A Comprehensive Structural Analysis of Clostridium botulinum Neurotoxin A Cell-Binding Domain from Different Subtypes (original) (raw)
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International Journal of Molecular Sciences
Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT’s high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determin...
FEBS Open Bio, 2020
Clostridium botulinum neurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (H C), a translocation domain (H N), and a catalytic domain (LC). Here we present high-resolution crystal structures of the binding domains of BoNT subtypes /A5 (H C /A5) and /A6 (H C /A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor binding sites.
Toxins, 2022
Botulinum neurotoxins (BoNT) cause the potentially fatal neuroparalytic disease botulism that arises due to proteolysis of a SNARE protein. Each BoNT is comprised of three domains: a cell binding domain (HC), a translocation domain (HN), and a catalytic (Zn2+ endopeptidase) domain (LC). The HC is responsible for neuronal specificity by targeting both a protein and ganglioside receptor at the neuromuscular junction. Although highly toxic, some BoNTs are commercially available as therapeutics for the treatment of a range of neuromuscular conditions. Here we present the crystal structures of two BoNT cell binding domains, HC/A4 and HC/A5, in a complex with the oligosaccharide of ganglioside, GD1a and GM1b, respectively. These structures, along with a detailed comparison with the previously reported apo-structures, reveal the conformational changes that occur upon ganglioside binding and the interactions involved.
Biochemistry, 2004
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 I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only 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 for understanding the physiological relevance of different Syt receptor isoforms in vivo.
Molecular Architecture of Botulinum Neurotoxin E Revealed by Single Particle Electron Microscopy
Journal of Biological Chemistry, 2007
Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of fulllength BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A. Botulinum neurotoxin (BoNT), 4 considered the most potent toxin known, causes botulism (1) by selectively inhibiting synaptic vesicle exocytosis (2). This conspicuously specific activity has transformed BoNT into the first bacterial toxin approved by the FDA for treatment of a number of diseases characterized by abnormal muscle contraction and as a blockbuster cosmeceutical and a most feared bioweapon (1, 3). Clostridium botulinum cells produce seven BoNT isoforms designated serotypes A to G (2). All BoNT serotypes are synthesized as a single polypeptide chain with molecular mass ϳ150 kDa. This precursor protein is cleaved by a clostridial protease into two polypeptides that remain linked by a disulfide bridge. The mature dichain toxin consists of a 50-kDa light chain (LC) Zn 2ϩ metalloprotease and a 100-kDa heavy chain (HC). The HC encompasses the translocation domain (TD) (the N-terminal half) and the receptor-binding domain (RBD) (the C-terminal half). BoNT/E is atypical in that it is not activated by proteolytic cleavage in the clostridial cells, thereby requiring unidentified proteases in the host cells to cleave the LC from the HC to achieve full toxicity (4, 5). BoNTs exert their neuroparalytic effect by a multistep mechanism (2, 6). RBD-mediated binding to protein and lipid receptors on the cell surface of peripheral nerve endings (7-11) triggers receptor-mediated endocytosis and traffic to the endosomes. The acidic pH of endosomes induces a conformational change of the toxin; the HC inserts into the lipid bilayer and forms a protein-conducting channel (12, 13). The HC channel then translocates the protease domain into the cytoplasm (13), colocalizing with its substrate SNARE (soluble NSF attachment protein receptor) (14-16). Because the SNARE core complex is essential for synaptic vesicle fusion with the presynaptic membrane (14-16), BoNTs efficiently block synaptic vesicle exocytosis. In contrast to BoNT/A (17), little is known about the molecular architecture of BoNT/E, making it a novel target for structural analysis. Here we report the