Intrinsic disorder is an essential characteristic of components in the conserved circadian circuit

Abstract

Introduction: The circadian circuit, a roughly 24 h molecular feedback loop, or clock, is conserved from bacteria to animals and allows for enhanced organismal survival by facilitating the anticipation of the day/night cycle. With circadian regulation reportedly impacting as high as 80% of protein coding genes in higher eukaryotes, the protein-based circadian clock broadly regulates physiology and behavior. Due to the extensive interconnection between the clock and other cellular systems, chronic disruption of these molecular rhythms leads to a decrease in organismal fitness as well as an increase of disease rates in humans. Importantly, recent research has demonstrated that proteins comprising the circadian clock network display a significant amount of intrinsic disorder.

Main body: In this work, we focus on the extent of intrinsic disorder in the circadian clock and its potential mechanistic role in circadian timing. We highlight the conservation of disorder by quantifying the extent of computationally-predicted protein disorder in the core clock of the key eukaryotic circadian model organisms Drosophila melanogaster, Neurospora crassa, and Mus musculus. We further examine previously published work, as well as feature novel experimental evidence, demonstrating that the core negative arm circadian period drivers FREQUENCY (Neurospora crassa) and PERIOD-2 (PER2) (Mus musculus), possess biochemical characteristics of intrinsically disordered proteins. Finally, we discuss the potential contributions of the inherent biophysical principals of intrinsically disordered proteins that may explain the vital mechanistic roles they play in the clock to drive their broad evolutionary conservation in circadian timekeeping.

Conclusion: The pervasive conservation of disorder amongst the clock in the crown eukaryotes suggests that disorder is essential for optimal circadian timing from fungi to animals, providing vital homeostatic cellular maintenance and coordinating organismal physiology across phylogenetic kingdoms. Video abstract.

Keywords: Circadian clock; FREQUENCY; Neurospora crassa; Oscillator mechanism; PERIOD-2.

Fig. 1

Conservation of the Core Clock Architecture. a The Transcription-Translation Negative Feedback Loop (TTFL) in fungi and animals comprises two main complexes. The first is a pair of heterodimeric activators known as the positive arm complex (green diamond) and the second, a repressing negative arm complex (red hexagon). The positive arm drives the expression of genes encoding negative arm components leading to the transcription and translation of the negative arm proteins. The negative arm proteins complex with kinases and are post-translationally modified by phosphorylation (shown as yellow stars), enabling negative arm repression of the positive arm. Distinct phospho-states trigger both positive-arm repression (blunted arrow) and negative-arm protein instability (represented by the faded red hexagon). Phosphorylation-related turnover is not necessary to close the loop in fungi, where this has been examined most closely, but instead represents cellular good housekeeping, cleaning up proteins that are no longer useful. When active, the positive arm also promotes the expression of clock-controlled genes (ccgs), which are predicted to control circadian output (reviewed [15]) b In the mammalian clock, positive arm-proteins Brain and Muscle ARNT-Like 1 (BMAL1, also known as ARNTL) and Circadian Locomotor Output Cycles Kaput (CLOCK) form a complex to activate the negative-arm PERIODs (PER1, PER2, and PER3) in addition to CRYPTOCHROMEs (CRY1 and CRY2). PERs and CRYs complex with several kinases to repress CLOCK:BMAL activity [16]. In the Neurospora clock, the positive heterodimer complex is comprised of White Collar-1 (WC-1) and White Collar-2 (WC-2) which together form the White Collar Complex (WCC). WCC drives the expression of the negative arm component FREQUENCY (FRQ), which, with its essential binding partner FREQUENCY-Interacting RNA Helicase (FRH), associates with the kinase Casein Kinase-1 (CK-1a) forming the FRQ-FRH Complex (FFC) [23, 24]. The positive-arm constituents in the Drosophila clock are CYCLE (CYC) and dClk, while the negative arm components are PERIOD (dPER), TIMELESS (TIM) and casein kinase 1, called DOUBLETIME (DBT) in Drosophila [25]. Figure 1b was created using BioRender.com

Fig. 2

Computational Analysis Demonstrates Disorder in the Negative-Arm Constituents. Plots of the disordered propensity scores vs the primary amino acid sequence of the negative-arm components from Murine, Neurospora, and Drosophila clocks. The propensity scores were calculated using four algorithms, the first three from the PONDR family VLXT (red line), VL3-BA (green line) and VSL2 (purple line) and the last from IUPred2A long (orange line). The mean is plotted as the blue line with the SD distribution shaded in light blue [–54]. Any residue scoring 0.5 or higher in the mean calculation was considered toward the percent disorder calculation

Fig. 3

Heat treatment of mPER2 demonstrates that mPER2 has the biochemical characteristics of an IDP. To demonstrate that mPER2 remained soluble after heat stability, murine liver lysates were subjected to heat treatment (∆H: 100 °C for 10 min) with a mock treatment on ice in parallel. Centrifugation was used to separate the soluble fraction from the aggregated proteins. For western blot visualization, 10 μg of total protein was loaded per lane for all treatments, with the Santa Cruz Per-2 H-90 (sc-25,363) antibody used for detection

Fig. 4

Computational Analysis Demonstrates Disorder in the Positive-Arm Constituents. Plots of the disordered propensity scores vs the primary amino acid sequence of the negative-arm components from murine, Neurospora, and Drosophila clocks. The propensity scores were calculated using four algorithms, the first three from the PONDR family VLXT (red line), VL3-BA (green line) and VSL2 (purple line) and the last from IUPred2A long (orange line). The mean is plotted as the blue line with the SD distribution shaded in light blue. Any residue scoring 0.5 or higher in the mean calculation was considered toward the percent disorder calculation