Publications (23) View all
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Article: Botulinum neurotoxin G binds synaptotagmin-II in a mode similar to serotype B: tyrosine-1186 and lysine-1191 cause its lower affinity.
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ABSTRACT: Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoform I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for the mosaic type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information for BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased binding of BoNT/G approximately to the level of BoNT/B affinity suggesting a similar synaptotagmin bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. Based on these data molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses.Biochemistry 05/2013; · 3.42 Impact Factor -
Article: Identification of the SV2-Protein Receptor Binding Site of Botulinum Neurotoxin Type E.
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ABSTRACT: The highly specific binding and uptake of botulinum neurotoxins (BoNT/A-G) into peripheral cholinergic motoneurons turns them into the most poisonous substances known. Interaction with gangliosides accumulates the neurotoxins on the plasma membrane and binding to a synaptic vesicle membrane protein leads to neurotoxin endocytosis. The synaptic vesicle (glyco-)protein 2 (SV2) mediates the uptake of BoNT/A and E, whereas synaptotagmin (Syt) is responsible for the endocytosis of BoNT/B and G. The Syt-binding site of the former was identified by co-crystallization and mutational analyses. Here we report the identification of the SV2‑binding interface of BoNT/E. Mutations interfering with SV2‑binding were located at a site that corresponds to the Syt‑binding site of BoNT/B and at an extended surface area located on the back side of the conserved ganglioside binding site, comprising the N- and C‑terminal half of the BoNT/E binding domain. Mutations impairing the affinity also reduced the neurotoxicity of full-length BoNT/E at mouse phrenic nerve hemidiaphragm preparations demonstrating the crucial role of the identified binding interface. Furthermore, we show that a monoclonal antibody neutralizes BoNT/E activity because it directly interferes with the BoNT/E-SV2 interaction. The data obtained suggest a novel mode of binding for BoNTs that exploit SV2 as cell surface receptor.Biochemical Journal 04/2013; · 4.90 Impact Factor -
Article: Double Receptor Anchorage of Botulinum Neurotoxins Accounts for their Exquisite Neurospecificity.
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ABSTRACT: The high potency of the botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT) is mainly due to their neurospecific binding which is mediated by the interaction with two receptor components. TeNT and all BoNT bind first to complex polysialo-gangliosides abundantly present on the outer leaflet of neuronal membranes. The ganglioside binding occurs in BoNT/A, B, E, F and G via a conserved ganglioside binding pocket within the most carboxyl-terminal 25 kDa domain H(CC) whereas TeNT, BoNT/C and D display two different ganglioside binding sites within their H(CC)-domain. Subsequently, upon exocytosis the intraluminal domains of synaptic vesicle proteins are exposed and can be accessed by the surface accumulated neurotoxins. BoNT/B and G bind with their H(CC)-domain to a 20-mer membrane juxtaposed segment of the intraluminal domain of synaptotagmin-I and -II, respectively. BoNT/A and E employ the intraluminal domain 4 of the synaptic vesicle glycoprotein 2 (SV2) as protein receptor. Whereas the 50 kDa cell binding domain H(C) of BoNT/A interacts with all three SV2 isoforms, BoNT/E H(C) only binds SV2A and SV2B. Also, BoNT/D, F, and TeNT employ SV2 for binding and uptake. Thereafter, the synaptic vesicle is recycled and the anchored neurotoxin is endocytosed. Acidification of the vesicle lumen triggers membrane insertion of the translocation domain followed by pore formation and finally translocation of the enzymatically active light chain to its site of action leading to block of neurotransmitter release.Current topics in microbiology and immunology 01/2013; 364:61-90. · 4.93 Impact Factor -
Article: Botulinum neurotoxin is shielded by NTNHA in an interlocked complex.
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ABSTRACT: Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologics.Science 02/2012; 335(6071):977-81. · 31.20 Impact Factor -
Article: Human synaptotagmin-II is not a high affinity receptor for botulinum neurotoxin B and G: increased therapeutic dosage and immunogenicity.
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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.FEBS letters 02/2012; 586(4):310-3. · 3.54 Impact Factor
