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Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A
Biochemical and Biophysical Research Communications 288, 1231–1237 (2001) doi:10.1006/bbrc.2001.5911, available online at http://www.idealibrary.com on
Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A Michela Rigoni,* Paola Caccin,* Eric A. Johnson,† Cesare Montecucco,* and Ornella Rossetto* ,1 *Dipartimento di Scienze Biomediche Sperimentali, Universita` di Padova, Viale G. Colombo, 3, 35121, Padua, Italy; and †Department of Food Microbiology and Toxicology, and Bacteriology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706
Received October 9, 2001
Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed. © 2001 Academic Press Key Words: botulinum neurotoxin type A; recombinant protein; site directed-mutagenesis; active site
Tetanus neurotoxin (TeNT) and the seven botulinum neurotoxins (BoNT/A to/G) form the family of clostridial neurotoxins metalloproteases, with unique biochemical and structural properties (1, 2). These neurotoxins are closely similar and are produced as single-chain proteins of approximately 150 kDa which are cleaved endogenously or exogenously resulting in a heavy chain (H, 100 kDa) and a light chain (L, 50 kDa) joined by a single inter-chain disulfide bond. The heavy chain mediates the neurotoxin The amino acid numbering used throughout the article is that of clostridial neurotoxins with methionine as the first residue. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹39-0498276049. E-mail: [email protected].
binding to a cell-surface receptor and the entry of the light chain into the neuronal cytosol. The L chains of the clostridial neurotoxins are zinc-dependent proteases very specific for three protein components of the neuroexocytosis apparatus, whose cleavage results in a sustained blockade of the release of neurotransmitters at the synapse (3, 4). TeNT, BoNT/B,/D,/F and/G cleave VAMP, each at different single sites. At variance, BoNT/A and/E cleave SNAP-25 at two different sites within its COOH-terminal, whereas BoNT/C cleaves both syntaxin and SNAP-25 (3, 4). Strikingly, TeNT and BoNT/B cleave VAMP at the same peptide bond (Gln76-Phe77), conclusively proving that the different symptoms of tetanus and botulism result from different intraneuronal targets, rather than from different biochemical activities. A characteristic feature of zinc-dependent endopeptidases is the presence of the zinc binding consensus sequence His-Glu-Xaa-Xaa-His within an active site ␣-helix and the catalytic zinc ion is coordinated by the two motif histidines, by a water molecule linked to glutamic acid and by one or two additional residues (1, 2, 5). The fourth ligand is a glutamic acid in the thermolysin family of metalloproteases (6), whereas one histidine and one tyrosine are implicated in the astacin family (7). The recently solved crystallographic structure of BoNT/A and BoNT/B (8 –10) revealed that in both molecules the zinc atom is coordinated by the residues of the motif and by Glu-262 in BoNT/A and Glu-268 in BoNT/B, which corresponds to Glu-271 of TeNT, a residue conserved among all clostridial neurotoxins. This arrangement is very similar to that in thermolysin even if there is little similarity between the secondary structure of the two enzymes at this site (10). The zinc coordinating residues identify a primary sphere of residues essential to the catalytic function. Site-directed mutagenesis of TeNT and BoNT/A residues of the zinc-binding motif (11–13) and of the conserved glutamic acid 271 of TeNT (14, 15) confirmed the essential role of these residues in catalysis. From
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the analysis of the available structures, it appears that a secondary layer of residues is present at the active site of BoNT/A and BoNT/B. Among these residues, Tyr-366 in BoNT/A (corresponding to Tyr-373 in BoNT/ B), points its phenolic ring inside the cleft-shaped active site of the toxin (8 –10). A mutant generated by the substitution of the corresponding Tyr-375 of TeNT with Ala was completely inactive (15) indicating the fundamental role of this residue in the biological function of TeNT. Hanson and Stevens (16) have proposed that Tyr-373 of BoNT/B assists the hydrolysis of the peptide bond by donating a proton to the amide nitrogen of Phe 77 of VAMP thus stabilizing the leaving group. Taken together, these data and considerations indicate that Tyr-366 of BoNT/A could play a major role in the proteolytic activity, which remains, however, to be proven. In addition, other fully conserved residues contained inside the active site pocket, such as Phe-266 of BoNT/A corresponding to Phe-272 of BoNT/B could play a role in the enzymatic activity of clostridial neurotoxins. Here, we present the results of an investigation of the active site of BoNT/A performed by site-directed mutagenesis of putative key residues for the L chain proteolytic activity. Glu-262, Phe-266 and Tyr-366 in BoNT/A were substituted with the nonconservative residue Ala (E262A, F266A and Y366A). The L chain mutants were expressed as GST fusion protein and were isolated in a correctly folded and active form. The analysis of the proteolytic activity of these mutants indicates that the active site tyrosine but also phenylalanine-266 play an important but not essential role in the enzymatic activity of BoNT/A neurotoxin. The characterization of the structure of the active site of BoNT/A is important for the design