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Reaction Mechanism of Drosophila Cryptochrome

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Reaction Mechanism of Drosophila Cryptochrome Reaction mechanism of Drosophila cryptochrome Nuri Ozturka, Christopher P. Selbya, Yunus Annayeva, Dongping Zhongb, and Aziz Sancara,1 aDepartment of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and bDepartments of Physics, Chemistry, and Biochemistry, The Ohio State University, Columbus, OH 43210 Contributed by Aziz Sancar, November 15, 2010 (sent for review October 12, 2010) Cryptochrome (CRY) is a blue-light sensitive flavoprotein that func- Cryptochrome is the primary circadian photoreceptor in tions as the primary circadian photoreceptor in Drosophila melano- Drosophila (17). Upon exposure to light it undergoes a change that gaster. The mechanism by which it transmits the light signal to the makes both CRY and its downstream partner Timeless (TIM) core clock circuitry is not known. We conducted in vitro studies on targets for ubiquitination (18) and proteolysis (19). TIM is a the light-induced conformational change in CRY and its effect on key component of the negative arm of the TTFL (Transcription- protein–protein interaction and performed in vivo analysis of the Translation-Feedback Loop) that constitutes the core circadian lifetime of the signaling state of the protein to gain some insight clock and degradation of TIM resets the clock. It has been into the mechanism of phototransduction. We find that exposure reported that Jetlag (JET) is the E3 ligase responsible for the of CRY to blue light induces a conformation similar to that of the light-dependent ubiquitination and proteolysis of both TIM and constitutively active CRY mutant with a C-terminal deletion (CRYΔ). CRY (18, 20). However, it was unclear how the absorption of a This light-induced conformation has a half-life of ∼15 min in the blue-light photon by the flavin cofactor made CRY a target for dark at 25 °C and is characterized by increased affinity to Jetlag JET. The isolation of a constitutively active CRY mutant, CRYΔ – E3 ligase. In vivo analysis reveals that in the Drosophila S2 cell line, (21 24), which is missing the C-terminal 22 amino acids, raised the the signaling state induced by a millisecond light exposure has a possibility that light causes a conformational change in CRYand half-life of 27 min in the dark at 0 °C during which period it is sus- thus affects its interaction with downstream partners enabling it ceptible to degradation by the ubiquitin-proteasome system. These to transduce the signal. In this study, through analyses of partial findings lead to a plausible model for circadian photoreception/ proteolysis patterns we demonstrate that light causes a confor- phototransduction in Drosophila. mational change in CRY. Moreover, we find that this signaling (or “Lit”) state conformation (henceforth we will use these two circadian clock ∣ photocycle ∣ proteolysis ∣ sensory flavoprotein terms interchangeably) that is remarkably long-lived in the dark is similar to the conformation of the constitutively active CRYΔ, ryptochrome (CRY) is a flavoprotein that regulates growth and exhibits enhanced affinity for JET. Finally, we demonstrate in vivo that a light pulse of a millisecond duration induces a Lit state and development in plants in response to blue light, functions C CRY with a half-life of 27 min at 0 °C min during which period it as a circadian photoreceptor in Drosophila and other insects, and remains susceptible to ubiquitination and degradation in the dark. acts as a core component of the molecular clock in mammalian Collectively, the data are consistent with a model whereby blue organisms (1–4). Despite extensive research on CRYs photosen- light induces a significant stable conformational change in CRY sory function in Arabidopsis and Drosophila, its mechanism of and this Lit state binds to JET E3 ligase with high affinity and in- photoreception/phototransduction is poorly understood. Even dependently of light, and promotes the ubiquitination and proteo- the redox status of the FAD cofactor is a matter of some debate lysis of both CRYand TIM, and thus resets the clock. (5–8). In the phylogenetically related protein, DNA photolyase, − photoinduced cyclic electron transfer from the FADH cofactor Results to a pyrimidine photodimer repairs the DNA damage and regen- Purification and Spectroscopic Properties of CRY and CRYΔ. We − – erates the FADH for new rounds of catalysis (9 11). However, wished to compare the photoresponse of CRY to that of the con- Arabi- there is no evidence so far for a similar reaction in either stitutively active CRYΔ with regard to light-inducible conforma- dopsis Drosophila CRY1 (AtCRY1) and CRY2 or CRY, which tional change to find out if light induced a conformation of CRY at present are the most extensively studied CRYs. The lack of evi- that might be similar to the CRYΔ conformation in the dark. To dence for a cyclic redox reaction in CRYs has led to consideration this end we constructed baculoviruses expressing CRYand CRYΔ of the mechanisms of other