Asymmetric coiled-coil structure with Guanine nucleotide exchange activity.
Asymmetric coiled-coil structure with Guanine nucleotide exchange activity.
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DOI：
10.1016/j.str.2007.01.003
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发表时间：
2007-02
期刊：
影响因子：
5.7
通讯作者：
Yusuke Sato;R. Shirakawa;H. Horiuchi;N. Dohmae;S. Fukai;O. Nureki
中科院分区：
文献类型：
--
作者：
Yusuke Sato;R. Shirakawa;H. Horiuchi;N. Dohmae;S. Fukai;O. Nureki
胞吐过程中的囊泡运输由酵母中的Rab GT3，Sec 4p调节，其由称为Sec 2 p的鸟嘌呤核苷酸交换因子（GEF）激活。GEF活性定位于Sec 2 p的N-末端160个残基，其与具有已知结构的任何其他GEF缺乏序列相似性，因此Sec 2 p的鸟嘌呤核苷酸交换机制仍然未知。在这里，我们报告的Sec 2 p GEF结构域的晶体结构在3.0 nm分辨率。该结构出乎意料地由延伸超过180 μ m的同源二聚体平行卷曲螺旋组成。对Sec 2 p的一系列缺失突变体和点突变体的下拉和鸟嘌呤核苷酸交换分析揭示了其GEF活性的催化残基以及Sec 4p结合位点，从而提出了简单卷曲螺旋的核苷酸交换机制。目前的功能分析使我们能够建立Sec 2 p：Sec 4p复合物模型，该模型解释了Rab GTP酶通过其各自的GEF蛋白的特异性。
Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 Å resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 Å. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.