Solid‐phase synthesis, conformational analysis and <i>in vitro</i> cleavage of synthetic human synaptobrevin II 1–93 by tetanus toxin L chain

Cornille, Fabrice; Goudreau, Nathalie; Ficheux, Damien; Niemann, Heiner; Roques, Bernárd P.

doi:10.1111/j.1432-1033.1994.tb18855.x

Cited by 42 publications

(31 citation statements)

References 46 publications

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“…Likewise, Snc2p appears to be unfolded as judged by CD spectroscopy (Fig. 2a) and amide proton exchange experiments (data not shown), in accord with previous reports for Snc2p homologs 26,33 . Unlike Sso1p and Sso2p, which display CD spectra indicative of substantial (~70%; see Methods) α-helix content, Sso1CT and Sso2CT are largely unfolded (Fig.…”

Section: Assembly-induced Folding Of Snare Domainssupporting

confidence: 90%

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Nicholson

Munson

Miller

et al. 1998

Nat Struct Mol Biol

210

257

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The fusion of intracellular transport vesicles with their target membranes requires the assembly of SNARE proteins anchored in the apposed membranes. Here we use recombinant cytoplasmic domains of the yeast SNAREs involved in Golgi to plasma membrane trafficking to examine this assembly process in vitro. Binary complexes form between the target membrane SNAREs Sso1p and Sec9p; these binary complexes can subsequently bind to the vesicle SNARE Snc2p to form ternary complexes. Binary and ternary complex assembly are accompanied by large increases in alpha-helical structure, indicating that folding and complex formation are linked. Surprisingly, we find that binary complex formation is extremely slow, with a second-order rate constant of approximately 3 M(-1) s(-1). An N-terminal regulatory domain of Sso1p accounts for slow assembly, since in its absence complexes assemble 2,000-fold more rapidly. Once binary complexes form, ternary complex formation is rapid and is not affected by the presence of the regulatory domain. Our results imply that proteins that accelerate SNARE assembly in vivo act by relieving inhibition by this regulatory domain.

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Section: Assembly-induced Folding Of Snare Domainssupporting

confidence: 90%

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Nicholson

Munson

Miller

et al. 1998

Nat Struct Mol Biol

210

257

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“…Indeed, the formation of a unique tertiary structure, comparable to that of S 27-93, by mixing the three short peptides S 27-55 (S 1 ), S 56 -81, and S 82-93 (S 2 ) is highly improbable. This is supported by the lack of a well defined tertiary structure for synaptobrevin in solution as previously observed by 1 H NMR (29). A mechanism which could account for the present results is the promotion of an active conformation of TeNT induced by the binding of the S 1 and S 2 domains of synaptobrevin to exosites present on the toxin surface, as in the case of allosteric enzymes (36 -38).…”

Section: Discussionsupporting

confidence: 71%

“…TeNT cleaves synaptobrevin selectively at one single peptide bond Gln 76 -Phe 77 (28). Successive truncations of synaptobrevin at its NH 2 and COOH terminus have shown that the removal of both the proline-rich NH 2 -terminal 1-25 domain and the 93-116 transmembrane anchor does not reduce the rate of hydrolysis by TeNT (29,30) (Fig. 1).…”

mentioning

confidence: 99%

Cooperative Exosite-dependent Cleavage of Synaptobrevin by Tetanus Toxin Light Chain

