HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 16, 10906-10911, April 21, 2006

This Article Abstract Full Text (PDF) All Versions of this Article: 281/16/10906    most recent M513032200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, S. Articles by Barbieri, J. T. Search for Related Content PubMed PubMed Citation Articles by Chen, S. Articles by Barbieri, J. T. Social Bookmarking                      What's this?

Unique Substrate Recognition by Botulinum Neurotoxins Serotypes A and E^*

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, December 6, 2005 , and in revised form, February 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A–G. BoNT serotype A and serotype E cleave SNAP25 at residues 197–198 and 180–181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156–202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167–186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The Clostridium botulinum neurotoxins (BoNTs)² are the most potent protein toxins for humans (1). There are seven distinguishable BoNT serotypes, A–G, with serotypes A, B, and E responsible for most natural human intoxications (2). BoNT serotypes are defined by the specificity of antibody neutralization where antibodies that neutralize one serotype fail to neutralize other serotypes. There are two licensed vaccines against botulism, a pentavalent vaccine against serotypes A–E (3) and a heptavalent vaccine against serotypes A–G (4). However, these vaccines are produced from chemically inactivated BoNT produced in C. botulinum and are currently in limited supply. Upon intoxication, BoNT intoxication has significant morbidity and mortality (5, 6). Thus, there is a need to develop more efficient vaccines and therapies against botulism.

BoNTs are zinc proteases that elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNTs are 150-kDa single chain proteins that are activated by proteolysis to generate disulfide-linked di-chain proteins. BoNTs are organized into three functional domains: an N-terminal catalytic domain (light chain, LC), an internal translocation domain (heavy chain, HCT), and a C-terminal receptor binding domain (heavy chain, HCR) (7, 8). BoNTs enter neurons via receptor-mediated endocytosis. The tropism for neurons is due to the affinity of BoNT for receptors on peripheral neurons (7). BoNT-receptor interactions are sometimes enhanced by gangliosides, which act as co-receptors (9). In the acidic endosome, the HCT inserts into the endosome membrane to generate a pore that allows the catalytic LC to translocate into the cytosol (10). LCs cleave neurotransmitter vesicle fusion proteins. BoNT/A cleaves SNAP25 between residues 197 and 198, and BoNT/E cleaves SNAP25 between residues 180 and 181, inactivating SNAP25 on the plasma membrane (11). BoNT/C cleaves both SNAP25 and syntaxin (12), whereas the other BoNT serotypes and tetanus toxin cleave the vesicle-associated membrane protein (13).

Previous studies have addressed the specificity of substrate recognition to define residues and regions of SNAP25 that are required for cleavage by BoNT. Several amino acids that surround the scissile bond contribute to efficient cleavage of SNAP25 (14). Montecucco and coworkers (15) defined an amino acid repeat motif within SNAP25, termed the SNARE motif, which correlated to substrate recognition, whereas Binz and coworkers (16) defined regions and residues of SNAP25 required for optimal cleavage by BoNT/A, BoNT/E, and BoNT/C. Although the first co-crystal of BoNT/B and vesicle-associated membrane protein did not provide electron density of substrate (17, 18), a recent co-crystal structure of a non-catalytic LC/A and SNAP25-(146–204) (19) defined an -exosite and -exosite within SNAP25 that were implicated in substrate recognition. This study provides a reference for the functional mapping of substrate determinants required for catalysis.

Relative to thermolysin, the BoNTs recognize a larger region of substrate for optimal cleavage. The large regions required for substrate recognition of vesicle fusion proteins, such as SNAP25, may be due to the limited structure of the SNARE proteins which may require an extended contact region for high affinity binding. There is limited understanding of how the SNAP25 is recognized for specific cleavage, which prompted a detailed study to identify how these BoNT/A and BoNT/E recognize SNAP25.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasmid Construction and Protein Expression
BoNT LC/A and LC/E were constructed by amplifying DNA encoding LC fragments from Clostridium botulinum serotype A1 Hall strain (20) and E_Begula (21), (E. A. Johnson, University of Wisconsin-Madison) respectively, and subcloning into pET-15b. Plasmids encoding LCs were transformed into Escherichia coli BL21(DE3) RIL (Stratagene). Protein expression was achieved by culturing E. coli at 16 °C overnight with 0.75 mM isopropyl 1-thio--D-galactopyranoside as previously described (22). Briefly, cells were broken with a French press. His-LCs were purified sequentially from cell lysates by nitrilotriacetic acid-affinity chromatography, gel filtration, and DEAE chromatography. SNAP25 was constructed by amplifying DNA encoding the indicated region of SNAP25 and subcloning into pGEX-2T. Plasmids encoding SNAP25 were transformed into E. coli TG1. Protein expression was achieved by culturing E. coli at 30 °C for 2 h with 0.75 mM isopropyl 1-thio--D-galactopyranoside. GST-SNAP25 was purified using glutathione-Sepharose 4B affinity chromatography (Amersham Biosciences).

