Structural Changes Are Associated with Soluble N-Ethylmaleimide-sensitive Fusion Protein Attachment Protein Receptor Complex Formation*
- From the ‡Howard Hughes Medical Institute,§Department of Pharmacology, Yale University School of Medicine, and ‡Department of Molecular Biochemistry and Biophysics, Yale University, New Haven, Connecticut 06510
Abstract
SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors α-, β-, and γ-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant α-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in α-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion.
Footnotes
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↵* This work was supported by National Institutes of Health Grants GM54160-01 (to A. T. B.) and 5 RO1 (to R. J.) NS33709-02.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.
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↵¶ Supported by a grant from the Deutsche Forschungsgemeinschaft.
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↵‖ Present address: Max-Planck-Institut für Biophysikalische Chemie, Abteilung Neurobiologie, Am Fassberg II, 37077 Göttingen, Germany.
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↵** Present address: Freie Universität Berlin, Institut für Biochemie, Thielallee 63, 14195 Berlin, Germany.
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↵§§ To whom correspondence should be addressed.
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↵1 The abbreviations used are: VAMP, vesicle-associated membrane protein; NSF,N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; SNAP-25, synaptosomal associated protein of 25 kDa; PAGE, polyacrylamide gel electrophoresis, PCR, polymerase chain reaction; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride); MALLS, multiangle laser light scattering.
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↵2 G. Warren and A. T. Brünger, unpublished results.
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- Received June 11, 1997.
- Revision received August 18, 1997.
