Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing

Fribourgh, Jennifer L.; Srivastava, Ashutosh; Sandate, Colby R.; Michael, Alicia K.; Hsu, Peter L.; Rakers, Christin; Nguyen, Leslee T.; Torgrimson, Megan R.; Parico, Gian Carlo G; Tripathi, Sarvind; Zheng, Ning; Lander, Gabriel C.; Hirota, Tsuyoshi; Tama, Florence; Partch, Carrie L.

doi:10.7554/elife.55275

Cited by 57 publications

(79 citation statements)

References 71 publications

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“…To understand the dramatic change in the function of CRY1 upon Arg293 to His293 mutation and to assess the role of Arg293 in CRY1, we employed molecular dynamics simulations. Computational analysis suggested that Arg293 might be a critical residue for the regulation of the serine loop in CRY1 which is known to be important for CLOCK binding to the secondary pocket (38). Replacing Arg293 with His changes a highly dynamic serine loop into a less dynamic state and closes the gate of the secondary pocket ( Figure 5B-E), which may be responsible for the reduced binding of CLOCK to the mutant CRY1.…”

Section: Discussionmentioning

confidence: 99%

“…This interaction allows the serine loop, located adjacent to the secondary pocket, to adapt different conformations. A recent study indicates that PER2 remodels the serine loop of CRY2 and, in turn, enhances its affinity towards BMAL1/CLOCK (38). In the same study, replacement of amino acid residues in the serine loop of CRY1 reduces its affinity to BMAL1/CLOCK.…”

mentioning

confidence: 80%

“…The secondary pocket of CRY1 is critical for its interaction with CLOCK. Subtle changes in this pocket can result in substantial differences in periodicity in cycling cells (28,38). For example, differential rhythms driven by CRY1 and CRY2 are the result of these changes (23).…”

Section: Parg293his Cry1 Variant Exhibits Similar Characteristics Tomentioning

confidence: 99%

“…To investigate this, we employed molecular dynamics (MD) simulations using CRY1 structural data. Recently, it has been shown that CRYs bind to the CLOCK PASB domain through the secondary pocket, and this is regulated by the serine loop (38). We, therefore, explored possible dynamical differences between WT CRY1 and p.Arg293His CRY1 around the secondary pocket via MD simulations.…”

Section: Understanding the Role Of Arg293 In Cry1 Functionmentioning

confidence: 99%

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The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Gül

Aydın

Ozcan

et al. 2020

Journal of Biological Chemistry

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Mammalian circadian clocks are driven by transcription/translation feedback loops composed of positive transcriptional activators (BMAL1 and CLOCK) and negative repressors (CRYPTOCHROMEs (CRYs) and PERIODs (PERs)). CRYs, in complex with PERs, bind to the BMAL1/CLOCK complex and repress E-box driven transcription of clock associated genes. There are two individual CRYs, with CRY1 exhibiting higher affinity to the BMAL1/CLOCK complex than CRY2. It is known that this differential binding is regulated by a dynamic serine-rich loop adjacent to the secondary pocket of both CRYs, but the underlying features controlling loop dynamics are not known. Here we report that allosteric regulation of the serine-rich loop is mediated by Arg293 of CRY1, identified as a rare CRY1 SNPs in the Ensembl and 1000 Genomes databases. The p.Arg293His CRY1 variant caused a shortened circadian period in a Cry1-/-Cry2-/-double knockout mouse embryonic fibroblast cell line. Moreover, the variant displayed reduced repressor activity on BMAL1/CLOCK driven transcription, which is explained by reduced affinity to BMAL1/CLOCK in the absence of PER2 compared to wild type CRY1. Molecular dynamics simulations revealed that the p.Arg293His CRY1 variant altered a communication pathway between Arg293 and the serine loop, by reducing its dynamicity. Collectively, this study provides direct evidence that allosterism in CRY1 is critical for the regulation of circadian rhythm.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 80%

Section: Parg293his Cry1 Variant Exhibits Similar Characteristics Tomentioning

confidence: 99%

Section: Understanding the Role Of Arg293 In Cry1 Functionmentioning

confidence: 99%

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The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Gül