either of a recombinant vaccine or of neurotoxin inhibitors of therapeutic value. MATERIALS AND METHODS Chemicals. Restriction endonucleases and T4 DNA ligase and other DNA-modifying enzymes were from New England Biolabs and used as recommended by the manufacturer. pGEX-4T-2 cloning vector, thrombin, GSH-Sepharose 4B and benzamidine resin were purchased from Amersham Pharmacia Biotech. pCR 2.1 vector was from Invitrogen. The plasmid purification kit was obtained from Qiagen Inc. Quickchange site-directed mutagenesis kit was from Stratagene. SNAP-25 was expressed in E. coli as glutathione S-transferase fusion protein and was purified by affinity chromatography on GSHSepharose 4B (Pharmacia). Anti-rabbit immunoglobulin conjugated to alkaline phosphatase was from Boehringer Mannheim. Native BoNT/A was purified as described (18). Cloning of BoNT/A L chain. The L chain gene segment (LC) was PCR amplified using, as primers, the synthetic oligonucleotides 5⬘-AAAGGATCCGGAGGAGGAATGCCATTTGTTAATAAACAATTTAAT-3⬘ and 5⬘-AAAGAATCCTTATTATTTAGAAGTTATTATCCCTCTTAC-3⬘. The latter were designed with the BamHI restriction site sequence at the 5⬘ end and the EcoRI restriction site sequence and stop codon at the 3⬘ end. Chromosomal DNA from C. botulinum
A (strain P64) served as a template in the PCR performed under the following conditions: initial denaturation for 4 min at 95°C, 2 cycles of 1 min at 95°C, 45 s at 52°C, 2 min at 72°C, 2 cycles of 1 min at 95°C, 45 s at 50°C, 2 min at 72°C and 26 cycles of 1 min at 95°C, 45 s at 48°C, 2 min at 72°C. The resultant product was purified by agarose gel electrophoresis and cloned in pCR 2.1 vector. The cloned L chain gene was verified by DNA sequencing and subcloned in pGEX-4T2 vector containing the GST coding sequence (17). Site-directed mutagenesis. For site-directed mutagenesis of BoNT/A the LC cloned in a pCR 2.1 vector was used as template in a PCR amplification performed using the Quickchange site-directed mutagenesis kit (Stratagene). For BoNT/A L-chain mutants E262A, F266A and Y366A, the forward primers were 5⬘-GGTTAGAAGTAAGCTTTGAGGCACTTAGAACATTTGG-3⬘; 5⬘-GGAACTTAGAACAGCAGGGGGACATG-3⬘ and 5⬘GTACTTAACAGAAAAACAGCCTTGAATTTTGATAAAGCCG-3⬘ respectively. The mutated codon is underlined. PCR was performed under the following conditions: initial denaturation for 30 s at 95°C, 16 cycles of 30 s at 95°C, 1 min at 55°C and 10.5 min at 68°C. After digestion of the parental DNA for 1 h at 37°C with DpnI, the amplified plasmids were transformed into E. coli XL-1 Blue competent cells. The presence of the mutation was confirmed by DNA sequencing. The mutated LC genes were digested with BamHI and EcoRI, ligated to pGEX-4T-2 and transformed into strain E. coli BL21. Expression and affinity purification of GST-LC and GST-LC mutants of BoNT/A. LC and its mutants were expressed as GST fusion proteins and isolated as previously described (17). Briefly expression of the BoNT/A LC wild type and its mutants was induced for 3 h at 30°C with IPTG (isopropyl -D-thiogalactopyranoside) and proteins were isolated on a glutathione–Sepharose 4B affinity column according to the manufacturer’s instructions. Resin-bound GST-LCs were cleaved with thrombin for 2 h at 23°C in PBS. The proteins were eluted with PBS and incubated for 1 h at 4°C with benzamidine resin to eliminate the residual thrombin. Protein purity was checked by SDS–PAGE on a 12% polyacrylamide gel (19). Analysis of zinc content. The zinc content of the mutants and the wild-type toxin was measured using a Perkin–Elmer 4000 atomic absorption flame spectrophotometer. After purification of the recombinant protein, metal content of samples containing 3 M of protein was measured after standardization in the linear range of 0 – 0.5 ppm of zinc and the buffer was used as blank. Analysis were performed in triplicate. Fluorescence spectroscopy. Fluorescence spectra were recorded on a Perkin–Elmer LS-50 spectrofluorometer using a 1-cm-pathlength quartz cell kept at 25°C and an excitation wavelength of 280 nm. Samples were prepared by dilution to a final concentration of 3 M in PBS, pH 7.4. The fluorescence spectra were the average of three accumulations and were corrected by subtracting the corresponding base line. Circular dichroic spectroscopy. CD spectra were recorded in the wavelength range of 240 to 190 nm on a Jasco J-715 spectropolarometer fitted with a thermostat cell holder and interfaced with a Neslab (Newington, NH) RTE-110 water bath. Measurements of optical ellipticity were made at 25°C in a 0.1-cm-path-length quartz cell at a protein concentration of 4 M in PBS previously filtrated with 0.45-m filters. At least eight reproducible scans were collected for each sample. Buffer alone was used for a control blank in these experiments. Ellipticities were normalized to residue concentration using the relationship [] ⫽ ( obs/10). (M/l 䡠 c), where obs is the observed ellipticity at a given wavelength, M is the mean residue mass, l is the cuvette pathlength in centimeters, and c is the protein concentration expressed as mg/ml. Secondary structure estimation of the proteins was carried out by using the method described previously (20).
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FIG. 1. Schematic structure of activated di-chain CNTs and amino-acidic sequence of TeNT, BoNT/B, and BoNT/A L-chains. The zinc-binding motif in the central part of the L chain is indicated by an asterisk. Amino acids E262, F266, and Y366 of BoNT/A, which are mutated here, are indicated by an arrow.
