Casein kinase 1a mediates a two-step subunit remodeling mechanism to regulate the FRQ-FRH circadian clock complex

The circadian clock of Neurospora operates through a negative feedback loop in which FREQUENCY (FRQ), along with FRQ-interacting RNA helicase (FRH) and casein kinase 1a (CK1a), inhibits its transcriptional activator, WHITE COLLAR COMPLEX (WCC), via phosphorylation. CK1a, anchored to FRQ, hyperphosphorylates FRQ at its intrinsically disordered regions in a slow, temperature-independent manner, forming a module suited for molecular timekeeping. However, the molecular processes triggered by FRQ's hyperphosphorylation have remained unclear. Here we show that FRH, the folded binding partner of disordered FRQ, decodes FRQ's time-dependent phosphorylation state by triggering a two-step remodeling of the FRQ-FRH complex: initially, two FRH molecules bind a FRQ dimer, keeping it inactive by blocking its interaction with WCC. Slow phosphorylation eventually triggers the dissociation of one FRH, thereby activating the complex by exposing a WCC-binding site. Due to the slow and stochastic nature of phosphorylation, the release of the second FRH occurs with a delay, promoting nuclear export and subsequent degradation of FRQ. This ensures precise activation and inactivation of FRQ and positions FRH as a hub for decoding temporal phosphorylation information.