Here, Molecular Cell talks to first author Jonathan Philpott and co-corresponding author Carrie Partch about their paper, “PERIOD phosphorylation leads to feedback inhibition of CK1 activity to ...control circadian period” (in this issue of Molecular Cell) and their scientific journeys until now.
Circadian clocks control gene expression to provide an internal representation of local time. We report reconstitution of a complete cyanobacterial circadian clock in vitro, including the central ...oscillator, signal transduction pathways, downstream transcription factor, and promoter DNA. The entire system oscillates autonomously and remains phase coherent for many days with a fluorescence-based readout that enables real-time observation of each component simultaneously without user intervention. We identified the molecular basis for loss of cycling in an arrhythmic mutant and explored fundamental mechanisms of timekeeping in the cyanobacterial clock. We find that SasA, a circadian sensor histidine kinase associated with clock output, engages directly with KaiB on the KaiC hexamer to regulate period and amplitude of the central oscillator. SasA uses structural mimicry to cooperatively recruit the rare, fold-switched conformation of KaiB to the KaiC hexamer to form the nighttime repressive complex and enhance rhythmicity of the oscillator, particularly under limiting concentrations of KaiB. Thus, the expanded in vitro clock reveals previously unknown mechanisms by which the circadian system of cyanobacteria maintains the pace and rhythmicity under variable protein concentrations.
The inner workings of an ancient biological clock Fang, Mingxu; LiWang, Andy; Golden, Susan S. ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
March 2024, 2024-03-00, 20240301, Volume:
49, Issue:
3
Journal Article
Peer reviewed
An in vitro circadian clock can be assembled from purified components to study clock input, timekeeping, and output in cyanobacteria.Phosphorylation and nucleotide binding are linked to allosteric ...regulation of clock protein interactions on the ATPase KaiC.Proteins that signal clock input and output also participate directly in timekeeping.Changes to the Kai protein network can convert a self-sustaining circadian clock to an hourglass timer that depends on regular environmental changes to keep time.
Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth’s rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.
Circadian clocks evolved in diverse organisms as an adaptation to the daily swings in ambient light and temperature that derive from Earth’s rotation. These timing systems, based on intracellular molecular oscillations, synchronize organisms' behavior and physiology with the 24-h environmental rhythm. The cyanobacterial clock serves as a special model for understanding circadian rhythms because it can be fully reconstituted in vitro. This review summarizes recent advances that leverage new biochemical, biophysical, and mathematical approaches to shed light on the molecular mechanisms of cyanobacterial Kai proteins that support the clock, and their homologues in other bacteria. Many questions remain in circadian biology, and the tools developed for the Kai system will bring us closer to the answers.
Cryptochrome (CRY) proteins play an essential role in regulating mammalian circadian rhythms. CRY is composed of a structured N-terminal domain known as the photolyase homology region (PHR), which is ...tethered to an intrinsically disordered C-terminal tail. The PHR domain is a critical hub for binding other circadian clock components such as CLOCK, BMAL1, PERIOD, or the ubiquitin ligases FBXL3 and FBXL21. While the isolated PHR domain is necessary and sufficient to generate circadian rhythms, removing or modifying the cryptochrome tails modulates the amplitude and/or periodicity of circadian rhythms, suggesting that they play important regulatory roles in the molecular circadian clock. In this commentary, we will discuss how recent studies of these intrinsically disordered tails are helping to establish a general and evolutionarily conserved model for CRY function, where the function of PHR domains is modulated by reversible interactions with their intrinsically disordered tails. Video abstract.
The C-terminal transactivation domain (TAD) of BMAL1 (brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and, consequently, ...contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Toward this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.
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•The highly flexible BMAL1 TAD possesses a slow binary conformational switch•The switch is generated by cis/trans isomerization about a Trp-Pro imide bond•Conformationally locking the switch into trans isomer shortens circadian period•Cyclophilins accelerate isomerization to modulate circadian period
Gustafson et al. identify a cis/trans isomerization switch in the BMAL1 transactivation domain that controls circadian rhythms. Conformationally locking the switch or inhibiting prolyl isomerases influences circadian period to suggest that slow protein dynamics play a role in timekeeping.
