoxin, we have undertaken a structural investigation of the L chain and tested with different methods a possible structural role played by the zinc atom in addition to its catalytic one.

MATERIALS AND METHODS

Materials. The Glu-C protease from Staphylococcus aureus V8, trypsin, chymotrypsin, thermolysin and papain were purchased from Boehringer (Mannheim). EDTA, o-phenanthro- line and teraethylenepentamine were from Fluka AG. Trifluoro- acetic acid and constant-boiling HC1 were purchased from Pierce Chem. Co. All reagents for SDSPAGE were from Bio-Rad.

62 De Filippis et al. ( E m J. Biochem. 229)

Solvents and reagents used for peptidelprotein sequence analysis were from Applied Biosystems. Analytical-grade solvents and salts were purchased from C. Erba or Merck.

Purification of L chain tetanus toxin. 22 1 Clostridium te- tuni culture filtrates were treated with 250 g/l ground solid am- monium sulfate at 4°C under stirring. After two days in the cold, the Supernatant was removed by aspiration. The bottom suspension (about 4 1) was centrifuged in a GSA Sorvall rotor at 10000 rpm for 8 min. All following operations were per- formed in the cold and deareated buffers were used. The pellet was resuspended in 500 ml 10 mM sodium phosphate, pH 7.4. The suspension was stirred for 30 min. After centrifugation as above, a cold saturated ammonium sulfate solution was added, under stirring, up to 46% saturation while maintaining the pH at 7.2 with the addition of ammonia. After centrifugation as above, the pellet was resuspended in 200 ml 10 mM sodium phosphate pH 7.4 and dialysed extensively against the same buffer. After centrifugation, the supernatant was applied to a col- umn (2.5X19 cm) of DEAE-cellulose (Whatman DE-52). The column was equilibrated with 10 mM sodium phosphate, pH 7.4 until base line was reached. The toxin was eluted with a linear gradient of 10-100 mM sodium phosphate pH 7.4 at a flow rate of 50 mlk and recording the absorbance of the eluted material at 280 nm. 5-ml fractions were collected and analysed by SDS/ PAGE (9% or 12% acrylamide). Two pools were made: one included those fractions containing TeNT and the second one was made with those fractions (last third of the protein peak) enriched in L chain. To both pools, a cold saturated solution of ammonium sulfate was added to a final saturation of 60%. The TeNT fraction was processed as previously described (Matsuda and Yoneda, 1975), while the L chain suspension was centri- fuged, resuspended in 10 mM sodium phosphate, pH 7.4 (final volume 7 ml) and loaded on a Ultrogel ACA-34 (Merck) gel- filtration column (3x130 cm). The column was eluted with the same phosphate buffer at a constant flow of 20 m l k and 8-ml fractions were collected. The fractions enriched in L chain and devoid of residual TeNT were collected and the protein was re- covered by ammonium sulfate precipitation as above. The pellet was resuspended in 100 mM sodium phosphate, pH 6.8, centri- fuged at 15.000 rpm for 15 min and 1-ml aliquots of the super- natant were loaded onto a TSK G-3000 SW HPLC gel-filtration column (7.5X600 mm; Pharmacia) eluted with 100 mM sodium phosphate pH 6.8 at a flow rate of 0.65 ml/min. Fractions still containing a low amount of degraded L chain were pooled and dialysed extensively against 20 mM Tris/HCl pH 8. The solution was centrifuged at 15000 rpm for 10 min and loaded onto a Mono-Q HR 5/5 column (Pharmacia) equilibrated in 20 mM TrisMCl pH 8.0 and eluted with a gradient of 1 M NaCl in the same equilbration buffer from 0 to 10% in 2 min, from 10% to 25% in 20 min, from 25% to 40% in 3 min and for 2 min at 40% at a flow rate of 1 ml/min and monitoring the absorbance of the effluent at 280nm. To these fractions, solid ammonium sulfate was added to a final saturation of 60%. The suspension was centrifuged, resuspended in 50 mM TrisMCl pH 7.4, centri- fuged again and the supernatant was frozen in small aliquots (protein concentrations varying over 0.35- 1 mglml) in liquid nitrogen and stored at -80°C.

Reverse-phase HPLC was conducted on a Vydac C4 column (4.6X150 mm, 10 pm particle size) purchased from The Separa- tion Group (Hesperia, CA, USA) and eluted with a linear gradi- ent of 20-60% acetonitrile/O.l% trifluoroacetic acid in 30 min. Electrophoresis was performed on 9 % or 12 % polyacrylamide gels according to Laemmli (1 970). N-terminal sequence analyses were performed by automated Edman degradation using a pulsed liquid-phase sequencer model 477A with on-line analyzer model 120A (Applied Biosystems) for the detection of phenylthiohy-

dantoin derivatives. Amino acids were analyzed on acid-hy- drolyzed samples (6 M HCI at 110°C for 24 h) using a C. Erba amino acid analyzer model 430-A-30 and following manufactur- er's procedure. Immunoblotting techinque was performed as re- ported by Towbin et al. (1979) with rabbit IgG anti-L chain revealed by mouse antibodies anti-rabbit IgG coupled to alkaline phosphatase. Proteolytic activity of the L chain of TeNT was assayed after incubation with small synaptic vescicles (SSVs) as previously described (Schiavo et al., 1992a, b).

