Reaction mechanism of

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 , November 15, 2010 (sent for review October 12, 2010)

Cryptochrome (CRY) is a blue-light sensitive 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 (TIM) core 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 (- –protein interaction and performed in vivo analysis of the -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 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 by the flavin 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 it is sus- thus affects its interaction with downstream partners enabling it ceptible to degradation by the ubiquitin- 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 ∣ 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 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 , 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 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 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 , − 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 (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 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 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 , 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 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 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. In with trypsin. Purified CRY and CRY , kept either in the dark addition to the marked bands, other faint bands are seen that are not af- or under blue light, were subjected to partial proteolysis with tryp- fected by light in either CRY or CRYΔ and are not considered further. Note sin. Then, the products were separated by SDS-PAGE and probed that cleavage at the C-terminal tail (IV site) is not detectable with the C-term- with the appropriate antibodies to identify the cleavage sites. inal probe because of its small size or removal of the tag by secondary clea- From the results shown in Fig. 2, we identify 5 cleavage sites in vage after the release of the C-terminal extension. (B) FH-CRY and FH-CRYΔ CRYand 4 in CRYΔ (Fig. 2) as described in SI Text: We designate partial digests were probed with anti-Flag antibodies. (Top) Immunoblot of these sites I, Ia-IV in CRYand I’,Ia’-III’ in CRYΔ, as appropri- the entire gel of a short run to reveal the overall tryptic cleavage pattern. ate. Site I in CRY (and I’ in CRYΔ) is at K289, and the resulting (Lower) Immunoblot of the high molecular size region to resolve closely spaced bands (asterisk) in the range of 50–65 kDa. Note the presence of fragments (intensity of cleavage at this site is unaffected by light) ’ A the additional light-insensitive Ia (Ia ) band not seen with C-terminal probe, are detectable with both N- and C-terminal probes (Fig. 2 , lanes presumably because it is the product of secondary cleavage of band I. In 2 and 3, and lanes 5 and 6, and Fig. 2B, lanes 2 and 3, and lanes 5 addition, the band corresponding to cleavage at site II is not detectable and 6). Site Ia and Ia’ is around 200 amino acids from the N-ter- with the N-terminal probe presumably because the very efficient cleavage BIOCHEMISTRY minus, and the resulting fragment is detectable only with the at site I depletes site II cleavage products. (C) Linear representation of N-terminal probe (Fig. 2B, lanes 2 and 3, and lanes 5 and 6). CRY and CRYΔ indicate the main trypsin cleavage sites detectable with The intensity of cleavage at this site is unaffected by light and C- and N-terminal probes. the fragment resulting from cleavage at this site is presumably the product of secondary cleavage of Fragment I (I’) because ary proteolysis. Cleavage at this site is only detectable with it is not detectable with the C-terminal probe. Site II (II’)isat high-resolution electrophoresis and using the N-terminal probe R430. Cleavage at this site is an important conformational signa- that reveals a light-induced proteolytic fragment of CRY that is ture: Cleavage of CRYat this site is induced by light and cleavage approximately 3 kDa smaller than the full-length protein and of CRYΔ is very efficient and it is not affected by light (Fig. 2A, corresponds to, essentially, the CRYΔ protein (Fig. 2B, Bottom, lanes 2 and 3, and lanes 5 and 6). The site II (II’) cleavage is not lanes 4–6). These cleavage sites are indicated on 1D and 3D detectable with the N-terminal probe presumably because of representations of the Drosophila CRY in Fig. 2C and SI Text. the much stronger cleavage efficiency at K289 site I (I’)(SI Text). Thus, the partial proteolysis experiments with CRY and CRYΔ It is also possible that cleavage at site II promotes a secondary yield complementary results consistent with the model that blue cleavage on the N-terminal side of the fragment. Site III (III’) light induces a conformation in CRY that is similar to the con- cleavage at K503 is light-enhanced in CRY (Fig. 2 A, lanes 2 formation of the