Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity

Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T.

doi:10.1038/nature05387

Cited by 225 publications

(342 citation statements)

References 29 publications

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“…Confirmation for our data was also achieved by the crystal structure of the H C B-Syt-II complex (32). This study revealed that pocket 4 accommodates the C-terminal 10 residues (K51-K60) of Syt-II and that the Syt binding interface of BoNT/B extended into a small neighboring hollow interacting with M46-L50 of Syt-II (32).…”

Section: Interference Of Ganglioside and Syt Interaction Sites Of Bonsupporting

confidence: 54%

“…This study revealed that pocket 4 accommodates the C-terminal 10 residues (K51-K60) of Syt-II and that the Syt binding interface of BoNT/B extended into a small neighboring hollow interacting with M46-L50 of Syt-II (32). The luminal domain of Syt-II displays an ␣-helical conformation, in which the membrane-juxtaposed region of Syt-II is oriented toward the ganglioside binding site.…”

Section: Interference Of Ganglioside and Syt Interaction Sites Of Bonmentioning

confidence: 96%

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Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Rummel

Eichner

Weil

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Botulinum neurotoxins (BoNTs) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery within motoneurons. Complex gangliosides initially bind into a pocket that is conserved among the seven BoNTs and tetanus neurotoxin. Productive neurotoxin uptake also requires protein receptors. The interaction site of the protein receptor within the neurotoxin is currently unknown. We report the identification and characterization of the protein receptor binding site of BoNT/B and BoNT/G. Their protein receptors, synaptotagmins I and II, bind to a pocket at the tip of their HCC (C-terminal domain of the C-terminal fragment of the heavy chain) that corresponds to the unique second carbohydrate binding site of tetanus neurotoxin, the sialic acid binding site. Substitution of amino acids in this region impaired binding to synaptotagmins and drastically decreased toxicity at mouse phrenic nerve preparations; CD-spectroscopic analyses evidenced that the secondary structure of the mutated neurotoxins was unaltered. Deactivation of the synaptotagmin binding site by single mutations led to virtually inactive BoNT/B and BoNT/G when assayed at phrenic nerve preparations of complex-ganglioside-deficient mice. Analogously, a BoNT B mutant with deactivated ganglioside and synaptotagmin binding sites lacked appreciable activity at wild-type mouse phrenic nerve preparations. Thus, these data exclude relevant contributions of any cell surface molecule other than one ganglioside and one protein receptor to the entry process of BoNTs, which substantiates the double-receptor concept. The molecular characterization of the synaptotagmin binding site provides the basis for designing a novel class of potent binding inhibitors.synaptotagmin ͉ tetanus B otulinum neurotoxins (BoNTs) (serotypes A-G) are the causative agents of the disease botulism. The neurotoxins are produced as Ϸ150-kDa single-chain proteins in Clostridium botulinum and subsequently cleaved by proteases, yielding an Ϸ100-kDa heavy chain (HC) and an Ϸ50-kDa light chain (LC). These chains remain connected via a single disulfide bridge, noncovalent interactions, and a HC-derived peptide loop wrapped around the LC. The LCs act as zinc metallopeptidases, which solely hydrolyze one of three SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins: vesicle associated membrane protein/synaptobrevin, synaptosomal-associated protein of 25 kDa, or syntaxin. Together, these substrate molecules constitute the core of the vesicular fusion machinery. Thus, cleavage of one of these proteins inhibits the release of neurotransmitters from synaptic vesicles into the synaptic cleft. The HCs mediate the neurospecific binding, uptake by receptor-mediated endocytosis, and transport of the LC across the endosomal membrane into the cytosol, where the LCs encounter their substrates. The Ϸ50-kDa

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Section: Interference Of Ganglioside and Syt Interaction Sites Of Bonsupporting

confidence: 54%

Section: Interference Of Ganglioside and Syt Interaction Sites Of Bonmentioning

confidence: 96%

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Rummel

Eichner

Weil

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

171

216

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“…The interaction of HCR with ganglioside is independent of pH ranging from 4.0 to 7.4 (data not shown), suggesting that the toxin remains bound to the membrane within the acidified endosome. Intriguingly, the interaction of BoNT/B with synaptotagmin II was also reported to be independent of pH, potentially signifying a shared mechanism among the CNTs (40). The need to orient the HCT relative to the membrane may be related to the large ␣-helices of the domain, which probably insert into the bilayer.…”

Section: Discussionmentioning

confidence: 99%

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

Burns

Baldwin

2014

Journal of Biological Chemistry

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Background: Tetanus neurotoxin enters the neuronal cytosol to block the release of neurotransmitters. Results: Channel formation is enhanced by receptor binding and dependent on acidic lipids. Conclusion: A two-step sequential process takes place: ganglioside-mediated membrane anchorage, followed by pH-triggered channel formation modulated by the membrane environment. Significance: This represents a new paradigm for how A-B toxins translocate enzymatic domains across cellular membranes.

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“…The monomer of HCR/T (Protein Data Bank code 1fv2 (7)) was used as the search model. The initial structure from molecular replacement was refined using the program CNS (19). Each round of refinement contained rigid body minimization, positional refinement, and slow cooling simulated annealing protocol beginning at 3000 K and a second positional refinement.…”

Section: Hcr/t Binding To Proteinase K-treated Pc12 Cells-pc12mentioning

confidence: 99%

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Chen

Kim

et al. 2009

Journal of Biological Chemistry

119

134

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Tetanus neurotoxin (TeNT) is an exotoxin produced byClostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ϳ150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT) 3 produced by the sporeforming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicleassociated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ϳ150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (ϳ50 kDa) and a C-terminal heavy chain domain (ϳ100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptorbinding domain (HCR/T, ϳ50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of ␣-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5-9). Current models implicate a dual receptor mechanism for the binding of t...

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Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity

Cited by 225 publications

References 29 publications

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept

Tetanus Neurotoxin Utilizes Two Sequential Membrane Interactions for Channel Formation

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

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