Molecular assembly of botulinum neurotoxin progenitor complexes (original) (raw)
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A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms
Journal of Biological Chemistry, 2007
The botulinum neurotoxins (BoNTs) are the most potent toxins known in nature, causing the lethal disease known as botulism in humans and animals. The BoNTs act by inhibiting neurotransmitter release from cholinergic synapses. Clostridium botulinum strains produce large BoNTs toxin complexes, which include auxiliary non-toxic proteins that appear not only to protect BoNTs from the hostile environment of the digestive tract but also to assist BoNT translocation across the intestinal mucosal layer. In this study, we visualize for the first time a series of botulinum serotype D toxin complexes using negative stain transmission electron microscopy (TEM). The complexes consist of the 150-kDa BoNT, 130-kDa nontoxic non-hemagglutinin (NTNHA), and three kinds of hemagglutinin (HA) subcomponents: 70-kDa HA-70, 33-kDa HA-33, and 17-kDa HA-17. These components assemble sequentially to form the complex. A novel TEM image of the mature L-TC revealed an ellipsoidal-shaped structure with "three arms" attached. The "body" section was comprised of a single BoNT, a single NTNHA and three HA-70 molecules. The arm section consisted of a complex of HA-33 and HA-17 molecules. We determined the x-ray crystal structure of the complex formed by two HA-33 plus one HA-17. On the basis of the TEM image and biochemical results, we propose a novel 14-mer subunit model for the botulinum toxin complex. This unique model suggests how non-toxic components make up a "delivery vehicle" for BoNT.
Molecular Architecture of Botulinum Neurotoxin E Revealed by Single Particle Electron Microscopy
Journal of Biological Chemistry, 2007
Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of fulllength BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A. Botulinum neurotoxin (BoNT), 4 considered the most potent toxin known, causes botulism (1) by selectively inhibiting synaptic vesicle exocytosis (2). This conspicuously specific activity has transformed BoNT into the first bacterial toxin approved by the FDA for treatment of a number of diseases characterized by abnormal muscle contraction and as a blockbuster cosmeceutical and a most feared bioweapon (1, 3). Clostridium botulinum cells produce seven BoNT isoforms designated serotypes A to G (2). All BoNT serotypes are synthesized as a single polypeptide chain with molecular mass ϳ150 kDa. This precursor protein is cleaved by a clostridial protease into two polypeptides that remain linked by a disulfide bridge. The mature dichain toxin consists of a 50-kDa light chain (LC) Zn 2ϩ metalloprotease and a 100-kDa heavy chain (HC). The HC encompasses the translocation domain (TD) (the N-terminal half) and the receptor-binding domain (RBD) (the C-terminal half). BoNT/E is atypical in that it is not activated by proteolytic cleavage in the clostridial cells, thereby requiring unidentified proteases in the host cells to cleave the LC from the HC to achieve full toxicity (4, 5). BoNTs exert their neuroparalytic effect by a multistep mechanism (2, 6). RBD-mediated binding to protein and lipid receptors on the cell surface of peripheral nerve endings (7-11) triggers receptor-mediated endocytosis and traffic to the endosomes. The acidic pH of endosomes induces a conformational change of the toxin; the HC inserts into the lipid bilayer and forms a protein-conducting channel (12, 13). The HC channel then translocates the protease domain into the cytoplasm (13), colocalizing with its substrate SNARE (soluble NSF attachment protein receptor) (14-16). Because the SNARE core complex is essential for synaptic vesicle fusion with the presynaptic membrane (14-16), BoNTs efficiently block synaptic vesicle exocytosis. In contrast to BoNT/A (17), little is known about the molecular architecture of BoNT/E, making it a novel target for structural analysis. Here we report the three-dimensional structure of BoNT/E holotoxin at ϳ30 Å resolution as determined by single particle electron microscopy (EM). Domains of BoNT/E were assigned to the globular features observed in the structure by labeling the toxin with functionally relevant monoclonal antibodies (mAbs). Although the individual domains of BoNT/E are similar to those of BoNT/A, their spatial arrangement within the global fold is unique. Analysis of the BoNT/E structure and structure-function correlation studies with mAbs bound to BoNT/A and BoNT/E define previously unrecognized biophysical characteristics that differ between these two BoNT isoforms. EXPERIMENTAL PROCEDURES Materials-Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich. Purified native BoNT serotypes A and E holotoxins were from Metabiologics. Di-chain BoNT/E holotoxin was generated by cleavage with trypsin: BoNT/E holotoxin (0.5 mg/ml) was incubated with 0.15 mg/ml trypsin in 20 mM HEPES, pH 7.0, for 30 min at 37°C. Thereaf
