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 represses expression of genes activated by the White-Collar complex (WCC). A key question concerns how FRQ orchestrates molecular interactions at the core of the clock despite containing little predicted tertiary structure. We present the reconstitution and biophysical characterization of FRQ and the FFC in unphosphorylated and highly phosphorylated states. Site-specific spin labeling and pulse-dipolar ESR spectroscopy provides domain-specific structural details on the full-length, 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous 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.…”

Allosteric regulation of kinase activity

“…Many of the open questions about the allosteric regulation of kinase activity concern the influence of intrinsic disorder, phase separation, and clustering. For example, Venkat et al report on the role of intrinsically disordered regions in a process called isoform-specific ‘supercharging’ in the DCLK family of kinases ( Venkat et al, 2023 ), and Tariq et al highlight the role of disorder and phase separation in controlling the phosphorylation events that organize the circadian clock in a fungal system ( Tariq et al, 2024 ). Elsewhere, in a review article, Gormal et al discuss the mechanisms responsible for kinase clustering, and how this phenomenon can drive various pathologies ( Gormal et al, 2023 ).…”