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 ␣-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 ␣-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.Intracellular membrane fusion events are mediated by conserved sets of membrane-bound proteins referred to as SNAREs 1 (acronym for soluble N-ethylmaleimide-sensitive factor attachment protein receptors) (1). SNAREs comprise a family of proteins that is distinguished by the presence of the SNARE motif, a homologous stretch of approximately 60 amino acids that is usually localized adjacent to the membrane anchor domains (2-4). Best characterized are SNARE proteins functioning in neuronal exocytosis. They include the synaptic vesicle protein synaptobrevin (also 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).Studies performed largely on recombinant proteins in solution have revealed a detailed picture of SNARE complex assembly. Of the monomeric SNAREs, only syntaxin 1 is partially ␣-helical, whereas both SNAP-25 and synaptobrevin are largely unstructured. Upon assembly, a dramatic increase in ␣-helical content is observed suggesting major conformational changes (6, 7). Interactions in the complex are largely confined to the SNARE motifs. X-ray crystallography revealed that the assembled core complex consists of an elongated bund...