Mutagenesis of SNAP25
Random and directed mutagenesis was performed to achieve saturation mutagenesis of SNAP25. Mutations are designated N(XXX)N, where the first N is the native amino acid, the XXX is the residue mutated, and the second N is the mutation. Random mutagenesis of SNAP25 was performed with GeneMorph II Random Mutagenesis Kit (Stratagene) by Mutazyme II DNA polymerase during DNA amplification. Briefly, amplification was performed in a 50-µl reaction containing 1 ng of pGEX-SNAP25-(141–206), 2.5 units of Mutazyme II DNA polymerase, 40 µM dNTPs, and 50 pmol of 5'-GTTCCGCGTGGATCGGAGCTC and 5'-AGTCACGATGAATTCGGATCC. The reaction was preheated at 95 °C for 2 min, and then incubated for 35 cycles at 95 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min, and then finishing by incubating at 72 °C for 10 min. Amplified products were purified, digested, and subcloned into pGEX-2T. Recombinant plasmids were sequenced to identify the mutations within SNAP25. Site-directed mutagenesis was performed to mutate DNA encoding residues that were not mutated by random mutagenesis. Site-directed mutagenesis was performed by QuikChange (Stratagene) following the manufacturer's instruction, using pGEX-SNAP25-(141–206) as a template. For example, the G168R, R176C, D179V, I181E, A195D, and R198E point mutations generated by random mutagenesis that had >50% inhibition of LC cleavage were engineered with more conserved mutations, G168A, R176A, D179A, I181A, A195S, and R198A, and tested for sensitivity to cleavage by LC/A and LC/E.

Cleavage of SNAP25 by BoNT-LCs
Linear Velocity Reactions—Reactions contained (10 µl): 5 µM of the indicated SNAP25 incubated with LC/A or LC/E in 10 mM Tris-HCl (pH 7.6) with 20 mM NaCl. Reactions were run at 37 °C for 10 min, stopped by adding SDS-PAGE buffer and heating for 5 min, and subjected to SDS-PAGE. Gels were stained, and the amount of SNAP25 cleavage was determined by densitometry.

Kinetic Parameters—K_m and k_cat determinations were made for LC/A with the indicated SNAP25-(146–206). LC was adjusted to cleave <10% substrate in various concentrations of substrates. Reaction velocity versus substrate concentration was fit to the Michaelis-Menten equation, and kinetic constants were derived, using the EnzFitter program (Elsevier, UK).

Molecular Modeling
Proteins were modeled with Swiss Model software from the Brookhaven National Laboratories (23).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Residues That Define Substrate Recognition of SNAP25 for LC/A and LC/E
Initial attempts to identify the molecular basis for the substrate recognition of BoNT, by exchanging the surface determinants within LC/A and LC/E were not successful. In these experiments, chimeric proteins were generated that contained complementary loops of the two LCs (at residues 50, 170, 250, and 370 of LC/A and the corresponding region of LC/E) but yielded chimeric proteins that were either inefficient or inactive in SNAP25 cleavage. This prompted the reciprocal strategy to define substrate specificity through the mutagenesis of SNAP25.

