Flavin reduction activates Drosophila cryptochrome

Abstract

Entrainment of circadian rhythms in higher organisms relies on light-sensing proteins that communicate to cellular oscillators composed of delayed transcriptional feedback loops. The principal photoreceptor of the fly circadian clock, Drosophila cryptochrome (dCRY), contains a C-terminal tail (CTT) helix that binds beside a FAD cofactor and is essential for light signaling. Light reduces the dCRY FAD to an anionic semiquinone (ASQ) radical and increases CTT proteolytic susceptibility but does not lead to CTT chemical modification. Additional changes in proteolytic sensitivity and small-angle X-ray scattering define a conformational response of the protein to light that centers at the CTT but also involves regions remote from the flavin center. Reduction of the flavin is kinetically coupled to CTT rearrangement. Chemical reduction to either the ASQ or the fully reduced hydroquinone state produces the same conformational response as does light. The oscillator protein Timeless (TIM) contains a sequence similar to the CTT; the corresponding peptide binds dCRY in light and protects the flavin from oxidation. However, TIM mutants therein still undergo dCRY-mediated degradation. Thus, photoreduction to the ASQ releases the dCRY CTT and promotes binding to at least one region of TIM. Flavin reduction by either light or cellular reductants may be a general mechanism of CRY activation.redox | photolyase | protein-protein interaction

Mechanism of Photosignaling by Drosophila Cryptochrome

“…While this manuscript was in preparation, a study was published claiming that flavin reduction either by light or chemically was necessary for the conformational change leading to the dCRY signaling state (34). Our data presented in this study unambiguously show that light excitation of dCRY FAD can cause functionally relevant conformational change in the absence of flavin reduction.…”

CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

“…Whether direct chemical redox reactions occur between CRY and Hk is unclear. For CRY, light or chemical reduction induces one-electron reduction of the FAD cofactor of CRY (39)(40)(41), whereas the reductive catalytic mechanism of AKRs (such as Hk) requires a hydride ion transferred from NADPH to a substrate carbonyl, then a solvent-donated proton reduces the substrate carbonyl to an alcohol (42). These differences in redox chemistry between CRY and Hk suggest that other intermediates, such as oxygen, are possibly required for redox coupling.…”

“…Spectroscopic analysis of animal and plant CRYs suggest that light activation causes reduction of the FAD oxidized base state (39)(40)(41)43). Light activation of Drosophila CRY also evokes conformational changes in the C terminus of CRY that clearly promotes CRY C-terminal access to proteolytic degradation and subsequent interactions with the TIMELESS clock protein, thus signaling degradation and circadian entrainment (44)(45)(46)(47).…”

“…Further distinguishing the distinct mechanisms of the downstream effects of light-activated CRY, the light-induced conformational changes that couple CRY to ubiquitin ligase binding (thus causing circadian entrainment) occur in oxidized and reduced states of CRY and are unaffected in CRY tryptophan mutants that presumably are responsible for intraprotein electron transfer reactions following light-evoked reduction of the FAD cofactor (47). Another recent study shows that light-or chemical-evoked reduction of Drosophila CRY FAD is coupled to conformational changes of the CRY C terminus (41), along with reporting a surprising negative result that DPI has no effect on the reoxidation of the reduced anionic semiquinone of purified Drosophila CRY. DPI could hypothetically influence the electrophysiological light response by blocking the pentose phosphate pathway (48) which Fig.…”

Resolving cryptic aspects of cryptochrome signaling

“…For example, in crystal structures of Drosophila CRY (dCRY) the CCE docks to a solvent-exposed cleft adjacent to the photocatalytic FAD under darkstate conditions (9). Photo-or chemical reduction of the FAD then releases the CCE to engage interaction partners (Timeless) and target CRY for degradation (10). Although only structures of the isolated PHR domain of AtCRY1 are currently available, biochemical studies suggest plant CRYs conserve a similar mechanism.…”