Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

Circadian clocks are composed from molecular oscillators that pace rhythms of gene expression to the diurnal cycle. Therein, transcriptional-translational negative feedback loops (TTFLs) generate oscillating levels of transcriptional repressor proteins that regulate their own gene expression. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and Casein-Kinase I (CK1) form the FFC complex that acts to repress expression of clock-controlled genes activated by the White-Collar complex (WCC). A key question concerns how the FRQ protein orchestrates molecular interactions at the core of the clock despite containing little tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC complex in unphosphorylated and highly phosphorylated states. An integrated structural and computational biology approach incorporating site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on FRQ and the FFC. FRQ, although substantially disordered, contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. Phosphorylation of FRQ increases conformational flexibility and alters oligomeric state but the resulting changes in structure and dynamics are non-uniform. FRQ undergoes liquid-liquid phase separation (LLPS) in a phosphorylation dependent manner to sequester FRH and CK1 and influence CK1 enzymatic activity. Live imaging of Neurospora hyphae reveals FRQ foci near the nuclear periphery characteristic of condensates. Analog clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino-acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic circadian clocks. Significance The central transcriptional repressor proteins of circadian clocks are often highly disordered, undergo extensive post-translational modification and have poorly defined structural properties. The fungal repressor protein Frequency (FRQ) nucleates a semi-ordered molecular assembly that despite conformational heterogeneity arranges its components in close proximity and facilitates their sequestration and enzymatic regulation through post-translational modification and liquid-liquid phase separation. Analogous properties are predicted by the similar amino acid compositions of clock proteins in insects and mammals.