constitutively “on” CRYΔ. and 3, and B, lanes 2 and 3) and constitutively trypsin-sensitive and unaffected by light in CRYΔ (Fig. 2A, lanes 5 and 6, and B, Mechanism of the Photoinduced Conformational Change of CRY. Next, lanes 5 and 6) and therefore cleavage at this site may also be we addressed the question of the photochemical mechanism that considered as a signature of the signaling state conformation. Site induces the signaling state conformation. Currently, there are two IV cleavage at position ∼520–542 (Fig. 2B, lanes 2 and 3) repre- models for the photosignaling reaction of Drosophila CRY (25). sents a light-induced conformational change in the C-terminal In one model (5, 6), the “dark state” CRY contains oxidized FAD, extension of CRY. It is not detectable with the C-terminal probe FADox. Upon blue-light exposure, the excited •− because of small size of the C-terminal product and most likely (FADox)* is converted to the FAD anion radical concomitant further degradation of the small unstructured peptide by second- with a conformational change in CRY that generates CRY‡, the

Ozturk et al. PNAS ∣ January 11, 2011 ∣ vol. 108 ∣ no. 2 ∣ 517 Downloaded by guest on October 1, 2021 •− signaling or Lit state; reoxidation of FAD to FADox in the dark causes the CRY conformation to change back to the dark state and turns off the signal:

1∶ ð Þ→hν ð Þ Model CRY FADox CRY FADox þ − − − →e ‡ð •−Þ →e ð Þ: CRY FAD CRY FADox

In the alternative model (7, 8), it is proposed that the Drosophila •− CRY contains FAD in the ground state in the dark (the FADox- form of CRY is presumed to be a purification artifact). According to this model, the excitation of FAD•− by light to (FAD•−)* and not the formation of FAD•− causes the conformational change responsible for signaling:

hν Model 2∶ CRYðFAD•−Þ→CRYðFAD•−Þ → CRY‡ðFAD•−Þ → CRYðFAD•−Þ:

According to this model, the decay of the signaling state confor- Fig. 3. Chemical reduction does not induce conformational change in CRY mation, CRY‡, back to the ground state conformation is not me- but light induces conformational change in chemically reduced CRY. CRY chanistically coupled with a change in the oxidation state of FAD. was reduced with dithionite and then subjected to partial proteolysis in The experiments described in Fig. 2, however, cannot discrimi- the dark or under blue light. Because the experiments relating to decay kinetics required shorter trypsinization times we treated CRY with a higher nate between these two models because during conditions of con- trypsin concentration (1∶100 w∶w) for 10 min for this and subsequent experi- tinuous irradiation, photoreduction by the first absorbed photon ments. CRY-V5H was used in these experiments and proteolysis was probed is followed by absorption of a second photon and the excitation of with anti-V5 antibodies. Under this proteolysis regimen the light-inducible the flavin anion radical: band II (cleavage at K430) is removed by secondary cleavage and hence the light-enhanced band III (cleavage at K503) is used for comparison of the effects − hν þe •− hν •− of flavin reduction and flavin excitation on trypsin sensitivity. (A) Proteolysis in FAD →ðFAD Þ → FAD →ðFAD Þ : •− ox ox the dark. (Top) Conversion of FADox (solid line) to FAD (dotted line) by treat- ing CRY with dithionite. The bottom panel shows tryptic digestion patterns of ð Þ ð •−Þ As a consequence, with the Fig. 2A experimental design it cannot CRY FADox and CRY FAD probed for the C-terminal tag. The upper and be determined whether the signaling state conformation was lower halves of the gel were subjected to short and long exposure to reveal þ − →e •− the light insensitive and light sensitive bands, respectively. (B) CRY reduced by induced by the FADox FAD photoreduction or by the dithionite was exposed to light where indicated (Top) and subjected to pro- hν FAD•−→ðFAD•−Þ photoreaction. teolysis and probed for the C-terminal tag as in (A, Bottom). The solid and To discriminate between models 1 and 2, we chemically re- dotted line in the top panel indicate absorption spectra of dithionite-reduced duced the flavin to FAD•− by incubation with dithionite (26) and dithionite-reduced and light-exposed (10 min) CRYs, respectively. The pro- tein in lanes 1 and 4 was not treated with trypsin. and then carried out comparative partial proteolysis of CRY ð •−Þ ð •−Þ (FADox), CRY FAD , and CRY FAD exposed to light using the readily quantifiable light-enhanced site III cleavage Quantitative analysis of the data from this and two additional experiments carried out under identical conditions is shown in to monitor conformational change. The results of these experi- B ments are shown in Fig. 3. The following conclusions emerge Fig. 4 . From this figure, a half-life of 15 min is calculated that from this figure. First, dithionite reduces the flavin in CRY to is in the range of half-lives of other flavin-based sensory