Previous deletion analysis resolved residues 141–202 as the minimal region of SNAP25 that can be efficiently cleaved by LC/A (15, 16). Initial mapping showed that deletion of the SNARE motif (residues 145–154) did not affect the ability of LC/E to cleave a SNAP25 and had 5-fold lower rate for cleavage by LC/A (Table 1). Further mapping showed that SNAP25-(167–202) was efficiently cleaved by LC/E, whereas LC/A cleavage was less efficient, suggesting that the binding domain for LC/E was shorter than LC/A. This also indicated that residues 146–155 played a limited role in SNAP25 recognition by LC/A or LC/E and prompted a saturation mutagenesis approach to residues 156–202 of SNAP25 to define cleavage properties of LC/A and LC/E. Fig. 1A shows the differential affect of representative single amino acids mutations within residues 156–202 of SNAP25 on the cleavage capacities of LC/A and LC/E. A composite of the effect of individual point mutations within residues 156–202 of SNAP25 on cleavage by LC/A and LC/E cleavage is presented in Fig. 1B. Mutation of 18 of 48 residues did not effect LC/A or LC/E cleavage of SNAP25, mutations of 11 of 48 residues inhibited the ability of LC/A and LC/E to cleave SNAP25, whereas 18 of 48 residues had differential effects on SNAP25 cleavage by LC/A or LC/E. Two regions were resolved that included mutated residues, which showed the greatest inhibitory action to LC/A, residues 193–202, termed the active site (AS) domain and residues 156–181, termed the binding (B) domain. Consistent with the co-crystal structure of a non-catalytic form of LC/A and SNAP25 (19), Ile¹⁵⁶, Asp¹⁶⁶, Glu¹⁷⁰, Ile¹⁷¹, Asp¹⁷², Arg¹⁷⁶, Ile¹⁷⁸, Ile¹⁸¹, Glu¹⁸³, and Asp¹⁹³, which showed side-chain interaction with LC/A, showed inhibitory effects on LC/A cleavage of the mutated SNAP25. Several additional residues (Ala¹⁹⁵, Arg¹⁹⁸, and Lys²⁰¹) also inhibited LC/A cleavage of the mutated SNAP25. In linear velocity reactions, mutation of R198E in SNAP25 abolished the ability of LC/A to cleave SNAP25, whereas the more conserved mutation, R198A, reduced LC/A cleavage efficiency 1000-fold. Mutation of A195S reduced LC/A cleavage efficiency 5000-fold.

Thus, two domains in SNAP25 were required for optimal LC/A and LC/E cleavage. The AS domain included the respective cleavage sites for LC/A and LC/E, and the B domain included residues that were 12 or 5 amino acids upstream of the respective cleavage site domain for LC/A and LC/E. Limited trypsin digestion patterns for point mutated SNAP25 that were cleaved by LC/A or LC/E by <50% cleavage relative to wild type yielded three identical SNAP25-dependent peptides for the mutated and wild-type SNAP25, suggesting that the point mutations that had the greatest effect on cleavage did not have gross conformational effects on SNAP25 structure (data not shown).

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Mutational analysis of the cleavage of residues 155–202 of SNAP25 by LC/A and LC/E. 5µM of the indicated SNAP25 was incubated with 20 nM LC/A or 2 nM LC/E at 37 °C for 10 min. The reaction was stopped, subjected to SDS-PAGE, stained, and subjected to densitometry. A, cleavage of representative mutated SNAP25 by LC/A and LC/E. B, cleavage of point-mutated SNAP25 by LC/A or LC/E. The top row is the primary amino acid sequence of SNAP25. The second and third rows show the cleavage efficiency of the indicated point mutated SNAP25 by LC/A and LC/E, respectively. Red indicates 75% inhibition of LC cleavage, blue indicates 50% inhibition of LC cleavage, and yellow indicates inhibition to 25%. Multiple mutations have been obtained at several residues, such as G168R (A) and G168A (B).

Deletion Mapping SNAP25 Peptides as Substrates for LC/A and LC/E
Saturation mutagenesis studies predicted optimal cleavage sites of SNAP25 that should support cleavage by LC/A or LC/E. Deletion mutations were engineered within SNAP25 to test the role of B domain and AS domain on efficiency and specificity of SNAP25 cleavage by LC/A and LC/E.

