AN ALPHA-HELICAL MINIMAL BINDING DOMAIN WITHIN THE H3 DOMAIN OF SYNTAXIN IS REQUIRED FOR SNAP-25 BINDING

The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 bin ding domain on syntaxin, we have prepared peptides that span the synta xin H3 domain (residues 191-266), the region previously shown to be im portant for binding to SNAP-25, and then determined the affinities of these peptides for binding to SNAP-25. A minimal binding domain was id entified within a region of 32 amino acids (residues 189-220). Its aff inity for SNAP-25 is substantially enhanced by C-terminal extension (r esidues 221-266). Circular dichroism revealed the presence of substant ial or-helicity in the H3 domain and in the 32-mer minimal binding dom ain, but not in 113 peptides that do not bind to SNAP-25. At temperatu res that denature the or-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserve d residues of the amphiphilic alpha-helix within the minimal binding d omain (e.g., residues 205 and 209) greatly reduce the affinity for SNA P-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The 113 domain pept ide and the minimal binding domain peptide inhibit norepinephrine rele ase from PC12 cells. These results suggest that specific amino acid re sidues in the 113 domain, positioned by the underlying ct-helical stru cture, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytos is.