photo- FAD•− (Fig. 3A, Top) in a manner indistinguishable from photo- receptors such as (12, 28). reduction (Fig. 1B). Second, the proteolysis pattern of CRY as – probed by the sensitivity of K503 (site III) to trypsin is not af- Effect of Light-induced Conformational Change on CRY JET Interac- þe− fected by theFAD → FAD•− reduction (Fig. 3A, Bottom). tion. If the conformational change detected by partial proteolysis ox is relevant to CRY function, then it is expected that light would Third, exposure of dithionite-reduced CRY to light does not Δ cause further change in the absorption spectrum of the flavin co- promote CRY-downstream target interaction whereas CRY factor (Fig. 3B, Top) but it causes a conformational change in would be expected to exhibit these interactions strongly and in- Δ CRY that sensitizes K503 to proteolytic attack (Fig. 3B, Bottom). dependently of light. To test these predictions CRY or CRY These data are consistent with Model 2. were added to JET bound to sepharose beads, and the mixtures were either kept in the dark or exposed to light for 10 min. Then, Lifetime of the Signaling State of CRY. The proposed model for the the beads were collected by centrifugation, washed extensively, action mechanism of CRY that the light-activated state must have and the bound proteins were visualized by immunoblotting. a longer lifetime than the photochemically excited state of flavin The results of such an experiment are shown in Fig. 5. As is ap- because the lifetime of the flavin excited state is in the nanose- parent from the figure, the binding of CRY to JET is essentially cond range (26, 27), and considering the intracellular concentra- light-dependent (Fig. 5, lanes 5 and 7). In contrast, CRYΔ bound tions of CRY and its known protein targets, JET and TIM, the to JET strongly, irrespective of light (Fig. 5, lanes 6 and 8). These probability of encounter within the lifetime of the excited state data are consistent with the model that deletion of the C-terminal is negligible. To measure the lifetime of the signaling state 20 amino acids causes a conformational change in CRY that en- conformation we exposed CRY to a light pulse and then kept ables it to bind to JETat a maximal level (20) and that exposure of it in the dark for various periods of time before subjecting it CRY to blue light induces a conformation similar to that of to tryptic proteolysis and monitoring the rate of disappearance CRYΔ with the consequent promotion of high affinity CRY– of light-enhanced cleavage at K503 (site III) of CRY. Fig. 4A JET interaction. Next, we wished to determine if the half-life shows an immunoblot used to measure the rate of decay of of the signaling state defined by the partial proteolysis profile the light-promoted hypersensitivity to trypsin cleavage at K503. matched the half-life determined by CRY–JET binding. To this

518 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017093108 Ozturk et al. Downloaded by guest on October 1, 2021 Fig. 4. Half-life of the signaling state of CRY. CRY-V5H was exposed to 366 nm light at a fluence of 1 mW cm−2 for 10 min. Then, it was kept in the dark for the indicated time periods before adding trypsin (1∶100 w∶w) and incubating in the dark at 25 °C for another 10 min. The products were analyzed by immunoblotting with anti-V5 antibodies. The intensity of Fig. 6. Decay kinetics of the signaling state of CRY in vitro. FH-CRY was ex- Band III was quantified to assess the decay kinetics of the signaling state. posed to 366 nm light at a fluence rate of 1 mW cm−2 for 10 min, and then (A) Representative immunoblot. (B) Quantitative analysis of the decay of incubated in the dark for the indicated time period before adding to GST-JET the light-induced signaling state. The values at different time points are ex- beads and incubating in the dark for additional 12.5 min. The beads were pressed relative to the intensity of band III from the sample that was treated washed three times with 1 mL TBS in the dark and the bound proteins were with trypsin immediately (“zero time”) after light exposure (panel A, lane 2). visualized by immunoblotting using anti-Flag antibodies. (A) Representative Bars indicate SEM of 3 experiments. Note that, because under these condi- immunoblot with anti-Flag antibodies. The D and L lanes in the “Input” re- tions photoreduced CRY is reoxidized back to FADox nearly completely in present the CRY samples that were kept in the dark or exposed to light, re- 4.5 min (8), it is concluded that the light-induced conformational change spectively, before mixing with GST-JET beads that were kept in the dark is maintained independent of the FAD redox status. The y-axis is in log scale. throughout the experiment. (B) Quantitative analysis of 3 experiments in- cluding the one shown in (A). The amount bound at time “zero” (CRY added end, we irradiated CRY with blue light and then kept it in the to GST-JET