Casein Kinase 1δ and PERIOD2 regulate circadian rhythms through a combination of substrate selectivity and feedback inhibition

Biological clocks such as circadian rhythms are perhaps among the most fundamentally conserved adaptations of organisms that have evolved under the light/dark cycle of earth. These rhythms confer an advantage to organisms by allowing them to anticipate daily environmental changes. While the genetic networks that govern circadian rhythms in mammals are now fairly well-established, a picture of the molecular mechanisms that regulate the intrinsic timing of circadian rhythms is only recently beginning to emerge. In this dissertation, I discuss the molecular level details of the mammalian clock and provide new insights that shed light on the biochemical mechanisms of period control within.Chapter 2 describes how Casein Kinase 1δ and ε (CK1) post- translationally control PERIOD (PER) stability. CK1 is a deeply conserved circadian protein, yet little is known about its regulation of functionally antagonistic sites in PER that control circadian timing. The balance of CK1 activity within these two regions defines a model of PER2 stability known as the phosphoswitch. In this chapter, we discover an allosteric conformational switch in the CK1 activation loop segment that influences substrate specificity on PER2 to directly regulate its stability. We further show that period-altering mutations of the kinase across organisms differentially modulate the activation loop switch and provide a framework to understand and manipulate CK1 regulation of circadian period. PER proteins are fundamental in defining the phase and timing of circadian rhythms, likely due to their role as stoichiometrically limiting factors in the assembly of repressive complexes that provide feedback inhibition of transcription within the clock. CK1-dependent changes in PER abundance are therefore central to circadian timing. CK1 phosphorylation of PER2 is mediated by the stable anchoring of CK1 to PER2 via the Casein Kinase 1 Binding Domain (CK1BD). This stable interaction is also required for CK1-mediated displacement of CLOCK from DNA. Chapter 3 describes the role of CK1 phosphorylation of the PER2 FASP region in the regulation of PER2 stability and repressive activity. We show that the phosphorylated FASP region (pFASP) directly interacts with and inhibits CK1δ, and that stable anchoring to the CK1BD increases the kinetics of FASP phosphorylation and product inhibition. We solve multiple crystal structures of CK1δ bound to pFASP and conduct accelerated molecular dynamics simulations to reveal a mechanism of inhibition where phosphoserines in pFASP anchor into conserved anion binding sites along the substrate binding cleft and active site of the kinase. We further show how limiting phosphorylation within the FASP region reduces product inhibition and find that feedback inhibition is a conserved mechanism within Drosophila PER. Much of the work in this dissertation focuses on the molecular determinants that regulate the stabilizing arm of the PER2 phosphoswitch. Chapter 4 discusses the molecular features of PER2 degradation and provides a survey of the current state of my contributions to this area. I provide a framework for extending previous studies using reagents from mPER2 into hPER2, and further discuss future directions to shed light on mechanisms of regulation for CK1 activity within the PER2 Degron and the recruitment of b- TrCP. In summary, throughout this dissertation I have used an integrative approach of utilizing biochemistry, biophysics, molecular dynamics, and tissue culture to describe how CK1 and PER2 form a critical regulatory nexus within the mammalian circadian clock. In addition to the findings discussed herein, this work has provided a framework for targeted mutations to further develop a molecular level understanding of circadian timekeeping, as well as an avenue to develop novel therapeutics to target the clock and modulate circadian period