Deciphering the contributions of protein disorder to the repressing complex of the circadian clock

Abstract

Circadian rhythms are biological oscillations that coordinate organismal physiology with the Earth’s 24 hr. light/dark cycle. These rhythms are orchestrated by a core set of activating and repressing proteins termed the “core clock”; with molecular architecture conserved from lower to higher eukaryotes. Recent evidence has emerged implicating a complex and unexplored layer of clock controlled post-transcriptional regulation. Another unexplained evolutionarily con-served feature found throughout core clock proteins are intrinsically disordered regions (IDRs), which lack a fixed 3D structure. IDRs can be thought of as possessing sequence-encoded “con-formational states” - collections of structures that have certain biases. IDR conformational state (and hence function) can be tuned by binding partners, post-translational modifications, and their environment. Therefore, we hypothesized that circadian post-transcriptional regulation and envi-ronmental responsiveness of the clock stems from conserved IDRs that function as modules for tunable molecular function. Using three classical circadian model organisms, Neurospora crassa, Drosophila mela-nogaster, and Homo sapiens, we assessed how IDR-dependent interactions influence the clock and found temporal changes in physiologically diverse macromolecular complex components correlated with changes in the conformational state of the IDRs. Our newly developed assay to assess the conformational state of FREQUENCY (FRQ), the key protein in the repressing com-plex of the fungi Neurospora crassa, was followed with co-immunoprecipitation and mass spec-trometry of FRQ over the circadian day. This approach was mirrored by examining the functional analog proteins in Drosophila and humans. As the repressing complexes interacted with several post-transcriptionally regulated proteins, it suggests the repressors tune cellular physiology and proteostasis via changes in the circadian repressor complexes. To explore the relationship of the repressing arm proteins, combined in silico, in vitro, and in vivo methodologies revealed that formation of a “fuzzy” complex is essential for clock timing and robustness, supporting a model in which dynamic protein-protein interactions driven by IDRs underlie tunable clock function. The conformational state of IDRs is inherently sensitive to changes in temperature, offering a potential biophysical link for the poorly understood observa-tion that circadian clocks are temperature compensated. Intriguingly, FRQ (and its higher eukar-yotic functional analogs) possess multiple isoforms, the relative expression of which are gov-erned by temperature-dependent alternative splicing. FRQ has two known isoforms that differ in their N-terminal IDRs and are known to play a role in temperature response and clock period homeostasis. We found that the temperature regulated repressing isoforms of the Neurospora core clock differ in their biochemical properties and have a differential interactome, suggesting that this IDR may function as a temperature-dependent rheostat for the repressive complex. Taken together, this work offers direct insight into the mechanistic role for protein disorder in regulation of the circadian clock as a function of time, partners, and environment.