Thermal stabilization of the catalytic domain of botulinum neurotoxin E by phosphorylation of a single tyrosine residue

The catalytic domain of clostridial neurotoxins is a substrate of tyrosine-specific protein kinases. The functional role of tyrosine phosphorylation and also the number and location of its (their) phosphorylation site(s) are yet elusive. We have used the recombinant catalytic domain of botulinum neurotoxin E (BoNT E) to examine these issues. Bacterially expressed and purified BoNT E catalytic domain was fully active, and was phosphorylated in vitro by the tyrosine-specific kinase Src. Tyrosine phosphorylation of the catalytic domain increased the protein thermal stability without affecting its proteolytic activity. Covalent modification of the endopeptidase promoted a disorder-to-order transition, as evidenced by the 35% increment of the alpha-helical content, which resulted in a 4 degrees C increase of its denaturation temperature. Site-directed replacement of tyrosine at position 67 completely abolished phosphate incorporation by Src. Constitutively unphosphorylated endopeptidase mutants exhibited functional properties virtually identical to those displayed by the nonphosphorylated wild-type catalytic domain. These findings indicate the presence of a single phosphorylation site in the catalytic domain of clostridial neurotoxins, and that its covalent modification primarily modulates the protein thermostability.     

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.     

Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins

In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum. Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin. Type A C. botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs). Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes. BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively. Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions. Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively. Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs. We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.     

Glycine insertion at protease cleavage site of SNAP25 resists cleavage but enhances affinity for botulinum neurotoxin serotype A

The light chain of botulinum neurotoxin A (BoNT/A‐LC) is a Zn‐dependent protease that specifically cleaves SNAP25 of the SNARE complex, thereby impairing vesicle fusion and neurotransmitter release at neuromuscular junctions. The C‐terminus of SNAP25 (residues 141–206) retains full activity for BoNT/A‐LC‐catalyzed cleavage at P1‐P1' (Gln197‐Arg198). Using the structure of a complex between the C‐terminus of SNAP25 and BoNT/A‐LC as a model to design SNAP25‐derived pseudosubstrate inhibitors (SNAPIs) that prevent presentation of the scissile bond to the active site, we introduced multiple His residues to replace Ala‐Asn‐Gln‐Arg (residues 195–198) at the substrate cleavage site, with the intent to identify possible side‐chain interactions with the active site Zn. We also introduced multiple Gly residues between the P1‐P1' residues to explore the spatial tolerance within the active‐site cleft. Using a FRET substrate YsCsY, we compared a series of SNAPIs for inhibition of BoNT/A‐LC. Among the SNAPIs tested, several known cleavage‐resistant, single‐point mutants of SNAP25 were poor inhibitors, with most of the mutants losing binding affinity. Replacement with His at the active site did not improve inhibition over wildtype substrate. In contrast, Gly‐insertion mutants were not only resistant to cleavage, but also surprisingly showed enhanced affinity for BoNT/A‐LC. Two of the Gly‐insertion mutants exhibited 10‐fold lower IC₅₀ values than the wildtype 66‐mer SNAP25 peptide. Our findings illustrate a scenario, where the induced fit between enzyme and bound pseudosubstrate fails to produce the strain and distortion required for catalysis to proceed.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

Non-toxic derivatives of botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150 kDa molecule consisting of a 50 kDa light chain (LC) and a 100 kDa heavy chain (HC) joined by a disulfide bond and rendered atoxic through the introduction of metalloprotease-inactivating point mutations in the light chain. Studies in neuronal cultures demonstrated that BoNT/A ad cannot cleave synaptosomal-associated protein 25 (SNAP25), the substrate of wt BoNT/A, and that it effectively competes with wt BoNT/A for binding to endogenous neuronal receptors. In vitro and in vivo studies indicate accumulation of BoNT/A ad at the neuromuscular junction of the mouse diaphragm. Immunoprecipitation studies indicate that the LC of BoNT/A ad forms a complex with SNAP25 present in the neuronal cytosolic fraction, demonstrating that the atoxic LC retains the SNAP25 binding capability of the wt toxin. Toxicity of BoNT/A ad was found to be reduced approximately 100,000-fold relative to wt BoNT/A.     

Src phosphorylates ezrin at tyrosine 477 and induces a phosphospecific association between ezrin and a kelch-repeat protein family member