Previous studies that mapped the minimal domain required for the LC/A and LC/E cleavage of SNAP25 (16) were performed using GST fusion proteins. Our initial studies determined that GST-SNAP25-(156–206) is cleaved by LC/A and LC/E at a slow rate when the cleavage site residues were aligned close to the GST component of the fusion protein (data not shown). This indicated that the GST component of GST-SNAP25 fusion proteins hindered cleavage by BoNTs when the cleavage site was aligned near GST. To circumvent these complications, an extended version of GST-SNAP25 was engineered to include residues 93–206 of SNAP25, which placed the SNAP25 cleavage site more distanced from the GST component of the fusion protein than used in the earlier studies. In this platform, LC/A cleaved SNAP25-(93–206)146–155 within 5-fold of the rate of SNAP25-(93–206) cleavage. Deletion of residues 146–166 reduced the ability of LC/A to cleave by 100-fold, whereas removal of residues 146–186 reduced the ability of LC/A to cleave substrate by 1000-fold relative to the cleavage of SNAP25-(93–206) (Table 1). Deletion of 199–206 of SNAP25 abolished LC/A cleavage, indicating the importance of C-terminal residues for substrate cleavage (Table 1). The efficiency of LC/A cleavage of deletion peptides of SNAP25 were consistent with the analysis of point mutations within SNAP25, where mutation to Ile¹⁵⁶, Leu¹⁶⁰, Met¹⁶³, and Ala¹⁶⁴ inhibited LC/A cleavage of SNAP25. This defined residues 156–202 of SNAP25 as an optimal cleavage site for LC/A.

LC/E cleaved SNAP25-(93–206)146–155 and SNAP25-(93–206)146–166 similar efficiencies, within 2-fold, as SNAP25-(93–206). LC/E cleaved SNAP25-(146–186) at a rate similar as SNAP25-(93–206). LC/E did not cleave SNAP25-(141–182), implicating a role for residues 183–186 in substrate cleavage (Table 1). This defined residues 167–186 of SNAP25 as an optimal cleavage site for LC/E.

Minimal AS Domains of SNAP25 That Can Be Cleaved by LC/A and LC/E
The cumulative data indicated the AS domain contributed to the catalytic capacity and substrate specificity of SNAP25 by LC/A and LC/E. To test this model, GST fusion proteins were engineered that included two minimal LC/A AS domains, ¹⁹²DEANQRATK²⁰⁰ and ¹⁹⁴ANQRATK²⁰⁰ termed as SN/A1 and SN/A2, respectively, and two minimal LC/E AS domains for LC/E, ¹⁷⁸IDRIMEKAD¹⁸⁶ and ¹⁷⁸IDRIME¹⁸³ termed SN/E1 or SN/E2, respectively (Fig. 2A). LC/A cleaved SN/A1, but did not cleave SN/A2 at the highest concentration of LC/A tested, 10 µM. This indicated that ¹⁹²DEANQRATK²⁰⁰ was a minimal substrate for LC/A. LC/E cleaved SN/E1 more efficiently than SN/E2, but cleavage of SN/E2 was detected at the highest concentration of LC/E, 10 µM (Fig. 2B). This indicated that residues 184–186 enhanced substrate cleavage, but that ¹⁷⁸IDRIME¹⁸³ was a minimal cleavable substrate for LC/E. Controls showed that 10 µM LC/A did not cleave SN/E1 and that 10 µM LC/E did not cleave SN/A1 (Fig. 2C).

To test a minimal cleavage domain of SNAP25 for LC/E, a peptide, Y¹⁷⁴NRQIDRIMEK¹⁸⁴ (1466.7 Da), comprising SNAP-(174–184) was tested as a substrate for LC/E. Mass spectrometry analysis detected two peptide products with masses of 965.5 Da and 520.3 Da, indicating that LC/E cleaved the peptide at the scissile bond, between Arg¹⁸⁰ and Ile¹⁸¹ of SNAP25.

A Peptide Comprising Residues 167–181 of SNAP25 Inhibits LC/A and LC/E Catalysis
Mutational analysis showed a common region (residues 167–181) that impacted the ability of LC/A and LC/E to cleave SNAP25 at their respective cleavage sites. A peptide comprising SNAP25-(167–181), MGNEIDTQNRQIDRI, was synthesized and tested for the ability to inhibit LC/A and LC/E cleavage of SNAP25. The peptide inhibited 50% cleavage of SNAP25 by LC/E at 400 µM and LC/A at 600 µM. The concentration of peptide required for inhibition was similar to the K_m of peptides that were previously identified as substrates for BoNT/A cleavage (14). The peptide did not inhibit cleavage of vesicle-associated membrane protein by LC/B, which indicated inhibition specificity. Mass spectrometry did not detect cleavage of the peptide by LC/E or LC/A (data not shown).