Preparation of the apo-L chain of tetanus toxin. Buffers were prepared with Milli-Q grade water (Millipore) with a con- ductance value lower than 10 MOhm and demetallized with Am- berlite MB-3 (Sigma Chem. Co.). All glassware, dialysis tubing and other materials were extensively rinsed with this water. EDTA in 1 M Tris was added to TeNT L chain preparation (0.5-1.0 mg/ml) to reach a final concentration of 10 mM EDTA. TeNT was incubated in these conditions for 1 h at 37°C followed by extensive dialysis at 4°C against 150 mM TrisMCl pH 7.4. In other samples the L chain was incubated under iden- tical conditions with 10 mM o-phenanthroline and then dialysed extensively. The zinc content was determined by atomic absorp- tion measurements with a Perkin-Elmer 4000 atomic absorption flame spectrophotometer, as previously reported (Schiavo et al., 1992a).

Ultraviolet absorption spectroscopy. Protein concentration was determined by absorbance at 280 nm using a Perkin-Elmer lambda-5 spectrophotometer. The absorbtion coefficient at 280 nm and 0.1 % (by mass) protein concentration for TeNT L chain was taken as 0.78 mg-' . cm2, determined according to the method of Gill and von Hippel (1989). The absence of light scattering in the wavelength region above 320nm was used as a criterion for the absence of large aggregates in solution (Ikeguchi and Sugai, 1989).

Second-derivative profiles of the absorbtion spectra were ob- tained using the software PECSS licenced by Perkin-Elmer. Ali- quots (100-200 pg) of purified TeNT L chain were dissolved in 300 pl 50 mM sodium phosphate pH 7.5 containing 100 mM NaCl. Measurements were carried out in 1-cm pathlength cu- vettes (0.5-ml inside volume) and thermostatted at 25 "C. In all experiments, the absorbance at 280 nm was in the range 0.20- 0.25.

Circular dichroic spectroscopy. CD spectra were recorded on a Jasco J-710 spectropolarimeter fitted with a thermostatted cell holder and interfaced with a Neslab (Newington, NH, USA) water bath RTE-110. The results were expressed as the mean residue ellipticity, [@I, = (@ob7/10) . (MA . c), where @lobs, is the observed ellipticity at a given wavelength, &I, is the mean residue mass, 1 is the cuvette pathlength in cm, and c is the protein concentration expressed as g/ml. The units of [@I, are deg . cmz . dmol-I. The mean residue molecular mass for L chain was calculated from its amino acid composition (Niemann, 1991) and taken as 114.6Da. Far-ultraviolet CD spectra were recorded at room temperature (22-24°C) in a 0.2-mm or 1-mm pathlength quartz cell at a protein concentration ranging over 5-10 pM. CD spectra between 350-240 nm were recorded at 20-30 pM protein concentration in a 1-cm pathlength rectangu- lar quartz cuvette thermostatted at 25 "C. All CD spectra resulted from averaging four scans and the final spectrum was corrected by subtracting the corresponding base line spectrum obtained under identical conditions. All measurements, except where ex- plicitly specified, were performed in 50 mM sodium phosphate pH 7.5 containing 100 mM NaC1. The far-ultraviolet CD spectra were analyzed to estimate the percentage of protein secondary structure with a computer program provided by Jasco and based on the method of Yang et al. (1986).

De Filippis et al. ( E m J. Biochern. 229) 63

Fluorescence spectroscopy. Measurements were performed on a Perkin-Elmer LS-50 spectrofluorimeter using a 1-cm path- length quartz cell thermostatted at 25 "C. Samples were prepared by diluting a stock solution of L chain TeNT to a final concen- tration of about 2 pM in 50 mM sodium phosphate pH 7.5, 100 mM NaCl. Fluorescence spectra resulted from averaging four accumulations and were corrected by subtracting the corre- sponding base line.

Stability measurements. The urea-induced unfolding pro- cess of the L chain of TeNT was followed by monitoring the CD signal at 220 nm as a function of increasing urea concentra- tions. A 10 M urea stock solution in 50 mM sodium phosphate pH 7.5, 100 mM NaCl was prepared gravimetrically in a 200-ml volumetric flask. Urea solutions (10 ml) at different concentra- tion were prepared by diluting the 10 M urea stock solution with the same buffer, divided into 900-pl aliquots, frozen in solid CO, and stored at -20°C. For each data point in the urea-in- duced unfolding experiment, 100 pl L chain solution (0.35 mg/ ml) was added to 900 pl of the appropriate urea solution. Before performing spectrosopic measurements, samples were incubated for about 2 h at room temperature. The ellipticity at 220 nm was recorded in a 0.5-cm pathlength rectangular quartz cu- vette thermostatted at 25°C. The reversibility of the unfolding process was checked by diluting protein samples to low urea concentrations (1 M) and measuring the recovery of ellipticity at 220 nm.