immediately after irradiation) was taken to be 100% and the dark for various periods of time before adding it to JET-GST amount of CRY bound at other time points was expressed relative to this va- lue. The y-axis is in log scale. beads and measuring the level of CRY–JET binding by immuno- blotting. The results of such an experiment are shown in Fig. 6A. ing ubiquitin ligase so that CRY could be marked for proteolysis. As is apparent from this figure, light greatly enhances the CRY– However, in that study the kinetics of conversion of the Lit state JET interaction even when CRY is preirradiated with blue light to the Dark state could not be determined because following the prior to mixing with JET. Importantly, this light-enhanced affinity light flash, cells were incubated at 25 °C, and during incubation in of CRY for JET decays with a half-life of ∼15 min (Fig. 6 A B the dark CRY was continuously being degraded. To overcome this and ). This half-life is essentially the same as the half-life of problem we used the experimental design illustrated in Fig. 7A: blue-light-induced conformational change as probed by proteoly- S2 cells expressing CRY were placed in an ice bath and exposed to sis, indicating that the two observations are intimately linked a camera flash lasting ∼1 ms. The cells were then kept on ice in and reflect different facets of the same phenomenon. In the next the dark for various periods before moving to a 25 °C bath to series of experiments we tested the in vivo implications of these allow enzymatic modification and proteolysis. This approach is observations. based on the fact that whereas photochemical reactions in gen- eral are insensitive to temperature over a wide temperature Lifetime of the Signaling State of CRY in Vivo. Upon exposure of Dro- sophila range, most chemical reactions, including enzymatic reactions, S2 cells to light CRY is proteolytically degraded by the are inhibited at 0 °C. Thus, to determine the half-life of the UPS (Ubiquitin/Proteasome system) (19, 29). It was reported that Lit state of CRY both the control (no light exposure) and the test proteolytic degradation of CRY stopped when the light was (∼1 ms flash) samples were kept on ice and at time intervals BIOCHEMISTRY turned off (23), suggesting that CRY had to be in a photochemi- samples were removed from the ice bath and placed at 25 °C cally excited state for enzymatic modification that ultimately and incubated for 60 min. Then, the S2 cells were lysed and the leads to its proteolysis. However, a subsequent study using cam- proteolytic degradation of CRY was assessed by immunoblotting. era flash photolysis revealed that proteolysis of CRY continues The results of a representative experiment are shown in Fig. 7B for 60 min after the light pulse (30), leading to the conclusion and data points from 2 experiments are plotted in Fig. 7C.Asis that light induces a long-lived signaling state conformation (Lit apparent from this figure, if a flashed sample is kept on ice for the state) that continues to interact with downstream partners includ- duration of the experiment, no CRY degradation occurs (Fig. 7B, lanes 1 and 10). In contrast, if immediately following the flash the cells are incubated at 25 °C for 60 min nearly all CRY is proteo- lyzed (Fig. 7B, lanes 1 and 2). However, the sensitivity of photo- flashed CRY to proteolysis decays in proportion to the time the sample is kept on ice before transfer to 25 °C. From the plot in Fig. 7C we calculate the half-life of the Lit state (signaling state) to be 27 min. This value is in remarkable agreement with the CRY Fig. 5. CRYΔ and light-activated CRY bind to JET similarly in vitro. Recom- Lit state values determined from partial proteolysis (Fig. 4) and binant GST-JET was bound to sepharose beads. Then, FH-CRY or FH-CRYΔ CRY–JET binding (Fig. 6) experiments. Taken together, the were added to the beads that were then incubated at 25 °C for 12.5 min decay data along with previous ultrafast kinetics on CRY (26) 1 −2 either in the dark (D) or under blue light (L, mW cm ). The beads were are consistent with the following reaction scheme: collected and washed three times with 1 mL TBS in the dark. Then, bound – hν proteins were separated on 4 12% SDS-PAGE and visualized by immunoblot- CRY ðFAD•−Þ → CRY ðFAD•−Þ Δ D 15 −1 D ting using anti-Flag antibodies for CRY and CRY and using anti-GST antibo- 10 s Δ dies for JET. Note that CRY (in D or L) binds slightly stronger to JET than CRY → ‡ð •−Þ → ð •−Þ: even after light exposure indicating that under our experimental conditions CRY FAD CRYD FAD 109 s−1 10−3 s−1 only a subpopulation of CRY is activated or a CRY subpopulation loses its ‡ active conformation at a fast rate. Where ðDÞ, (*), and ð Þ represent “dark state,”“excited state,”

