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The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live‐cell super‐resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor‐binding‐deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG‐synaptotagmin‐1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1‐SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A‐ and BoNT/E‐induced neurointoxication as quantified by SNAP‐25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting. Synopsis: Synaptic targeting of botulinum neurotoxins is mediated by interaction with toxin‐type‐specific receptors and complex gangliosides, such as polysialoganglioside (PSG). This study shows that botulinum neurotoxin type A (BoNT/A) uptake and sorting into synaptic vesicles requires a tripartite nanocluster on the neuronal plasma membrane. Live‐cell super‐resolution and electron microscopy in primary hippocampal neurons shows BoNT/A synaptic vesicle targeting depends on a tripartite PSG‐synaptotagmin 1 (Syt1)‐synaptic vesicle protein 2 (SV2) nanocluster on the plasma membrane.Coincidental binding of BoNT/A to a preassembled PSG‐Syt1 complex and SV2 facilitates Syt1‐SV2 nanoclustering on the neuronal plasma membrane and endocytic sorting of the toxin into synaptic vesicles.Syt1 is required for the BoNT/A and BoNT/E neurointoxication, suggesting a general mechanism of entry for clostridial botulinum neurotoxins. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) are among the most lethal toxins known to humans, comprising seven established serotypes termed BoNT/A–G encoded in two types of gene clusters (ha and orfX) in BoNT‐producing clostridia. The ha cluster encodes four non‐toxic neurotoxin‐associated proteins (NAPs) that assemble with BoNTs to protect and enhance their oral toxicity. However, the structure and function of the orfX‐type NAPs remain largely unknown. Here, we report the crystal structures for OrfX1, OrfX2, and an OrfX1–OrfX3 complex, which are encoded in the orfX cluster of a BoNT/E1‐producing Clostridium botulinum strain associated with human foodborne botulism. These structures lay the foundation for future studies on the potential roles of OrfX proteins in oral intoxication and pathogenesis of BoNTs. [ABSTRACT FROM AUTHOR]

Highlights: [•] Neutralising mechanism of potent therapeutic monoclonal BoNT/A antibody is exhibited. [•] Single mutation G1292R eliminates interaction of BoNT/A with all three SV2 isoforms. [•] Proof for neuronal uptake of BoNT/A by a double receptor mechanism. [•] Inactive quintuple BoNT/A mutant represents ideal antigen for vaccination. [ABSTRACT FROM AUTHOR]

Highlights: [•] Three specific surface carboxylates of BoNT/B were neutralised by mutation to amide. [•] The triple mutant expedites entry of the enzymatic domain into neurons. [•] The enzymatic domain of the triple mutant translocates at less acidic pH values. [•] The triple mutant exhibits higher neurotoxicity at cholinergic nerve terminals. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins essential for exocytosis. The synaptic vesicle protein synaptotagmin-II of rat and mouse acts as neuronal high affinity receptor for BoNT/B and BoNT/G. Here, we show that human synaptotagmin-II is not a high affinity receptor for BoNT/B and G due to a phenylalanine to leucine mutation in its luminal domain present only in humans and chimpanzees. It eliminates one of three major interactions between synaptotagmin-II and BoNT/B and hereby explains the disparity in potency of BoNT/B in humans and mice as well as the 40-fold higher dosage of rimabotulinumtoxinB versus onabotulinumtoxinA. Structured summary of protein interactions: rSyt-II binds to BoNT/G by pull down (View Interaction: 1 , 2 ) rSyt-II binds to BoNT/B by pull down (View Interaction: 1 , 2 ) [Copyright &y& Elsevier]

The four-domain structure of botulinum neurotoxins (BoNTs) reflects their multistep intoxication process. The high toxicity of BoNTs primarily results from specific binding and uptake into neurons mediated by their 50-kDa cell-binding fragment (HC). X-ray crystallography data have revealed that the HC fragment consists of two domains of equal size, named the 25-kDa N-terminal half of HC (HCN) and the 25-kDa C-terminal half of HC (HCC). In recent years, the ganglioside-binding sites of all seven BoNT serotypes have been allocated to the HCC domain. For BoNT/A, BoNT/B and BoNT/G, the protein receptor-binding site has been also been localized to the HCC domain. Here, we demonstrate that the HCC serotype can modulate the affinity of the HC fragment for neuronal membranes as well as the potency of full-length BoNT by replacing the BoNT/A HCC domain with the BoNT/B HCC, BoNT/C HCC and BoNT/E HCC domains, which exhibit higher affinity for synaptosomes. Indeed, the hybrids HCAB and HCAC display a higher affinity than wild-type HCA. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the HCCB domain (AAAB; letters represent the serotype origin of the four domains) was quadrupled as compared with wild-type BoNT/A. Analogously, exchange of the HC fragment (AABB) yielded a neurotoxin with four-fold higher potency. As BoNT/A and BoNT/B are extensively used to treat neurological disorders, thereby facing the problem of BoNT neutralizing antibody formation, a BoNT with increased potency would lower the repeatedly administered protein dosage while maintaining the clinical benefit. Such a lowered protein load will delay the onset of neurotoxin antibody formation in patients. [ABSTRACT FROM AUTHOR]

