Electron Density Projection Map of the Botulinum Neurotoxin 900-kilodalton Complex by Electron Crystallography

Burkard, F.; Chen, Flora; Kuziemko, Geoffrey M.; Stevens, Raymond C.

doi:10.1006/jsbi.1997.3910

Cited by 15 publications

(8 citation statements)

References 21 publications

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“…In an earlier study, based on negative stain electron microscopy of the serotype A BoNT and analysis of a separated non-toxic complex, Boroff et al (32) designed a model for the complex that allowed the BoNT strand to fit within the coils of the HAs. Likewise, Burkard et al (33) proposed a model for the serotype A 900-kDa LL-TC (dimer of L-TC) that was triangular in shape, with six lobes apparent in the electron crystallographic images that were obtained using a lipid-layer technique. However, both models provided neither the stoichiometry of the components nor their structural organization.…”

Section: Resultsmentioning

confidence: 99%

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Hasegawa

Watanabe

Suzuki

et al. 2007

Journal of Biological Chemistry

104

108

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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.Different strains of Clostridium botulinum produce seven distinct serotypes of neurotoxins (BoNTs), 2 classified A through G. BoNT has attracted much interest in recent years due to extensive research on its biochemistry, determination of its crystal structure, and investigations into the pharmacology and applications of BoNTs as therapeutic agents for the treatment of human disease (1-3). After ingestion of BoNT, the BoNT is absorbed from intestinal epithelial cells into the bloodstream, after which it consequently reaches the neuromuscular junctions. BoNT enters nerve cells via receptor-mediated endocytosis, where it cleaves specific sites on target proteins, inhibiting release of neurotransmitters from peripheral cholinergic synapses through its zinc protease activity (4 -6). This process causes muscular paralysis in humans and animals, leading to the disease botulism.Toxins with serotypes A-D and G are encoded by two gene clusters in close proximity to each other; cluster 1 contains the bont and ntnha genes, and cluster 2 contains three genes : ha-70, ha-33, and ha-17 (7, 8). Therefore, botulinum TC consists of five components: BoNT, non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, HA-33, and HA-17). All serotypes of BoNT associate non-covalently with auxiliary non-toxic proteins, thereby forming large toxin complexes (TCs). Serotype A-D strains produce the M-TC (BoNT⅐NTNHA complex) and L-TC (BoNT⅐NTNHA⅐HAs complex) in the culture medium, while serotype E and F strains produce only M-TC. The major biological function of t...

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Section: Resultsmentioning

confidence: 99%

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Hasegawa

Watanabe

Suzuki

et al. 2007

Journal of Biological Chemistry

104

108

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“…Prior to this work, the best structural information available on a PTC was a projection map of the BoNT/A LL-PTC at 30 Å resolution (Burkard et al 1997) and a negative stain transmission microscopic image of BoNT/D L-PTC ).…”

Section: Architecture Of the M-ptcmentioning

confidence: 99%

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

Gu¹,

Jin²

2012

Current Topics in Microbiology and Immunology

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Botulinum neurotoxins (BoNTs) are among the most poisonous substances known to man, but paradoxically, BoNT-containing medicines and cosmetics have been used with great success in the clinic. Accidental BoNT poisoning mainly occurs through oral ingestion of food contaminated with Clostridium botulinum. BoNTs are naturally produced in the form of progenitor toxin complexes, which are high molecular weight (up to ~900 kDa) multi-protein complexes composed of BoNT and several non-toxic neurotoxin-associated proteins (NAPs). NAPs protect the inherently fragile BoNTs against the hostile environment of the gastrointestinal tract and help BoNTs pass through the intestinal epithelial barrier before they are released into the general circulation. These events are essential for ingested BoNTs to gain access to motoneurons, where they inhibit neurotransmitter release and cause muscle paralysis. In this review, we discuss the structural basis for assembly of NAPs and BoNT into the progenitor toxin complexes that protect BoNT and facilitate its delivery into the bloodstream.

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“…Once in the circulation, free BoNT travels to neuromuscular junctions where it invades neurons. The molecular architecture of PTC is largely unknown, with the exception of two low-resolution structures revealed by electron crystallography (30 Å) and electron microscopy (7, 8). …”

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confidence: 99%

Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex

Rumpel

Zhou

et al. 2012

Science

203

256

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Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial non-toxic non-hemagglutinin (NTNHA) protein at 2.7 angstrom. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication, and of delivery vehicles for oral administration of biologics.

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Electron Density Projection Map of the Botulinum Neurotoxin 900-kilodalton Complex by Electron Crystallography

Cited by 15 publications

References 21 publications

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

A Novel Subunit Structure of Clostridium botulinum Serotype D Toxin Complex with Three Extended Arms

Assembly and Function of the Botulinum Neurotoxin Progenitor Complex

Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex

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