Thermal denaturation of the L chain of TeNT was monitored by recording the decrease of the CD signal at 220 nm as a func- tion of sample temperature. The experiments were performed in 50mM sodium phosphate pH7.5, 1OOmM NaCl at a protein concentration of 50 pg/ml under stirring. The temperature in- side the cuvette (1 cm) and CD signal were acquired using a temperature scan program provided by Jasco. The reversibility of the unfolding process was checked by measuring the restora- tion of the CD signal upon cooling to the starting temperature (20°C).

Predictive methods. Secondary structure location of the TeNT L chain was predicted with the program GOR (Garnier et al., 1978). The method uses known structures and collects information on significant pair-wise dependence of an amino acid in position i with each of eight residues on either side of it in a particular secondary structural setting. The hydrophilicity profile was computed on a sliding window average of six amino acids using the program ANTIGEN (Hopp and Woods, 1981). The protein flexibility profile was obtained using the program FLEXPRO included in the PC/GENETM package (IntelliGene- tics, CA, USA). This method makes use of the temperature B factors of Ca atoms for proteins of known crystal structure (Kar- plus and Schulz, 1985). Average flexibility was calculated with a moving window of seven amino acids and the values plotted with respect to the central residue.

Limited proteolysis. The holo- and apo-forms of the L chain of TeNT were subjected to proteolysis reaction under identical conditions (1 mg/ml protein concentration in 50 mM sodium phosphate pH 7.8 for 4.5 h, 37°C) at a proteasehbstrate ratio of 1 : 10 (by mass) with Glu-C protease and 1 :20 with trypsin and chymotrypsin. Digestion with papain was performed as with the Glu-C protease, but in the presence of 0.5 mM EDTA and 10 mM cystine. Digestion with thermolysin was conducted in 50 mM Tris/HCl pH 7.8 containing 5 mM CaC1,. Proteolysis re- actions were stopped by diluting 10- pl aliquots of digestion mixture with 0.1 % aqueous trifluoroacetic acid (50 pl). Samples were dried in a Speedvac concentrator (Farmingdale, NY, USA) and subject to SDSPAGE as previously described (Schiavo et al., 1992b).

RESULTS AND DISCUSSION

Purification of the L chain of tetanus neurotoxin. The L chain is the one part of the entire tetanus neurotoxin responsible for the intraneuronal specific proteolytic activity which leads to the blockade of exocytosis (Bittner and Holz, 1988 ; Ahnert-Hilger et al., 1989; Mochida et al., 1989; Stecher et al., 1989). The L chain of TeNT can be released and isolated from the H chain by reduction of the single interchain disulfide bond in the presence of urea, which lessens non-covalent interactions that would otherwise keep the two chains together (Matsuda and Yoneda, 1975). These agents may cause loss of catalytic activity. In order to preserve the full catalytic activity of the L chain and also to perform reliable structural studies, we developed an alternative procedure that does not involve the use of denaturating agents. Cultures of Clostridium tetani contain free L chain that is likely to be derived from a preferential proteolytic digestion of the H chain in the bacterial culture.

The purification procedure described here leads to a prepara- tion of TeNT L chain essentially free of contaminants, as judged from overloaded SDSPAGE gels and from the appearance of a single symmetric peak on a Vydac C4 reverse-phase HPLC col- umn (results not shown). In particular, the elimination of the largest part of TeNT was attained by gel filtration chromatogra- phy. The remaining traces of TeNT were removed in the second gel filtration step, and low-molecular-mass peptide material in the final ion-exchange chromatographic step (see Materials and Methods). The identity of the purified TeNT L chain was estabi- lished by N-terminal sequence analysis of the first 30 residues (not shown) and by immunoreactivity with rabbit anti-(L chain) IgG as previously described by Schiavo et al. (1992a). The analysis of the amino acid composition (not shown) of L chain was found to be in reasonable agreement with data deduced from cDNA (Fairweather and Lyness, 1986). Moreover, the purified TeNT L chain contains 0.98 2 0.04 zinc atomlL chain, as ob- tained by atomic absorption measurements (not shown), and its proteolytic activity, assayed on small synaptic vescicles (SSVs) (Schiavo et al., 1992b), is as high as that of the native dithio- threitol-treated tetanus neurotoxin. Nevertheless, on the basis of the purity criteria employed in this study, the presence of C- terminal heterogeneity cannot be excuded. In fact, previous studies (Krieglstein et al., 1991) have demonstrated that in vivo processing of TeNT leads to formation of L chain species with ragged C-terminals differing by only a few residues, which have been shown to be irrelevant to toxicity (Kurazono et al., 1992).