Protein denaturation. The guanidinium-induced unfolding process of the LC of BoNT/A and its mutants was followed by monitoring the fluorescence signal at 280 nm as a function of increasing guanidinium concentrations. Guanidinium solutions at different concentration were prepared by diluting a 8 M stock solution in PBS. For each data point LC and mutants were treated at 3 M final concentration with the appropriate guanidinium solution and fluorescence spectra recorded in a 1-cm-path-length quartz cell kept at 25°C.
Assay of proteolytic activity. The proteolytic activity of wildtype-LC and mutants was determined as described previously (21). Briefly, 1.9 M of recombinant GST-SNAP25 (expressed and purified as described above for recombinant LCs) were incubated with recombinant LC or mutants of BoNT/A, in a toxin:substrate ratio of 1:400, at 37°C for various incubation time between 15 and 120 min. In one experiment recombinant GST-SNAP25 was treated in the same conditions with whole native BoNT/A previously reduced as described
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Zinc Content of the Wild Type Recombinant LC and of the Mutants Protein
Zn 2⫹ (mol/mol of protein)
LC E262A F266A Y366A
1.02 ⫾ 0.25 0.57 ⫾ 0.04 0.95 ⫾ 0.22 0.71 ⫾ 0.15
Note. Data are presented as means ⫾ SD of three different preparation.
RESULTS FIG. 2. CD spectroscopy of the recombinant L chain of BoNT/A neurotoxin and its mutants E262A, F266A, and Y366A. Farultraviolet CD emission spectra were recorded in PBS (see also Materials and Methods).
(21). The buffer used for the cleavage reaction was 50 mM Hepes, pH 7.4, containing 150 mM NaCl. The cleavage reaction was stopped by adding SDS–PAGE sample buffer to the mixture. Samples were immunoblotted and revealed with a polyclonal antibody against the COOH-terminal of SNAP25 (1:500 dilution). Antibody reactivity was monitored with an anti-rabbit immunoglobulin conjugated to alkaline phosphatase (1:5000) and quantified by densitometric scanning using a Gel Doc 2000 Bio-Rad apparatus. Rates of proteolysis were estimated by determining the decrease of intact SNAP-25. To ensure that the cleavage of GST-SNAP25 was due to the specific zincendopeptidase activity of the toxin, the recombinant proteins were pre-incubated for 30 min at 37°C in the presence of 10 mM EDTA and then the substrate was added. Under these conditions no cleavage was observed. Protein determination. Protein concentration was determined following the method of Bradford (22) with BSA as standard.
Site-Specific Mutagenesis of BoNT/A L Chain The structures of BoNT/A and BoNT/B have revealed that, in addition to the three residues of the zinc-binding motif, another glutamic acid (Glu-262 in BoNT/A and Glu-268 in BoNT/B, corresponding to Glu-271 in TeNT) acts as fourth ligand (8 –10). In addition, a secondary layer of residues, less close to the zinc atom, is present at the active site and includes Tyr-366 in BoNT/A and Tyr373 in BoNT/B. This tyrosine is about 4.5 Å away from zinc and points its phenolic ring inside the cleft-shaped active site of the toxin; in the isolated BoNT/B L chain, this Tyr residue is closer (3.6 Å) to the zinc atom (16). Moreover, the conserved Phe-272 of BoNT/B (corresponding to Phe-266 in BoNT/A) partially shields the opposite opening of the mouth of the active site cavity (10). Accordingly, Glu-262, Tyr-366 and Phe-266 of BoNT/A L chain were selected as targets for mutagenesis, and were replaced with Ala using a PCR-based mutagenesis (Fig. 1). The resulting circular plasmid containing the point mutations were verified by sequencing and used to transform E. coli BL21 cells. Protein Expression and Purification The BoNT/A wild type LC and its mutants were expressed in E. coli as fusion proteins with GST, affinity purified by GSH-agarose, and the L chain was released with thrombin. Wild type and mutant proteins were subjected to SDS–PAGE under reducing conditions. Results indicate that a purification to homogeneity was achieved and that all proteins have identical electrophoretic mobilities. This latter result, coupled with the similar expression yields observed for the proteins examined argues against errant protein folding and stability during expression. To further prove this point, a spectroscopic analysis was performed. Spectroscopic Analysis
FIG. 3. Fluorescence of the recombinant L chain of BoNT/A neurotoxin and its mutants E262A, F266A and Y366A. Fluorescence emission spectra were recorded in PBS, pH 7.3 (see also Materials and Methods).
