Conformational changes in the negative arm of the circadian clock correlate with dynamic interactomes involved in post-transcriptional regulation.

Biology is tuned to the Earth's diurnal cycle by the circadian clock, a transcriptional/translational negative feedback loop that regulates physiology via transcriptional activation and other post-transcriptional mechanisms. We hypothesize that circadian post-transcriptional regulation might stem from conformational shifts in the intrinsically disordered proteins that comprise the negative arm of the feedback loop to coordinate variation in negative-arm-centered macromolecular complexes. This work demonstrates temporal conformational fluidity in the negative arm that correlates with 24-h variation in physiologically diverse macromolecular complex components in eukaryotic clock proteins. Short linear motifs on the negative-arm proteins that correspond with the interactors localized to disordered regions and known temporal phosphorylation sites suggesting changes in these macromolecular complexes could be due to conformational changes imparted by the temporal phospho-state. Interactors that oscillate in the macromolecular complexes over circadian time correlate with post-transcriptionally regulated proteins, highlighting how time-of-day variation in the negative-arm protein complexes may tune cellular physiology.

Circadian Interactomics: How Research Into Protein-Protein Interactions Beyond the Core Clock Has Influenced the Model of Circadian Timekeeping.

The circadian clock is the broadly conserved, protein-based, timekeeping mechanism that synchronizes biology to the Earth's 24-h light-dark cycle. Studies of the mechanisms of circadian timekeeping have placed great focus on the role that individual protein-protein interactions play in the creation of the timekeeping loop. However, research has shown that clock proteins most commonly act as part of large macromolecular protein complexes to facilitate circadian control over physiology. The formation of these complexes has led to the large-scale study of the proteins that comprise these complexes, termed here "circadian interactomics." Circadian interactomic studies of the macromolecular protein complexes that comprise the circadian clock have uncovered many basic principles of circadian timekeeping as well as mechanisms of circadian control over cellular physiology. In this review, we examine the wealth of knowledge accumulated using circadian interactomics approaches to investigate the macromolecular complexes of the core circadian clock, including insights into the core mechanisms that impart circadian timing and the clock's regulation of many physiological processes. We examine data acquired from the investigation of the macromolecular complexes centered on both the activating and repressing arm of the circadian clock and from many circadian model organisms.

Characterizing Time-of-Day Conformational Changes in the Intrinsically Disordered Proteins of the Circadian Clock.

Circadian rhythms are 24-h oscillations conserved in nearly all living organisms that allow for the anticipation of daily environmental changes. These rhythms are maintained by a molecular clock comprised of a transcriptional/translational negative feedback loop. Many of the proteins that organize this feedback loop are intrinsically disordered proteins (IDPs), which lack a fixed or ordered three-dimensional structure. Little is known about the impact of intrinsic disorder in clock proteins and this lack of comprehension is compounded by the fact that sophisticated techniques to understand the inherent nature of IDPs are only now emerging. Here, we add to that conversation by describing our novel protocol to track the conformation of a core clock protein (FREQUENCY) in a vital clock model organism (Neurospora crassa). Our protocol, CiRcadian nAtive FasT parallel proteolYsis (CRAFTY), utilizes a parallel proteolysis approach in native conditions to determine the conformational shifts in FREQUENCY over time, providing biologically relevant information and contributing to our understanding of the importance of disorder in the circadian clock.