Conformational characterization of the L chain of tetanus neurotoxin. Circular dichroism. The far-ultraviolet CD spectrum of the purified TeNT L chain (Fig. 1 A) resembles that previously reported by Robinson et al. (1982), although substan- tial differences exist in the shape of the spectrum as well as in the signal intensity. The spectrum shown in Fig. 1A indicates that the L chain of TeNT possesses a considerable amount of both a-helical and P-sheet secondary structure with two minima at 220 nm and 208 nm and a maximum at 193 nm (Brahms and Brahms, 1980). Under the limitations of the computing method employed (Yang et al., 1986), the secondary structure content estimated here for the L chain of TeNT (27 % a-helix and 43 % P-structure) compares favourably to that previously reported by Robinson et al. (1982) (24% a-helix and 40% P-sheet). Interest- ingly, the figure of secondary structure content, obtained by uti- lizing the GOR method (Gamier et al., 1978), predicts 25% a- helix and 49% P-sheet. The analysis of the distribution of sec- ondary structure elements along the L chain of TeNT sequence (Fig. 2 A) suggests that the 80-residue-long N-terminal region, highly conserved among clostridial neurotoxins (Niemann,

64

I I 1 I 6 0 - -

C Y -

-

-

- e x c a t 2 9 5 nrn

-

De Filippis et al. ( E m J. Biochern. 229)

A -

-

a - 5 I O D

1 0 I I I I I 1 I 1 9 0 2 1 0 2 3 0 2 5 0

W A V E L E N G T H , n m

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0

W A V E L E N G T H . nm

Fig.l. Conformational characterization of the L chain of tetanus neurotoxin. (A) Far-ultraviolet CD, (B) near-ultraviolet CD and (C) fluorescence emission spectra of TeNT L chain. All spectra were re- corded in SO mM sodium phosphate, pH 7.5 containing 100 mM NaCl (see also Materials and Methods).

1991 ; Minton, 1995), adopts to a great extent a &structure. The rest of the molecule is predicted to possess a substantial amount of both a- and p-structure with four stretches of sequence char- acterized by high a-helix probability. One of them, in analogy with other zinc-dependent metalloproteases (Jiang and Bond, 1992, and references therein cited), includes the zinc-binding motif of the L chain of TeNT His-Glu-Leu-Ile-His- (Schiavo et al., 1992a; Wright et al., 1992) (Fig. 2A). This is also expected on the basis of the geometrical requirements necessary for a correct coordination of the zinc ion in the active-site cleft (Chris- tianson, 1991).

1 0 2 0 3 0 4 0 50

P I T I N N F R Y S V P V N N V T I I M M E P P Y C K G L D I Y Y K A F K I T D R I W I V P E R Y E

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1 1 0 1 2 0 1 3 0 1 4 0 150 V A G E A L L D K I I N A I P Y L G N S Y S L L D K F V T N S N S V U E Q V P S G A T T K S

1 6 0 1 7 0 1 8 0 1 9 0 200

A M L T N L I I F G P G P V L N K N E V R G I V L R V D N K N ~ D G F G S I M Q M A F C P E 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0

Y V P T F D N V I E N I T S L T _ L G K S K Y F Q V P A L L L M H E L I H V L H G L Y G M Q V S S H E 2 6 0 2 1 0 2 0 0 2 9 0 3 0 0

I I P S K Q E I Y M Q H T Y P I S A E E L F T F G G Q V A N L ~ I S I V I K N D L Y E K T L N D Y K A 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0

I A N K L S Q V T S C N D P N I ~ I D S Y K Q I Y Q Q K Y Q F V K ~ S N G ~ N E D K F Q ~ 3 6 0 3 7 0 3 6 0 3 9 0 4 0 0

N S I M Y G F T E I E L G K K F N I K T R L S Y P S M N H D P V K I P N L L D D T I Y N D T E G F N 4 1 0 4 2 0 4 3 0 4 4 0

I E S K D L K S E Y K G Q N M R V N T N A F R N V ~ G ~ G ~ I G L C K K I I P P T N I R E

Trp43

0.8 I I I I 1 I ' I I 50 150 250 350 450

RESIDUE NUMBER

1 I I I I I I I I

1 1 1

I ' I I I I I I I

50 150 250 350 450

RESIDUE NUMBER

Fig. 2. Prediction of the (A) secondary structure content, (B) flexibil- ity and (C) hydrophilicity profiles of the L chain of tetanus neuro- toxin. The amino acid residues predicted to be in a-helix are in bold, whereas those in 8-structure are underlined (Gamier et al., 1978). The flexibility plot was calculated according to Karplus and Schulz (1985). The hydrophilicity profile was computed with the method of Hopp and Woods (1981) (see also Materials and Methods).