three-dimensional structure of BoNT/E holotoxin at ϳ30 Å resolution as determined by single particle electron microscopy (EM). Domains of BoNT/E were assigned to the globular features observed in the structure by labeling the toxin with functionally relevant monoclonal antibodies (mAbs). Although the individual domains of BoNT/E are similar to those of BoNT/A, their spatial arrangement within the global fold is unique. Analysis of the BoNT/E structure and structure-function correlation studies with mAbs bound to BoNT/A and BoNT/E define previously unrecognized biophysical characteristics that differ between these two BoNT isoforms. EXPERIMENTAL PROCEDURES Materials-Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich. Purified native BoNT serotypes A and E holotoxins were from Metabiologics. Di-chain BoNT/E holotoxin was generated by cleavage with trypsin: BoNT/E holotoxin (0.5 mg/ml) was incubated with 0.15 mg/ml trypsin in 20 mM HEPES, pH 7.0, for 30 min at 37°C. Thereaf
Scientific Reports, 2021
Clostridium botulinum neurotoxin serotype A (BoNT/A) is a potent neurotoxin that serves as an effective therapeutic for several neuromuscular disorders via induction of temporary muscular paralysis. Specific binding and internalization of BoNT/A into neuronal cells is mediated by its binding domain (H C /A), which binds to gangliosides, including GT1b, and protein cell surface receptors, including SV2. Previously, recombinant H C /A was also shown to bind to FGFR3. As FGFR dimerization is an indirect measure of ligand-receptor binding, an FCS & TIRF receptor dimerization assay was developed to measure rH C /A-induced dimerization of fluorescently tagged FGFR subtypes (FGFR1-3) in cells. rH C /A dimerized FGFR subtypes in the rank order FGFR3c (EC 50 ≈ 27 nM) > FGFR2b (EC 50 ≈ 70 nM) > FGFR1c (EC 50 ≈ 163 nM); rH C /A dimerized FGFR3c with similar potency as the native FGFR3c ligand, FGF9 (EC 50 ≈ 18 nM). Mutating the ganglioside binding site in H C /A, or removal of GT1b from the media, resulted in decreased dimerization. Interestingly, reduced dimerization was also observed with an SV2 mutant variant of H C/ A. Overall, the results suggest that the FCS & TIRF receptor dimerization assay can assess FGFR dimerization with known and novel ligands and support a model wherein H C /A, either directly or indirectly, interacts with FGFRs and induces receptor dimerization. Botulinum neurotoxin type A (BoNT/A) is a 150 kDa metalloenzyme belonging to the family of neurotoxins produced by Clostridium botulinum. The toxin causes temporary muscle paralysis by inhibiting acetylcholine release at the neuromuscular junction 1-4. The neuronal specificity and high potency of BoNT/A has allowed its use in the treatment of a large number of medical and aesthetic conditions 3,5-7 , relying on injection of picomolar (pM) concentrations of the toxin. Though BoNT/A has been the subject of extensive study, greater understanding of the complex mechanism associated with BoNT/A's neuronal specificity and cellular entry could lead to further therapeutic applications. BoNT/A is a single-chain protein activated by proteolytic cleavage to form a 150 kDa di-chain molecule. The di-chain is composed of a light chain (L C /A), which encodes a Zn 2+-dependent endopeptidase (~ 50 kDa), linked by a single disulfide bond and non-covalent interactions to a ~ 100 kDa heavy chain (HC) containing the receptor binding and translocation domains 8. The 50 kDa receptor binding domain, H C /A, is located at the C-terminal half of the HC and mediates specific binding and internalization of the toxin into neurons. Following internalization, the translocation domain (H N) of BoNT/A, residing at the N-terminal half of the HC, facilitates the translocation of L C /A from the endocytic vesicle into the cytosol. Once in the cytosol, L C /A enzymatically cleaves the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptosomal-associated protein 25 (SNAP-25) 9,10 , which is essential for mediating vesicular fusion and exocytosis. Cleavage of SNAP-25 leads to inhibition of neurotransmitter/neuropeptide release, including acetylcholine, from neuronal cells and is responsible for BoNT/A's observed pharmacological effects on smooth and skeletal muscles and glands 1,11,12 .