Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD ox ) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD ox to the red anionic FAD . radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD ox corresponds to the ground state, whereas the FAD . radical represents the light-activated state that mediates resetting of the Drosophila circadian clock. Cryptochromes (CRYs)4 constitute a family of flavoproteins that use flavin adenine dinucleotide (FAD) and sometimes an additional pterin derivative (methenyltetrahydrofolate, MTHF) as noncovalently bound cofactors and blue light absorbing chromophores (1). CRYs share moderate sequence, but significant structural homology with DNA photolyases, which repair UV-damaged DNA in a blue light dependent manner (2). Cyclobutane pyrimidine dimer (CPD) photolyases repair UV light-induced pyrimidine-dimer (PyrϽϾPyr) DNA lesions by intermolecular redox reactions between the catalytically active fully reduced flavin chromophore (FADH Ϫ ) and the PyrϽϾPyr substrate (2). A similar reaction mechanism is presumed for the (6-4)-photolyases, which repair pyrimidinepyrimidone (6-4) photoproducts (Pyr (6-4) Pyr), a second class of UV light-induced DNA lesions (3, 4). Cryptochromes do not exhibit any DNA repair activities, despite significant sequence homology of plant cryptochromes to CPD-photolyases and animal cryptochromes to (6-4)-photolyases (5, 6). Blue light absorption, phosphorylation, and effector interactions of plant cryptochromes control fundamental biological processes such as de-etiolation and flowering onset (7). Action spectra (8) and spectroscopic studies (9, 10) on Arabidopsis thaliana cryptochrome 1 (AtCRY1) suggest that its native and functionally active chromophore is oxidized FAD (FAD ox ), in contrast to photolyases, where the active chromophore is the two electronreduced FADH Ϫ . However, in both AtCRY1 (11, 12) and photolyases (2), blue light activation leads to the formation of a neutral blue FADH ⅐ radical. Whereas in AtCRY1 FAD ox is photoreduced to FADH ⅐ upon blue light illumination, the FADH ⅐ radical in photolyases is produced after a blue lightactivated electron t...

The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537 mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.In mammals many physiological processes are regulated in a day-time-dependent manner. These circadian (24 h) rhythms are generated by circadian clocks, which are operated by transcriptional and translational feedback loops. In the central feedback loop, the bHLH-PAS (basic Helix-Loop-Helix-PER-ARNT-SIM) transcription factors mBMAL1 (brain and muscle ARNT-like protein) and mCLOCK (circadian locomotor output cycle kaput) activate the transcription of three period genes (mper1,2,3) and two cryptochromes (mcry1,2) (1). The mPER proteins and (even more potently) the mCRY proteins feedback-repress their own transcription by regulating the activity of mBMAL1 and mCLOCK (2, 3). Notably, the mBMAL1-mCLOCK transcription factor complex not only regulates the mper and mcry genes but also a large number of clock controlled genes, including genes involved in cell cycle regulation, cellular detoxification, and metabolism (4). Hence, the regulation of these transcription factors is of relevance for many body functions and associated diseases (e.g. sleep and depressive disorders, metabolic syndrome, cardiovascular diseases, and tumor formation) that are under the control of the circadian clock (5). The importance of mBMAL1 for clock function is clearly demonstrated by the fact that mBMAL1 Ϫ/Ϫ knock-out mice show an immediate and complete loss of circadian rhythmicity at a behavio...

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