Ozturk et al. PNAS ∣ January 11, 2011 ∣ vol. 108 ∣ no. 2 ∣ 519 Downloaded by guest on October 1, 2021 Fig. 7. Decay Kinetics of signaling state of CRY in vivo. Drosophila S2 cells were transfected with vectors expressing CRY-V5H and β-galactosidase-V5H. The cells were split into several culture dishes, and cul- tured in the dark overnight. For the light activation experiment, the dishes were cooled on ice water and were individually exposed to a single camera flash. After the flash, the dishes were incubated for various periods of time on ice in the dark. Each dish was then incubated at 25 °C for 60 min. Then, the cells were collected, and the amounts of β-gal (Control) and CRY were probed by immunoblotting with anti-V5 antibodies. (A) Outline of the experimental design. (B) Results of a representative experiment. The (−) controls were kept in dark for the duration of the experiment. (C) Quantitative analysis of data from two experiments. The data points for the indivi- dual experiments are indicated with squares and tri- angles, respectively. The level of CRY degradation was normalized to β-gal (as a light-insensitive loading con- trol) and is expressed relative to the zero time point that is the sample exposed to a camera flash and im- mediately incubated at 25 °C. The y-axis is in log scale. The light sensitization of CRY to proteolysis is calcu- lated to have a half-life of ∼27 min from this graph.

and “signaling (Lit) state” CRY, respectively, and the values be- cofactor is transmitted through a change in the H-bonding net- low the reaction arrows indicate the approximate values for the work leading to the displacement of an α-helix folded upon the first-order rate constants of the respective reactions. LOV domain (32, 33). The resulting conformational change sig- Some caution is warranted in interpreting light-induced con- nal is transmitted to the rest of the molecule with functional con- formational changes and their mechanistic relevance. For exam- sequences such as inducing the latent activity associated ple, undergoes light-induced photoreduction with an with the photoreceptor polypeptide, changing the quaternary accompanying conformational change (31). However, flavodoxin structure of the photoreceptor and potentially changing its inter- has no known photosensory function. Conversely, not all flavo- acting partners, and the subcellular localization of the protein. proteins that carry out phototosignaling exhibit a detectable In BLUF domain proteins, the FAD excited state singlet that is light-induced conformational change (31). For example, as shown formed by absorption of a blue-light photon abstracts an electron in SI Text, we find that the zebrafish CRY4 does not exhibit any from a nearby Trp residue to form FAD•−, which rapidly takes up light-induced conformational change under the conditions of par- a proton to form FADH•. Finally, a H-atom abstraction from the tial proteolysis used with Drosophila CRY. This is in spite of the latter generates the long-lived (3–2,000 s) flavin that is, like the fact that zebrafish CRY4 is thought to be a photosensory CRYand ground-state flavin, 2-electron oxidized; but now is 10-nm red- the purified protein possesses a full complement of flavin that is shifted relative to the ground-state flavin (28). The formation photoreduced in vitro. The results with zebrafish CRY4 show that of this red-shifted flavin, FADRED, is associated with H-bond re- the light-induced conformational change in Drosophila CRY is arrangement around the FAD binding site and causes a confor- not a response that is common to all CRYs and support the con- mational change in the photoreceptor that may induce a latent clusion that a light-induced conformational change is the me- enzymatic activity (adenylyl cyclase) or alter protein–protein in- chanistic basis for the photosensory role of Drosophila CRY. teractions that are involved in regulation (AppA). However, the greatest support for this conclusion is provided In contrast to the photophysics/photochemistry of LOV and by the structural similarity between the constitutively active BLUF proteins we have only limited knowledge of CRY photo- CRYΔ and the Lit state CRY‡, the light-dependent (CRY) reception/phototransduction mechanisms. If we don’t take into and -independent (CRYΔ) interactions with JET, and in addition account the so-called CRY-DASH proteins, which turned out to the finding that not only is a long-lived, Lit state of CRYobserved be specific for pyrimidine dimers in single-stranded in vitro, but a long-lived, active state of CRY, which is a substrate DNA (34), the current state of knowledge regarding CRYaction for the UPS system, is formed by light in vivo. mechanism might be summarized as follows: (i) Photolyase, which evolutionarily is related to CRY contains the flavin cofac- Discussion tor in the two-electron reduced and deprotonated FADH− form Currently, 3 classes of photosensory flavoproteins are known and carries out catalysis by a nonreductive cyclic electron transfer (12, 28, 31): Cryptochrome, LOV