Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.
Structural and functional analyses of PAS domain interactions of the clock proteins Drosophila PERIOD and mouse PERIOD2.
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DOI：
10.1371/journal.pbio.1000094
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发表时间：
2009-04-28
期刊：
影响因子：
9.8
通讯作者：
Wolf E
中科院分区：
文献类型：
--
作者：
Hennig S;Strauss HM;Vanselow K;Yildiz O;Schulze S;Arens J;Kramer A;Wolf E
PERIOD蛋白是果蝇和哺乳动物生物钟的核心组成部分。果蝇PERIOD（dPER）片段含有两个PER-ARNT-SIM（PAS）结构域（PAS-A和PAS-B）和两个额外的C-末端α-螺旋（αE和αF），其晶体结构揭示了PAS-A与PAS-B和α-螺旋中的色氨酸482分子间相互作用介导的同源二聚体。在这里，我们提出了一个单体PAS结构域片段的dPER缺乏αF螺旋的晶体结构。此外，我们已经解决了PAS结构域片段的小鼠PERIOD同系物mPER 2的晶体结构。mPER 2结构显示出与dPER不同的二聚体界面，其通过PAS-B β-折叠表面（包括色氨酸419（相当于Trp 482 dPER））的相互作用而稳定。我们已经验证和定量分析的同源二聚体的相互作用dPER和mPER 2的定点诱变，使用分析凝胶过滤，分析ultracenthesis，和免疫共沉淀实验。此外，我们通过酵母双杂交实验证明，dPER的PAS-B β-折叠表面介导与TIMELESS（dTIM）的相互作用。我们的研究揭示了定量和定性的差异之间的同源二聚体PAS结构域的相互作用dPER和哺乳动物同源mPER 2。此外，我们确定PAS-B β折叠表面是一个多功能的相互作用位点，在哺乳动物系统中介导mPER 2同源二聚化，在果蝇系统中介导dPER-dTIM异源二聚体形成。大多数生物体都有日常活动周期（昼夜节律），这是由生物钟产生的。昼夜节律周期性是由特定的时钟蛋白相互作用和翻译后修饰以及它们的细胞定位、表达和稳定性的变化产生的。为了更多地了解果蝇和小鼠生物钟运作的分子过程，我们分析了时钟蛋白果蝇PERIOD（dPER）和小鼠PERIOD 2（mPER 2）的同源和异源二聚体相互作用。我们发现，dPER和mPER 2使用不同的相互作用表面的同二聚体的形成，这是与不同的二聚亲和力。此外，我们提出了一个基于结构的生化分析dPER与其合作伙伴果蝇TIMELESS（dTIM）的异二聚体相互作用。我们确定了一个通用的分子表面的PERIOD蛋白，介导同源二聚体形成的mPER 2，但用于dPER-dTIM异源二聚体形成在果蝇。我们的研究结果揭示了苍蝇和哺乳动物中PERIOD时钟蛋白分子相互作用的定量和定性差异，使它们能够适应这些不同生物体中不同的结合伴侣和调节功能。果蝇PERIOD和小鼠PERIOD 2生物钟蛋白的晶体结构和基于结构的生物化学研究揭示了不同的同源二聚体相互作用，并确定了一个多功能的分子表面，介导小鼠PERIOD 2的同源二聚体化，但参与果蝇PERIOD与TIMELESS的异源二聚体相互作用。
PERIOD proteins are central components of the Drosophila and mammalian circadian clocks. The crystal structure of a Drosophila PERIOD (dPER) fragment comprising two PER-ARNT-SIM (PAS) domains (PAS-A and PAS-B) and two additional C-terminal α-helices (αE and αF) has revealed a homodimer mediated by intermolecular interactions of PAS-A with tryptophane 482 in PAS-B and helix αF. Here we present the crystal structure of a monomeric PAS domain fragment of dPER lacking the αF helix. Moreover, we have solved the crystal structure of a PAS domain fragment of the mouse PERIOD homologue mPER2. The mPER2 structure shows a different dimer interface than dPER, which is stabilized by interactions of the PAS-B β-sheet surface including tryptophane 419 (equivalent to Trp482dPER). We have validated and quantitatively analysed the homodimer interactions of dPER and mPER2 by site-directed mutagenesis using analytical gel filtration, analytical ultracentrifugation, and co-immunoprecipitation experiments. Furthermore we show, by yeast-two-hybrid experiments, that the PAS-B β-sheet surface of dPER mediates interactions with TIMELESS (dTIM). Our study reveals quantitative and qualitative differences between the homodimeric PAS domain interactions of dPER and its mammalian homologue mPER2. In addition, we identify the PAS-B β-sheet surface as a versatile interaction site mediating mPER2 homodimerization in the mammalian system and dPER-dTIM heterodimer formation in the Drosophila system. Most organisms have daily activity cycles (circadian rhythms), which are generated by circadian clocks. Circadian periodicity is produced by specific clock protein interactions and posttranslational modifications as well as changes in their cellular localization, expression, and stability. To learn more about the molecular processes underlying circadian clock operation in fruit flies and mouse, we analysed the homo- and heterodimeric interactions of the clock proteins Drosophila PERIOD (dPER) and mouse PERIOD2 (mPER2). We show that dPER and mPER2 use different interaction surfaces for homodimer formation, which are associated with different dimerization affinities. In addition, we present a structure-based biochemical analysis of the heterodimeric interaction of dPER with its partner Drosophila TIMELESS (dTIM). We identify a versatile molecular surface of the PERIOD proteins, which mediates homodimer formation of mPER2 but is used for dPER-dTIM heterodimer formation in Drosophila. Our results reveal quantitative and qualitative differences in the molecular interactions of PERIOD clock proteins in flies and mammals, allowing them to adjust to their different binding partners and regulatory functions in these different organisms. Crystal structures and structure-based biochemical studies ofDrosophila PERIOD and mouse PERIOD2 circadian clock proteins reveal different homodimer interactions and identify a versatile molecular surface that mediates homodimerization of mouse PERIOD2 but is involved in heterodimeric interactions ofDrosophila PERIOD with TIMELESS.
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