biostudies-literatureUnknownSchneps CMNational Institute of General Medical SciencesNIGMS NIH HHSNIH HHSNational Science FoundationCircadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of <i>Drosophila melanogaster</i>, 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.Biochemistryhttps://www.ebi.ac.uk/biostudies/studies/S-EPMC11289166biostudies-literatureDissecting the Interaction between Cryptochrome and Timeless Reveals Underpinnings of Light-Dependent Recognition.PMC11289166MCB2129728R24 GM146107P30 GM124166P41 GM103521R35 GM122535R24GM146107R35122535S10 OD021543Crane BRDunleavy RSchneps CMfalseDissecting the Interaction between Cryptochrome and Timeless Reveals Underpinnings of Light-Dependent Recognition.Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of <i>Drosophila melanogaster</i>, 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.2024-01-01T00:00:00Z2024 Jan2026-07-02T03:21:36.622Z2025-08-17T03:06:02.432ZS-EPMC112891663829488010.1021/acs.biochem.3c00630