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Katsuhiko Mikoshiba - One of the best experts on this subject based on the ideXlab platform.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse
2013Co-Authors: On The Endoplasmic Reticulum, Mitsunori Fukuda, Katsuhiko Mikoshiba, Yuichiro Kida, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Abstract. Synaptotagmin II is a type I signal-anchor protein, in which the NH 2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity. Key words: membrane topology • membrane protein • signal-anchor sequence • protein translocation • transloco
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membrane topogenesis of a type i signal anchor protein mouse Synaptotagmin II on the endoplasmic reticulum
Journal of Cell Biology, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin II, on the Endoplasmic Reticulum
Rockefeller University Press, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
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functional diversity of c2 domains of Synaptotagmin family mutational analysis of inositol high polyphosphate binding domain
Journal of Biological Chemistry, 1995Co-Authors: Mitsunori Fukuda, Toshio Kojima, Jun Aruga, Michio Niinobe, Katsuhiko MikoshibaAbstract:Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple Synaptotagmins (II-IV). The IP4 binding domain of Synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in Synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among Synaptotagmins I through IV. However, Synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all Synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.
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inositol 1 3 4 5 tetrakisphosphate binding to c2b domain of ip4bp Synaptotagmin II
Journal of Biological Chemistry, 1994Co-Authors: Mitsunori Fukuda, Jun Aruga, Michio Niinobe, Saburo Aimoto, Katsuhiko MikoshibaAbstract:IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/Synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of Synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.
Shunji Kozaki - One of the best experts on this subject based on the ideXlab platform.
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sequence of the gene for clostridium botulinum type b neurotoxin associated with infant botulism expression of the c terminal half of heavy chain and its binding activity
Biochimica et Biophysica Acta, 2003Co-Authors: Hideshi Ihara, Tomoko Kohda, Fumihiro Morimoto, Kentaro Tsukamoto, Tadahiro Karasawa, Shinichi Nakamura, Masafumi Mukamoto, Shunji KozakiAbstract:Abstract Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (HC) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the HC, recombinant genes for the HC and two hybrid HC in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant HC of 111/NT to the protein receptor Synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid HC revealed that mutation of 23 residues in carboxy terminal half of HC (1029–1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.
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the high affinity binding of clostridium botulinum type b neurotoxin to Synaptotagmin II associated with gangliosides gt1b gd1a
FEBS Letters, 1996Co-Authors: Teiichi Nishiki, Masami Takahashi, Yoshimi Tokuyama, Yoichi Kamata, Yasuo Nemoto, Akira Yoshida, Kazuki Sato, Mariko Sekiguchi, Shunji KozakiAbstract:125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat Synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for Synaptotagmin II and Synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of Synaptotagmin II. These results suggest that this region of Synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides.
Katsuyoshi Mihara - One of the best experts on this subject based on the ideXlab platform.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse
2013Co-Authors: On The Endoplasmic Reticulum, Mitsunori Fukuda, Katsuhiko Mikoshiba, Yuichiro Kida, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Abstract. Synaptotagmin II is a type I signal-anchor protein, in which the NH 2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity. Key words: membrane topology • membrane protein • signal-anchor sequence • protein translocation • transloco
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translocation of a long amino terminal domain through er membrane by following signal anchor sequence
The EMBO Journal, 2005Co-Authors: Yuichiro Kida, Katsuyoshi Mihara, Masao SakaguchiAbstract:Type I signal-anchor sequences mediate translocation of the N-terminal domain (N-domain) across the endoplasmic reticulum (ER) membrane. To examine the translocation in detail, dihydrofolate reductase (DHFR) was fused to the N-terminus of Synaptotagmin II as a long N-domain. Translocation was arrested by the DHFR ligand methotrexate, which stabilizes the folding of the DHFR domain, and resumed after depletion of methotrexate. The targeting of the ribosome–nascent chain complex to the ER requires GTP, whereas N-domain translocation does not require any nucleotide triphosphates. Significant translocation was observed even in the absence of a lumenal hsp70 (BiP). When the nascent polypeptide was released from the ribosomes after the membrane targeting, the N-domain translocation was suppressed and the nascent chain was released from the translocon. Ribosomes have a crucial role in maintaining the translocation-intermediate state. The translocation of the DHFR domain was greatly impaired when it was separated from the signal-anchor sequence. Unfolding and translocation of the DHFR domain must be driven by the stroke of the signal-anchor sequence into translocon.