The near-ultraviolet CD spectrum (Fig. IS) of the L chain of TeNT reveals the presence of extensive fine structure in the absorption region of aromatic amino acids. In particular, the spectral region between 255 -270 nm is dominated by the ab- sorption of the 23 Phe residues in the L chain sequence (Nie- mann, 1991), with two characteristic negative bands at 262 nm and 268 nm corresponding to the 0-0 and 0-(0+930)cm-' vib- rionc transitions, respectively (Strickland, 1974). The contribu- tion of the single Trp43 appears as two positive bands at 288 nm and 280 nm corresponding to the 0-0 and 0-(0+850)cm-' vibri- onic components of Trp 'L, electronic transition superimposed to the negative background of the other aromatic residues. The

De Filippis et al. (Eul: J. Biochem. 229) 65

band centered at about 295 nm likely derives from the overlap- ping of 'L, and 'La Trp bands cancelling each other out (Strick- land, 1974; Lindsay and Pain, 1990). The presence of a well defined fine structure in the 280-290 nm spectral region indi- cates that the single Trp43 has a restricted set of allowed confor- mations, suggesting that it is located into an asymmetric and rigid environment of the L-chain three-dimensional structure (Strickland, 1974). This is in keeping with the result of the analysis of the flexibility profile of the L chain (Fig. 2B) calcu- lated according to the method of Karplus and Schulz (1985), where Trp43 falls exactly in one of the lowest minima of the flexibility plot.

Fluorescence. Fig. 1 C shows the emission fluorescence spectra of TeNT L chain in sodium phosphate pH 7.5. The spectrum obtained after exciting the sample at 280 nm shows a maximum of fluorescence intensity at about 315 nm. In this spectrum the contribution of the single Trp43 is shadowed by the emission of the 27 Tyr residues. In order to selectively moni- tor the fluorescence emission of the single Trp43, the sample was also excited at 295 nm and 300 nm (Lakowicz, 1986). The resulting wavelength of the maximum fluorescence intensity, A,,, is centered at about 320 nm. This unusually short value of A,,, strongly suggests that Trp43 is buried inside the protein in a hydrophobic environment (Lakowicz, 1986). A similar indica- tion is provided by the analysis of the hydrophilicity profile (Hopp and Woods, 1981) of the L chain, showing that Trp43 is located into a minimum of the hydrophylicity plot (Fig. 2 C).

Considering that the interior of proteins has a prevalent hy- drophobic character (Chothia, 1974) with amino acid side chains tightly packed in a highly dense and compact structure (Ponder and Richards, 1987), the results of the fluorescence experiments are consistent with the localization of Trp43 in a rigid environ- ment, as previously revealed by near-ultraviolet circular dichro- ism (Fig. I B).

Stability of the L chain of tetanus neurotoxin. In view of a general physico-chemical characterization, as well as to derive possible correlations between stability properties and functional activity, we studied the stability of TeNT L chain as a function of several parameters such as pH, temperature and urea concen- tration.

pH. To penetrate into neurons, TeNT has to interact with different cell compartments, characterized by different physico- chemical parameters (pH, ionic strength, viscosity, etc.) (Mon- tecucco and Schiavo, 1994). In particular, there is evidence that TeNT enters the cytosol from a low-pH intracellular compart- ment (Williamson and Neale, 1994).

The pH-induced denaturation of TeNT L chain, monitored by recording the CD signal variation at 220 nm, shows a bell- shaped profile with a maximum at about pH 7 (Fig. 3A). A closer analysis of CD spectra recorded at different pH values reveals that, on going from pH 7 to approximately pH 5 (a lower limit never approached inside the lumen of endosomal compart- ments), the ellipticity value at 220 nm slightly decreases, but the overall shape of the spectrum remains roughly constant with two minima at 220 nm and 208 nm, indicating that the overall se- condary structure content of the L chain at pH 5 is essentially unchanged (Fig. 3 B). For lower pH values (pH 3 or lower), CD spectra show a single minimum centered at 215 nm, indicative of an increased amount of P-like secondary structure (Brahms and Brahms, 1980), possibly due to the aggregation of partially denatured L chain monomers. At alkaline pH values (> pH lo), the introduction of a large number of negative charges, also de- rived from the ionization of the 27 Tyr residues, accounts for a CD spectrum characteristic of a polypeptide chain in a rather

1 . 2

1 .o

0 - 0 T 0 . 8

0 - -

2

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P - 2

- 0 E

N

E

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- 1 0

0 2 4 6 8 1 0 1 2 1 4 P H

I I I

B

2 0 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0

W A V E L E N G T H , n m

Fig. 3. pH-induced denaturation of the L chain of tetanus neurotoxin monitored by far-ultraviolet CD spectroscopy. (A) The unfolding pro- cess was followed by monitoring the decrease of the CD signal at 220 nm as a function of pH. Ellipticity values are reported as the ratio [@]I[@],, where [@I, is the CD signal at pH 7.0. (B) Far-ultraviolet CD spectra of TeNT L chain at different pH values. All measurements were performed at 25°C in 5 mM sodium citrate, 5 mM sodium borate and 5 mM sodium phosphate, 100 mM NaCl buffered with aqueous phospho- ric acid at the indicated pH value.

denatured state, even though substantial ellipticity at 220 nm still persists (Fig. 3 B).