Botulinum neurotoxins: genetic, structural and mechanistic insights
Nature Reviews Microbiology, 2014
Clostridium is a genus of sporulating and anaerobic Gram-positive, rod-shaped bacteria that includes more than 150 species. These bacteria are widely distributed in the environment and in anaerobic regions of the intestines of several animals, where they are typically found as spores, which are resistant to physical and chemical stresses and can persist for long periods of time until favourable conditions enable germination 1,2 . Under appropriate environmental conditions (such as humidity, nutrients and the absence of oxygen), spores germinate into vegetative cells; conversely, exposure to oxygen, as well as water and nutrient deprivation, trigger sporulation. Several clostridia, including Clostridium difficile, Clostridium perfringens and Clostridium sordelli, are pathogenic, owing to the release of protein toxins, but only a few species are neurotoxigenic. For example, Clostridium tetani produces tetanus neurotoxin, which blocks neurotransmitter release in spinal cord interneurons and causes the spastic paralysis of tetanus 3 . In addition, six phylogenetically distinct clostridia produce more than 40 different botulinum neurotoxins (BoNTs) (BOX 1). BoNTs consist of three primary domains: two of these domains enable binding to nerve terminals and translocation of the toxin into the neuronal cytosol, and the third domain comprises a metalloprotease that inhibits the release of neurotransmitter by peripheral nerve terminals (BOX 2), which causes the flaccid paralysis and autonomic dysfunctions that are typical of botulism 2,4 . The neurospecificity and toxic potency of BoNTs make them the most powerful known toxins, and they are potential bioterrorism weapons 5,6 . By contrast, their absolute neurospecificity has enabled BoNTs to be used as effective therapeutic agents for human diseases that Neurotransmitter An endogenous chemical that transmits signals across a synapse from a neuron to a postsynaptic cell.
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.
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,
Current Microbiology, 2016
A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the ''toxin complex.'' Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the ''toxin complex'' produced by C-N71. The ''toxin complex'' was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the ''toxin complex.'' Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic ''toxin complex'' of C-N71 may be valuable for the development of an oral drug delivery system.
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.
Toxins
Botulinum neurotoxins (BoNTs) cause flaccid neuromuscular paralysis by cleaving one of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex proteins. BoNTs display high affinity and specificity for neuromuscular junctions, making them one of the most potent neurotoxins known to date. There are seven serologically distinct BoNTs (serotypes BoNT/A to BoNT/G) which can be further divided into subtypes (e.g., BoNT/A1, BoNT/A2…) based on small changes in their amino acid sequence. Of these, BoNT/A1 and BoNT/B1 have been utilised to treat various diseases associated with spasticity and hypersecretion. There are potentially many more BoNT variants with differing toxicological profiles that may display other therapeutic benefits. This review is focused on the structural analysis of the cell-binding domain from BoNT/A1 to BoNT/A6 subtypes (HC/A1 to HC/A6), including features such as a ganglioside binding site (GBS), a dynamic loop, a synaptic vesicle glyc...
PLOS One, 2007
Background. Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression. Methodology/ Principal Findings. Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid. Conclusions/Significance. Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum. Citation: Smith TJ, Hill KK, Foley BT, Detter JC, Munk AC, et al (2007) Analysis of the Neurotoxin Complex Genes in Clostridium botulinum A1-A4 and B1 Strains: BoNT/A3, /Ba4 and /B1 Clusters Are Located within Plasmids. PLoS ONE 2(12): e1271.
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