To examine whether the mutants are correctly folded, far-UV CD and fluorescence spectroscopes were employed. CD spectra of the mutants are indistin-
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guishable from that of the wild type LC. The amount of ␣-helix is in agreement with the one derived from the crystal structure and all CD spectra show the characteristic double minimum at 208 and 222 nm, indicating their highly helical nature (Fig. 2) (23). Figure 3 shows the emission fluorescence spectra of BoNT/A LC and mutants obtained after excitation at 280 nm. The spectra of the mutants and of the wild type LC show a maximum of fluorescence intensity at about 327 nm. In addition, the guanidinium-induced denaturation curves of wild type LC and mutants were found to be virtually identical (not shown). All together, these data indicate that the active site mutants produced here have a tertiary structure not appreciably different from that of the wild type BoNT/A L chain. Zinc Content of the BoNT/A-L Chains The purified recombinant proteins were analyzed for their zinc content using atomic absorption spectrometry (Table 1). The recombinant LC contains 1 mole of zinc per mole of protein. The F266A mutant has the same amount of zinc of the wild type LC, while the E262A and Y366A mutants of BoNT/A L chain(s) have a reduced zinc content. In particular E262A mutant retains about 60% of zinc, a value similar to that found in a previous experiment of mutagenesis of the corresponding glutamyl residue of TeNT (15). This result confirms the function of Glu-262 as fourth ligand of zinc in BoNT/A.
FIG. 4. Time course of the proteolytic activity of recombinant BoNT/A L chain and of its mutants. (A) Western blot of the time course of the proteolytic activity of the recombinant BoNT/A L chain with an anti C-terminal SNAP25 polyclonal antibody. Lanes 1 and 7: 0.5 and 1 g of control GST-SNAP25, respectively. Lanes 2– 6: GSTSNAP25 after 0, 15, 30, 60, and 120 min of incubation with recombinant BoNT/A L chain. (B) Rates of proteolysis of GST-SNAP25 by the recombinant BoNT/A L chain and by its mutants were measured at 37°C at an enzyme/substrate ratio of 1:400 as detailed under Materials and Methods. Bars are the ⫾ SD of three different experiments.
Proteolytic Activity of BoNT/A Mutants The zinc-dependent endopeptidase activity of BoNT/A can be assayed conveniently with quantitative SDS– PAGE by following the decrease of the SNAP-25 band estimated by immunoblotting with an antibody specific for the C-terminal (21). Figure 4 shows that the wild type recombinant LC has a high activity (50% of hydrolysis after 15 min), which is higher than that of the native whole BoNT/A (not shown), and is completely abolished by 10 mM EDTA. As expected from its coordinating role, the substitution of Glu-262 with Ala in the BoNT/A L chain dramatically impairs its endopeptidase activity. Figure 4 also shows that the Y366A and F266A mutants, after one hour of incubation, had cleaved only 40% and 50% of the substrate hydrolyzed by the native enzyme, respectively. DISCUSSION The crystallographic structure of BoNT/A (8) and of BoNT/B (10, 16), provides a firm structural basis for a rational design of mutants addressing the problem of the relation between structure and function in these
molecules of paramount importance for human therapy and for scientific research (4). The present study shows that mutation of the third zinc ligand of BoNT/A (Glu-262) to an alanine results in a drastic decrease of its metallo-proteolytic activity. The substitution of the corresponding residue (Glu271) in TeNT was found to produce a completely inactive mutant (15). The alanine substitution of the conserved Y375 (TeNT) has a different impact on the activities of TeNT and of BoNT/A. In fact, while the Y366A mutant of BoNT/A L chain produced here still retains little activity, the corresponding mutant of TeNT is inactive (15). This different effect of the same active site mutations in the two related metalloproteases may be explained in terms of their different protein substrates, which may be accommodated in the active site with slightly different geometries. Alternatively, the coordination sphere of TeNT may be more rigid than that of BoNT/A, with more rigid requirements for the spatial positioning of the active site zinc and of the peptide bond to be hydrolyzed. Phenylalanine-266 of BoNT/A L chain was chosen here for site-directed mutagenesis because it is strictly
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conserved among all clostridial neurotoxins and in BoNT/B LC crystal structure it appears to shield partly the mouth of active site cavity (10). The replacement of Phe-266 by an alanine leads to a reduction of the rate of hydrolysis and it is possible that this mutation alters the distances between the bound substrate, the Arg363 (postulated to stabilize the transition state complex) and the Tyr-366, which appear to be critical for catalysis (16). The alternative possibility that mutants have a lowered affinity for the catalytically essential zinc can be discarded on the basis of the finding that the F266A mutant and the wild type L chain have similar contents of zinc. On the contrary, the replacement of glutamate 262 by an alanine leads to a reduction of bound zinc, similarly to what found previously with the related mutant of TeNT (15). Such decrease of zinc binding is however lower than that found in corresponding glutamate mutants of thermolysin (6), neutral endopeptidase NEP (24) and angiotensin I-converting enzyme ACE (25). This probably reflects some differences in the spatial disposition of zinc ligands in BoNT/A and thermolysin (9). The generation of the mutants described here is very relevant to the problem of designing a genetically inactivated mutant of BoNT/A to be used as a component in a vaccine against botulism (26, 27). In fact, the present study shows that enzymatically inactive double mutants can be produced in E. coli in high amounts in a soluble form. ACKNOWLEDGMENTS Work performed in the authors’ laboratory is supported by Telethon-Italia Grant GP0272/01, Progetto Strategico MURST 01.00459.ST97, and MURST Grant 990698133.