Aydın

Ozcan

et al. 2020

Journal of Biological Chemistry

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“…Kd should be at least on the order of 0.1. In fact, Fribourgh et al(41), who recently studied the docking of PER2:CRY1/2 to the core PAS domain of BMAL:CLOCK, measured Kd ≈ 400 nM, or in dimensionless terms, Kd ≈ 40. This estimate may easily be off by a factor of 10, because the measurements were made in vitro with purified protein domains (not with full length proteins).…”

mentioning

confidence: 99%

Mathematical Analysis of Robustness of Oscillations in Models of the Mammalian Circadian Clock

Heidebrecht

Chen

Tyson

2020

Preprint

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A wide variety of organisms possess endogenous circadian rhythms (~24 h period), which coordinate many physiological functions with the day-night cycle. These rhythms are mediated by a molecular mechanism based on transcription-translation feedback. A number of mathematical models have been developed to study features of the circadian clock in a variety of organisms. In this paper, we use bifurcation theory to explore properties of mathematical models based on Kim & Forger’s interpretation of the circadian clock in mammals. Their models are based on a simple negative feedback (SNF) loop between a regulatory protein (PER) and its transcriptional activator (BMAL). In their model, PER binds to BMAL to form a stoichiometric complex (PER:BMAL) that is inactive as a transcription factor. However, for oscillations to occur in the SNF model, the dissociation constant of the PER:BMAL complex, Kd, must be smaller than 10−3 nM, orders of magnitude below the limit set by the biophysics of protein binding. We have relaxed this constraint by introducing two modifications to Kim & Forger’s SNF model: (1) replacing the first-order rate law for degradation of PER in the nucleus by a Michaelis-Menten rate law, and (2) introducing a multistep reaction chain for posttranslational modifications of PER. These modifications significantly increase the robustness of oscillations, and increase the maximum allowable Kd to more reasonable values, 1—100 nM. In a third modification, we considered alternative rate laws for gene transcription to resolve an unrealistically large rate of PER transcription at very low levels of BMAL transcription factor. Additionally, we studied Kim & Forger’s extensions of the SNF model to include a second negative feedback loop (involving REV-ERB) and a supplementary positive feedback loop (involving ROR). We found that the supplementary positive feedback loop—but not the supplementary negative feedback loop— provides additional robustness to the clock model.AUTHOR SUMMARYThe circadian rhythm aligns bodily functions to the day/night cycle and is important for our health. The rhythm originates from an intracellular, molecular clock mechanism that mediates rhythmic gene expression. It is long understood that transcriptional negative feedback with sufficient time delay is key to generating circadian oscillations. However, some of the most widely cited mathematical models for the circadian clock suffer from problems of parameter “fragilities”. That is, sustained oscillations are possible only for physically unrealistic parameter values. A recent model by Kim and Forger nicely incorporates the inhibitory binding of PER, a key clock protein, to its transcription activator BMAL, but oscillations in their model require a binding affinity between PER and BMAL that is orders of magnitude lower than the physical limit of protein-protein binding. To rectify this problem, we make several physiologically credible modifications to the Kim-Forger model, which allow oscillations to occur with realistic binding affinity. The modified model is further extended to explore the potential roles of supplementary feedback loops in the mammalian clock mechanism. Ultimately, accurate models of the circadian clock will provide predictive tools for chronotherapy and chrono-pharmacology studies.

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How circadian clocks keep time: the discovery of slowness

Partch

Brunner

2022

FEBS Letters

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Circadian clocks are molecular timers that measure 24‐hour periods through a series of slow but precise biochemical processes. While circadian clocks of cyanobacteria are driven by interactions and ordered phosphorylation of highly structured protein assemblies, timekeeping by circadian clocks of eukaryotes involves slow progressive hyperphosphorylation of a large number of redundant sites in intrinsically disordered regions of clock proteins.

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Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing

Cited by 57 publications

References 71 publications

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

The Arg-293 of Cryptochrome1 is responsible for the allosteric regulation of CLOCK-CRY1 binding in circadian rhythm

Mathematical Analysis of Robustness of Oscillations in Models of the Mammalian Circadian Clock

How circadian clocks keep time: the discovery of slowness

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