View larger version (55K): [in this window] [in a new window]   FIGURE 2. Cleavage of peptide substrates by LC/A and LC/E. A, schematic showing the structure and sequence of SN/A1, SN/A2, SN/E1, and SN/E2. SNAP25-(93–146) was used as platform for the indicated peptide. Residues in the filled box are the indicated sequences of SNAP25 that include the scissile cleavage site for LC/A and LC/E. Residues in the C-terminal open box are a C-terminal extension to allow detection of substrate cleavage. B, cleavage of SN/A1, SN/A2, SN/E1, and SN/E2 by LC/E and LC/A. 5 µM or 10 µM of LC/E (B1) or LC/E (B2) were incubated with SN/A1, SN/A2 or SN/E1, and SN/E2 protein at room temperature for 12 h. The reaction was stopped and subjected to SDS-PAGE. The arrow shows the cleaved products. C, cleavage of SN/A1 by LC/E and SN/E1 by LC/A. LC/A was incubated with SN/E1 and LC/E with SN/A1 as described in B. The reaction was stopped and subjected to SDS-PAGE.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

SNAP25 Cleavage by LC/A—Previous studies implicated several regions and residues of SNAP25 that play a role in substrate recognition by LC/A. Three motifs have been implicated in substrate recognition, the SNARE motif, -exosite, and -exosite (15, 19). The SNARE motif and -exosite lie outside residues 156–202, suggesting that neither play major roles substrate recognition, while several residues that are components of the -exosite were identified as contributing to substrate recognition. The inability to detect mutations that influenced catalysis in residues throughout the -exosite indicated that this region is localized and that discontinuous residues were utilized for substrate recognition. This is supported by the co-crystal of LC/A and SNAP25 (19), which showed that an extended region of SNAP25 in the -exosite aligned, but that several residues were not bonded to LC/A. Non-contact residues within the -exosite may provide proper alignment for rather than contact points in the association where Gln¹⁵² and Asp¹⁶⁶ in the N-terminal portion of the -exosite show direct interactions with LC/A.

View larger version (61K): [in this window] [in a new window]   FIGURE 3. Model of active-site pocket mediated substrate recognition for LC/A and LC/E. Schematic of the B domain and AS domain of SNAP25 for LC/A and LC/E and the important residues in those domains. Point size reflects the importance of each residue for SNAP25 cleavage by LC/A (upper sequence) or LC/E (lower sequence). The lower panel is a model of active site-mediated substrate recognition by LC/A and LC/E. The model was generated by SWISS-MODEL software using the co-crystal structure of LCA-SNAP25 (PDB 1XTG) as a template. The red ribbon structure represents SNAP25 AS domain aligned within the active site cavity of LC/A and LC/E. The red-white-blue color gradations of the LCs represent the acidic-neutral-basic, respectively, nature of the amino acids within the active site cavity.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Alignment of human SNAP25, human SNAP23, and mouse SNAP23. Underlined residues are the B and AS regions of human SNAP25 for LC/A (upper panel) and LC/E (lower panel). Bold residues in human SNAP25 are the important residues involved in LC/E and LC/A binding and recognition. Bold residues in human and mouse SNAP23 were located within the B and AS regions of the human SNAP25 and differed from the respective residues in human SNAP25.

Recognition of SNAP25 Isoforms by LC/A and LC/E—Human SNAP25 is a substrate of LC/A and LC/E, whereas human SNAP23 is not cleaved by LC/A and LC/E and mouse SNAP23 is a poor substrate for LC/A and LC/E (16, 30). Sequence alignment shows that the B domain for LC/E binding is identical between SNAP25 and the two isoforms (Fig. 4). However, a Lys in human SNAP23 disrupts the "IDRI" sequence of the AS domain to interfere with LC/E cleavage. The mutation of K185D makes human SNAP23 a good substrate of LC/E. The presence of Gln¹⁸⁴ in mouse SNAP23 shows less effect than Lys in this position because both Gln and Asp have similar sized R-groups (16). This explains why mouse SNAP23 is cleaved more efficiently by LC/E than human SNAP23.

