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Clostridium botulinum neurotoxin (BoNT) causes flaccid paralysis by disabling synaptic exocytosis. Intoxication requires the tri-modular protein to undergo conformational changes in response to pH and redox gradients across endosomes, leading to the formation of a protein-conducting channel. The ∼50 kDa light chain (LC) protease is translocated into the cytosol by the ∼100 kDa heavy chain (HC), which consists of two modules: the N-terminal translocation domain (TD) and the C-terminal Receptor Binding Domain (RBD). Here we exploited the BoNT modular design to identify the minimal requirements for channel activity and LC translocation in neurons. Using the combined detection of substrate proteolysis and single-channel currents, we showed that a di-modular protein consisting only of LC and TD was sufficient to translocate active protease into the cytosol of target cells. The RBD is dispensable for cell entry, channel activity, or LC translocation; however, it determined a pH threshold for channel formation. These findings indicate that, in addition to its individual functions, each module acts as a chaperone for the others, working in concert to achieve productive intoxication.

Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of Synaptotagmin (Syt)-I and SytII, homologous synaptic vesicle proteins. We observe conditions in which BoNT/B binds both Syt isoforms, but BoNT/G only binds SytI. Both serotypes bind ganglioside G T1b . The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform to understand the physiological relevance of different Syt receptor isoforms in vivo.Botulinum neurotoxin (BoNT) is the causative agent of botulism, a potentially lethal neuroparalytic condition in humans (1). The extreme potency of BoNT (LD 50 value ∼ 0.1 ng kg -1 ) stems from the toxin's high affinity for neuronal receptors at the neuromuscular junction and enzymatic inhibition of neurotransmitter release (2). BoNT's are produced as single chain proteins in seven antigenically distinct forms (serotypes A-G). Most BoNT serotypes undergo post-translational cleavage to form a dichain molecule composed of a light-and heavy-chain linked by a disulfide bond. The light chain (LC) is a zinc metalloprotease that cleaves SNARE proteins to inhibit neurotransmitter vesicle fusion to the plasma membrane (3). The N-terminal half of the heavy chain (HCT) is involved in translocation of the LC across the endosomal membrane, and the C-terminal half of the heavy chain (HCR) is involved in binding receptors (4).BoNT targets the neuromuscular junction through specific interactions with both ganglioside and protein receptors (5). BoNTs bind G D1a and gangliosides in the G 1b series and show the highest affinity for the trisialoganglioside, G T1b (6). The protein receptor can vary with BoNT serotype. Synaptotagmin (Syt)-I and SytII mediate the internalization of BoNT/B and/G, but not BoNT/A or/E, into neuronal cells (7-9). SytI and SytII are homologous calcium sensors that couple neuronal calcium influx to the fast phase of neurotransmitter release (10). BoNT/B and/G bind to the luminal domains of SytI and SytII following the fusion of synaptic vesicles with the plasma membrane. The ability of a peptide, corresponding to 20 amino acids of the SytII luminal domain, in conjunction with gangliosides, to neutralize BoNT/B toxicity in mice is consistent with the SytII luminal domain being the neuronal receptor for BoNT/B (7).* To whom correspondence should be addressed. jtb01@mcw.edu; phone (414) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBoNT/G is a recently discovered serotype whose HCR shares a high degree of primary amino acid conservation with BoNT/B (50% identical, 71% similar). Despite the homology between BoNT/B and BoNT/G and between SytI and SytII, several differences exist in their interactions with neurons. BoNT/B binds SytII independent of ganglioside but requires gangliosid...

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...