A fuzzy complex in the negative arm regulates circadian clock robustness.

Abstract Organismal physiology is widely regulated by the circadian clock, a molecular circuit composed of a Transcription-Translation Feedback Loop 1,2. Protein components of the molecular clock are enriched in intrinsically disordered regions, inherently flexible regions that interact with other proteins via short linear binding motifs (SLiMs) 3–5. SLiM-driven interactions contribute to circadian timing and the circadian regulation of the cell. However, the mechanism that allows the formation of dynamic clock complexes remains unclear as structural analysis of these protein-protein interactions has been limited due to inherent protein disorder. Here, we apply a synthetic peptide microarray approach to demonstrate that the core clock forms a fuzzy complex to support circadian robustness 6,7. We found positively charged islands on the clock protein FREQUENCY (FRQ) drove a multi-valent interaction between FRQ and its partner FRQ-interacting RNA Helicase (FRH) that enabled clock robustness rather than the previously-reported feedback 8. We found these positively charged islands were a conserved molecular feature throughout clocks in fungi, insects, and mammals, and may enable the formation of fuzzy complexes. This study constitutes the first mechanistic reason for the uniquely-broad conservation of intrinsic disorder in circadian negative-arm proteins and will aid in the development of the molecular model of clock protein interactions. Furthermore, we anticipate the application of synthetic peptide microarrays to study disordered clock proteins and will be useful in characterizing sites of interaction for clock-specific drug discovery 9.