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Site-Directed Mutagenesis Identifies Active-Site Residues of the Light Chain of Botulinum Neurotoxin Type A Michela Rigoni,* Paola Caccin,* Eric A. Johnson,† Cesare Montecucco,* and Ornella Rossetto* ,1 *Dipartimento di Scienze Biomediche Sperimentali, Universita` di Padova, Viale G. Colombo, 3, 35121, Padua, Italy; and †Department of Food Microbiology and Toxicology, and Bacteriology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706
Received October 9, 2001
Botulinum neurotoxins (BoNTs) are metalloproteases which block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such proteolysis accounts for the extreme toxicity of these neurotoxins and of their prolonged effect. The recently determined structures of BoNT/A and/B allows one to design active-site mutants to probe the role of specific residues in the proteolysis of SNARE proteins. Here we present the results of mutations of the second glutamyl residue involved in zinc coordination and of a tyrosine and a phenylalanine residues that occupy critical positions within the active site of BoNT/A. The spectroscopic properties of the purified mutants are closely similar to those of the wild-type molecule indicating the acquisition of a correct tertiary structure. Mutation of the Glu-262* nearly abolishes SNAP-25 hydrolysis as expected for a residue involved in zinc coordination. The Phe-266 and Tyr-366 mutants have reduced proteolytic activity indicating a direct participation in the proteolytic reaction, and their possible role in catalysis is discussed. © 2001 Academic Press Key Words: botulinum neurotoxin type A; recombinant protein; site directed-mutagenesis; active site
Tetanus neurotoxin (TeNT) and the seven botulinum neurotoxins (BoNT/A to/G) form the family of clostridial neurotoxins metalloproteases, with unique biochemical and structural properties (1, 2). These neurotoxins are closely similar and are produced as single-chain proteins of approximately 150 kDa which are cleaved endogenously or exogenously resulting in a heavy chain (H, 100 kDa) and a light chain (L, 50 kDa) joined by a single inter-chain disulfide bond. The heavy chain mediates the neurotoxin The amino acid numbering used throughout the article is that of clostridial neurotoxins with methionine as the first residue. 1 To whom correspondence and reprint requests should be addressed. Fax: ⫹39-0498276049. E-mail: [email protected].
binding to a cell-surface receptor and the entry of the light chain into the neuronal cytosol. The L chains of the clostridial neurotoxins are zinc-dependent proteases very specific for three protein components of the neuroexocytosis apparatus, whose cleavage results in a sustained blockade of the release of neurotransmitters at the synapse (3, 4). TeNT, BoNT/B,/D,/F and/G cleave VAMP, each at different single sites. At variance, BoNT/A and/E cleave SNAP-25 at two different sites within its COOH-terminal, whereas BoNT/C cleaves both syntaxin and SNAP-25 (3, 4). Strikingly, TeNT and BoNT/B cleave VAMP at the same peptide bond (Gln76-Phe77), conclusively proving that the different symptoms of tetanus and botulism result from different intraneuronal targets, rather than from different biochemical activities. A characteristic feature of zinc-dependent endopeptidases is the presence of the zinc binding consensus sequence His-Glu-Xaa-Xaa-His within an active site ␣-helix and the catalytic zinc ion is coordinated by the two motif histidines, by a water molecule linked to glutamic acid and by one or two additional residues (1, 2, 5). The fourth ligand is a glutamic acid in the thermolysin family of metalloproteases (6), whereas one histidine and one tyrosine are implicated in the astacin family (7). The recently solved crystallographic structure of BoNT/A and BoNT/B (8 –10) revealed that in both molecules the zinc atom is coordinated by the residues of the motif and by Glu-262 in BoNT/A and Glu-268 in BoNT/B, which corresponds to Glu-271 of TeNT, a residue conserved among all clostridial neurotoxins. This arrangement is very similar to that in thermolysin even if there is little similarity between the secondary structure of the two enzymes at this site (10). The zinc coordinating residues identify a primary sphere of residues essential to the catalytic function. Site-directed mutagenesis of TeNT and BoNT/A residues of the zinc-binding motif (11–13) and of the conserved glutamic acid 271 of TeNT (14, 15) confirmed the essential role of these residues in catalysis. From
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the analysis of the available structures, it appears that a secondary layer of residues is present at the active site of BoNT/A and BoNT/B. Among these residues, Tyr-366 in BoNT/A (corresponding to Tyr-373 in BoNT/ B), points its phenolic ring inside the cleft-shaped active site of the toxin (8 –10). A mutant generated by the substitution of the corresponding Tyr-375 of TeNT with Ala was completely inactive (15) indicating the fundamental role of this residue in the biological function of TeNT. Hanson and Stevens (16) have proposed that Tyr-373 of BoNT/B assists the hydrolysis of the peptide bond by donating a proton to the amide nitrogen of Phe 77 of VAMP thus stabilizing the leaving group. Taken together, these data and considerations indicate that Tyr-366 of BoNT/A could play a major role in the proteolytic activity, which remains, however, to be proven. In addition, other fully conserved residues contained inside the active site pocket, such as Phe-266 of BoNT/A corresponding to Phe-272 of BoNT/B could play a role in the enzymatic activity of clostridial neurotoxins. Here, we present the results of an investigation of the active site of BoNT/A performed by site-directed