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

doi:10.1074/jbc.272.7.4582

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

From the ^§ Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06510 and the
^‡ Department of Pharmacology, Yale University School of Medicine, Yale University, New Haven, Connecticut 06510 and the
^¶ Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, Connecticut 06510

^∥To whom correspondence should be sent:
Dept. Molecular Biophysics and Biochemistry, Bass Center, 266 Whitney Ave., New Haven, CT 06520
. Tel.: 203-432-6143; Fax: 203-432-6946; E-mail: brunger{at}laplace.csb.yale.edu

Abstract

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.

Footnotes

↵* This work was supported by a grant from the Deutsche Forschungsgemeinschaft (to D. F.) and by National Institutes of Health Grant GM54160-01 (to A. T. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SNAP(s)

soluble NSF attachment protein(s

NSF

N-ethylmaleimide-sensitive fusion protein

SNAP-25

synaptosomal associated protein of 25 kDa

SNARE

SNAP receptor

DTT

dithiothreitol

GST

glutathione S-transferase.
↵² Bruns, D., Engers, S., Yang, C., Ossig, R., Jeromin, A., and Jahn, R. (1997) J. Neurosci., in press.
↵³ Programs are available on the World Wide Web (URLs: http://ulrec3.unil.ch/software/COILS_form.html and http://ostrich.lcs.mit/. edu/cgi-bin/score).
- Received September 8, 1996.
- Revision received November 13, 1996.