Genetically encoded biological clocks are found broadly throughout eukaryotes and in cyanobacteria, where they generate circadian (about a day) rhythms that allow organisms to anticipate regular ...environmental changes and align their physiology and behavior with Earth's daily light/dark cycle. In recent years, many have sought to expand our biochemical and structural understanding of the clock proteins that constitute the molecular “cogs” of these biological clocks. These new studies are beginning to reveal how macromolecular assemblies of dedicated clock proteins form and evolve to contribute to the generation of clocks that function over the timescale of a day. This review will highlight structural and biochemical studies that provide important insight into the molecular mechanisms of cyanobacterial and vertebrate animal clocks. Collectively, these studies demonstrate emerging biochemical properties that appear to be shared by these different clocks, suggesting that there may be some conservation in the regulation and assembly of circadian macromolecular assemblies.
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•Most clocks rely on transcriptional cycles, but some can occur posttranslationally.•Some biochemical principles may be shared by all clocks.•Clocks rely on the remodeling of core clock protein complexes throughout the day.•Slow biochemical or enzymatic steps are crucial for timekeeping in all clock types.•Clocks depend on competition for mutually exclusive binding at key interfaces.
Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use ...of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.
Highlights • The circadian clock generates molecular rhythms with 24 h periodicity. • Circadian control of physiology is distributed to peripheral tissues. • 24 h timing arises from the ordered ...recruitment of clock proteins to promoters. • Many mechanisms are used to generate rhythmic output from the core molecular clock. • Clocks integrate with systemic cues to give flexibility to circadian physiology.
Mammalian circadian rhythms drive ∼24 h periodicity in a wide range of cellular processes, temporally coordinating physiology and behaviour within an organism, and synchronising this with the ...external day-night cycle. The canonical model for this timekeeping consists of a delayed negative-feedback loop, containing transcriptional activator complex CLOCK-BMAL1 (BMAL1 is also known as ARNTL) and repressors period 1, 2 and 3 (PER1, PER2 and PER3) and cryptochrome 1 and 2 (CRY1 and CRY2), along with a number of accessory factors. Although the broad strokes of this system are defined, the exact molecular mechanisms by which these proteins generate a self-sustained rhythm with such periodicity and fidelity remains a topic of much research. Recent studies have identified prominent roles for a number of crucial post-transcriptional, translational and, particularly, post-translational events within the mammalian circadian oscillator, providing an increasingly complex understanding of the activities and interactions of the core clock proteins. In this Review, we highlight such contemporary work on non-transcriptional events and set it within our current understanding of cellular circadian timekeeping.
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•Circadian rhythms are generated by periodic oscillations in clock genes within a negative feedback loop.•PAS domains represent the most abundant well-defined structured regions in ...clock proteins.•PAS domains mediate homodimerization and heterodimerization of clock proteins.•Mutations in the PAS-PAS interface alter or disrupt circadian rhythms.
Circadian rhythms are genetically encoded molecular clocks for internal biological timekeeping. Organisms from single-cell bacteria to humans use these clocks to adapt to the external environment and synchronize their physiology and behavior to solar light/dark cycles. Although the proteins that constitute the molecular ‘cogs’ and give rise to circadian rhythms are now known, we still lack a detailed understanding of how these proteins interact to generate and sustain the ∼24-hour circadian clock. Structural studies have helped to expand the architecture of clock proteins and have revealed the abundance of the only well-defined structured regions in the mammalian clock called Per-ARNT-Sim (PAS) domains. PAS domains are modular, evolutionarily conserved sensory and signaling domains that typically mediate protein–protein interactions. In the mammalian circadian clock, PAS domains modulate homo and heterodimerization of several core clock proteins that assemble into transcription factors or repressors. This review will focus on the functional importance of the PAS domains in the circadian clock from a biophysical and biochemical standpoint and describe their roles in clock protein interactions and circadian timekeeping.