Similar results on the stability of TeNT L chain were ob- tained by following the variation of Trp fluorescence at 325 nm as a function of pH, after excitation at 295 nm (Fig. 4). At acidic as well as alkaline pH values, there is a decrease of the intensity of the single Trp43 fluorescence and the wavelength of the max- imum fluorescence intensity is red-shifted from 320 nm to about 350 nm (not shown), suggesting that at pH extremes the protein is extensively denaturated (Lakowicz, 1986).

Heat. Temperature-induced unfolding of the L chain of TeNT was monitored by recording the decrease of the ellipticity value at 220 nm for increasing temperature. The unfoiding pro- cess follows a sigmoidal profile (Fig. 5A) with a temperature of half-denaturation of about 55 "C. Interestingly, the CD signal at 220 nm and 74°C still mantains about 80% of the initial elliptic- ity value (recorded at 20 "C) (Fig. 5 A). In addition, the analysis of the CD spectra recorded at different temperatures (Fig. 5B) clearly indicates that, even at high temperatures, the L chain retains a considerable amount of P-like secondary structure. The denaturation process is fully irreversible, as judged by the ab-

66 De Filippis et al. ( E m J. Biochem. 229)

1 . 2 I I I 1 I I I

0 . 2 L 0 2 4 6 8 1 0 1 2 1 4

P H

Fig. 4. pH-induced denaturation of the holo- and apo-L chain of teta- nus neurotoxin monitored by fluorescence spectroscopy. The unfold- ing process was monitored by recording the decrease of fluorescence emission intensity at 325 nm (after excitation at 295 nm) as a function of pH. Fluorescence data are reported as the ratio FIF,, where Fo is the fluorescence intensity value at pH7.0. (0) Holo-L chain; (V) apo-L chain of TeNT obtained after incubation of the holo-protein with 10 mM o-phenanthroline for 1 h at 37°C; (V) apo-L chain of TeNT obtained after incubation of the holo-protein with 10 mM EDTA for 1 h at 37°C (see Materials and Methods). All measurements were performed in the same buffer and temperature conditions as reported in the legend to Fig. 3.

sence of any restoration of the CD signal upon cooling the sam- ple at the starting temperature (Fig. 5 B). Nevertheless, during the melting experiment, the protein solution remains clear with- out any visible precipitate.

Urea. The urea-mediated unfolding process of TeNT L chain was moniored at 25 "C by following the decrease of the elliptic- ity at 220 nni on increasing the urea concentration. The process was found to be substantially irreversible, highly cooperative, with a midpoint of denaturation at about 4.5 M urea (Fig. 6). At variance from pH- and heat-induced unfolding experiments, in the presence of urea, the L chain undergoes a rather complete unfolding, as documented by the low value of residual ellipticity at 9 M urea.

Comparison of the conformational and stability properties of holo- and apo-L chain. It has been shown that the binding of metal ions can substantially affect the conformational stability of naturally occurring globular proteins by enthalpic as well as entropic contributions (Hiraoka et et a]., 1980; Pace and Grim- sley, 1988 and refereneces therein cited). These observations have been confirmed by the fact that substantial protein stabili- zation can be attained by engineering metal chelation sites into the native three-dimensional structure of proteins (Kuroki et al., 1989; Toma et al., 1991; Kellis et al., 1991; Braxton and Wells, 1992).

In the case of TeNT L chain, the zinc ion is linked to the rest of the protein via the two His of the motif and to one or two as yet unidentified residues located in unknown position(s) toward the C-terminus of the chain (Schiavo et al., 1994, 1995). Hence, by anchoring a segment of the L chain not contiguous to the motif, the zinc atom could play a structural role, in addi- tion to its catalytic one.

Therefore, in order to investigate the contribution of the bound zinc ion (essential for the functional activity), as well as that of a possible binding of other metal ions (i.e. Ca+*, Mg+Z), to the conformation and stability of TeNT L chain, we studied the L chain molecule by different and complementary techniques

1 .1 I I I I I 1

"t 0.6 1 I I I I 1

5 0 6 0 7 0 3 0 40 T E M P E R A T U R E . O C

4 1 I I I I I

h

- 2 - 0

73

N

€ 0

- 2

0)

a, D - 4

rn v

b - 6 7

X r-7

0 - 8 Y

R B

2 0 0 2 1 0 220 2 3 0 2 4 0 2 5 0 W A V E L E N G T H , n m

Fig. 5. Thermal unfolding of holo- and apo-L-chain of tetanus neuro- toxin. (A) Heat-induced denaturation of the protein was assayed by mon- itoring the decrease of the CD signal at 220 nm on increasing temper- ature (see Materials and Methods). (0) Holo-L chain; (V) apo-L chain of TeNT obtained after incubation of the holo-protein at 37°C with 10 mM o-phenantroline for 1 h. (B) Far-ultraviolet CD spectra of TeNT L chain at 20°C (-), 74°C (---) and after cooling the sample at the starting temperature (20 "C) (- . -).

in the presence of chelating agents selective for Zn+* (o-phen- antroline and tetraethylenepentamine) as well as for other diva- lent cations like Caf2 and Mg" (EDTA).

