Structure and Function of the Mammalian Circadian Clock

Abstract

Circadian clocks are essential in all kingdoms of life to maintain appropriate synchronization with their environment. Robust, 24-hr rhythms in eukaryotes are generated by a transcription-translation negative feedback loop. Proper oscillation of the mammalian clock depends on the complex formation and periodic turnover of the Period (PER) and Cryptochrome (CRY) proteins, which together inhibit their own transcriptional activator complex, CLOCK-BMAL1. While these four proteins form the core of the clock, post-translational modifications are essential for generating the oscillations. Phosphorylation and ubiquitination are necessary for localization, complex formation, and degradation of each component. Ubiquitin ligase FBXL3 is required for the nuclear degradation of CRY and competes with both PER and a novel clock-modulating compound KL001 for CRY binding. We present the mechanism of action of this small molecule showing that it occupies the same pocket as FBXL3 thereby stabilizing CRY and lengthening the circadian period. We also report structural and functional evidence for the diverse roles of PERs with respect to CRYs. Briefly, the PER CRY-binding domain adopts a highly-extended conformation, embracing CRY2 with a sinuous binding mode. Unexpectedly, a strictly-conserved intermolecular zinc finger, whose integrity is important for rhythmicity, stabilizes PER-CRY. This work has provided the foundation to explore the assemblies of the core clock machinery. Preliminary progress has been made in establishing the CRY-CLOCK-BMAL1 repression complex.