title: An alpha-helical minimal binding domain within the H3 domain of syntaxin is required for SNAP-25 binding.
abstract: 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 binding domain on syntaxin, we have prepared peptides that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25, and then determined the affinities of these peptides for binding to SNAP-25. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism revealed the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis.
authors.affiliation: Neurex Corporation, Menlo Park, California 94025, USA.
authors.forename: P
authors.initials: P
authors.lastname: Zhong
authors.forename: Y A
authors.initials: YA
authors.lastname: Chen
authors.forename: D
authors.initials: D
authors.lastname: Tam
authors.forename: D
authors.initials: D
authors.lastname: Chung
authors.forename: R H
authors.initials: RH
authors.lastname: Scheller
authors.forename: G P
authors.initials: GP
authors.lastname: Miljanich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002942:Circular Dichroism; D005089:Exocytosis / Q000502:physiology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism*; D009638:Norepinephrine / Q000037:antagonists & inhibitors / Q000378:metabolism; D016716:PC12 Cells / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013572:Synaptic Vesicles / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D013696:Temperature
chemical_list: D000954:Antigens, Surface; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D009638:Norepinephrine