CK1a-Mediated Two-Step Subunit Remodeling of the Circadian FRQ-FRH Complex

Abstract The circadian clock of Neurospora operates through a negative feedback loop in which FRQ, along with FRH and CK1a, inhibits its transcriptional activator, WCC, via phosphorylation. CK1a, anchored to FRQ, hyperphosphorylates FRQ at its IDRs 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. 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. Gradual phosphorylation induces with a delay the dissociation of one FRH, exposing a binding site for WCC and activating the complex. Following a time delay, attributable to the slow and stochastic nature of phosphorylation, the release of the second FRH promotes nuclear export and subsequent degradation of FRQ. This stepwise remodeling ensures precise activation and inactivation of FRQ and positions FRH as a hub for decoding temporal phosphorylation information.