Abstract
Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and thus may regulate SNARE function. Using purified proteins, we have performed a detailed analysis of the structure of complexin and of its interaction with SNARE proteins. NMR spectroscopy revealed that isolated complexins have no tertiary structure but contain an unusual alpha-helical middle domain of approximately 58 amino acids that overlaps with the most highly conserved region of the molecules. Complexins form a stable stoichiometric complex with the central domain of the ternary SNARE complex, whereas no binding was observed to monomeric SNAREs. Using a combination of limited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substitution of SNAP-25. In addition, a stretch of 12 amino acids in the middle of the SNARE motif of syntaxin 1A was able to confer binding activity to the non-binding relative syntaxin 4. Furthermore, disassembly of ternary complexes is not affected by complexins. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central alpha-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. Complexins may regulate the function of ternary complexes and control membrane fusion through this interaction.
Highlights
Complexins are evolutionarily conserved proteins that bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and may regulate SNARE function
Using a combination of limited proteolysis, deletion mutagenesis, and nuclear magnetic resonance (NMR) spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex
We conclude that complexins are specific ligands of the neuronal core complex that bind with a central ␣-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin
Summary
Complexins are evolutionarily conserved proteins that bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and may regulate SNARE function. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central ␣-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. They include the synaptic vesicle protein synaptobrevin ( referred to as vesicle-associated membrane protein), and the synaptic plasma membrane proteins SNAP-25 and syntaxin 1 These proteins form a stable ternary complex, the core complex, that can be reversibly disassembled by the ATPase NSF (N-ethylmaleimide-sensitive factor) and additional cofactors termed SNAPs (for soluble NSF attachment proteins) [5]. It is unusually heat-stable and only denatures above 80 °C [10] These and other findings suggested that SNARE complex assembly is the essential step in initiating membrane fusion. The SNAREs relax into a complex in which all transmembrane domains are aligned in parallel (“cis” complex) and which is re-energized by disassembly involving NSF and SNAPs [11]
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