The human CRY1 tail controls circadian timing by regulating its association with CLOCK:BMAL1

Abstract

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.

The tail of cryptochromes: an intrinsically disordered cog within the mammalian circadian clock

“…a The hCRY1 tail is composed of exons 10 (residues 496-529), exon 11 (residues 530-553), and exon 12 (residues 554-586). NMR chemical shift mapping identified distinct linear motifs in exons 10 and 11 (pink) that may be involved in binding directly to the PHR domain [35]. DNA-PK-dependent phosphorylation sites in exons 11 and 12 are marked below a circled 'P' [36].…”

“…c A possible model for how the tail (magenta) might bind to both the FAD-binding pocket and the secondary pocket on the PHR domain in the absence of PER proteins. Exon 10 has been implicated at binding near the FAD-binding pocket [37], while exon 11 inhibits CLOCK PAS-B binding at the secondary pocket and deletion of exon 12 has no effect on affinity of the tail for the PHR domain [35] These findings are consistent with the emerging model that tighter binding of CRY leads to a longer circadian period [30]. Because the mutant allele directly modifies only the intrinsically disordered C-terminal tail, and not the PHR domain where cryptochromes have been shown to directly interact with CLOCK:BMAL1, these data suggest that loss of exon 11 relieves some type of autoinhibitory role of the tail between the CRY1 PHR domain and CLOCK:BMAL1, thus making CRY1Δ11 a more effective repressor.…”

“…Experimental evidence for a direct interaction between the PHR domain of a mammalian CRY and its disordered tail was first published in 2005 [18]. A recent study revisited intramolecular interactions of mammalian cryptochromes in more depth with fluorescence polarization (FP) binding assays and nuclear magnetic resonance (NMR) spectroscopy to identify the molecular determinants by which the CRY1 tail directly interacts with its PHR domain [35].…”

“…It can be quite challenging to study long IDPs at atomic resolution with conventional protein NMR methods such as the 1 H-15 N heteronuclear single quantum coherence (HSQC) experiment due to the lack of unique chemical environments in the disordered protein that severely restrict peak dispersion in the 1 H dimension of HSQC spectra [18,35]. By contrast, innovative direct 13 C-detected experiments such as the 13 C-15 N CON not only increase peak dispersion, but also allow for detection of proline residues that are typically enriched in IDPs, thus improving biophysical studies of long, native-like IDPs [49].…”

“…Related 13 C-detected triple resonance experiments such as the 3D (HACA) N (CA) NCO and (HACA) N (CA) CON aid in assigning peaks for individual residues for long IDPs [49]. The use of CON NMR methods was critical for enabling the identification of residues on the CRY1 tail that exhibit chemical shift perturbations upon addition of the PHR domain; these residues are primarily centered in short linear motifs in exons 10 and 11, which also correspond to the minimal regions on the tail needed for binding to the PHR domain by FP-based binding assays [35] (Fig. 3a).…”