Determining the molecular details of the PER2-CK1 delta interaction in the mammalian circadian clock

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Project summary Circadian rhythms govern most behavioral and physiological processes to coincide with the 24-hour solar day. Disruption of circadian rhythms leads to increased risk for depression, cardiovascular disease, and cancer. The protein Period 2 (PER2) sits at the center of the mammalian molecular clock, and the levels of PER2 are tightly regulated through phosphorylation by casein kinase 1 delta (CK1). Mutation of a phosphorylation site destabilizes PER2, shortens circadian period, and leads to familial advanced sleep phase (FASP) syndrome in humans. Additionally, phosphorylation of PER2 here by CK1 may underlie temperature compensation, which allows the clock to generate ~24-hour timing independently of temperature. The molecular details of how CK1 interacts with PER2 to promote this stabilizing phosphorylation and how this interaction contributes to temperature compensation are poorly understood. The ​long-term objective​ of my research is to understand how CK1 phosphorylation of PER2 regulates the mammalian circadian clock. This proposal focuses on determining how CK1 interacts with PER2 to form a stable, stoichiometric complex and how it contributes to temperature compensation. My ​central​ ​hypothesis​ is that temperature compensation in the mammalian circadian clock is achieved via changes in dynamics in CK1 that influence its ability to form a stable complex with PER2 and/or phosphorylate the FASP region. The first aim of this project is to determine the molecular details of the PER2- CK1 interaction that contribute to complex formation. Using biophysical techniques, I will structurally characterize the complex and the binding of a new small molecule inhibitor that disrupts the PER2- CK1 interaction. The second aim of this project is to determine how altered protein dynamics contribute to temperature compensation in the clock. The dynamics may be occurring on several timescales, which makes it an appropriate target to study using solution NMR spectroscopy. This work outlined in this proposal will define how PER2 and CK1 form a stable complex and determine the molecular origins of how regulation of this complex is impacted by temperature. This project will provide me with training in cell culture and small molecule inhibitor characterization, while also building on skills in NMR initially developed during graduate school to better prepare me for a career as an independent scientist. The interaction between CK1 and PER2 is crucial to a properly functioning clock, so identifying the details of this regulatory mechanism has the potential to inform new therapeutic strategies to treat people with circadian rhythms disrupted by genetic disorders, jet lag, and shift work.