domain photoreceptors, and reaction (9, 35). In contrast, the redox state of FAD in CRY is not BLUF domain photoreceptors. Although CRY was the first fla- known with certainty. Although when and CRYs, vin-based sensory photoreceptor to be discovered, more progress expressed in heterologous systems, are purified they contain has been made in understanding the signaling mechanisms of flavin in the two-electron oxidized state (36, 37) they are readily LOV and BLUF proteins. reduced by light to FADH• (36) and FAD•− (5–7) and there In LOV domain proteins blue light generates an excited singlet are some experimental data indicating that the FADox-form is an state of FMN that by intersystem crossing produces a long-lived artifact generated by exposure to air during purification (8). (ii) (1–2 μs) flavin triplet state. The 3FMN reacts with a cysteine in Some CRYs (Arabidopsis CRY1 and human CRY1 and CRY2) the flavin binding site, leading to the formation of a covalent cy- but not others (Arabidopsis CRY2 and insect Type 1 CRYs) have steinyl-C(4a) adduct of flavin in which the flavin is formally in a autokinase activity (30, 38–40). Furthermore, the crystal structure two-electron reduced state. This adduct formation is accompa- of Arabidopsis CRY1 crystallized in the presence of ATP reveals nied by a blue shift of the absorption maximum from 450 to that the ATP is located in the cavity leading to flavin, which 390 nm. This change in the redox state and the covalent structure corresponds to the photodimer binding site in DNA photolyases of FMN, and the accompanying conformational change of the (41, 42). Whether the autokinase activity is stimulated by light

520 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1017093108 Ozturk et al. Downloaded by guest on October 1, 2021 and whether the ATP detected in the crystal structure is the one the PHR domain in a manner analogous to the N-terminal cap of used for the kinase reaction are not known with any certainty. (iii) the LOV domain photoreceptor VIVID (44, 45). Upon absorp- In Arabidopsis, CRY binds to COP1 E3 ligase (which ubiquiti- tion of a blue-light photon the FAD•− goes through its photophy- nates and inactivates transcription factors) independently of light sical cycle in <1 ns; however, the excited (FAD•−)* doublet or but affects (inhibits) COP1 activity only upon light exposure (15, quartet state causes a modest bending motion around the N5– Drosophila 16). In contrast, in , CRY activates/binds to JET E3 N10 axis (27, 31) leading to a significant change in the H-bonding ligase essentially in a light-dependent manner (20) and light ac- network around the flavin binding site that is transmitted to the tivates the ubiquitination and destruction of CRYand its binding “ ” partner TIM (17). (iv) All bona fide CRYs have C-terminal ex- C-terminal cap, causing opening of the cap (as evidenced by the tensions beyond the photolyase region (PHR), which increased sensitivity of the C-terminal hinge regions to trypsin). range from ∼20 amino acids in Drosophila CRY to ∼250 amino This conformation is relatively stable and it is retained even after acids in Arabidopsis CRY1 (2, 14, 36). (v) Light causes a signifi- the flavin excited state is deactivated by internal conversion in cant conformational change in the C-terminal extensions of Ara- <1 ns. The active (signaling state) conformation of the protein bidopsis CRY1 (14, 43) and Drosophila CRY (this work) as decays with a much longer half-life of approximately 15 min, dur- revealed by the effect of light on the partial proteolysis profile. ing which time CRY does interact with JET strongly as shown (vi) The C-terminal extensions of Arabidopsis CRYs when sepa- here and with TIM as has been shown in previous studies “ ” rated from the PHR domain confer a constitutive light-on phe- (21, 46). These interactions lead to the ubiquitination and even- Drosophila notype (15). In contrast, the CRY PHR domain when tual proteolytic degradation of both CRYand TIM, relieving the separated from the C-terminal 20 amino acid extension confers a repression of clock controlled and thus resetting the clock. constitutive light-on phenotype (21–23). Considering the current knowledge of CRY structure/function Materials and Methods with the findings reported in this paper, we offer the following The plasmids used in this study, protein expression, purification, and immu- model (Model 2) for CRY action mechanism in Drosophila that noblotting as well as detailed description of biochemical and spectroscopic is illustrated in SI Text: The photoreceptor contains the flavin co- experiments are given in SI Text. factor in the FAD•− form and in this form it either does not or only weakly interacts with JET possibly because the C-terminal 20 ACKNOWLEDGMENTS. This work was supported by National Institutes of Health amino acid extension functions as a C-terminal cap folded upon Grant GM31082.