Spectroscopic studies. Far- and near-ultraviolet CD and fluo- rescence spectra of apo-L chain at pH 7.5 are superimposable, within the limits of the experimental error, to those of the holo- protein (not shown). These results clearly indicate that the loss of the zinc atom from the active site does not alter the secondary structure content and the overall three-dimensional fold of the L chain. Of note, irrespective of the chelating agent used to re- move the zinc ion, the shape of the far-ultraviolet CD spectrum and the ellipticity value at 220 nm were approximately constant: 8.230 deg . cm2 . dmol-' in the presence of o-phenantroline, and 8.065 deg . cmz . dmol-' when EDTA was used.

Second-derivative absorption spectroscopy is particularly sensitive to the three-dimensional environment in which the aro- matic side chains are located. In particular, this technique can be used as a fingerprinting of the aromatic side-chain topology for a given protein structure (Craig et al., 1989; Breton et al., 1995). In our case, the close correspondence of the vibrionic

De Filippis et al. ( E m J. Biochem. 229) 67

1 .OWV

0 . 8 - - m A 0 . 6 - m I

- 0.4 -

0 . 2 -

V

d

V V

1 0 1 2 3 4 5 6 7 8 9

U R E A , M o l a r i t y

Fig. 6. Urea-induced unfolding of the holo-and apo-L chain of teta- nus neurotoxin. The conformational transition of the protein was fol- lowed by monitoring the decrease of the CD signal at 220nm for increasing urea concentrations. Ellipticity values vs urea concentration are given as [O]l[O],, where [O], is the ellipticity value measured in the absence of urea. (0) Holo-L chain; (V) apo-L chain obtained after incubation of the L chain molecule with 10 mM o-phenanthroline for 1 h at 37°C. All measurements were performed at 25°C in 50 mM sodium phosphate pH 7.5, 100 mM NaC1. Protein solutions were kept at room temperature for 2 h before measurements (see Materials and Methods).

0 ' 4 0 1 \ I I I

I \ .

N 7 3 -

0 . 0 2 t t - 0.1 6

T h o l o - L c h a i n 4

\"! a p o - L c h a i n 1 ?i : ! i J

1 0 . 3 0 I I 1 I I

2 4 0 2 6 0 2 8 0 3 0 0 3 2 0 W A V E L E N G T H , n m

Fig. 7. Second-derivative absorption spectra of holo- (-) and apo- L chain of tetanus neurotoxin (-*-). Measurements were performed at 25°C in 50mM sodium phosphate pH7.5, 100mM NaCI. Apo-L chain was prepared as reported in the legend to Fig. 6.

fine structure in the two spectra reported in Fig. 7 strongly sug- gests that the environment sorrounding the aromatic side-chains (23 Phe, 27 Tyr and 1 Trp) is essentially unchanged upon zinc removal.

Apo- and holo-toxin share common stability properties. The unfolding of the secondary structure followed by monitoring the decrease of the CD signal as a function of temperature (Fig. 5A) or by increasing urea concentration (Fig. 6) shows similar dena- turation profiles. Moreover, the unfolding of the tertiary struc- ture monitored by recording the decrease of Trp43 fluorescence at 325 nm in the pH range 1 - 13, is also very similar for apo- and holo-toxin (Fig. 4).

These results indicate that the removal of the zinc ion from the active site of this metallo-proteinase does not appreciably alter its conformation and stability. However, it cannot be ex- cluded that the zinc removal could induce minor structural changes, particularly at the active-site region, that are below the

Fig. 8. Limited proteolysis of holo- and apo-L chain of tetanus neuro- toxin. Given the scope of this experiment, a TeNT L chain preparation still containing small traces (less than 1 %, as judged by Coomassie blue staining) of degraded L chain was used (lane 1). Holo- and apo-L chain were run in lanes identified as a and b, respectively. Lanes 2; trypsin at an enzymelsubstrate ratio of 1 : 20 in 50 mM sodium phosphate buffer; lanes 3, chymotrypsin at an enzymelsubstrate ratio of 1 : 20 in 50 mM sodium phosphate buffer; lanes 4, thermolysin at an enzymelsubstrate ratio of 1 : 20 in 25 mM Tris/HCI, 25 mM sodium phosphate, 5 mM CaCI,; lanes 5, V8 protease at an enzymelsubstrate ratio of 1:lO in 50 mM sodium phosphate; lanes 6, papain at an enzymehbstrate ratio of 1:lO in 50mM sodium phosphate, 10mM cystine and 0.5 mM EDTA. All enzymic digestions were performed at pH 7.8, for 4.5 h at 37°C (see also Materials and Methods). The left lane shows molecular mass markers with values in m a .

detection limits of the spectroscopic methods used here in the analysis of a protein containing as many as 449 amino acid resi- dues (Fainveather and Lyness, 1986).

