Reaction mechanism of Drosophila cryptochrome

Abstract

Cryptochrome (CRY) is a blue-light sensitive flavoprotein that functions as the primary circadian photoreceptor in Drosophila melanogaster. The mechanism by which it transmits the light signal to the core clock circuitry is not known. We conducted in vitro studies on the light-induced conformational change in CRY and its effect on protein-protein interaction and performed in vivo analysis of the lifetime of the signaling state of the protein to gain some insight into the mechanism of phototransduction. We find that exposure of CRY to blue light induces a conformation similar to that of the constitutively active CRY mutant with a C-terminal deletion (CRYΔ). This light-induced conformation has a half-life of ∼15 min in the dark at 25°C and is characterized by increased affinity to Jetlag E3 ligase. In vivo analysis reveals that in the Drosophila S2 cell line, the signaling state induced by a millisecond light exposure has a half-life of 27 min in the dark at 0°C during which period it is susceptible to degradation by the ubiquitin-proteasome system. These findings lead to a plausible model for circadian photoreception/ phototransduction in Drosophila.circadian clock | photocycle | proteolysis | sensory flavoprotein C ryptochrome (CRY) is a flavoprotein that regulates growth and development in plants in response to blue light, functions as a circadian photoreceptor in Drosophila and other insects, and acts as a core component of the molecular clock in mammalian organisms (1-4). Despite extensive research on CRYs photosensory function in Arabidopsis and Drosophila, its mechanism of photoreception/phototransduction is poorly understood. Even the redox status of the FAD cofactor is a matter of some debate (5-8). In the phylogenetically related protein, DNA photolyase, photoinduced cyclic electron transfer from the FADH − cofactor to a pyrimidine photodimer repairs the DNA damage and regenerates the FADH − for new rounds of catalysis (9-11). However, there is no evidence so far for a similar reaction in either Arabidopsis CRY1 (AtCRY1) and CRY2 or Drosophila CRY, which at present are the most extensively studied CRYs. The lack of evidence for a cyclic redox reaction in CRYs has led to consideration of the mechanisms of other photosensory flavoproteins as potential models for CRYs in general and Drosophila CRY in particular.Currently, three types of photosensory flavoproteins are known (12): the photolyase/CRY family, the LOV domain proteins such as phototropin, and the BLUF domain proteins such as the photoactivated adenylyl cyclase. Whereas photolyase, as noted above, carries out catalysis by light-induced cyclic electron transfer, LOV and BLUF domain proteins initiate photosignaling by a lightinduced conformational change. Failing to obtain any evidence for CRY signaling by a photolyase-like mechanism, we considered the possibility that CRY also may carry out its light signaling by a light-induced conformational change that would affect the interaction of CRY with downstream signal transduction partners (13,14). Inde...

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

“…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). However, all existing evidence suggests that light activated CRY- In contrast, LNvdirected expression of the redox-disabled D260N-Hk mutant in hk −/− (1.12 ± 0.03, n = 13) is significantly different from control (P = 0.036) but not hk −/− (P = 0.58) recordings, indicating failure to rescue.…”

Determining complete electron flow in the cofactor photoreduction of oxidized photolyase

“…protein electron transfer | flavin photoreduction | femtosecond dynamics | electron flow directionality | reduction potential funnel P hotolyase and cryptochrome are evolutionally related and contain a flavin adenine dinucleotide (FAD) as the catalytic cofactor with a unique bent structure in the active sites, but the two perform different functions: photolyase repairs UV-damaged DNA and cryptochrome functions as a photoreceptor for regulation of plant growth or synchronization of circadian rhythm (1)(2)(3)(4)(5). The active state of the cofactor in vivo is in the anionic hydroquinone form (FADH -) in photolyase (6), but currently the redox status of flavin in cryptochrome is under debate with some studies suggesting flavin to be in oxidized (FAD), whereas others claiming anionic (FAD -/FADH -) states for the functional form in vivo (7)(8)(9)(10).…”

Fly cryptochrome and the visual system

“…dCRY interacts also with the kinase shaggy/GSK3 (SGG), and the cryptochrome's stability in light is considerably increased by this interaction whereas the inactivation of the kinase leads to the degradation of dCRY in darkness (10). The molecular mechanism by which dCRY is activated by light is still not fully understood, but a regulatory role for the C terminus of the protein has been demonstrated by several studies (3,5,(11)(12)(13). The activation of dCRY by light requires a conformational change (13), but the release of a putative repressor cannot be excluded (11).…”

“…The molecular mechanism by which dCRY is activated by light is still not fully understood, but a regulatory role for the C terminus of the protein has been demonstrated by several studies (3,5,(11)(12)(13). The activation of dCRY by light requires a conformational change (13), but the release of a putative repressor cannot be excluded (11). In fact, it has been hypothesized that the activation of dCRY by light is mediated also by specific "regulators" that bind its C terminus, known to regulate the light dependence of dCRY activity (13).…”

“…The activation of dCRY by light requires a conformational change (13), but the release of a putative repressor cannot be excluded (11). In fact, it has been hypothesized that the activation of dCRY by light is mediated also by specific "regulators" that bind its C terminus, known to regulate the light dependence of dCRY activity (13). This hypothesis was supported by the observation that the C terminus of dCRY is a hotspot for molecular interactions: by in silico analysis and experimental validation, we could identify several proteinprotein interaction motifs in this small region and, among them, two class III PDZ-binding motifs (3).…”