1. Lin C, Shalitin D (2003) Cryptochrome structure and signal transduction. Annu Rev 24. Hemsley MJ, et al. (2007) Linear motifs in the C-terminus of D. melanogaster crypto- Plant Biol 54:469–496. chrome. Biochem Biophys Res Commun 355:531–537. 2. Cashmore AR (2003) : Enabling plants and to determine 25. Ozturk N, et al. (2007) Structure and function of cryptochromes. Cold Spring circadian time. Cell 114:537–543. Harb Symp 72:119–131. 3. Sancar A (2004) Regulation of the mammalian circadian clock by cryptochrome. J Biol 26. Kao YT, et al. (2008) Ultrafast dynamics and anionic active states of the flavin cofactor Chem 279:34079–34082. in cryptochrome and photolyase. J Am Chem Soc 130:7695–7701. 4. Partch CL, Sancar A (2005) Photochemistry and photobiology of cryptochrome 27. Kao YT, et al. (2008) Ultrafast dynamics of flavins in five redox states. J Am Chem Soc blue-light : The search for a photocycle. Photochem Photobiol 130:13132–13139. 81:1291–1304. 28. Kennis JT, Groot ML (2007) Ultrafast of biological photoreceptors. Curr 5. Bouly JP, et al. (2007) Cryptochrome blue light photoreceptors are activated through Opin Struct Biol 17:623–630. – interconversion of flavin redox states. J Biol Chem 282:9383 9391. 29. VanVickle-Chavez SJ, Van Gelder RN (2007) Action spectrum of Drosophila crypto- 6. Berndt A, et al. (2007) A novel photoreaction mechanism for the circadian blue light chrome. J Biol Chem 282:10561–10566. – photoreceptor Drosophila cryptochrome. J Biol Chem 282:13011 13021. 30. Ozturk N, et al. (2009) Comparative photochemistry of animal type 1 and type 4 7. Song SH, et al. (2007) Formation and function of flavin anion radical in cryptochrome 1 cryptochromes. Biochemistry 48:8585–8593. – blue-light photoreceptor of . J Biol Chem 282:17608 17612. 31. Senda T, Senda M, Kimura S, Ishida T (2009) Redox control of protein conformation in 8. Ozturk N, Song SH, Selby CP, Sancar A (2008) Animal type 1 cryptochromes. Analysis of flavoproteins. Antioxid Redox Sign 11:1741–1766. the redox state of the flavin cofactor by site-directed mutagenesis. J Biol Chem 32. Harper SM, Neil LC, Gardner KH (2003) Structural basis of a phototropin light switch. – 283:3256 3263. Science 301:1541–1544. 9. Sancar A (2003) Structure and function of DNA photolyase and cryptochrome

33. Harper SM, Christie JM, Gardner KH (2004) Disruption of the LOV-Jalpha helix BIOCHEMISTRY blue-light photoreceptors. Chem Rev 103:2203–2237. interaction activates phototropin kinase activity. Biochemistry 43:16184–16192. 10. Kao YT, Saxena C, Wang L, Sancar A, Zhong D (2005) Direct observation of thymine 34. Selby CP, Sancar A (2006) A cryptochrome/photolyase class of with single- dimer repair in DNA by photolyase. Proc Natl Acad Sci USA 102:16128–16132. stranded DNA-specific photolyase activity. Proc Natl Acad Sci USA 103:17696–17700. 11. Li J, et al. (2010) Dynamics and mechanism of repair of ultraviolet-induced (6-4) 35. Sancar A (2008) Structure and function of photolyase and in vivo enzymology: 50th photoproduct by photolyase. Nature 466:887–890. anniversary. J Biol Chem 283:32153–32157. 12. Losi A (2007) Flavin-based Blue-Light photosensors: A photobiophysics update. Photo- 36. Lin C, et al. (1995) Association of flavin adenine dinucleotide with the Arabidopsis blue chem Photobiol 83:1283–1300. light receptor CRY1. Science 269:968–970. 