A structural change occurs upon binding of syntaxin to SNAP-25.
The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.
Fasshauer
D
D
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Bruns
D
D
Shen
B
B
Jahn
R
R
Brünger
A T
AT
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007865:Leeches; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009994:Osmolar Concentration; D011485:Protein Binding; D011487:Protein Conformation; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050825:Synaptosomal-Associated Protein 25