国家自然科学基金面上资助项目(31070643)
This work was supported by a grant from The National Natural Science Foundation of China (31070643)
Dvl(Dishevelled)是Wnt信号通路传递的核心分子,无论内源的还是过表达的Dvl在细胞体内都能因自聚而形成puncta.研究已报道,Dvl主要通过其DIX结构域上的三个作用区域来介导自聚:SiteⅠ、SiteⅡ和SiteⅢ,其中SiteⅠ和SiteⅡ还参与了Dvl-DIX与Ccd1-DIX的异聚.为了进一步得到Dvl2-DIX上SiteⅠ和SiteⅡ的直接三维结构,本研究设计了一系列的SiteⅢ突变体.通过体内和体外实验进一步证实了这些突变氨基酸确实参与了Dvl2-DIX的自聚,然后对这些SiteⅢ突变体蛋白成功地进行了纯化和结晶,最终得到3.1Å的Dvl2-DIX(G65A)晶体数据.分析表明该晶体存在片层位移现象,需对数据进行一定修正后才能进行后续的结构分析.体外实验又证实了这些突变氨基酸不影响Dvl2-DIX与Ccd1-DIX的异聚,为了进一步研究Dvl2-DIX与Ccd1-DIX相互作用,我们对这些SiteⅢ突变体蛋白与Ccd1-DIX进行共结晶.最终获得Dvl2-DIX(G65A)与Ccd1-DIX复合物的初晶,利于进一步的晶体优化及数据收集.
Dvl (Dishevelled) is a key effector molecule of the Wnt signaling pathway. The DIX domain of Dvl (Dvl-DIX) can homo-oligomerize into cytoplasmic puncta via the intra-filament SiteⅠ, SiteⅡ and the inter- filament SiteⅢ, and form hetero-complex with the DIX domain of Ccd1 (Ccd1-DIX) through SiteⅠ and SiteⅡ. Since all efforts to solve the wildtype Dvl2-DIX structure were unsuccessful, we carried out crystallization trials with the SiteⅢ mutants of Dvl2-DIX that display impaired homo-oligomerization and Wnt activity. Crystals of Dvl2-DIX(G65A) protein were obtained, but the diffraction data encountered an unexpected lattice-translocation defect. These SiteⅢ mutations of Dvl2-DIX retained the ability to hetero-interact with Ccd1-DIX, and we thus successfully generated various complexes between Dvl2-DIX mutants and wildtype Ccd1-DIX. After extensive trials, crystals of the Dvl2-DIX(G65A)-Ccd1-DIX complex were produced, but of poor quality and unsuitable for diffraction studies. Optimization is under way.
但琼洁,刘奕彤,吴嘉炜,王志新. Dvl2-DIX结构域的SiteⅢ相关突变体结晶及其与Ccd1-DIX的共结晶[J].生物化学与生物物理进展,2012,39(7):647-654
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