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

Abstract

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.

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation

“…Previous studies of MBD2 and MBD3 have characterized truncated forms of MBD2 and MBD3, but still suffered from low yields due to degradation and aggregation (30,31,56). We have also encountered extensive protein degradation but have adapted a protocol effective for intrinsically disordered proteins and optimized it for MBD2 and MBD3 (32,57). As a result, we have obtained large amounts of protein suitable for phase separation studies (See Expression and purification of MBD2 and MBD3 and their respective truncations under Materials & Methods).…”

“…Their intrinsic disorder has posed challenges for biophysical studies thus far, primarily due to poor protein yields. However, we have optimized expression and purification methods tailored specifically for intrinsically disordered proteins (IDPs), enabling us to produce sufficient quantities of full-length (FL) protein for in vitro characterization (32). Determining how MBD2 and MBD3 are involved in condensate formation will be important for understanding how aberrations in their ability to interact with themselves and other binding partners lead to dysregulation in condensate dynamics and, consequently, their roles in heterochromatin formation and transcriptional repression.…”