The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions.
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
We report an improved model of the Drosophila cryptochrome structure that corrects errors in the original coordinates (3TVS.pdb). Further refinement of the structure with automated rebuilding algorithms in PHENIX 1 followed by manual building, indicated that a model of dCRY could be produced with excellent refinement statistics without taking into account the non-merohedral twinning originally reported (Table 1). The rebuilt structure has an RMSD on Cα positions of 2.4 Å compared to the deposited coordinates with most differences found in the conformation of surface loops (Fig. 1). However, the new analysis also indicates that the sequence register of the C-terminal tail helix (CTT) is displaced by two residues (Fig. 2). This change in sequence register offsets the invariant FFW motif along the helix axis such that Phe534, and not Trp536, approaches closest to the flavin ring (Fig. 3). In the new model, the three residues composing the FFW motif continue to make extensive interactions with the photolyase homology domain. This new position of the FFW motif is more consistent with the cellular data of Fig. S6, which shows that substitution of FFW to three alanine residues has a dramatic effect on dCRY stability, but that the W536A substitution alone, does not. The configuration of the flavin center is similar between the old and new models, with the largest difference in the angle of the ribityl-to-flavin (N10) bond (Fig. 4). Phosphorylation of Thr518 is not apparent in the new electron density maps despite identification of this modification by mass spectrometry. The errors in the original structure stemmed from model bias introduced during the detwinning procedure. Lower resolution data sets to which the original dCRY structure was built appear to suffer more from twinning than the 2.3 Å resolution data that the final model was refined against. Although the high-resolution data does contain indications of non-merohedral twinning, including intensity oscillations along the reciprocal space l axis and spurious Patterson peaks, a model that agrees well with the diffraction data as collected can be produced without compensation for these effects (Table 1). The new coordinates have been deposited in the PDB as 4GU5. B.R.C. apologizes for these errors.
Cryptochromes (CRYs) entrain the circadian clocks of plants and animals to light. Irradiation of the cryptochrome (dCRY) causes reduction of an oxidized flavin cofactor by a chain of conserved tryptophan (Trp) residues. However, it is unclear how redox chemistry within the Trp chain couples to dCRY-mediated signaling. Here, we show that substitutions of four key Trp residues to redox-active tyrosine and redox-inactive phenylalanine tune the light sensitivity of dCRY photoreduction, conformational activation, cellular stability, and targeted degradation of the clock protein timeless (TIM). An essential surface Trp gates electron flow into the flavin cofactor, but can be relocated for enhanced photoactivation. Differential effects of Trp-mediated flavin photoreduction on cellular turnover of TIM and dCRY indicate that these activities are separated in time and space. Overall, the dCRY Trp chain has evolutionary importance for light sensing, and its manipulation has implications for optogenetic applications of CRYs.
Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pK a . Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.light sensing | flavoprotein | photochemistry | redox | molecular dynamics C ryptochromes (CRYs) are flavin-binding proteins that perform a variety of sensory and catalytic functions in all kingdoms of life (1, 2). CRYs are closely related to the DNA photolyases (PLs), which catalyze light-driven redox reactions to break apart pyrimidine dimers in UV-damaged DNA (1, 2). CRYs and PLs share a conserved photolyase homology region that consists of an α-helical domain, which binds flavin adenine dinucleotide (FAD) and an α/β Rossman-fold domain, which sometimes binds a pteridine or deazaflavin antenna cofactor. CRYs also contain C-terminal extensions of variable sizes that contribute to their specific functions. The range of activities found for CRYs and PLs require that their flavin cofactors assume a broad range of redox, protonation, and excited states (1, 2).In the fruit fly Drosophila melanogaster, a type I cryptochrome (dCRY) is the primary light receptor of the circadian clock (1, 3). In response to blue light, dCRY coordinates interactions between Timeless (TIM) and the E3-ubiquitin ligase Jetlag (JET) (4). JETmediated proteolysis of TIM destabilizes its partner Period (PER). PER serves as the principal repressor of circadian gene expression and its degradation phase-shifts the clock (3). dCRY also catalyzes light-induced self-degradation that involves another E3-ligase: Brwd3 or RAMSHACKLE (5)...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.