The high toxicity of clostridial neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven botulinum neurotoxin serotypes A–G (BoNT/A–G) inhibit acetylcholine release, leading to flaccid paralysis, while tetanus neurotoxin blocks neurotransmitter release in inhibitory neurons, resulting in spastic paralysis. Uptake of BoNT/A, B, E and G requires a dual interaction with gangliosides and the synaptic vesicle (SV) proteins synaptotagmin or SV2, whereas little is known about the entry mechanisms of the remaining serotypes. Here, we demonstrate that BoNT/F as wells depends on the presence of gangliosides, by employing phrenic nerve hemidiaphragm preparations derived from mice expressing GM3, GM2, GM1 and GD1a or only GM3. Subsequent site-directed mutagenesis based on homology models identified the ganglioside binding site at a conserved location in BoNT/E and F. Using the mice phrenic nerve hemidiaphragm assay as a physiological model system, cross-competition of full-length neurotoxin binding by recombinant binding fragments, plus accelerated neurotoxin uptake upon increased electrical stimulation, indicate that BoNT/F employs SV2 as protein receptor, whereas BoNT/C and D utilise different SV receptor structures. The co-precipitation of SV2A, B and C from Triton-solubilised SVs by BoNT/F underlines this conclusion. [ABSTRACT FROM AUTHOR]

Tetanus neurotoxin and botulinum neurotoxins are the causative agents of tetanus and botulism. They block the release of neurotransmitters from synaptic vesicles in susceptible animals and man and act in nanogram quantities because of their ability to specifically attack motoneurons. They developed an ingenious strategy to enter neurons. This involves a concentration step via complex polysialo gangliosides at the plasma membrane and the uptake and ride in recycling synaptic vesicles initiated by binding to a specific protein receptor. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had misused before to enter their target cells, via specific cleavage of protein core components of the cellular membrane fusion machinery. The uptake of four out of seven known botulinum neurotoxins into synaptic vesicles has been demonstrated to rely on binding to intravesicular segments of the synaptic vesicle proteins synaptotagmin or synaptic vesicle protein 2. This review summarizes the present knowledge about the cell receptor molecules and the mode of toxin-receptor interaction that enables the toxins’ sophisticated access to their site of action. [ABSTRACT FROM AUTHOR]

Abstract: Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by selectively cleaving core components of the vesicular fusion machinery. The synaptic vesicle proteins Synaptotagmin-I and -II act as receptors for BoNT/B and BoNT/G. Here we show that BoNT/A also interacts with a synaptic vesicle protein, the synaptic vesicle glycoprotein 2C (SV2C), but not with the homologous proteins SV2A and SV2B. Binding of BoNT/A occurs at the membrane juxtaposed region preceding transmembrane domain 8. A peptide comprising the intravesicular domain between transmembrane domains 7 and 8 specifically reduces the neurotoxicity of BoNT/A at phrenic nerve preparations demonstrating the physiological relevance of this interaction. [Copyright &y& Elsevier]

Multi-domain bacterial protein toxins are being explored as potential carriers for targeted delivery of biomolecules. Previous approaches employing isolated receptor binding subunits disallow entry into the cytosol. Strategies in which catalytic domains are replaced with cargo molecules are presumably inefficient due to co-operation of domains during the endosomal translocation step. Here, we characterize a novel transport vehicle in which cargo proteins are attached to the amino terminus of the full-length botulinum neurotoxin type D (BoNT/D). The intrinsic enzymatic activity of the neurotoxin allowed quantification of the efficacy of cargo delivery to the cytosol. Dihydrofolate reductase and BoNT type A (BoNT/A) light chain (LC) were efficiently conveyed into the cytosol, whereas attachment of firefly luciferase or green fluorescent protein drastically reduced the toxicity. Luciferase and BoNT/A LC retained their catalytic activity as evidenced by luciferin conversion or SNAP-25 hydrolysis in the cytosol of synaptosomes, respectively. Conformationally stabilized dihydrofolate reductase as cargo considerably decreased the toxicity indicative for the requirement of partial unfolding of cargo protein and catalytic domain as prerequisite for efficient translocation across the endosomal membrane. Thus, enzymatically inactive clostridial neurotoxins may serve as effective, safe carriers for delivering proteins in functionally active form to the cytosol of neurones. [ABSTRACT FROM AUTHOR]

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors. [ABSTRACT FROM AUTHOR]

Summary The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. [ABSTRACT FROM AUTHOR]