Abstract
Circadian rhythms in mammals are tightly regulated through phosphorylation of period (PER) proteins by casein kinase 1 (CK1, subtypes δ and ε). CK1 acts on at least two different regions of PER with opposing effects: phosphorylation of phosphodegron regions leads to PER degradation, whereas phosphorylation of the familial advanced sleep phase (FASP) region leads to PER stabilization. To investigate how substrate selectivity is encoded by the conformational dynamics of CK1, we performed a large set of independent molecular dynamics simulations of wild-type CK1 and the tau mutant (R178C) that biases kinase activity toward a phosphodegron. We used Markovian state models to integrate the simulations into a single model of the conformational landscape of CK1 and used Gaussian accelerated molecular dynamics to build the first molecular model of CK1 and the unphosphorylated FASP motif. These findings were biochemically validated using in vitro kinase assays and provide a mechanistic view of CK1, establishing how the activation loop acts as a key molecular switch to control substrate selectivity. We show that the wild-type CK1 prefers a "loop down" conformation that binds FASP, whereas the tau mutant favors an alternative conformation of the activation loop and significantly accelerates the dynamics of CK1. This reshapes the binding cleft in a way that impairs FASP binding and would ultimately lead to PER destabilization. Finally, we identified a potential binding pocket that could be targeted to influence the conformational state of this molecular switch and lead to predictable changes in circadian period. Our integrated approach offers a detailed model of CK1's conformational landscape and its relevance to normal, mutant, and druggable circadian timekeeping.