Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6
Computer modelling in combination with in vitro studies reveals similar binding affinities of Drosophila Crumbs for the PDZ domains of Stardust and DmPar-6
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DOI：
10.1016/j.ejcb.2006.03.003
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发表时间：
2006-08-01
影响因子：
6.6
通讯作者：
Knust, Elisabeth
中科院分区：
文献类型：
--
作者：
Kempkens, Ozlem;Medina, Ernmanuelle;Knust, Elisabeth
多蛋白复合物的形成是细胞图案化的常见主题，从而在特定区域产生空间和功能上不同的实体。这些复合物的核心成分是支架蛋白，它包含多个蛋白质-蛋白质相互作用结构域，并提供一个招募各种其他成分的平台。越来越多的证据表明蛋白质复合物是动态结构，并且它们的成分可以根据细胞环境进行各种相互作用。然而，迄今为止，人们对调节这种行为的因素知之甚少。果蝇和哺乳动物上皮细胞中都存在一种进化上保守的蛋白质复合物，它分别由跨膜蛋白 Crumbs/Crb3 和支架蛋白 Stardust/Pals1 和 DPATJ/PATJ 组成，位于粘连带的顶部。在这里，我们通过体外分析表明，与脊椎动物类似，果蝇DmPar-6的单个PDZ结构域可以与跨膜蛋白Crumbs的四个C端氨基酸（ERLI）结合。为了进一步评估 Crumbs 与 DmPar-6 和 MAGUK 蛋白 Stardust 的结合能力，对 PDZ 结构数据库进行了分析，并对 Crumbs 的 C 末端与这两种蛋白的 PDZ 结构域之间的相互作用进行了建模。结果表明，两个 PDZ 结构域都以相似的亲和力结合 Crumbs。这些数据得到定量酵母两种杂交体相互作用的支持。在细胞培养物和果蝇胚胎中进行的体内分析表明，Crumbs 的细胞质结构域可以将 DmPar-6 和 DaPKC 募集到质膜上。这里提供的数据是针对这些蛋白质之间可能的动态相互作用进行讨论的。 (c) 2006 年爱思唯尔有限公司。版权所有。
Formation of multiprotein complexes is a common theme to pattern a cell, thereby generating spatially and functionally distinct entities at specialised regions. Central components of these complexes are scaffold proteins, which contain several protein-protein interaction domains and provide a platform to recruit a variety of additional components. There is increasing evidence that protein complexes are dynamic structures and that their components can undergo various interactions depending on the cellular context. However, little is known so far about the factors regulating this behaviour. One evolutionarily conserved protein complex, which can be found both in Drosophila and mammalian epithelial cells, is composed of the transmembrane protein Crumbs/Crb3 and the scaffolding proteins Stardust/Pals1 and DPATJ/PATJ, respectively, and localises apically to the zonula adherens. Here we show by in vitro analysis that, similar as in vertebrates, the single PDZ domain of Drosophila DmPar-6 can bind to the four C-terminal amino acids (ERLI) of the transmembrane protein Crumbs. To further evaluate the binding capability of Crumbs to DmPar-6 and the MAGUK protein Stardust, analysis of the PDZ structural database and modelling of the interactions between the C-terminus of Crumbs and the PDZ domains of these two proteins were performed. The results suggest that both PDZ domains bind Crumbs with similar affinities. These data are supported by quantitative yeast two-hybrid interactions. In vivo analysis performed in cell cultures and in the Drosophila embryo show that the cytoplasmic domain of Crumbs can recruit DmPar-6 and DaPKC to the plasma membrane. The data presented here are discussed with respect to possible dynamic interactions between these proteins. (c) 2006 Elsevier GmbH. All rights reserved.