Mammalian circadian timekeeping arises from a transcription-based feedback loop driven by a set of dedicated clock proteins. At its core, the heterodimeric transcription factor CLOCK:BMAL1 activates ...expression of Period, Cryptochrome, and Rev-Erb genes, which feed back to repress transcription and create oscillations in gene expression that confer circadian timing cues to cellular processes. The formation of different clock protein complexes throughout this transcriptional cycle helps to establish the intrinsic ∼24 h periodicity of the clock; however, current models of circadian timekeeping lack the explanatory power to fully describe this process. Recent studies confirm the presence of at least three distinct regulatory complexes: a transcriptionally active state comprising the CLOCK:BMAL1 heterodimer with its coactivator CBP/p300, an early repressive state containing PER:CRY complexes, and a late repressive state marked by a poised but inactive, DNA-bound CLOCK:BMAL1:CRY1 complex. In this review, we analyze high-resolution structures of core circadian transcriptional regulators and integrate biochemical data to suggest how remodeling of clock protein complexes may be achieved throughout the 24 h cycle. Defining these detailed mechanisms will provide a foundation for understanding the molecular basis of circadian timing and help to establish new platforms for the discovery of therapeutics to manipulate the clock.
Circadian rhythms enable cells and organisms to coordinate their physiology with the cyclic environmental changes that come as a result of Earth's light/dark cycles. Cyanobacteria make use of a ...post-translational oscillator to maintain circadian rhythms, and this elegant system has become an important model for circadian timekeeping mechanisms. Composed of three proteins, the KaiABC system undergoes an oscillatory biochemical cycle that provides timing cues to achieve a 24-h molecular clock. Together with the input/output proteins SasA, CikA, and RpaA, these six gene products account for the timekeeping, entrainment, and output signaling functions in cyanobacterial circadian rhythms. This Minireview summarizes the current structural, functional and mechanistic insights into the cyanobacterial circadian clock.
In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how ...inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.
Human body-surface epithelia coexist in close association with complex bacterial communities and are protected by a variety of antibacterial proteins. C-type lectins of the RegIII family are ...bactericidal proteins that limit direct contact between bacteria and the intestinal epithelium and thus promote tolerance to the intestinal microbiota. RegIII lectins recognize their bacterial targets by binding peptidoglycan carbohydrate, but the mechanism by which they kill bacteria is unknown. Here we elucidate the mechanistic basis for RegIII bactericidal activity. We show that human RegIIIα (also known as HIP/PAP) binds membrane phospholipids and kills bacteria by forming a hexameric membrane-permeabilizing oligomeric pore. We derive a three-dimensional model of the RegIIIα pore by docking the RegIIIα crystal structure into a cryo-electron microscopic map of the pore complex, and show that the model accords with experimentally determined properties of the pore. Lipopolysaccharide inhibits RegIIIα pore-forming activity, explaining why RegIIIα is bactericidal for Gram-positive but not Gram-negative bacteria. Our findings identify C-type lectins as mediators of membrane attack in the mucosal immune system, and provide detailed insight into an antibacterial mechanism that promotes mutualism with the resident microbiota.
Cryptochromes are evolutionarily related to the light‐dependent DNA repair enzyme photolyase, serving as major regulators of circadian rhythms in insects and vertebrate animals. There are two types ...of cryptochromes in the animal kingdom: Drosophila‐like CRYs that act as nonvisual photopigments linking circadian rhythms to the environmental light/dark cycle, and vertebrate‐like CRYs that do not appear to sense light directly, but control the generation of circadian rhythms by acting as transcriptional repressors. Some animals have both types of CRYs, while others possess only one. Cryptochromes have two domains, the photolyase homology region (PHR) and an extended, intrinsically disordered C‐terminus. While all animal CRYs share a high degree of sequence and structural homology in their PHR domains, the C‐termini are divergent in both length and sequence identity. Recently, cryptochrome function has been shown to extend beyond its pivotal role in circadian clocks, participating in regulation of the DNA damage response, cancer progression and glucocorticoid signaling, as well as being implicated as possible magnetoreceptors. In this review, we provide a historical perspective on the discovery of animal cryptochromes, examine similarities and differences of the two types of animal cryptochromes and explore some of the divergent roles for this class of proteins.
Structurally similar to photolyase, cryptochromes regulate circadian rhythms in animals. Left, light absorption leads to a conformational change in Drosophila CRY (dCRY) that regulates its interaction with TIM for circadian photoentrainment. Mouse cryptochromes (mCRY) repress CLOCK:BMAL1 independently of light to control the circadian transcription–translation feedback loop. Right, CRYs have also been proposed to act as light‐dependent magnetoreceptors.
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