Architecture of the Mammalian Circadian Repressive Complex

Intricate timing systems have evolved to help organisms in all walks of life organize their physiology to the solar day. Mammalian circadian clocks are driven by a transcription/translation feedback loop composed of positive regulators (CLOCK/BMAL1) and repressors (CRY1/2 and PER1/2). To understand what drives periodicity within this clock, I took structural approaches with the hope of identifying atomic-level details that inform behavioral outputs. Despite high sequence identity, null mutations of Cry1 or Cry2 have divergent effects on periodicity, accelerating and decelerating the clock speed, respectively. To understand the unique roles of CRY1 and CRY2, we used statistical coupling analysis to identify co-evolving residues within the CRY protein family. We identified an evolutionary hotspot, an ancestral secondary cofactor-binding pocket, which has been repurposed for direct interaction with CLOCK and BMAL1. Mutations weakening binding between CLOCK/BMAL1 and CRY1 lead to acceleration of the clock, revealing a novel mode of period regulation in the mammalian clock. Subtle divergence between CRY1 and CRY2 at the secondary pocket underlies differences in affinity for CLOCK/BMAL1. The lower affinity interaction with CRY2 is strengthened by co-expression of PER2, suggesting that PER expression limits the length of the repressive phase in CRY2-driven rhythms. In order to better understand PER's role, we collaborated with another lab to solve and validate a structure of CRY2 bound to a fragment of PER. In so doing, we discovered that interaction between PER and CRY is necessary for rhythmic derepression, providing insight into the role of a key interaction in the molecular clockwork.