A Structural Change Occurs upon Binding of Syntaxin to SNAP-25*

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the α-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% α-helical content. In contrast, the α-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in α-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in α-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

[1]  J. Rothman,et al.  Protein Sorting by Transport Vesicles , 1996, Science.

[2]  R. Scheller,et al.  Synaptic vesicle biogenesis, docking, and fusion: a molecular description. , 1996, Physiological reviews.

[3]  A. Lupas.  Prediction and analysis of coiled-coil structures. , 1996, Methods in enzymology.

[4]  V. O'Connor,et al.  Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion. , 1995, The EMBO journal.

[5]  D. Bruns,et al.  Real-time measurement of transmitter release from single synaptic vesicles , 1995, Nature.

[6]  B. Berger,et al.  Predicting coiled coils by use of pairwise residue correlations. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Hanson,et al.  The N-Ethylmaleimide-sensitive Fusion Protein and α-SNAP Induce a Conformational Change in Syntaxin (*) , 1995, The Journal of Biological Chemistry.

[8]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[9]  S. Nauenburg,et al.  Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro. , 1995, The EMBO journal.

[10]  R. Scheller,et al.  Distinct domains of syntaxin are required for synaptic vesicle fusion complex formation and dissociation , 1995, Neuron.

[11]  N. Barton,et al.  SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils. , 1994, The Journal of biological chemistry.

[12]  T. Südhof,et al.  Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. , 1994, The EMBO journal.

[13]  P. Brennwald,et al.  Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis , 1994, Cell.

[14]  Jonathan Pevsner,et al.  Specificity and regulation of a synaptic vesicle docking complex , 1994, Neuron.

[15]  Reinhard Jahn,et al.  Vesicle fusion from yeast to man , 1994, Nature.

[16]  R. Hodges,et al.  The role of interhelical ionic interactions in controlling protein folding and stability. De novo designed synthetic two-stranded alpha-helical coiled-coils. , 1994, Journal of molecular biology.

[17]  R. Scheller,et al.  Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking. , 1994, Science.

[18]  Mark K. Bennett,et al.  A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion , 1993, Cell.

[19]  Paul Tempst,et al.  SNAP receptors implicated in vesicle targeting and fusion , 1993, Nature.

[20]  A. Lupas,et al.  Predicting coiled coils from protein sequences , 1991, Science.

[21]  M. Weiss,et al.  Thermal unfolding studies of a leucine zipper domain and its specific DNA complex: implications for scissor's grip recognition. , 1990, Biochemistry.

[22]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[23]  D. Gelfand,et al.  1 – OPTIMIZATION OF PCRs , 1990 .

[24]  S. McKnight,et al.  The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. , 1988, Science.

[25]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[26]  Y H Chen,et al.  Determination of the helix and beta form of proteins in aqueous solution by circular dichroism. , 1974, Biochemistry.

[27]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[28]  G. Fasman,et al.  Computed circular dichroism spectra for the evaluation of protein conformation. , 1969, Biochemistry.

[29]  F. Crick,et al.  The packing of α‐helices: simple coiled‐coils , 1953 .