13. Lin C, Todo T (2005) The cryptochromes. Genome Biol 6:220. 37. Malhotra K, Kim ST, Batschauer A, Dawut L, Sancar A (1995) Putative blue-light photo- 14. Partch CL, Clarkson MW, Ozgur S, Lee AL, Sancar A (2005) Role of structural plasticity in receptors from and Sinapis alba with a high degree of sequence signal transduction by the cryptochrome blue-light photoreceptor. Biochemistry homology to DNA photolyase contain the two photolyase cofactors but lack DNA 44:3795–3805. repair activity. Biochemistry 34:6892–6899. 15. Yang HQ, et al. (2000) The C termini of Arabidopsis cryptochromes mediate a consti- tutive light response. Cell 103:815–827. 38. Bouly JP, et al. (2003) Novel ATP-binding and autophosphorylation activity associated – 16. Yang HQ, Tang RH, Cashmore AR (2001) The signaling mechanism of Arabidopsis CRY1 with Arabidopsis and human cryptochrome-1. Eur J Biochem 270:2921 2928. involves direct interaction with COP1. Plant Cell 13:2573–2587. 39. Shalitin D, Yu X, Maymon M, Mockler T, Lin C (2003) Blue light-dependent in vivo and – 17. Stanewsky R, et al. (1998) The cryb mutation identifies cryptochrome as a circadian in vitro of Arabidopsis cryptochrome 1. Plant Cell 15:2421 2429. photoreceptor in Drosophila. Cell 95:681–692. 40. Ozgur S, Sancar A (2006) Analysis of autophosphorylating kinase activities of Arabi- – 18. Koh K, Zheng X, Sehgal A (2006) JETLAG resets the Drosophila circadian clock by dopsis and human cryptochromes. Biochemistry 45:13369 13374. promoting light-induced degradation of TIMELESS. Science 312:1809–1812. 41. Brautigam CA, et al. (2004) Structure of the photolyase-like domain of cryptochrome 1 – 19. Naidoo N, Song W, Hunter-Ensor M, Sehgal A (1999) A role for the proteasome in the from Arabidopsis thaliana. Proc Natl Acad Sci USA 101:12142 12147. light response of the timeless clock protein. Science 285:1737–1741. 42. Park HW, Kim ST, Sancar A, Deisenhofer J (1995) Crystal structure of DNA photolyase 20. Peschel N, Chen KF, Szabo G, Stanewsky R (2009) Light-dependent interactions from Escherichia coli. Science 268:1866–1872. between the Drosophila circadian clock factors cryptochrome, jetlag, and timeless. 43. Yu X, et al. (2007) Derepression of the NC80 motif is critical for the photoactivation of Curr Biol 19:241–247. Arabidopsis CRY2. Proc Natl Acad Sci USA 104:7289–7294. 21. Rosato E, et al. (2001) Light-dependent interaction between Drosophila CRY and the 44. Zoltowski BD, et al. (2007) Conformational switching in the fungal light sensor Vivid. clock protein PER mediated by the carboxy terminus of CRY. Curr Biol 11:909–917. Science 316:1054–1057. 22. Dissel S, et al. (2004) A constitutively active cryptochrome in Drosophila melanogaster. 45. Zoltowski BD, Vaccaro B, Crane BR (2009) Mechanism-based tuning of a LOV domain Nat Neurosci 7:834–840. photoreceptor. Nat Chem Biol 5:827–834. 23. Busza A, Emery-Le M, Rosbash M, Emery P (2004) Roles of the two Drosophila CRYP- 46. Ceriani MF, et al. (1999) Light-dependent sequestration of TIMELESS by CRYPTO- TOCHROME structural domains in circadian photoreception. Science 304:1503–1506. CHROME. Science 285:553–556.

Ozturk et al. PNAS ∣ January 11, 2011 ∣ vol. 108 ∣ no. 2 ∣ 521 Downloaded by guest on October 1, 2021