Structures of Drosophila Cryptochrome and Mouse Cryptochrome1 Provide Insight into Circadian Function

Abstract

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.

Mechanism of Photosignaling by Drosophila Cryptochrome

“…Our results appear to be contradictory to the recent finding that the W397F mutation impaired the conformational change of dCRY (22). However, in that study, the conformational change was probed with antibody directed to the C terminus of dCRY, which might have interfered with the proteolytic probe for conformational change.…”

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…This model is supported by recent crystal structures, which show that the C-terminal helix docks in a groove of the PHR domain that is analogous to the DNA-binding groove in photolyase (13)(14)(15). Mammalian CRYs are not activated by light, and their C-terminal tails are not conserved with the Drosophila CRY C-terminal tail.…”

“…It is unknown whether the tails play an analagous regulatory role. Recent crystal structures of mammalian CRY1 and CRY2 do not include the tail regions (13,16).…”

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

“…The repressive activity of CRY1 is essential to generate circadian rhythms (13)(14)(15); one way that CRY1 does this is by binding the BMAL1 TAD to sequester it from coactivators (11,16). However, CRY1 has only moderate affinity (K d ∼1 μM) for the isolated TAD (11,17), suggesting that it makes at least one other interaction with CLOCK:BMAL1 that allows it to serve as a potent repressor when expressed to near stoichiometric levels (18). Previous studies suggest the CLOCK PAS-B domain is important for repression by CRY1 (11,19,20), but evidence for a direct interaction is lacking.…”