Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation

被引:282
作者
Fasshauer, D
Otto, H
Eliason, WK
Jahn, R
Brunger, AT
机构
[1] YALE UNIV,SCH MED,HOWARD HUGHES MED INST,NEW HAVEN,CT 06510
[2] YALE UNIV,SCH MED,DEPT PHARMACOL,NEW HAVEN,CT 06510
[3] YALE UNIV,DEPT BIOCHEM & MOL BIOPHYS,NEW HAVEN,CT 06510
关键词
D O I
10.1074/jbc.272.44.28036
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 alpha-, beta-, and gamma-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 alpha-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in alpha-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.
引用
收藏
页码:28036 / 28041
页数:6
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