Cornille

Martin

Lenoir

et al. 1997

Journal of Biological Chemistry

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The light chain (L chain) of tetanus neurotoxin (TeNT) has been shown to have been endowed with zinc endopeptidase activity, selectively directed toward the Gln 76 -Phe 77 bond of synaptobrevin, a vesicle-associated membrane protein (VAMP) critically involved in neuroexocytosis. In previous reports, truncations at the NH 2 and COOH terminus of synaptobrevin have shown that the sequence 39 -88 of synaptobrevin is the minimum substrate of TeNT, suggesting either the requirement of a well defined three-dimensional structure of synaptobrevin or a role in the mechanism of substrate hydrolysis for residues distal from the cleavage site. In this study, the addition of NH 2 -and COOH-terminal peptides of synaptobrevin, S 27-55 (S 1 ) and S 82-93 (S 2 ), to the synaptobrevin fragment S 56 -81 allowed the cleavage of this latter peptide by TeNT to occur. This appears to result from an activation process mediated by the simultaneous binding of S 1 and S 2 with complementary sites present on TeNT as shown by surface plasmon resonance experiments and the determination of kinetic constants. All these results favor an exosite-controlled hydrolysis of synaptobrevin by TeNT, probably involving a conformational change of the toxin. This could account for the high degree of substrate specificity of TeNT and, probably, botulinum neurotoxins.

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“…The ability of this latter toxin to inhibit TeNT and BoNT/B proteolysis of VAMP, documented in Fig. 3b, was reduced when VAMP-2 D64S,D65S mutant and the VAMP-2 Asp 40 and Glu 41 were assayed (not shown). However, results did not allow us to draw a clear conclusion of a possible preference of BoNT/A between V1 and V2.…”

Section: Mutation Of the Negatively Charged Residues Of V1 Affects Vamentioning

confidence: 91%

Structural Determinants of the Specificity for Synaptic Vesicle-associated Membrane Protein/Synaptobrevin of Tetanus and Botulinum Type B and G Neurotoxins

Pellizzari

Rossetto

Lozzi

et al. 1996

Journal of Biological Chemistry

113

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Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by sitedirected mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.Tetanus neurotoxin (TeNT) 1 and botulinum neurotoxins (BoNTs, seven types from A to G) are three-domain protein toxins that bind selectively to the neuronal presynaptic membrane. They are internalized inside intracellular compartments from which the amino-terminal 50-kDa domain (termed L chain) enters into the cytosol (1-4). The L chains of TeNT and BoNTs are zinc-endopeptidases that cleave specifically three proteins of the neuroexocytosis apparatus, thereby blocking neurotransmitter release (4 -7). TeNT and BoNT/B, BoNT/D, BoNT/F, and BoNT/G recognize and cleave specifically a synaptic vesicle-associated membrane protein (VAMP, also referred to as synaptobrevin) at different single peptide bonds (4, 8 -12). BoNT/A and BoNT/E specifically recognize and cut SNAP-25 (synaptosomal-associated protein of 25 kDa) at two different peptide bonds near the COOH terminus (10, 13, 14), whereas BoNT/C cleaves syntaxin (15, 16) and . VAMP, SNAP-25, and syntaxin are collectively termed SNARE proteins, because they act as receptors of soluble Nethylmaleimide-sensitive factor accessory proteins, involved in vesicle-membrane fusion (5-7).Sequence comparison of the L chains of the eight clostridial neurotoxins show strong similarities (20), which are even more extensive at the level of predicted secondary structure (21). These similarities suggest that they derive from a common ancestral metalloproteinase. On this basis, to account for their different substrate specificity, we considered the possibility that the three SNAREs contain a common neurotoxin recognition site in addition to the cleavage sites specific for each neurotoxin type. We identified a nine-residue-long motif (SNARE motif) present in eukaryotes only in the three proteins known to be proteolytic substrates of the neurotoxins (22). The SNARE motif is included within regions...

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Solid‐phase synthesis, conformational analysis and in vitro cleavage of synthetic human synaptobrevin II 1–93 by tetanus toxin L chain

Cited by 42 publications

References 46 publications

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p

Cooperative Exosite-dependent Cleavage of Synaptobrevin by Tetanus Toxin Light Chain

Structural Determinants of the Specificity for Synaptic Vesicle-associated Membrane Protein/Synaptobrevin of Tetanus and Botulinum Type B and G Neurotoxins

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