Limited proteolysis. Limited proteolysis is an alternative and complementary approach to the classical spectroscopic tech- niques to gain more insight into the structure and dynamics of globular proteins (Neurath, 1980; Fontana et al., 1989, 1993). It has been shown that, for globular proteins, there is a close corre- lation between surface exposure, chain flexibility and the sites of limited proteolysis (Fontana et al., 1986; Hubbard et al., 1991). On this basis, proteolytic enzymes can be effectively used as probes of the global three-dimensional structure as well as to reveal subtle local changes in protein conformation and dynam- ics. Limited proteolysis was conducted in parallel on the holo- and apo-L chain of TeNT with a number of proteases having different substrate specificity. Chymotrypsin (Wilcox, 1970) and thermolysin (Heinrikson, 1977) preferentially cleave peptide bonds encompassing hydrophobic amino acids. Trypsin (Walsh, 1970) and Staphylococcus aureus V8 protease (Houmard and Drapeau, 1972) hydrolyse peptide bonds involving hydrophylic residues (Lys and Arg for trypsin; Glu for V8 protease). Papain has a wider substrate specificity, cleaving bonds to basic hydro- philic and hydrophobic amino acid residues including Gly, Leu and Tyr (Arnon, 1970). Therefore, the different substrate specifi- cities of the proteolytic enzymes employed in this study allowed us to map different regions of the L chain characterized by a different hydophobichydrophylic balance, thus obtaining a reli- able 'conformational' fingerprinting of both the holo- and apo- toxin. As reported in Fig. 8, holo- and apo-L chains show closely similar reactivity under identical conditions (4.5-h incubation at 37 "C) towards the proteolytic enzymes used. This finding fur- ther substantiates the structural similarity of holo- and apo-forms of the TeNT L chain documented above. Moreover, considering that there is a tight correlation between the conformational sta- bility of a given protein and the rate of its proteolytic degrada-

68 De Filippis et al. ( E m J. Biochem. 229)

tion (Parsell and Sauer, 1989), the identity of the fragmentation patterns of the holo- and apo-L chain is additional evidence that the conformational stability of the protein is unaffected by the removal of the catalytic zinc ion.

The results presented here parallel those previously obtained on thermolysin, a zinc-dependent neutral endoprotease of known three-dimensional structure (Holmes and Matthews, 1982), iso- lated from Bacillus thermoproteolyticus. In the case of thermoly- sin, in fact, the metal-free apoenzyme retains similar conforma- tional properties with respect to the holo-protein, and the re- moval of the zinc ion does not seem to contribute to the stability of the protein towards heat denaturation (Holmquist and Vallee, 1974).

CONCLUSIONS The present paper describes the preparation and conforma-

tional characterization of the light chain of tetanus neurotoxin from cultures of Clostridium tetani which will be useful to those researchers interested in probing the role of VAMP/synapto- brevin in a variety of cellular processes (Huttner, 1993; Roth- man and Warren, 1994; Schiavo et al., 1994; Niemann et al., 1994). A major achievement of this study is the preparation of L chain in a homogeneous form and in its fully active functional state. Moreover, the results described here indicate that the pro- tein has an N-terminal part (closely similar among all clostridial neurotoxins) rich in p-structure with the conserved unique Trp residue buried in the hydrophobic interior of the protein, distant from the active-site zinc atom.

The L chain of TeNT has stability properties similar to those reported for other globular proteins (Pace, 1986) and the zinc atom plays little, if any, role in the conformation and stability of the protein. In addition, the L chain of TeNT, that has to cross the membrane of a low-pH intracellular compartment to reach the neuronal cytosol, preserves its secondary structure at the lowest pH values recorded inside endosomal compartments. This result has a bearing on the development of a model for the membrane translocation of this neurotoxin across the hydropho- bic membrane barrier.

The contribution of Prof. Angelo Fontana to this work is gratefully acknowledged. We thank Dr Patrizia Polverino de Laureto for protein sequence analyses. The support and helpful discussions of Dr Olmetta De Filippis are also acknowledged. This work was supported by grants from Consiglio Nazionale della Ricerche Target Project Biotecnologia e Biostrumentazione and from Telethon-Italia.

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Note added in proof (Received 24 February 1995): After this article was submitted, a paper entitled Secondary Structural Predictions for the Clostridial Neurotoxins by Lebeda, F. J. and Olson, M. A. was published in Proteins Struct. Funct. Genet. 20, 293-300 (1994).