Ezrin, a linker between plasma membrane and actin cytoskeleton possesses morphogenic properties and can promote dissemination of tumor cells. Ezrin is phosphorylated on tyrosine, but a detailed picture of the signaling pathways involved in this modification is lacking. The transforming tyrosine kinase Src has various cytoskeletal substrates and is involved in regulation of cellular adhesion. We studied the role of Src in tyrosine phosphorylation of ezrin in adherent cells. We show that ezrin is phosphorylated in human embryonic kidney 293 cells in a Src family-dependent way. In SYF cells lacking Src, Yes, and Fyn, ezrin was not tyrosine-phosphorylated but reintroduction of wild-type Src followed by Src activation or introduction of active Src restored phosphorylation. Mapping of the Src-catalyzed tyrosine in vitro and in vivo by site-directed mutagenesis demonstrated Tyr(477) as the primary target residue. We generated a pTyr(477)-phosphospecific antibody, which confirmed that Tyr(477) becomes phosphorylated in cells in a Src-dependent manner. Tyr(477) phosphorylation did not affect ezrin head-to-tail association or phosphorylation of ezrin on threonine 566, indicating that the function of Tyr(477) phosphorylation is not related to the intramolecular regulation of ezrin. A modified yeast two-hybrid screen in which ezrin bait was phosphorylated by Src identified a novel interaction with a kelch-repeat protein family member, KBTBD2 (Kelch-repeat and BTB/POZ domain containing 2). The Src dependence of the interaction was further verified by affinity precipitation assays. Identification of a functional interplay with Src opens novel avenues for further characterization of the biological activities of ezrin.     

Characterization of in vivo keratin 19 phosphorylation on tyrosine-391

Background

Keratin polypeptide 19 (K19) is a type I intermediate filament protein that is expressed in stratified and simple-type epithelia. Although K19 is known to be phosphorylated on tyrosine residue(s), conclusive site-specific characterization of these residue(s) and identification potential kinases that may be involved has not been reported.

Methodology/Principal Findings

In this study, biochemical, molecular and immunological approaches were undertaken in order to identify and characterize K19 tyrosine phosphorylation. Upon treatment with pervanadate, a tyrosine phosphatase inhibitor, human K19 (hK19) was phosphorylated on tyrosine 391, located in the ‘tail’ domain of the protein. K19 Y391 phosphorylation was confirmed using site-directed mutagenesis and cell transfection coupled with the generation of a K19 phospho (p)-Y391-specific rabbit antibody. The antibody also recognized mouse phospho-K19 (K19 pY394). This tyrosine residue is not phosphorylated under basal conditions, but becomes phosphorylated in the presence of Src kinase in vitro and in cells expressing constitutively-active Src. Pervanadate treatment in vivo resulted in phosphorylation of K19 Y394 and Y391 in colonic epithelial cells of non-transgenic mice and hK19-overexpressing mice, respectively.

Conclusions/Significance

Human K19 tyrosine 391 is phosphorylated, potentially by Src kinase, and is the first well-defined tyrosine phosphorylation site of any keratin protein. The lack of detection of K19 pY391 in the absence of tyrosine phosphatase inhibition suggests that its phosphorylation is highly dynamic.     

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.     

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (H_N and H_C) differ by 13%. The LC acts as a zinc-dependent endopeptidase, H_N as the translocation domain, and H_C as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, H_N of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 H_N is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.     

A novel mechanism of modulation of hyperpolarization-activated cyclic nucleotide-gated channels by Src kinase

Hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) play a crucial role in the regulation of cell excitability. Importantly, they contribute to spontaneous rhythmic activity in brain and heart. HCN channels are principally activated by membrane hyperpolarization and binding of cAMP. Here, we identify tyrosine phosphorylation by Src kinase as another mechanism affecting channel gating. Inhibition of Src by specific blockers slowed down activation kinetics of native and heterologously expressed HCN channels. The same effect on HCN channel activation was observed in cells cotransfected with a dominant-negative Src mutant. Immunoprecipitation demonstrated that Src binds to and phosphorylates native and heterologously expressed HCN2. Src interacts via its SH3 domain with a sequence of HCN2 encompassing part of the C-linker and the cyclic nucleotide binding domain. We identified a highly conserved tyrosine residue in the C-linker of HCN channels (Tyr476 in HCN2) that confers modulation by Src. Replacement of this tyrosine by phenylalanine in HCN2 or HCN4 abolished sensitivity to Src inhibitors. Mass spectrometry confirmed that Tyr476 is phosphorylated by Src. Our results have functional implications for HCN channel gating. Furthermore, they indicate that tyrosine phosphorylation contributes in vivo to the fine tuning of HCN channel activity.     

Botulinum neurotoxin inhibitor binding dynamics and kinetics relevant for drug design

BackgroundA natural product analog, 3-(4-nitrophenyl)-7H-furo[3,2-g]chromen-7-one, which is a nitrophenyl psoralen (NPP) was found to be an effective inhibitor of botulinum neurotoxin type A (BoNT/A).MethodsIn this work, we performed enzyme inhibition kinetics and employed biochemical techniques such as isothermal calorimetry (ITC) and fluorescence spectroscopy as well as molecular modeling to examine the kinetics and binding mechanism of NPP inhibitor with BoNT/A LC.ResultsStudies of inhibition mechanism and binding dynamics of NPP to BoNT/A light chain (BoNT/A LC) showed that NPP is a mixed type inhibitor for the zinc endopeptidase activity, implying that at least part of the inhibitor-enzyme binding site may be different from the substrate-enzyme binding site. By using biochemical techniques, we demonstrated NPP forms a stable complex with BoNT/A LC. These observations were confirmed by Molecular Dynamics (MD) simulation, which demonstrates that NPP binds to the site near the active site.ConclusionThe NPP binding interferes with BoNT/A LC binding to the SNAP-25, hence, inhibits its cleavage. Based on these results, we propose a modified strategy for designing a molecule to enhance the efficiency of the inhibition against the neurotoxic effect of BoNT.General significanceInsights into the interactions of NPP with BoNT/A LC using biochemical and computational approaches will aid in the future development of effective countermeasures and better pharmacological strategies against botulism.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability

Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade. BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids). Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25. Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery. LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli. LC 1-448 was purified, but precipitated upon storage. Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430. Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility. Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage. Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat. Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.     