Circadian rhythms are generated by interlocked transcription–translation feedback loops that establish cell-autonomous biological timing of ∼24 h. Mutations in core clock genes that alter their ...stability or affinity for one another lead to changes in circadian period. The human CRY1Δ11 mutant lengthens circadian period to cause delayed sleep phase disorder (DSPD), characterized by a very late onset of sleep. CRY1 is a repressor that binds to the transcription factor CLOCK:BMAL1 to inhibit its activity and close the core feedback loop. We previously showed how the PHR (photolyase homology region) domain of CRY1 interacts with distinct sites on CLOCK and BMAL1 to sequester the transactivation domain from coactivators. However, the Δ11 variant alters an intrinsically disordered tail in CRY1 downstream of the PHR. We show here that the CRY1 tail, and in particular the region encoded by exon 11, modulates the affinity of the PHR domain for CLOCK:BMAL1. The PHR-binding epitope in exon 11 is necessary and sufficient to disrupt the interaction between CRY1 and the subunit CLOCK. Moreover, PHR–tail interactions are conserved in the paralog CRY2 and reduced when either CRY is bound to the circadian corepressor PERIOD2. Discovery of this autoregulatory role for the mammalian CRY1 tail and conservation of PHR–tail interactions in both mammalian cryptochromes highlights functional conservation with plant and insect cryptochromes, which also utilize PHR–tail interactions to reversibly control their activity.
Dimerization Rules of Mammalian PAS Proteins Rojas, Brenda L.; Vazquez-Rivera, Emmanuel; Partch, Carrie L. ...
Journal of molecular biology,
02/2024, Volume:
436, Issue:
3
Journal Article
Peer reviewed
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•We attempt to synthesize work from multiple laboratories to devise common rules for PAS domain pairing in the mammalian system. We conclude that all alpha–beta partnerships employ ...common mechanisms of dimerization, and that PASA-PASA interactions are distinct from PASB-PASB interactions.
The PAS (PER, ARNT, SIM) protein family plays a vital role in mammalian biology and human disease. This analysis arose from an interest in the signaling mechanics by the Ah receptor (AHR) and the Ah receptor nuclear translocator (ARNT). After more than fifty years by studying this and related mammalian sensor systems, describing the role of PAS domains in signal transduction is still challenging. In this perspective, we attempt to interpret recent studies of mammalian PAS protein structure and consider how this new insight might explain how these domains are employed in human signal transduction with an eye towards developing strategies to target and engineer these molecules for a new generation of therapeutics. Our approach is to integrate our understanding of PAS protein history, cell biology, and molecular biology with recent structural discoveries to help explain the mechanics of mammalian PAS protein signaling. As a learning set, we focus on sequences and crystal structures of mammalian PAS protein dimers that can be visualized using readily available software.
Multisite phosphorylation of the PERIOD 2 (PER2) protein is the key step that determines the period of the mammalian circadian clock. Previous studies concluded that an unidentified kinase is ...required to prime PER2 for subsequent phosphorylation by casein kinase 1 (CK1), an essential clock component that is conserved from algae to humans. These subsequent phosphorylations stabilize PER2, delay its degradation, and lengthen the period of the circadian clock. Here, we perform a comprehensive biochemical and biophysical analysis of mouse PER2 (mPER2) priming phosphorylation and demonstrate, surprisingly, that CK1δ/ε is indeed the priming kinase. We find that both CK1ε and a recently characterized CK1δ2 splice variant more efficiently prime mPER2 for downstream phosphorylation in cells than the well-studied splice variant CK1δ1. While CK1 phosphorylation of PER2 was previously shown to be robust to changes in the cellular environment, our phosphoswitch mathematical model of circadian rhythms shows that the CK1 carboxyl-terminal tail can allow the period of the clock to be sensitive to cellular signaling. These studies implicate the extreme carboxyl terminus of CK1 as a key regulator of circadian timing.
Assembly and function of bHLH–PAS complexes Fribourgh, Jennifer L.; Partch, Carrie L.
Proceedings of the National Academy of Sciences - PNAS,
05/2017, Volume:
114, Issue:
21
Journal Article
RegIII proteins are secreted C-type lectins that kill Gram-positive bacteria and play a vital role in antimicrobial protection of the mammalian gut. RegIII proteins bind their bacterial targets via ...interactions with cell wall peptidoglycan but lack the canonical sequences that support calcium-dependent carbohydrate binding in other C-type lectins. Here, we use NMR spectroscopy to determine the molecular basis for peptidoglycan recognition by HIP/PAP, a human RegIII lectin. We show that HIP/PAP recognizes the peptidoglycan carbohydrate backbone in a calcium-independent manner via a conserved "EPN" motif that is critical for bacterial killing. While EPN sequences govern calcium-dependent carbohydrate recognition in other C-type lectins, the unusual location and calcium-independent functionality of the HIP/PAP EPN motif suggest that this sequence is a versatile functional module that can support both calcium-dependent and calcium-independent carbohydrate binding. Further, we show HIP/PAP binding affinity for carbohydrate ligands depends on carbohydrate chain length, supporting a binding model in which HIP/PAP molecules "bind and jump" along the extended polysaccharide chains of peptidoglycan, reducing dissociation rates and increasing binding affinity. We propose that dynamic recognition of highly clustered carbohydrate epitopes in native peptidoglycan is an essential mechanism governing high-affinity interactions between HIP/PAP and the bacterial cell wall.
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