Abstract
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.
Highlights
In adaptation to daily environmental changes most organisms ranging from cyanobacteria to humans display 24-h day-night activity cycles, so called circadian rhythms
We show that Drosophila PERIOD (dPER) and mouse PERIOD2 (mPER2) use different interaction surfaces for homodimer formation, which are associated with different dimerization affinities
We identify a versatile molecular surface of the PERIOD proteins, which mediates homodimer formation of mPER2 but is used for dPER-dPER mediates interactions with TIMELESS (dTIM) heterodimer formation in Drosophila
Summary
In adaptation to daily environmental changes most organisms ranging from cyanobacteria to humans display 24-h day-night activity cycles, so called circadian rhythms. In the circadian oscillator of D. melanogaster, expression of the clock protein PERIOD (dPER) [6] and its interaction partner TIMELESS (dTIM) is activated by the heterodimeric bHLH-PAS transcription factors dCLOCK and dCYCLE. Daytime dependent changes in concentration, cellular localization, and transcriptional repressor activity of dPER are essential for maintaining circadian rhythmicity. These changes are critically influenced by Doubletime (DBT)- [7,8,9] and casein kinase II (CKII)- [10,11] mediated phosphorylation of dPER as well as the interaction of dPER with dTIM [12,13,14,15]. While DBT-dependent phosphorylation of dPER on Ser enhances its proteasomal degradation [16], dTIM binding [15] as well as dephosphorylation by the phosphatases PP2A and PP1 [17,18] increase dPER stability. dPER-dTIM interactions are reported to promote nuclear entry of the complex due to specific interactions of dTIM
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