mutagenesis of putative key residues for the L chain proteolytic activity. Glu-262, Phe-266 and Tyr-366 in BoNT/A were substituted with the nonconservative residue Ala (E262A, F266A and Y366A). The L chain mutants were expressed as GST fusion protein and were isolated in a correctly folded and active form. The analysis of the proteolytic activity of these mutants indicates that the active site tyrosine but also phenylalanine-266 play an important but not essential role in the enzymatic activity of BoNT/A neurotoxin. The characterization of the structure of the active site of BoNT/A is important for the design either of a recombinant vaccine or of neurotoxin inhibitors of therapeutic value. MATERIALS AND METHODS Chemicals. Restriction endonucleases and T4 DNA ligase and other DNA-modifying enzymes were from New England Biolabs and used as recommended by the manufacturer. pGEX-4T-2 cloning vector, thrombin, GSH-Sepharose 4B and benzamidine resin were purchased from Amersham Pharmacia Biotech. pCR 2.1 vector was from Invitrogen. The plasmid purification kit was obtained from Qiagen Inc. Quickchange site-directed mutagenesis kit was from Stratagene. SNAP-25 was expressed in E. coli as glutathione S-transferase fusion protein and was purified by affinity chromatography on GSHSepharose 4B (Pharmacia). Anti-rabbit immunoglobulin conjugated to alkaline phosphatase was from Boehringer Mannheim. Native BoNT/A was purified as described (18). Cloning of BoNT/A L chain. The L chain gene segment (LC) was PCR amplified using, as primers, the synthetic oligonucleotides 5⬘-AAAGGATCCGGAGGAGGAATGCCATTTGTTAATAAACAATTTAAT-3⬘ and 5⬘-AAAGAATCCTTATTATTTAGAAGTTATTATCCCTCTTAC-3⬘. The latter were designed with the BamHI restriction site sequence at the 5⬘ end and the EcoRI restriction site sequence and stop codon at the 3⬘ end. Chromosomal DNA from C. botulinum
A (strain P64) served as a template in the PCR performed under the following conditions: initial denaturation for 4 min at 95°C, 2 cycles of 1 min at 95°C, 45 s at 52°C, 2 min at 72°C, 2 cycles of 1 min at 95°C, 45 s at 50°C, 2 min at 72°C and 26 cycles of 1 min at 95°C, 45 s at 48°C, 2 min at 72°C. The resultant product was purified by agarose gel electrophoresis and cloned in pCR 2.1 vector. The cloned L chain gene was verified by DNA sequencing and subcloned in pGEX-4T2 vector containing the GST coding sequence (17). Site-directed mutagenesis. For site-directed mutagenesis of BoNT/A the LC cloned in a pCR 2.1 vector was used as template in a PCR amplification performed using the Quickchange site-directed mutagenesis kit (Stratagene). For BoNT/A L-chain mutants E262A, F266A and Y366A, the forward primers were 5⬘-GGTTAGAAGTAAGCTTTGAGGCACTTAGAACATTTGG-3⬘; 5⬘-GGAACTTAGAACAGCAGGGGGACATG-3⬘ and 5⬘GTACTTAACAGAAAAACAGCCTTGAATTTTGATAAAGCCG-3⬘ respectively. The mutated codon is underlined. PCR was performed under the following conditions: initial denaturation for 30 s at 95°C, 16 cycles of 30 s at 95°C, 1 min at 55°C and 10.5 min at 68°C. After digestion of the parental DNA for 1 h at 37°C with DpnI, the amplified plasmids were transformed into E. coli XL-1 Blue competent cells. The presence of the mutation was confirmed by DNA sequencing. The mutated LC genes were digested with BamHI and EcoRI, ligated to pGEX-4T-2 and transformed into strain E. coli BL21. Expression and affinity purification of GST-LC and GST-LC mutants of BoNT/A. LC and its mutants were expressed as GST fusion proteins and isolated as previously described (17). Briefly expression of the BoNT/A LC wild type and its mutants was induced for 3 h at 30°C with IPTG (isopropyl -D-thiogalactopyranoside) and proteins were isolated on a glutathione–Sepharose 4B affinity column according to the manufacturer’s instructions. Resin-bound GST-LCs were cleaved with thrombin for 2 h at 23°C in PBS. The proteins were eluted with PBS and incubated for 1 h at 4°C with benzamidine resin to eliminate the residual thrombin. Protein purity was checked by SDS–PAGE on a 12% polyacrylamide gel (19). Analysis of zinc content. The zinc content of the mutants and the wild-type toxin was measured using a Perkin–Elmer 4000 atomic absorption flame spectrophotometer. After purification of the recombinant protein, metal content of samples containing 3 M of protein was measured after standardization in the linear range of 0 – 0.5 ppm of zinc and the buffer was used as blank. Analysis were performed in triplicate. Fluorescence spectroscopy. Fluorescence spectra were recorded on a Perkin–Elmer LS-50 spectrofluorometer using a 1-cm-pathlength quartz cell kept at 25°C and an excitation wavelength of 280 nm. Samples were prepared by dilution to a final concentration of 3 M in PBS, pH 7.4. The fluorescence spectra were the average of three accumulations and were corrected by subtracting the corresponding base line. Circular dichroic spectroscopy. CD spectra were recorded in the wavelength range of 240 to 190 nm on a Jasco J-715 spectropolarometer fitted with a thermostat cell holder and interfaced with a Neslab (Newington, NH) RTE-110 water bath. Measurements of optical ellipticity were made at 25°C in a 0.1-cm-path-length quartz cell at a protein concentration of 4 M in PBS previously filtrated with 0.45-m filters. At least eight reproducible scans were collected for each sample. Buffer alone was used for a control blank in these experiments. Ellipticities were normalized to residue concentration using the relationship [] ⫽ ( obs/10). (M/l 䡠 c), where obs is the observed ellipticity at a given wavelength, M is the mean residue mass, l is the cuvette pathlength in centimeters, and c is the protein concentration expressed as mg/ml. Secondary structure estimation of the proteins was carried out by using the method described previously (20).
