Selective interaction of complexin with the neuronal SNARE complex - Determination of the binding regions

Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAR E) 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 isolat ed complexins have no tertiary structure but contain an unusual alpha-helic al middle domain of approximately 58 amino acids that overlaps with the mos t highly conserved region of the molecules. Complexins form a stable stoich iometric complex with the central domain of the ternary SNARE complex, wher eas no binding was observed to monomeric SNAREs. Using a combination of lim ited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the S NARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substituti on of SNAP-25. In addition, a stretch of 12 amino acids in the middle of th e 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 li gands of the neuronal core complex that bind with a central alpha-helical d omain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin, Complexins may regulate the function of ternary complexes an d control membrane fusion through this interaction.