LC/A does not cleave human SNAP23 and cleaves mouse SNAP23 poorly (16). While AS recognition of LC/A is identical between human SNAP25 and these isoforms, residues within the B domain of human SNAP25 vary with respect to human SNAP23 and mouse SNAP23, including Ile¹⁷³, Arg¹⁸², Glu¹⁸⁹, Lys¹⁹⁵, and Thr¹⁹⁶ of human SNAP23. Previous studies observed that the I173M and the R182P mutation enhanced the binding and cleavage of human SNAP23 by LC/A, which supports a role for these residues in SNAP recognition by LCs. The higher homology within the B domains of mouse SNAP23 and human SNAP25 than human SNAP23 and SNAP25 may be responsible for the relatively better cleavage of mouse SNAP23 by LC/A than human SNAP23.

AS Site Interactions of LC/A and LC/E with SNAP25—The known structures for BoNT LCs and tetanus toxin, along with the structure of thermolysin in complex with ZFPLA containing leucine at the P1' residue (31) provided an opportunity to identify the putative S1' of LC/A and LC/E, using the assumption that the S1' pocket of each LCs align with thermolysin and was complementary in size and hydrophobicity to the cognate P1' residue (31, 32). In case LC/A (Fig. 3), the S1' pocket, is formed by Phe¹⁶³, Phe¹⁹⁴, Thr²²⁰, Asp³⁷⁰, and P1' Arg¹⁹⁸ and may include salt bridge with Asp³⁷⁰ within the pocket. Insertion of P1' side chain into S1' pocket and the formation of hydrogen bond of LC/A Arg³⁶³ with carbonyl oxygen of P1' R198 may stabilize SNAP25 docking and contribute to substrate specificity. P3, Ala¹⁹⁵, may form a main-chain interaction to contribute to catalysis or Ala¹⁹⁵ function as a bridging molecule, since Arg²³¹ is at the corner of the S3 pocket. The S5 pocket of LC/A may also contribute to substrate recognition where insertion of P5 Asp¹⁹³ into the basic S5 pocket may stabilize substrate-LC/A interactions.

A similar pocket model can be predicted for LC/E cleavage of SNAP25 (Fig. 3). The S1' pocket of LC/E is formed by Thr¹⁵⁹, Phe¹⁹¹, Thr²⁰⁸, and Tyr³⁵⁴ and is shallower and more hydrophobic than the S1' pocket of LC/A, which may reflect the nature of the P1' Ile¹⁸¹ of SNAP25 for LC/E cleavage. Insertion of the P1' Ile¹⁸¹ side chain into the S1' pocket of LC/E where a hydrogen bond may form between LC/E Arg³⁴⁷and main-chain carbonyl oxygen of P1' to stabilize the substrate docking and contribute to substrate specificity. P2 Asp¹⁷⁹ of SNAP25 may contribute to substrate specificity through ionic interactions with Lys²²²in the S2 pocket of LC/E. The smaller overall cavity of LC/E relative to LC/A may be explained by the P1' and P2 sites specify of LC/E versus the P1', P3, and P5 site specify of LC/A. Relative to thermolysin, several of the AS interactions of LC/A and LC/E are unique and possibly a property common to the BoNT family proteins that can be exploited in designing small molecule inhibitors that specifically target BoNTs.

	FOOTNOTES

 
^* This work was supported by the Great Lakes Regional Center of Excellence from NIAID, National Institutes of Health Grant U54 AI057153. Assistance of the Protein-Nucleic Acid Facility at the Medical College of Wisconsin for MALDI-MS analyses is acknowledged. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Medical College of Wisconsin, Microbiology and Molecular Genetics, 8701 Watertown Plank Rd., Milwaukee, Wisconsin 53226. Tel.: 414-456-8412; Fax: 414-456-6535; E-mail: jtb01{at}mcw.edu.

² The abbreviations used are: BoNT, botulinum neurotoxin; SNARE, soluble NSF attachment protein receptors; SNAP25, soluble NSF attachment protein 25; LC, light chain; GST, glutathione S-transferase.

