Botulinum Neurotoxin Heavy Chain Belt as an Intramolecular Chaperone for the Light Chain

Brunger, Axel T.; Breidenbach, Mark A.; Jin, Rongsheng; Fischer, Audrey; Santos, Jose S.; Montal, Mauricio

doi:10.1371/journal.ppat.0030113

Cited by 99 publications

(88 citation statements)

References 38 publications

(75 reference statements)

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“…In vitro studies have shown that BoNT (and the isolated BoNT HCT) undergoes pH-dependent membrane insertion and pore formation (7)(8)(9)(10)(11)(12)(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).…”

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confidence: 60%

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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confidence: 60%

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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“…To achieve this effect, BoNTs undergo a cascade of cellular events (Simpson, 1981) that begin with HC binding to specific extracellular receptors followed by holotoxin endocytosis into the nerve terminal (Yowler et al, 2002;Dong et al, 2006). The HC forms a pore in the endosomal membrane which enables translocation of the LC zinc endoprotease into the cytosol (Montal et al, 1992;Brunger et al, 2007). The LC chain cleaves specific Soluble Nethylmaleimide-sensitive factor Attachment REceptor complex (SNARE) proteins that are essential to exocytosis of ACh-enriched vesicles.…”

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confidence: 99%

Capsaicin Protects Mouse Neuromuscular Junctions from the Neuroparalytic Effects of Botulinum Neurotoxin A

Thyagarajan

Krivitskaya²,

Potian³

et al. 2009

J Pharmacol Exp Ther

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“…Another interesting parameter potentially influencing the translocation process has recently been raised (20). It consists of the "belt" region of the translocation domain.…”

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confidence: 99%

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

Galloux

Vitrac

Montagner

et al. 2008

Journal of Biological Chemistry

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The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

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Botulinum Neurotoxin Heavy Chain Belt as an Intramolecular Chaperone for the Light Chain

Cited by 99 publications

References 38 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

Capsaicin Protects Mouse Neuromuscular Junctions from the Neuroparalytic Effects of Botulinum Neurotoxin A

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

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