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membrane topogenesis of a type i signal anchor protein mouse Synaptotagmin II on the endoplasmic reticulum
Journal of Cell Biology, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin II, on the Endoplasmic Reticulum
Rockefeller University Press, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
Mitsunori Fukuda - One of the best experts on this subject based on the ideXlab platform.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse
2013Co-Authors: On The Endoplasmic Reticulum, Mitsunori Fukuda, Katsuhiko Mikoshiba, Yuichiro Kida, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Abstract. Synaptotagmin II is a type I signal-anchor protein, in which the NH 2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity. Key words: membrane topology • membrane protein • signal-anchor sequence • protein translocation • transloco
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membrane topogenesis of a type i signal anchor protein mouse Synaptotagmin II on the endoplasmic reticulum
Journal of Cell Biology, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
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Membrane Topogenesis of a Type I Signal-Anchor Protein, Mouse Synaptotagmin II, on the Endoplasmic Reticulum
Rockefeller University Press, 2000Co-Authors: Yuichiro Kida, Mitsunori Fukuda, Katsuhiko Mikoshiba, Masao Sakaguchi, Katsuyoshi MiharaAbstract:Synaptotagmin II is a type I signal-anchor protein, in which the NH2-terminal domain of 60 residues (N-domain) is located within the lumenal space of the membrane and the following hydrophobic region (H-region) shows transmembrane topology. We explored the early steps of cotranslational integration of this molecule on the endoplasmic reticulum membrane and demonstrated the following: (a) The translocation of the N-domain occurs immediately after the H-region and the successive positively charged residues emerge from the ribosome. (b) Positively charged residues that follow the H-region are essential for maintaining the correct topology. (c) It is possible to dissect the lengths of the nascent polypeptide chains which are required for ER targeting of the ribosome and for translocation of the N-domain, thereby demonstrating that different nascent polypeptide chain lengths are required for membrane targeting and N-domain translocation. (d) The H-region is sufficiently long for membrane integration. (e) Proline residues preceding H-region are critical for N-domain translocation, but not for ER targeting. The proline can be replaced with amino acid with low helical propensity.
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functional diversity of c2 domains of Synaptotagmin family mutational analysis of inositol high polyphosphate binding domain
Journal of Biological Chemistry, 1995Co-Authors: Mitsunori Fukuda, Toshio Kojima, Jun Aruga, Michio Niinobe, Katsuhiko MikoshibaAbstract:Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca(2+)-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP4 binding properties of the C2B domains of multiple Synaptotagmins (II-IV). The IP4 binding domain of Synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP4 binding in Synaptotagmin II. When these lysine residues were altered to glutamine, the IP4 binding ability was completely abolished. The primary structures of the IP4 binding sites are highly conserved among Synaptotagmins I through IV. However, Synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all Synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.
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inositol 1 3 4 5 tetrakisphosphate binding to c2b domain of ip4bp Synaptotagmin II
Journal of Biological Chemistry, 1994Co-Authors: Mitsunori Fukuda, Jun Aruga, Michio Niinobe, Saburo Aimoto, Katsuhiko MikoshibaAbstract:IP4BP/Synaptotagmin II is an inositol-1,3,4,5-tetrakisphosphate (IP4) or inositol polyphosphate-binding protein, which is accumulated at nerve terminals. Here we report a novel function of the C2B domain, which was originally thought to be responsible for Ca(2+)-dependent binding to phospholipid membranes. A study of deletion mutants showed that about 30 amino acids of the central region of the C2B domain of mouse IP4BP/Synaptotagmin II (315 IHLMQNGKRLKKKKTTVKKKTLNPYFNESFSF 346) are essential for inositol polyphosphate binding. This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is also known to be essential for neurotransmitter release (Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163-165), suggesting that inositol polyphosphate has some effect on neurotransmitter release. Rabphilin 3A, another neuronal protein containing C2 domains, cannot bind IP4, indicating that the IP4 binding property is specific to the C2B domain of Synaptotagmin. Phospholipid and IP4 binding experiments clearly indicated that the C2A and C2B domains have different functions. The C2A domain binds phospholipid in a Ca(2+)-dependent manner, but the C2B domain binds inositol polyphosphate and phospholipid irrespective of the presence of Ca2+. Our data suggest that the C2B domain of synaptotogamin is the inositol polyphosphate sensor at the synaptic vesicle and may be involved in synaptic function.