photosensory flavoproteins as poten- carrying affinity tags either at the N- or C-termini to aid in pur- tial models for CRYs in general and Drosophila CRY in particular. ification and in analyzing the proteolysis patterns by immunoblot- Currently, three types of photosensory flavoproteins are known ting. Fig. 1A shows that the proteins are of high purity and Fig. 1B (12): the photolyase/CRY family, the LOV domain proteins such shows that their absorption spectra in the near UV-visible range as phototropin, and the BLUF domain proteins such as the photo- are essentially identical. Importantly, upon exposure to blue light activated adenylyl cyclase. Whereas photolyase, as noted above, both CRY and CRYΔ flavins undergo the same photoreduction •− carries out catalysis by light-induced cyclic electron transfer, LOV reaction whereby FADox is converted to FAD through intra- and BLUF domain proteins initiate photosignaling by a light- protein electron transfer (5–7). induced conformational change. Failing to obtain any evidence for CRY signaling by a photolyase-like mechanism, we considered Effect of Blue Light on CRY and CRYΔ. A widely held model for CRY the possibility that CRYalso may carry out its light signaling by a photoreception/phototransduction posits that light induces a con- light-induced conformational change that would affect the interaction of CRY with downstream signal transduction partners Author contributions: N.O. and A.S. designed research; N.O. and C.P.S. performed research; (13, 14). Indeed, a preliminary study with AtCRY1 revealed Y.A. contributed new reagents/analytic tools; N.O. and C.P.S. analyzed data; and N.O., that light induced a significant conformational change in the C.P.S., D.Z., and A.S. wrote the paper. C-terminal extension (14) of the protein that is considered to be The authors declare no conflict of interest. the signaling domain of this cryptochrome (15, 16). Hence, we en- Freely available online through the PNAS open access option. Drosophila tertained the possibility that CRY may also initiate 1To whom correspondence should be addressed. E-mail: [email protected]. photosignaling by a light-induced conformational change that This article contains supporting information online at www.pnas.org/lookup/suppl/ would affect its interactions with known signaling partners. doi:10.1073/pnas.1017093108/-/DCSupplemental. 516–521 ∣ PNAS ∣ January 11, 2011 ∣ vol. 108 ∣ no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1017093108 Downloaded by guest on October 1, 2021 Fig. 1. Purification and spectroscopic properties of CRY and CRYΔ proteins. (A) Analysis of purified proteins by 4–12% gradient SDS-PAGE and Coomassie Blue Staining. The numbers on the left indicate size markers in kilodaltons. Sample lanes contained approximately 1 μg of the indicated proteins. Lane 1, FH-CRY; Lane 2, FH-CRYΔ; Lane 3, CRY-V5H; Lane 4, CRYΔ-V5H; and Lane 5 contains Zebrafish CRY4-V5H that was used as a control in some of the ex- periments. H, F, and V5 indicate His, Flag, and V5 tags, respectively, and the placement of the tag symbols before or after the protein symbol indicate the position of the tag at the N- or C-terminus of the protein, respectively. (B) Spectroscopic and photochemical properties of CRY and CRYΔ. Near UV-visible spectra are shown. Solid line, purified proteins kept in the dark Δ exhibit characteristic FADox spectrum. Dashed line, absorption spectra after Fig. 2. Effect of blue light on the conformations of CRY and CRY . The con- exposure to blue light reveal that both CRY and CRYΔ are converted to the formations were probed by partial proteolysis with trypsin at 25 °C for 30 min FAD•− anion radical forms. and 1∶800 (trypsin∶CRY) ratio (w∶w). The digested proteins were separated by 4–12% gradient SDS-PAGE and probed with the appropriate antibodies against the tags at the N- or C-termini. C, control sample with no protease formational change in CRY that enables it to interact with down- treatment; D, protein that was treated with trypsin in the dark; L, protein stream targets and initiate the signaling cascade (2, 13, 14). In treated with trypsin under blue light. (A) CRY-V5H and CRYΔ-V5H partial di- particular, a change in the conformation of the C-terminal exten- gests were probed with anti-V5 antibodies. The bands designated by Roman sion is thought to be of special significance because mutations numerals indicate the cleavage at a hinge point in CRY (I) and CRYΔ (I’) and that delete the C-terminal ∼20 amino acids render CRY consti- cleavages affected by light in CRY (II-IV) but not in CRYΔ (II’-III’). The cleavage tutively active (21–23). To test this model, we used CRY and at K289 was much more efficient than cleavages at other sites. Therefore a CRYΔ (missing the C-terminal 22 amino acids) in an assay in short exposure of the immunoblot of the upper half of the gel is shown for which protein conformation was probed by partial proteolysis comparison of the light effect on cleavage at this site and a longer exposure Δ of the bottom half is shown for comparison to cleavages at other sites.
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