Drosophila cryptochrome (dCRY) is a FAD-dependent circadian photoreceptor, whereas mammalian cryptochromes (CRY1/2) are integral clock components that repress mCLOCK/mBMAL1-dependent transcription. We report crystal structures of full-length dCRY, a dCRY loop deletion construct, and the photolyase homology region of mouse CRY1 (mCRY1). Our dCRY structures depict Phe534 of the regulatory tail in the same location as the photolesion in DNA-repairing photolyases and reveal that the sulfur loop and tail residue Cys523 plays key roles in the dCRY photoreaction. Our mCRY1 structure visualizes previously characterized mutations, an NLS, and MAPK and AMPK phosphorylation sites. We show that the FAD and antenna chromophore-binding regions, a predicted coiled-coil helix, the C-terminal lid, and charged surfaces are involved in FAD-independent mPER2 and FBXL3 binding and mCLOCK/mBMAL1 transcriptional repression. The structure of a mammalian cryptochrome1 protein may catalyze the development of CRY chemical probes and the design of therapeutic metabolic modulators.

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...

Deregulation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in numerous pathologies like cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small molecule and ATP-competitive PI3K inhibitors with a wide range of selectivities have entered clinical development. In order to understand mechanisms underlying isoform selectivity of these inhibitors, we developed a novel expression strategy that enabled us to determine the first crystal structure of the catalytic subunit of the class IA PI3K p110δ. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI3K inhibitors reveal that selectivity towards p110δ can be achieved by exploiting its conformational flexibility and the sequence diversity of active-site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110δ with greatly improved potencies.

Cryptochromes are a class of flavoprotein blue-light signaling receptors found in plants, animals, and humans that control plant development and the entrainment of circadian rhythms. In plant cryptochromes, light activation is proposed to result from photoreduction of a protein-bound flavin chromophore through intramolecular electron transfer. However, although similar in structure to plant cryptochromes, the light-response mechanism of animal cryptochromes remains entirely unknown. To complicate matters further, there is currently a debate on whether mammalian cryptochromes respond to light at all or are instead activated by non–light-dependent mechanisms. To resolve these questions, we have expressed both human and Drosophila cryptochrome proteins to high levels in living Sf21 insect cells using a baculovirus-derived expression system. Intact cells are irradiated with blue light, and the resulting cryptochrome photoconversion is monitored by fluorescence and electron paramagnetic resonance spectroscopic techniques. We demonstrate that light induces a change in the redox state of flavin bound to the receptor in both human and Drosophila cryptochromes. Photoreduction from oxidized flavin and subsequent accumulation of a semiquinone intermediate signaling state occurs by a conserved mechanism that has been previously identified for plant cryptochromes. These results provide the first evidence of how animal-type cryptochromes are activated by light in living cells. Furthermore, human cryptochrome is also shown to undergo this light response. Therefore, human cryptochromes in exposed peripheral and/or visual tissues may have novel light-sensing roles that remain to be elucidated.