Min Dong - One of the best experts on this subject based on the ideXlab platform.
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Structure of dual receptor binding to botulinum neurotoxin B
2016Co-Authors: Ronnie P-a Berntsson, Min Dong, Lisheng Peng, Pål StenmarkAbstract:Botulinum neurotoxins are highly toxic, and bind two receptors to achieve their high affinity and specificity for neurons. Here we present the first structure of a botulinum neurotoxin bound to both its receptors. We determine the 2.3 Å structure of a ternary complex of botulinum neurotoxin type B bound to both its protein receptor Synaptotagmin II and its ganglioside receptor GD1a. We show that there is no direct contact between the two receptors, and that the binding affinity towards Synaptotagmin II is not influenced by the presence of GD1a. The interactions of botulinum neurotoxin type B with the sialic acid 5 moiety of GD1a are important for the ganglioside selectivity. The structure demonstrates that the protein receptor and the ganglioside receptor occupy nearby but separate binding sites, thus providing two independent anchoring points
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botulinum neurotoxin d c uses Synaptotagmin i and II as receptors and human Synaptotagmin II is not an effective receptor for type b d c and g toxins
Journal of Cell Science, 2012Co-Authors: Lisheng Peng, William H Tepp, Eric A Johnson, Ronnie P-a Berntsson, Rose M Pitkin, P Stenmark, Min DongAbstract:Botulinum neurotoxins (BoNTs) are classified into seven types (A–G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins Synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.
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structural basis of cell surface receptor recognition by botulinum neurotoxin b
Nature, 2006Co-Authors: Qing Chai, Joseph W Arndt, Min Dong, William H Tepp, Eric A Johnson, Edwin R Chapman, Raymond C StevensAbstract:Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations by blocking neurotransmitter release. A 'double receptor' model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry. Of seven BoNTs (subtypes A-G), the putative receptors for BoNT/A, BoNT/B and BoNT/G have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the Synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside G(T1b) indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and Synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin-receptor recognition.
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structural basis of cell surface receptor recognition by botulinum neurotoxin b
Nature, 2006Co-Authors: Qing Chai, Joseph W Arndt, Min Dong, William H Tepp, Eric A Johnson, Edwin R Chapman, Raymond C StevensAbstract:Botulinum toxins, produced by Clostridia botulinum, are a potential biological hazard to humans and a potential bioweapons threat. The toxins are potent inhibitors of neurotransmitter release at synapses, and it is this property that causes the neuroparalytic syndrome known as botulism. Two related papers now report the crystal structure of botulinum toxin B bound to its receptor on the exposed surface of the neuron. This will provide insight into the high affinity and specificity of this interaction, and aid in the development of antibotulism vaccines and drugs. One of two papers that describe how botulinum toxins produced by Clostridium botulinum are potent inhibitors of neurotransmitter release by elucidating the crystal structure of botulinum toxin B bound to its receptor. Botulinum neurotoxins (BoNTs) are potent bacterial toxins that cause paralysis at femtomolar concentrations1 by blocking neurotransmitter release. A ‘double receptor’ model has been proposed in which BoNTs recognize nerve terminals via interactions with both gangliosides and protein receptors that mediate their entry2. Of seven BoNTs (subtypes A–G), the putative receptors for BoNT/A3,4, BoNT/B5,6 and BoNT/G7 have been identified, but the molecular details that govern recognition remain undefined. Here we report the crystal structure of full-length BoNT/B in complex with the Synaptotagmin II (Syt-II) recognition domain at 2.6 A resolution. The structure of the complex reveals that Syt-II forms a short helix that binds to a hydrophobic groove within the binding domain of BoNT/B. In addition, mutagenesis of amino acid residues within this interface on Syt-II affects binding of BoNT/B. Structural and sequence analysis reveals that this hydrophobic groove is conserved in the BoNT/G and BoNT/B subtypes, but varies in other clostridial neurotoxins. Furthermore, molecular docking studies using the ganglioside GT1b indicate that its binding site is more extensive than previously proposed and might form contacts with both BoNT/B and Synaptotagmin. The results provide structural insights into how BoNTs recognize protein receptors and reveal a promising target for blocking toxin–receptor recognition.
