Crystal structure of the receptor binding domain of the botulinum C–D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions (original) (raw)
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Separation of the components of type A botulinum neurotoxin complex by electrophoresis
Toxicon, 2003
Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A-G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS -PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS -PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS -PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field. q
2010
Botulinum neurotoxins (BoNTs) are highly potent poisons produced by seven serotypes of Clostridium botulinum. The mechanism of neurotoxin action is a multistep process which leads to the cleavage of one of three different SNARE proteins essential for synaptic vesicle fusion and transmission of the nerve signals to muscles: synaptobrevin, syntaxin, or SNAP-25. In order to understand the precise mechanism of neurotoxin in a host, the domain structure of the neurotoxin was analyzed among different serotypes of C. botulinum. The results indicate that neurotoxins type A, C, D, E and F contain a coiled-coil domain while types B and type G neurotoxin do not. Interestingly, phylogenetic analysis based on neurotoxin sequences has further confirmed that serotypes B and G are closely related. These results suggest that neurotoxin has multi-domain structure, and coiled-coil domain plays an important role in oligomerisation of the neurotoxin. Domain analysis may help to identify effective antibodies to treat Botulinum toxin intoxication.
The Botulinum Neurotoxin Complex and the Role of Ancillary Proteins
Molecular Aspects of Botulinum Neurotoxin, 2014
All seven known serotypes of botulinum neurotoxin (BoNT) are produced in the form of a complex with a group of neurotoxin-associated proteins (NAPs). The BoNT complex is encoded by a gene cluster regulated by its own transcription factor, and the proteins coded by polycistronic messenger ribonucleic acid (mRNA) self-assemble into complexes of 300-900 kDa. Types A, B, C, D, and G complexes contain hemagglutinin (HA), whereas types E and F complexes do not contain HA. Sequence homology among respective BoNTs and NAPs range from 55.3 to 98.5 %, and all the proteins in the BoNT complex belong to a stable class of protein with high longevity inside mammalian cells. A new 250-kDa protein (P-250) with high immunogenicity has been identified in the BoNT/A complex which is not part of the neurotoxin gene cluster. The 33-kDa hemagglutinin (HA-33) is the most abundant NAP. The HA-33 is protease resistant and is highly immunogenic. HA-33 appears to play an important role in the translocation of the neurotoxin across the gut wall, enhancing the endopeptidase activity of BoNT and protection of BoNT against proteases. The role of other NAPs is not as clear, and their role in the biology of the bacteria is not understood at all. BoNT complexes are used as therapeutic products, although a therapeutic product without NAPs appears to retain the properties of the complex-based products. NAPs in therapeutic products may have other subtle long-term effects which need to be investigated. Keywords Botulinum • Botox • Clostridium • Dysport • Complex • Neurotoxin • NAPs • Protein stability • Serotypes • Therapeutic • Toxin • Xeomin • Botulinum neurotoxin • Neurotoxin-associated proteins • Hemagglutinin • Progenitor neurotoxin • Gene cluster • Operon • Polycistronic • Molecular stoichiometry • Endopeptidase K. A. Foster (ed.), Molecular Aspects of Botulinum Neurotoxin, Current Topics in Neurotoxicity 4,
2010
Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D_HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D_HCR was obtained. The recombinant BoNT/ D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å . Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.
A historical and proteomic analysis of botulinum neurotoxin type/G
BMC Microbiology, 2011
Background: Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results: An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs-nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.
