Molecular assembly of the period-cryptochrome circadian transcriptional repressor complex.
Bottom Line: Its N-terminal end tucks into CRY adjacent to a large pocket critical for CLOCK-BMAL1 binding, while its C-terminal half flanks the CRY2 C-terminal helix and sterically hinders the recognition of CRY2 by the FBXL3 ubiquitin ligase.Unexpectedly, a strictly conserved intermolecular zinc finger, whose integrity is important for clock rhythmicity, further stabilizes the complex.Our structure-guided analyses show that these interspersed CRY-interacting regions represent multiple functional modules of PERs at the CRY-binding interface.
Affiliation: Department of Pharmacology, University of Washington, Seattle, United States.Show MeSH
Related in: MedlinePlus
Mentions: During the preparation of this manuscript, the complex structure of mammalian CRY1-PHR and PER2-CBD was reported (Schmalen et al., 2014). With high sequence conservation between CRY1 and CRY2, PER2-CBD adopts a similar CRY-binding mode with a tetrahedral coordination of a zinc ion by an intermolecular CCCH zinc-binding motif. The major structural difference lies at the interface of the N-terminal region of PER2-CBD and the CRY secondary pocket. The CRY1-bound PER2-CBD fragment contains a residual fusion-protein sequence, which forms an artifactual ß-hairpin with the first five amino acids of the PER2-CBD (Figure 4—figure supplement 3). In contrast to the PER2-bound CRY2 serine loop, but reminiscent of the Drosophila CRY antenna loop (Zoltowski et al., 2011), the otherwise disordered (Czarna et al., 2013) CRY1 serine loop adopts an inward conformation and occludes the secondary pocket. This conformational difference reveals a substantial degree of structural plasticity, which might be necessary for differential binding and regulation at this site. Interestingly, Schmalen et al. (2014) identified a potential redox sensor involving a disulfide bond near the zinc finger between Cys412 and Cys363, which modulates CRY1-PER2 binding. However, in our circadian reporter assay, we did not detect any difference between the CRY1 wild type and C412A mutant (Figure 5D). More in-depth analyses can now exploit the specific structural differences between the two complexes to explain the non-redundant roles of the two Cryptochrome proteins.
Affiliation: Department of Pharmacology, University of Washington, Seattle, United States.