Zhong P, Chen Y A, Tam D, Chung D, Scheller R H, Miljanich G P
Neurex Corporation, Menlo Park, California 94025, USA.
Biochemistry. 1997 Apr 8;36(14):4317-26. doi: 10.1021/bi9625408.
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.
蛋白质 syntaxin 1A 与 SNAP-25 之间的相互作用是突触小泡对接与融合的关键步骤。为了确定 syntaxin 上的 SNAP-25 结合结构域，我们制备了跨越 syntaxin H3 结构域（第 191 - 266 位氨基酸残基）的肽段，该区域先前已显示对与 SNAP-25 的结合很重要，然后测定了这些肽段与 SNAP-25 结合的亲和力。在 32 个氨基酸（第 189 - 220 位氨基酸残基）的区域内鉴定出一个最小结合结构域。其 C 末端延伸（第 221 - 266 位氨基酸残基）显著增强了它与 SNAP-25 的亲和力。圆二色性显示 H3 结构域和 32 肽最小结合结构域中存在大量α-螺旋，但不与 SNAP-25 结合的 H3 肽段中不存在。在使最小结合结构域肽段的α-螺旋变性的温度下，SNAP-25 结合丧失。最小结合结构域内两亲性α-螺旋的进化保守残基（例如第 205 和 209 位氨基酸残基）的选定突变极大地降低了与 SNAP-25 的亲和力，但对二级结构没有重大影响，这表明这些残基可能直接与 SNAP-25 相互作用。H3 结构域肽段和最小结合结构域肽段抑制 PC12 细胞中去甲肾上腺素的释放。这些结果表明，由潜在的α-螺旋结构定位的 H3 结构域中的特定氨基酸残基对其与 SNAP-25 的结合很重要，并支持这种相互作用对突触前囊泡胞吐作用很重要的观点。
