Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

Oblatt-Montal, M.; Yamazaki, Masahito; Nelson, Richard M.; Montal, Mauricio

doi:10.1002/pro.5560040806

Cited by 59 publications

(40 citation statements)

References 63 publications

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“…NBD-labeled proteins with a special emphasis on two regions; sequence 659 -681 is the extended hydrophobic sequence identified by previous primary sequence analyses (22), and sequences 805-820 and 826 -835 were shown to be proteaseresistant in the context of proteoliposomes (Fig. 2).…”

Section: Steady-state Fluorescence Of Nbd-labeled Lc-hct In Soluble Vmentioning

confidence: 94%

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Studies of the Mechanistic Details of the pH-dependent Association of Botulinum Neurotoxin with Membranes

Mushrush

Koteiche

Sammons

et al. 2011

Journal of Biological Chemistry

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Botulinum neurotoxin (BoNT) belongs to a large class of toxic proteins that act by enzymatically modifying cytosolic substrates within eukaryotic cells. The process by which a catalytic moiety is transferred across a membrane to enter the cytosol is not understood for any such toxin. BoNT is known to form pHdependent pores important for the translocation of the catalytic domain into the cytosol. As a first step toward understanding this process, we investigated the mechanism by which the translocation domain of BoNT associates with a model liposome membrane. We report conditions that allow pH-dependent proteoliposome formation and identify a sequence at the translocation domain C terminus that is protected from proteolytic degradation in the context of the proteoliposome. Fluorescence quenching experiments suggest that residues within this sequence move to a hydrophobic environment upon association with liposomes. EPR analyses of spin-labeled mutants reveal major conformational changes in a distinct region of the structure upon association and indicate the formation of an oligomeric membrane-associated intermediate. Together, these data support a model of how BoNT orients with membranes in response to low pH. Botulinum neurotoxin (BoNT)3 inhibits the release of acetylcholine at peripheral cholinergic nerve terminals and causes the potentially lethal, flaccid paralytic condition known as botulism (1). It is produced by Clostridium botulinum as a single chain 150-kDa protein in one of seven antigenically distinct forms (serotypes A-G) (2). It is then cleaved to form a dichain molecule in which a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC) remain linked by a disulfide bond. The LC is a zinc metalloprotease that cleaves components of the synaptic membrane fusion complex and blocks neuronal exocytosis (3). The C-terminal half of the HC (HC receptor-binding domain (HCR)) binds neuronal receptors (4), whereas the N-terminal half of the HC (HC translocation domain (HCT)) mediates the translocation of the LC into the cytosol (5).After binding to its receptors, BoNT undergoes receptormediated endocytosis and is transported to the endosomal compartment. It is thought that the acidic pH of the endosome triggers HCT pore formation and LC translocation (6). In vitro studies have shown that BoNT (and the isolated BoNT HCT) undergoes pH-dependent membrane insertion and pore formation (7-13), and single molecule translocation events have been observed in excised patches of neuronal cells (14,15). These studies support a model in which the HCT acts as both a conduit and a chaperone for the transit of the LC protease across the membrane (5, 11, 16).The HCT structure, visualized in x-ray crystal structures of BoNT/A, BoNT/B, and BoNT/E (17-21), is unique and bears no resemblance to structures observed in other toxins known to mediate pH-dependent translocation events (e.g. anthrax toxin and diphtheria toxin). The HCT contains a pair of kinked ␣-helices (Ͼ100 Å in length) surrounded by several loops and shorter helical re...

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Section: Steady-state Fluorescence Of Nbd-labeled Lc-hct In Soluble Vmentioning

confidence: 94%

“…The HCT also contains a Ͼ50-amino acid "belt" that wraps around the LC. A sequence with amphipathic character (BoNT/A residues 659 -681) has been proposed as a putative transmembrane helix in the pore structure of BoNT (22) and has been observed as an extended structure on the surface of the BoNT HCT (see Fig. 1A).…”

mentioning

confidence: 99%

Studies of the Mechanistic Details of the pH-dependent Association of Botulinum Neurotoxin with Membranes

Mushrush

Koteiche

Sammons

et al. 2011

Journal of Biological Chemistry

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“…Once engulfed inside a neuronal cell, the N-terminal half of the HC (H N ) facilitates translocation of the LC into the cytosol. [4] and [5] The LC domain is a group of Zinc-dependent endoproteases6 that specifically cleave SNARE proteins (SNAP-25, VAMP and syntaxin) that are essential for release of the neurotransmitter acetylcholine.…”

mentioning

confidence: 99%

Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Zhou

Pellett

et al. 2009

Bioorganic & Medicinal Chemistry Letters

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Botulinum neurotoxins (BoNTs) are causative agents for botulism and are identified as a category A bioterror agents by the Centers for Disease Control and Prevention (CDC). Current antitoxins against BoNTs intoxication have some limitations including side effects or limited supply. As an alternative, neutralizing monoclonal antibodies will play an increasing role as BoNTs therapeutics. To date, no human anti-BoNT/B neutralizing monoclonal antibodies have yet to be reported. Herein, we describe an improved selection approach and characterization of a human monoclonal antibody, F2, which is capable of binding BoNT/B with high specificity and displays neutralizing activity in an in vitro cell-based assay. Through surface plasmon resonance studies, we have determined its association and dissociation rate constants. In sum, our data demonstrate that monoclonal antibody F2 is a promising BoNT/B therapeutic lead for further development.

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“…Cleavage of the SNARE components by BoNT disrupts membrane fusion and neurotransmitter release 282 . The heavy chains of the toxins were shown to form tetramers 339 and to insert into the lipid membranes, forming cation-selective channels 340 permeable to small molecules (< 700 Da) 337 . The mechanism of BoNT translocation is still not completely understood 14 .…”

Section: Channel-forming Bacterial Toxinsmentioning

confidence: 99%

“…The mechanism of BoNT translocation is still not completely understood 14 . However, the essential molecular details of the mechanism underlying BoNT translocation across endosomal membranes were obtained from single-molecule studies in planar lipid bilayers 340–348 . The nicked BoNT molecule is believed to act as a nanomachine 349–353 where the B-domain formed by the heavy-chain fragment acts as a specific protein-translocation chaperone for the light chain protease 282, 342–345, 351, 354, 355 .…”

Section: Channel-forming Bacterial Toxinsmentioning

confidence: 99%

Obstructing Toxin Pathways by Targeted Pore Blockage

Nestorovich

Bezrukov

2012

Chem. Rev.

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Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A

Cited by 59 publications

References 63 publications

Studies of the Mechanistic Details of the pH-dependent Association of Botulinum Neurotoxin with Membranes

Studies of the Mechanistic Details of the pH-dependent Association of Botulinum Neurotoxin with Membranes

Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Obstructing Toxin Pathways by Targeted Pore Blockage

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