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Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of Drosophila melanogaster, the clock is set by the light-induced degradation of the core oscillator protein timeless (TIM) by the principal light-sensor cryptochrome (CRY). The cryo-EM structure of CRY bound to TIM revealed that within the extensive CRY:TIM interface, the TIM N-terminus binds into the CRY FAD pocket, in which FAD and the associated phosphate-binding loop (PBL) undergo substantial rearrangement. The TIM N-terminus involved in CRY binding varies in isoforms that facilitate the adaptation of flies to different light environments. Herein, we demonstrate, through peptide binding assays and pulsed-dipolar electron spin resonance (ESR) spectroscopy, that the TIM N-terminal peptide alone exhibits light-dependent binding to CRY and that the affinity of the interaction depends on the initiating methionine residue. Extensions to the TIM N-terminus that mimic less light-sensitive variants have substantially reduced interactions with CRY. Substitutions of CRY residues that couple to the flavin rearrangement in the CRY:TIM complex have dramatic effects on CRY light activation. CRY residues Arg237 on α8, Asn253, and Gln254 on the PBL are critical for the release of the CRY autoinhibitory C-terminal tail (CTT) and subsequent TIM binding. These key light-responsive elements of CRY are well conserved throughout Type I cryptochromes of invertebrates but not by cryptochromes of chordates and plants, which likely utilize a distinct light-activation mechanism.
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Effect of temperature on the Arabidopsis cryptochrome photocycle
Abstract Cryptochromes are blue light‐absorbing photoreceptors found in plants and animals with many important signalling functions. These include control of plant growth, development, and the entrainment of the circadian clock. Plant cryptochromes have recently been implicated in adaptations to temperature variation, including temperature compensation of the circadian clock. However, the effect of temperature directly on the photochemical properties of the cryptochrome photoreceptor remains unknown. Here we show that the response to light of purifiedArabidopsisCry1 and Cry2 proteins was significantly altered by temperature. Spectral analysis at 15°C showed a pronounced decrease in flavin reoxidation rates from the biologically active, light‐induced (FADH°) signalling state of cryptochrome to the inactive (FADox) resting redox state as compared to ambient (25°C) temperature. This result indicates that at low temperatures, the concentration of the biologically active FADH° redox form of Cry is increased,leading to the counterintuitive prediction that there should be an increased biological activity of Cry at lower temperatures. This was confirmed using Cry1 cryptochrome C‐terminal phosphorylation as a direct biological assay for Cry activationin vivo. We conclude that enhanced cryptochrome functionin vivoat low temperature is consistent with modulation by temperature of the cryptochrome photocycle.
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- Award ID(s):
- 1658640
- PAR ID:
- 10450855
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Physiologia Plantarum
- Volume:
- 172
- Issue:
- 3
- ISSN:
- 0031-9317
- Page Range / eLocation ID:
- p. 1653-1661
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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