	ACKNOWLEDGMENTS

 
We acknowledge Michael Baldwin and the other members of the Barbieri laboratory for helpful discussion. We also acknowledge C. Pier and E. A. Johnson (University of Wisconsin-Madison) for supplying the LC constructs for subcloning.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Schiavo, G., Rossetto, O., Tonello, F., and Montecucco, C. (1995) Curr. Top. Microbiol. Immunol. 195, 257–274[Medline] [Order article via Infotrieve]
2. Marvaud, J. C., Raffestin, S., and Popoff, M. R. (2002) C. R. Biol. 325, 863–878; discussion 879–883[Medline] [Order article via Infotrieve]
3. Middlebrook, J. L. (1995) Adv. Exp. Med. Biol. 383, 93–98[Medline] [Order article via Infotrieve]
4. Robinson, R. F., and Nahata, M. C. (2003) Ann. Pharmacother. 37, 127–131[Abstract/Free Full Text]
5. Shapiro, R. L., Hatheway, C., and Swerdlow, D. L. (1998) Ann. Intern Med. 129, 221–228[Abstract/Free Full Text]
6. Arnon, S. S., Midura, T. F., Damus, K., Wood, R. M., and Chin, J. (1978) Lancet 1, 1273–1277[CrossRef][Medline] [Order article via Infotrieve]
7. Montecucco, C., and Schiavo, G. (1994) Mol. Microbiol. 13, 1–8[Medline] [Order article via Infotrieve]
8. Poulain, B., and Humeau, Y. (2003) Ann. Readapt. Med. Phys. 46, 265–275[Medline] [Order article via Infotrieve]
9. Dong, M., Richards, D. A., Goodnough, M. C., Tepp, W. H., Johnson, E. A., and Chapman, E. R. (2003) J. Cell Biol. 162, 1293–1303[Abstract/Free Full Text]
10. Koriazova, L. K., and Montal, M. (2003) Nat. Struct. Biol. 10, 13–18[CrossRef][Medline] [Order article via Infotrieve]
11. Tonello, F., Morante, S., Rossetto, O., Schiavo, G., and Montecucco, C. (1996) Adv. Exp. Med. Biol. 389, 251–260[Medline] [Order article via Infotrieve]
12. Foran, P., Lawrence, G. W., Shone, C. C., Foster, K. A., and Dolly, J. O. (1996) Biochemistry 35, 2630–2636[CrossRef][Medline] [Order article via Infotrieve]
13. Schiavo, G., Rossetto, O., Benfenati, F., Poulain, B., and Montecucco, C. (1994) Ann. N. Y. Acad. Sci. 710, 65–75[Medline] [Order article via Infotrieve]
14. Schmidt, J. J., and Bostian, K. A. (1997) J. Protein Chem. 16, 19–26[CrossRef][Medline] [Order article via Infotrieve]
15. Washbourne, P., Pellizzari, R., Baldini, G., Wilson, M. C., and Montecucco, C. (1997) FEBS Lett. 418, 1–5[CrossRef][Medline] [Order article via Infotrieve]
16. Vaidyanathan, V. V., Yoshino, K., Jahnz, M., Dorries, C., Bade, S., Nauenburg, S., Niemann, H., and Binz, T. (1999) J. Neurochem. 72, 327–337[CrossRef][Medline] [Order article via Infotrieve]
17. Hanson, M. A., and Stevens, R. C. (2000) Nat. Struct. Biol. 7, 687–692[CrossRef][Medline] [Order article via Infotrieve]
18. Rupp, B., and Segelke, B. (2001) Nat. Struct. Biol. 8, 663–664[Medline] [Order article via Infotrieve]
19. Breidenbach, M. A., and Brunger, A. T. (2004) Nature 432, 925–929[CrossRef][Medline] [Order article via Infotrieve]
20. Siegel, L. S., and Metzger, J. F. (1979) Appl. Environ. Microbiol. 38, 606–611[Abstract/Free Full Text]
21. Poulet, S., Hauser, D., Quanz, M., Niemann, H., and Popoff, M. R. (1992) Biochem. Biophys. Res. Commun. 183, 107–113[CrossRef][Medline] [Order article via Infotrieve]
22. Baldwin, M. R., Bradshaw, M., Johnson, E. A., and Barbieri, J. T. (2004) Protein Expr. Purif. 37, 187–195[CrossRef][Medline] [Order article via Infotrieve]
23. Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) Nucleic Acids Res. 31, 3381–3385[Abstract/Free Full Text]
24. Breidenbach, M. A., and Brunger, A. T. (2005) Trends Mol. Med. 11, 377–381[CrossRef][Medline] [Order article via Infotrieve]
25. Agarwal, R., Binz, T., and Swaminathan, S. (2005) Biochemistry 44, 11758–11765[CrossRef][Medline] [Order article via Infotrieve]
26. Agarwal, R., Binz, T., and Swaminathan, S. (2005) Biochemistry 44, 8291–8302[CrossRef][Medline] [Order article via Infotrieve]
27. Shone, C. C., and Roberts, A. K. (1994) Eur. J. Biochem. 225, 263–270[Medline] [Order article via Infotrieve]
28. Schmidt, J. J., and Stafford, R. G. (2005) Biochemistry 44, 4067–4073[CrossRef][Medline] [Order article via Infotrieve]
29. Schmidt, J. J., Stafford, R. G., and Bostian, K. A. (1998) FEBS Lett. 435, 61–64[CrossRef][Medline] [Order article via Infotrieve]
30. Sadoul, K., Berger, A., Niemann, H., Weller, U., Roche, P. A., Klip, A., Trimble, W. S., Regazzi, R., Catsicas, S., and Halban, P. A. (1997) J. Biol. Chem. 272, 33023–33027[Abstract/Free Full Text]
31. Holden, H. M., Tronrud, D. E., Monzingo, A. F., Weaver, L. H., and Matthews, B. W. (1987) Biochemistry 26, 8542–8553[CrossRef][Medline] [Order article via Infotrieve]
32. Arndt, J. W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M. A., Lebeda, F. J., and Stevens, R. C. (2005) J. Mol. Biol. 346, 1083–1093[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				H. Poras, T. Ouimet, S.-V. Orng, M.-C. Fournie-Zaluski, M. R. Popoff, and B. P. Roques Detection and Quantification of Botulinum Neurotoxin Type A by a Novel Rapid In Vitro Fluorimetric Assay Appl. Envir. Microbiol., July 1, 2009; 75(13): 4382 - 4390. [Abstract] [Full Text] [PDF]