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FIG. 1. Schematic structure of activated di-chain CNTs and amino-acidic sequence of TeNT, BoNT/B, and BoNT/A L-chains. The zinc-binding motif in the central part of the L chain is indicated by an asterisk. Amino acids E262, F266, and Y366 of BoNT/A, which are mutated here, are indicated by an arrow.
Protein denaturation. The guanidinium-induced unfolding process of the LC of BoNT/A and its mutants was followed by monitoring the fluorescence signal at 280 nm as a function of increasing guanidinium concentrations. Guanidinium solutions at different concentration were prepared by diluting a 8 M stock solution in PBS. For each data point LC and mutants were treated at 3 M final concentration with the appropriate guanidinium solution and fluorescence spectra recorded in a 1-cm-path-length quartz cell kept at 25°C.
Assay of proteolytic activity. The proteolytic activity of wildtype-LC and mutants was determined as described previously (21). Briefly, 1.9 M of recombinant GST-SNAP25 (expressed and purified as described above for recombinant LCs) were incubated with recombinant LC or mutants of BoNT/A, in a toxin:substrate ratio of 1:400, at 37°C for various incubation time between 15 and 120 min. In one experiment recombinant GST-SNAP25 was treated in the same conditions with whole native BoNT/A previously reduced as described
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Zinc Content of the Wild Type Recombinant LC and of the Mutants Protein
Zn 2⫹ (mol/mol of protein)
LC E262A F266A Y366A
1.02 ⫾ 0.25 0.57 ⫾ 0.04 0.95 ⫾ 0.22 0.71 ⫾ 0.15
Note. Data are presented as means ⫾ SD of three different preparation.
RESULTS FIG. 2. CD spectroscopy of the recombinant L chain of BoNT/A neurotoxin and its mutants E262A, F266A, and Y366A. Farultraviolet CD emission spectra were recorded in PBS (see also Materials and Methods).
(21). The buffer used for the cleavage reaction was 50 mM Hepes, pH 7.4, containing 150 mM NaCl. The cleavage reaction was stopped by adding SDS–PAGE sample buffer to the mixture. Samples were immunoblotted and revealed with a polyclonal antibody against the COOH-terminal of SNAP25 (1:500 dilution). Antibody reactivity was monitored with an anti-rabbit immunoglobulin conjugated to alkaline phosphatase (1:5000) and quantified by densitometric scanning using a Gel Doc 2000 Bio-Rad apparatus. Rates of proteolysis were estimated by determining the decrease of intact SNAP-25. To ensure that the cleavage of GST-SNAP25 was due to the specific zincendopeptidase activity of the toxin, the recombinant proteins were pre-incubated for 30 min at 37°C in the presence of 10 mM EDTA and then the substrate was added. Under these conditions no cleavage was observed. Protein determination. Protein concentration was determined following the method of Bradford (22) with BSA as standard.
Site-Specific Mutagenesis of BoNT/A L Chain The structures of BoNT/A and BoNT/B have revealed that, in addition to the three residues of the zinc-binding motif, another glutamic acid (Glu-262 in BoNT/A and Glu-268 in BoNT/B, corresponding to Glu-271 in TeNT) acts as fourth ligand (8 –10). In addition, a secondary layer of residues, less close to the zinc atom, is present at the active site and includes Tyr-366 in BoNT/A and Tyr373 in BoNT/B. This tyrosine is about 4.5 Å away from zinc and points its phenolic ring inside the cleft-shaped active site of the toxin; in the isolated BoNT/B L chain, this Tyr residue is closer (3.6 Å) to the zinc atom (16). Moreover, the conserved Phe-272 of BoNT/B (corresponding to Phe-266 in BoNT/A) partially shields the opposite opening of the mouth of the active site cavity (10). Accordingly, Glu-262, Tyr-366 and Phe-266 of BoNT/A L chain were selected as targets for mutagenesis, and were replaced with Ala using a PCR-based mutagenesis (Fig. 1). The resulting circular plasmid containing the point mutations were verified by sequencing and used to transform E. coli BL21 cells. Protein Expression and Purification The BoNT/A wild type LC and its mutants were expressed in E. coli as fusion proteins with GST, affinity purified by GSH-agarose, and the L chain was released with thrombin. Wild type and mutant proteins were subjected to SDS–PAGE under reducing conditions. Results indicate that a purification to homogeneity was achieved and that all proteins have identical electrophoretic mobilities. This latter result, coupled with the similar expression yields observed for the proteins examined argues against errant protein folding and stability during expression. To further prove this point, a spectroscopic analysis was performed. Spectroscopic Analysis
FIG. 3. Fluorescence of the recombinant L chain of BoNT/A neurotoxin and its mutants E262A, F266A and Y366A. Fluorescence emission spectra were recorded in PBS, pH 7.3 (see also Materials and Methods).
