Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function
Unwinding the differences of the mammalian PERIOD clock proteins from crystal structure to cellular function
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DOI：
10.1073/pnas.1113280109
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发表时间：
2012-02-28
影响因子：
11.1
通讯作者：
Wolf, Eva
中科院分区：
文献类型：
--
作者：
Kucera, Nicole;Schmalen, Ira;Wolf, Eva
三个PERIOD同源物mPER 1、mPER 2和mPER 3构成哺乳动物昼夜节律钟的中心组件。它们含有两个PAS（PER-ARNT-SIM）结构域（PAS-A和PAS-B），其介导同源和异源二聚体mPER-mPER相互作用以及与转录因子和激酶的相互作用。在这里，我们提出的PAS结构域片段的mPER 1和mPER 3的晶体结构，并比较它们与以前报道的mPER 2结构。结构显示同源二聚体，其由PAS-B β折叠表面的相互作用介导，包括高度保守的色氨酸（Trp 448（mPER 1）、Trp 419（mPER 2）、Trp 359（mPER 3））。mPER 1同源二聚体还通过PAS-A结构域和mPER 3同源二聚体之间的相互作用稳定，所述相互作用通过包括预测的螺旋-环-螺旋基序的N-末端区域进行。我们已经验证了这些同源二聚体接口的存在，在溶液中和细胞内，使用分析凝胶过滤和荧光素酶互补测定和定量的同源二聚体的稳定性，通过分析ultracentravation的贡献。我们还表明，通过光漂白分析后的荧光恢复，PAS-B/色氨酸二聚体界面的不稳定导致在人U2 OS细胞中含有mPER 2的复合物的更快的流动性。我们的研究揭示了三种小鼠PERIOD同源物的同二聚体相互作用之间的结构和数量差异，这可能有助于其独特的时钟功能。
The three PERIOD homologues mPER1, mPER2, and mPER3 constitute central components of the mammalian circadian clock. They contain two PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B), which mediate homo- and heterodimeric mPER-mPER interactions as well as interactions with transcription factors and kinases. Here we present crystal structures of PAS domain fragments of mPER1 and mPER3 and compare them with the previously reported mPER2 structure. The structures reveal homodimers, which are mediated by interactions of the PAS-B beta-sheet surface including a highly conserved tryptophan (Trp448(mPER1), Trp419(mPER2), Trp359(mPER3)). mPER1 homodimers are additionally stabilized by interactions between the PAS-A domains and mPER3 homodimers by an N-terminal region including a predicted helix-loop-helix motive. We have verified the existence of these homodimer interfaces in solution and inside cells using analytical gel filtration and luciferase complementation assays and quantified their contributions to homodimer stability by analytical ultracentrifugation. We also show by fluorescence recovery after photobleaching analyses that destabilization of the PAS-B/tryptophan dimer interface leads to a faster mobility of mPER2 containing complexes in human U2OS cells. Our study reveals structural and quantitative differences between the homodimeric interactions of the three mouse PERIOD homologues, which are likely to contribute to their distinct clock functions.