				S. Chen and J. T. Barbieri From the Cover: Engineering botulinum neurotoxin to extend therapeutic intervention PNAS, June 9, 2009; 106(23): 9180 - 9184. [Abstract] [Full Text] [PDF]

				R. Agarwal and S. Swaminathan SNAP-25 Substrate Peptide (Residues 180-183) Binds to but Bypasses Cleavage by Catalytically Active Clostridium botulinum Neurotoxin E J. Biol. Chem., September 19, 2008; 283(38): 25944 - 25951. [Abstract] [Full Text] [PDF]

				S. Sikorra, T. Henke, T. Galli, and T. Binz Substrate Recognition Mechanism of VAMP/Synaptobrevin-cleaving Clostridial Neurotoxins J. Biol. Chem., July 25, 2008; 283(30): 21145 - 21152. [Abstract] [Full Text] [PDF]

				S. Chen, C. Hall, and J. T. Barbieri Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin J. Biol. Chem., July 25, 2008; 283(30): 21153 - 21159. [Abstract] [Full Text] [PDF]

				S. Chen and J. T. Barbieri Multiple Pocket Recognition of SNAP25 by Botulinum Neurotoxin Serotype E J. Biol. Chem., August 31, 2007; 282(35): 25540 - 25547. [Abstract] [Full Text] [PDF]

				S. Chen, J.-J. P. Kim, and J. T. Barbieri Mechanism of Substrate Recognition by Botulinum Neurotoxin Serotype A J. Biol. Chem., March 30, 2007; 282(13): 9621 - 9627. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 281/16/10906    most recent M513032200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chen, S. Articles by Barbieri, J. T. Search for Related Content PubMed PubMed Citation Articles by Chen, S. Articles by Barbieri, J. T. Social Bookmarking                      What's this?