Allosteric regulation and functional dynamics of CRY1 in the mammalian circadian clock: Insights from mutational and small molecule interactions

Cryptochromes (CRYs) are essential components of the molecular clock that governs circadian rhythms in mammals, functioning as inhibitors of BMAL1/CLOCK-driven transcription. While mammals possess two CRYs with distinct roles in the circadian clock, the mechanisms underlying their differential functions remain unclear. This study elucidates how a specific mutation in CRY1 allosterically alters its dynamic behavior. Molecular dynamics simulations were conducted on eight CRY1 mutants experimentally shown to exhibit reduced repressor activity. My findings reveal that mutations in CRY1 affect the dynamic behavior of the serine loop and the accessibility of the secondary pocket, changes not observed in CRY2. Further analysis highlights that the flexibility of the serine loop influences secondary pocket volume, with S44 and S45 playing crucial roles through their interactions with E382. Additionally, I explored the potential for small molecules to modulate CRY1 allosterically, demonstrating both in silico and in vitro that this regulation impacts CRY1's affinity to CLOCK. This discovery provides a promising avenue for developing therapeutics targeting CRY1, though it also raises concerns about unforeseen side effects of existing small molecules. To address this, virtual screening was performed to identify compounds targeting the CRY1 secondary pocket. Subsequent phenotypic analysis revealed five distinct effects on circadian rhythm, underscoring the complexity of this regulatory mechanism. This study offers critical insights into CRY1 dynamics, its interaction with CLOCK, and the potential for small-molecule modulation, paving the way for advancements in circadian biology and drug design.