Tyrosine phosphorylation of CD45 phosphotyrosine phosphatase by p50csk kinase creates a binding site for p56lck tyrosine kinase and activates the phosphatase.

Src family protein tyrosine kinases (PTKs) play an essential role in antigen receptor-initiated lymphocyte activation. Their activity is largely regulated by a negative regulatory tyrosine which is a substrate for the activating action of the CD45 phosphotyrosine phosphatase (PTPase) or, conversely, the suppressing action of the cytosolic p50csk PTK. Here we report that CD45 was phosphorylated by p50csk on two tyrosine residues, one of them identified as Tyr-1193. This residue was not phosphorylated by T-cell PTKs p56lck and p59fyn. Tyr-1193 was phosphorylated in intact T cells, and phosphorylation increased upon treatment with PTPase inhibitors, indicating that this tyrosine is a target for a constitutively active PTK. Cotransfection of CD45 and csk into COS-1 cells caused tyrosine phosphorylation of CD45 in the intact cells. Tyrosine-phosphorylated CD45 bound p56lck through the SH2 domain of the kinase. Finally, p50csk-mediated phosphorylation of CD45 caused a severalfold increase in its PTPase activity. Our results show that direct tyrosine phosphorylation of CD45 can affect its activity and association with Src family PTKs and that this phosphorylation could be mediated by p50csk. If this is also true in the intact cells, it adds a new dimension to the physiological function of p50csk in T lymphocytes.     

Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity.

Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form. The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain. By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure. This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid. Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure. A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C.     

A second SNARE role for exocytic SNAP25 in endosome fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play key roles in membrane fusion, but their sorting to specific membranes is poorly understood. Moreover, individual SNARE proteins can function in multiple membrane fusion events dependent upon their trafficking itinerary. Synaptosome-associated protein of 25 kDa (SNAP25) is a plasma membrane Q (containing glutamate)-SNARE essential for Ca2+-dependent secretory vesicle-plasma membrane fusion in neuroendocrine cells. However, a substantial intracellular pool of SNAP25 is maintained by endocytosis. To assess the role of endosomal SNAP25, we expressed botulinum neurotoxin E (BoNT E) light chain in PC12 cells, which specifically cleaves SNAP25. BoNT E expression altered the intracellular distribution of SNAP25, shifting it from a perinuclear recycling endosome to sorting endosomes, which indicates that SNAP25 is required for its own endocytic trafficking. The trafficking of syntaxin 13 and endocytosed cargo was similarly disrupted by BoNT E expression as was an endosomal SNARE complex comprised of SNAP25/syntaxin 13/vesicle-associated membrane protein 2. The small-interfering RNA-mediated down-regulation of SNAP25 exerted effects similar to those of BoNT E expression. Our results indicate that SNAP25 has a second function as an endosomal Q-SNARE in trafficking from the sorting endosome to the recycling endosome and that BoNT E has effects linked to disruption of the endosome recycling pathway.     

Comparative role of neurotoxin-associated proteins in the structural stability and endopeptidase activity of botulinum neurotoxin complex types A and E

Seven serotypes of botulinum neurotoxins, the most toxic substances known to mankind, are each produced by different strains of Clostridium botulinum along with a group of neurotoxin-associated proteins (NAPs). NAPs play a critical role in the toxicoinfection process of botulism in addition to their role in protecting the neurotoxin from proteolytic digestion in the GI tract as well as from adverse environmental conditions. In this study we have investigated the effect of temperature on the structural and functional stability of BoNT/A complex (BoNT/AC) and BoNT/E complex (BoNT/EC). Although the NAPs in the two complexes are quite different, both groups of NAPs activate the endopeptidase activities of their BoNTs without any need to reduce the disulfide bonds between light and heavy chains of respective BoNTs. BoNT/AC attains optimum enzyme activity at the physiological temperature of 37 degrees C whereas BoNT/EC is maximally active at 45 degrees C, and this is accompanied by conformational alterations in its polypeptide folding at this temperature, leading to favorable binding with its intracellular substrate, SNAP-25, and subsequent cleavage of the latter. BoNT/A in its complex form is found to be structurally more stable against temperature whereas BoNT/E in its complex form is functionally better protected against temperature. Based on the analysis of isolated NAPs we have observed that the structural stability of the BoNT/AC is contributed by the NAPs. In addition to the unique structural conditions in which the enzyme remains active, functional stability of botulinum neurotoxins against temperature plays a critical role in the survival of the agent in cooked food and in food-borne botulism.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

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. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.