To examine whether the mutants are correctly folded, far-UV CD and fluorescence spectroscopes were employed. CD spectra of the mutants are indistin-
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guishable from that of the wild type LC. The amount of ␣-helix is in agreement with the one derived from the crystal structure and all CD spectra show the characteristic double minimum at 208 and 222 nm, indicating their highly helical nature (Fig. 2) (23). Figure 3 shows the emission fluorescence spectra of BoNT/A LC and mutants obtained after excitation at 280 nm. The spectra of the mutants and of the wild type LC show a maximum of fluorescence intensity at about 327 nm. In addition, the guanidinium-induced denaturation curves of wild type LC and mutants were found to be virtually identical (not shown). All together, these data indicate that the active site mutants produced here have a tertiary structure not appreciably different from that of the wild type BoNT/A L chain. Zinc Content of the BoNT/A-L Chains The purified recombinant proteins were analyzed for their zinc content using atomic absorption spectrometry (Table 1). The recombinant LC contains 1 mole of zinc per mole of protein. The F266A mutant has the same amount of zinc of the wild type LC, while the E262A and Y366A mutants of BoNT/A L chain(s) have a reduced zinc content. In particular E262A mutant retains about 60% of zinc, a value similar to that found in a previous experiment of mutagenesis of the corresponding glutamyl residue of TeNT (15). This result confirms the function of Glu-262 as fourth ligand of zinc in BoNT/A.
FIG. 4. Time course of the proteolytic activity of recombinant BoNT/A L chain and of its mutants. (A) Western blot of the time course of the proteolytic activity of the recombinant BoNT/A L chain with an anti C-terminal SNAP25 polyclonal antibody. Lanes 1 and 7: 0.5 and 1 g of control GST-SNAP25, respectively. Lanes 2– 6: GSTSNAP25 after 0, 15, 30, 60, and 120 min of incubation with recombinant BoNT/A L chain. (B) Rates of proteolysis of GST-SNAP25 by the recombinant BoNT/A L chain and by its mutants were measured at 37°C at an enzyme/substrate ratio of 1:400 as detailed under Materials and Methods. Bars are the ⫾ SD of three different experiments.
Proteolytic Activity of BoNT/A Mutants The zinc-dependent endopeptidase activity of BoNT/A can be assayed conveniently with quantitative SDS– PAGE by following the decrease of the SNAP-25 band estimated by immunoblotting with an antibody specific for the C-terminal (21). Figure 4 shows that the wild type recombinant LC has a high activity (50% of hydrolysis after 15 min), which is higher than that of the native whole BoNT/A (not shown), and is completely abolished by 10 mM EDTA. As expected from its coordinating role, the substitution of Glu-262 with Ala in the BoNT/A L chain dramatically impairs its endopeptidase activity. Figure 4 also shows that the Y366A and F266A mutants, after one hour of incubation, had cleaved only 40% and 50% of the substrate hydrolyzed by the native enzyme, respectively. DISCUSSION The crystallographic structure of BoNT/A (8) and of BoNT/B (10, 16), provides a firm structural basis for a rational design of mutants addressing the problem of the relation between structure and function in these
molecules of paramount importance for human therapy and for scientific research (4). The present study shows that mutation of the third zinc ligand of BoNT/A (Glu-262) to an alanine results in a drastic decrease of its metallo-proteolytic activity. The substitution of the corresponding residue (Glu271) in TeNT was found to produce a completely inactive mutant (15). The alanine substitution of the conserved Y375 (TeNT) has a different impact on the activities of TeNT and of BoNT/A. In fact, while the Y366A mutant of BoNT/A L chain produced here still retains little activity, the corresponding mutant of TeNT is inactive (15). This different effect of the same active site mutations in the two related metalloproteases may be explained in terms of their different protein substrates, which may be accommodated in the active site with slightly different geometries. Alternatively, the coordination sphere of TeNT may be more rigid than that of BoNT/A, with more rigid requirements for the spatial positioning of the active site zinc and of the peptide bond to be hydrolyzed. Phenylalanine-266 of BoNT/A L chain was chosen here for site-directed mutagenesis because it is strictly
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conserved among all clostridial neurotoxins and in BoNT/B LC crystal structure it appears to shield partly the mouth of active site cavity (10). The replacement of Phe-266 by an alanine leads to a reduction of the rate of hydrolysis and it is possible that this mutation alters the distances between the bound substrate, the Arg363 (postulated to stabilize the transition state complex) and the Tyr-366, which appear to be critical for catalysis (16). The alternative possibility that mutants have a lowered affinity for the catalytically essential zinc can be discarded on the basis of the finding that the F266A mutant and the wild type L chain have similar contents of zinc. On the contrary, the replacement of glutamate 262 by an alanine leads to a reduction of bound zinc, similarly to what found previously with the related mutant of TeNT (15). Such decrease of zinc binding is however lower than that found in corresponding glutamate mutants of thermolysin (6), neutral endopeptidase NEP (24) and angiotensin I-converting enzyme ACE (25). This probably reflects some differences in the spatial disposition of zinc ligands in BoNT/A and thermolysin (9). The generation of the mutants described here is very relevant to the problem of designing a genetically inactivated mutant of BoNT/A to be used as a component in a vaccine against botulism (26, 27). In fact, the present study shows that enzymatically inactive double mutants can be produced in E. coli in high amounts in a soluble form. ACKNOWLEDGMENTS Work performed in the authors’ laboratory is supported by Telethon-Italia Grant GP0272/01, Progetto Strategico MURST 01.00459.ST97, and MURST Grant 990698133.
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