Journal of Biological Chemistry, 2013
Background: The function of C terminus of botulinum neurotoxin catalytic domain is unknown. Results: Synthetic C-terminal peptides competitively inhibited but at stoichiometric concentrations stimulated serotype A proteolytic activity. Conclusion: C terminus interacts with the active site and may function by removing a product. Significance: The inhibition and product removal appear to be a unique feature of type A botulinum neurotoxin among catalytic proteins. Botulinum neurotoxins are the most toxic of all compounds. The toxicity is related to a poor zinc endopeptidase activity located in a 50-kDa domain known as light chain (Lc) of the toxin. The C-terminal tail of Lc is not visible in any of the currently available x-ray structures, and it has no known function but undergoes autocatalytic truncations during purification and storage. By synthesizing C-terminal peptides of various lengths, in this study, we have shown that these peptides competitively inhibit the normal catalytic activity of Lc of serotype A (LcA) and have defined the length of the mature LcA to consist of the first 444 residues. Two catalytically inactive mutants also inhibited LcA activity. Our results suggested that the C terminus of LcA might interact at or near its own active site. By using synthetic C-terminal peptides from LcB, LcC1, LcD, LcE, and LcF and their respective substrate peptides, we have shown that the inhibition of activity is specific only for LcA. Although a potent inhibitor with a K i of 4.5 M, the largest of our LcA C-terminal peptides stimulated LcA activity when added at near-stoichiometric concentration to three versions of LcA differing in their C-terminal lengths. The result suggested a product removal role of the LcA C terminus. This suggestion is supported by a weak but specific interaction determined by isothermal titration calorimetry between an LcA C-terminal peptide and N-terminal product from a peptide substrate of LcA. Our results also underscore the importance of using a mature LcA as an inhibitor screening target. Functions of catalytic and regulatory proteins are largely dictated by their three-dimensional structures, which are dependent on their primary sequences. Recent years have witnessed a tremendous proliferation of three-dimensional structure determinations by the advent of high throughput x-ray crystallography soon after the sequence and adequate expression of a protein became available (1, 2). In some proteins, no electron density can be observed for stretches of the amino acid sequence especially at the N or C terminus (3-8). Thus, a functional role for such regions is not always discernible from their three-dimensional structures. The catalytic domain of botulinum neurotoxin (BoNT) 3 belongs to this category of proteins. BoNT and tetanus neurotoxins are a unique class of zinc endopeptidases that act selectively at discrete sites on three synaptosomal proteins of the neuroexocytotic apparatus (for reviews, see Refs. 45 and 48). These neurotoxins are the most potent of all known toxins. Seven serotypes of BoNT, designated A-G, produced by immunologically distinct strains of Clostridium botulinum may cause death by flaccid muscle paralysis at the neuromuscular junction. These neurotoxins are expressed as 150-kDa single chain polypeptides. Posttranslational proteolytic cleavage generates a dichain molecule consisting of a 100-kDa C-terminal heavy chain and a 50-kDa N-terminal light chain (LC or Lc) of ϳ450 amino acids connected by a disulfide bond. The LC contains the zinc endopeptidase catalytic domain. The 100-kDa heavy chain can be further proteolyzed into a 50-kDa N-terminal membrane-spanning domain (Hn) and a 50-kDa C-terminal receptor-binding domain (Hc). The first x-ray structure determined for the 150-kDa BoNT/A accounted for only the first 431 amino acids only of * This work was supported in part by Defense Threat Reduction Agency-Joint Science and Technology Office for Chemical and Biological Defense Grant JSTOCBD3.10012_06_RD_B (to S. A. A.).
The Botulinum J., 2008
Botulinum Neurotoxin (BoNT) is responsible for botulism, a severe and often deadly disease. Light chain of BoNT behaves as endopeptidase, cleaving the SNARE proteins, and inhibits the neurotransmitter release. A double-mutant E224A/E262A full-length BoNT Type A was successfully cloned and expressed in E. coli. The purified protein lacks the endopeptidase activity involved in the toxic action, and is structurally compatible with the native BoNT/A. Thus this molecule not only can be used as a safe surrogate for study of BoNT, but also can be potentially used as an antidote against botulism, and as a vaccine candidate for botulism.
Botolinum toxins: their structure, properties, and genetics
2016
Clostridium botulinum is Gram positive, spore-forming anaerobic bacteria, which can produce botulinum neurotoxins (BoNTs). The toxins block the release of neurotransmitter, acetylcholine, at peripheral cholinergic nerve terminal and cause flaccid paralysis of muscle in human and animals, a condition known as botulism. BoNTs are classified into seven different serotypes (designated as BoNT/ A-BoNT/G), in which serotype A, B, E, and F cause botulism in human. BoNTs are comprised of one domain of light chain (L-chain) at N-terminus and two domains of heavy chains (H-chain) at C-terminus. The function of L-chain is to cleave SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins that involve in the exocytosis of neurotransmitter whereas H-chain is responsible for binding of toxin with nerve terminal and translocating of L-chain into cytosol from synaptic vesicle. The BoNTs are usually produced as complexes called progenitor toxin complex (PTC). They bind...