The C2B domain of synaptotagmin I is a Ca2+-binding module.
The C2B domain of synaptotagmin I is a Ca2+-binding module.
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DOI：
10.1021/bi010340c
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发表时间：
2001-05
期刊：
影响因子：
2.9
通讯作者：
J. Ubach;Y. Lao;I. Fernández;D. Araç;T. Südhof;J. Rizo
中科院分区：
文献类型：
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作者：
J. Ubach;Y. Lao;I. Fernández;D. Araç;T. Südhof;J. Rizo
Synaptotagmin I 是一种突触小泡蛋白，包含两个 C(2) 结构域，在神经递质释放中充当 Ca(2+) 传感器。突触结合蛋白 I C(2)A 结构域的 Ca(2+) 结合特性已得到充分表征，但 C(2)B 结构域的 Ca(2+) 结合特性尚不清楚。先前发现 C(2)B 结构域以 Ca(2+) 依赖性方式从脑匀浆中拉下突触结合蛋白 I，从而产生了一个有吸引力的模型，其中突触结合蛋白 I 通过 C(2)B 结构域的 Ca(2+) 依赖性多聚化参与融合孔的形成。然而，Ca(2+) 依赖性 C(2)B 结构域二聚化的研究以及其他 C(2)B 结构域相互作用的分析中描述了矛盾的结果。为了阐明这些问题，现在已经使用生物物理技术对 C(2)B 结构域进行了研究。表达为 GST 融合蛋白并通过亲和层析分离的重组 C(2)B 结构域尽管是电泳纯的，但仍含有紧密结合的细菌污染物。这些污染物与多碱基序列结合，该多碱基序列先前与多种 C(2)B 结构域相互作用有关，包括 Ca(2+) 依赖性二聚化。 NMR 实验表明，纯重组 C(2)B 结构域直接结合 Ca(2+)，但在 Ca(2+) 结合后不会二聚化。相比之下，来自脑匀浆的天然突触结合蛋白 I 的细胞质片段（包括 C(2)A 和 C(2)B 结构域）参与作为 Ca(2+) 功能的高分子量复合物。这些结果表明突触结合蛋白 I 的重组 C(2)B 结构域是单体、自主折叠的 Ca(2+) 结合模块，表明突触结合蛋白 I 多聚化在融合孔形成中的潜在功能不涉及 C(2)B 结构域之间的直接相互作用或需要翻译后修饰。
Synaptotagmin I is a synaptic vesicle protein that contains two C(2) domains and acts as a Ca(2+) sensor in neurotransmitter release. The Ca(2+)-binding properties of the synaptotagmin I C(2)A domain have been well characterized, but those of the C(2)B domain are unclear. The C(2)B domain was previously found to pull down synaptotagmin I from brain homogenates in a Ca(2+)-dependent manner, leading to an attractive model whereby Ca(2+)-dependent multimerization of synaptotagmin I via the C(2)B domain participates in fusion pore formation. However, contradictory results have been described in studies of Ca(2+)-dependent C(2)B domain dimerization, as well as in analyses of other C(2)B domain interactions. To shed light on these issues, the C(2)B domain has now been studied using biophysical techniques. The recombinant C(2)B domain expressed as a GST fusion protein and isolated by affinity chromatography contains tightly bound bacterial contaminants despite being electrophoretically pure. The contaminants bind to a polybasic sequence that has been previously implicated in several C(2)B domain interactions, including Ca(2+)-dependent dimerization. NMR experiments show that the pure recombinant C(2)B domain binds Ca(2+) directly but does not dimerize upon Ca(2+) binding. In contrast, a cytoplasmic fragment of native synaptotagmin I from brain homogenates, which includes the C(2)A and C(2)B domains, participates in a high molecular weight complex as a function of Ca(2+). These results show that the recombinant C(2)B domain of synaptotagmin I is a monomeric, autonomously folded Ca(2+)-binding module and suggest that a potential function of synaptotagmin I multimerization in fusion pore formation does not involve a direct interaction between C(2)B domains or requires a posttranslational modification.