Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Strotmeier, Jasmin; Mahrhold, Stefan; Krez, Nadja; Janzen, Constantin; Lou, Jianlong; Marks, James D.; Binz, Thomas; Rummel, Andreas

doi:10.1016/j.febslet.2014.02.034

Cited by 42 publications

(65 citation statements)

References 39 publications

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“…According to a well-accepted dual-receptor binding model [28,32,33], BoNTs bind to neuronal cell surface through subsequent associations with two receptors: polysialogangliosides and a protein receptor. Sequence analysis and structural alignment show that the ganglioside-binding site (GBS) motif ‘E…H…SXWY…G’, which is highly conserved in BoNT/A, B, E (residue H is replaced by a K), F, and G (residue E is replaced by a Q) [34], is strictly conserved in H C HA (E1195, H1245, S1256, W1258, Y1259, and G1271) (Figure 2A, green stars).…”

Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

Yao

Lam

Perry

et al. 2017

Toxins

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Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

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Section: Resultsmentioning

confidence: 99%

Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

Yao

Lam

Perry

et al. 2017

Toxins

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“…The ganglioside binding site resides in the C-terminal subdomain of the H C (residues 1090 to 1296), while the SV2 binding site resides within the C-terminal subdomain on an adjacent face of the H C and close to the N-terminal jellyroll subdomain (residues 870 to 1090) (41)(42)(43). Contributions of subdomains in neuron entry were examined with H C A1 and H C A2 chimeras, fusing the N-terminal subdomain with the C-terminal subdomain of reciprocal H C .…”

Section: Fig 1 H C A2mentioning

confidence: 99%

Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons

et al. 2017

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Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans but also are common therapies for neurological diseases. BoNTs are dichain toxins, comprising an N-terminal catalytic domain (LC) disulfide bond linked to a C-terminal heavy chain (HC) which includes a translocation domain (H N ) and a receptor binding domain (H C ). Recently, the BoNT serotype A (BoNT/A) subtypes A1 and A2 were reported to possess similar potencies but different rates of cellular intoxication and pathology in a mouse model of botulism. The current study measured H C A1 and H C A2 entry into rat primary neurons and cultured Neuro2A cells. We found that there were two sequential steps during the association of BoNT/A with neurons. The initial step was ganglioside dependent, while the subsequent step involved association with synaptic vesicles. H C A1 and H C A2 entered the same population of synaptic vesicles and entered cells at similar rates. The primary difference was that H C A2 had a higher degree of receptor occupancy for cells and neurons than HcA1. Thus, H C A2 and H C A1 share receptors and entry pathway but differ in their affinity for receptor. The initial interaction of H C A1 and H C A2 with neurons may contribute to the unique pathologies of BoNT/A1 and BoNT/A2 in mouse models.KEYWORDS botulinum toxin, gangliosides, synaptic vesicles, synaptic vesicle protein 2, TIRF microscopy, clostridial neurotoxins, Clostridium botulinum, toxins B otulinum neurotoxins (BoNTs) are AB exotoxins secreted by several species of the genus Clostridium. BoNTs are single-chain 150-kDa proteins cleaved by either bacterial or host proteases to a dichain comprising a 50-kDa light chain (LC) and a 100-kDa heavy chain (HC) linked by an interchain disulfide bond. The HC includes a translocation domain (H N ) and receptor binding domain (H C ) (1). The H C includes an N-terminal subdomain (H CN ) of limited known function and a C-terminal subdomain (H CC ) that confers neuron specificity by binding dual neuron-specific receptors. There are seven BoNT serotypes (A to G) (2). BoNT serotype A (BoNT/A) binds a ganglioside and synaptic vesicle glycoprotein 2 (SV2) (3-6), which allows rapid toxin entry via synaptic vesicles. Acidification of the synaptic vesicle lumen triggers H N to form a channel to facilitate LC translocation into the cytosol. Intracellular LC cleaves a SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) protein (7-12). SNARE protein cleavage inhibits exocytosis of cholinergic synaptic vesicles at neuromuscular junctions. BoNT LCs are long-lived proteases, sustaining paralysis in humans for several months depending on the serotype (2), which led to the licensing of BoNT/A and BoNT/B for human therapies (13,14).BoNT serotypes include subtypes neutralized by serotype-specific antisera (15-17). Informatics has classified eight BoNT/A subtypes (A1 to A8), which vary by ϳ10 to 15% in amino acid identity (15,16,(18)(19)(20)(21). While BoNT/A1 and BoNT/A2 cleave SNAP25 with similar kinetics (22-2...

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“…Because nature typically avoids such aggregation-prone open b-strands in proteins, these findings indicate that C. botulinum has exploited a weakness in the structure of SV2 proteins to transport BoNT/A into host cells. The specificity of the interaction was confirmed by site-directed mutagenesis [42,47]. Mutation of BoNT/A1 Arg1294, a residue that prominently contributes to the positive charge of the toxin surface, or Arg1156, which forms a cation-p-stacking interaction with Phe563 in SV2C, significantly reduced the binding to SV2C ( Figure 3D).…”

Section: Reviewmentioning

confidence: 82%

“…A second, nonconserved binding site that binds different receptors has been identified in several BoNTs. BoNT/A, BoNT/E and BoNT/F bind to synaptic vesicle glycoprotein 2 (SV2) (see Figure 1C in main text) [26,27,[42][43][44]47,65,73]. SV2 proteins are putative transporter proteins of unknown function that are predicted to have 12 transmembrane domains [74].…”

Section: Box 2 Bonts and Their Receptors: The Dual-receptor Modelmentioning

confidence: 99%