A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome

Abstract

Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FADox) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FADox to the red anionic FAD⋅¯ radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FADox corresponds to the ground state, whereas the FAD⋅¯ radical represents the light-activated state that mediates resetting of the Drosophila circadian clock. Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FADox) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FADox to the red anionic FAD⋅¯ radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FADox corresponds to the ground state, whereas the FAD⋅¯ radical represents the light-activated state that mediates resetting of the Drosophila circadian clock. Cryptochromes (CRYs) 4The abbreviations used are: CRY, cryptochrome; FAD, flavine adenine dinucleotide; MTHF, methenyltetrahydrofolate; β-ME, β-mercaptoethanol; CPD, cyclobutane pyrimidine dimer; PHR, photolyase homology region; RP, reversed phase; HPLC, high pressure liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; CHCA, cyano-4-hydroxycinnamic acid. constitute a family of flavoproteins that use flavin adenine dinucleotide (FAD) and sometimes an additional pterin derivative (methenyltetrahydrofolate, MTHF) as noncovalently bound cofactors and blue light absorbing chromophores (1Cashmore A.R. Jarillo J.A. Wu Y.J. Liu D. Science. 1999; 284: 760-765Crossref PubMed Scopus (810) Google Scholar). CRYs share moderate sequence, but significant structural homology with DNA photolyases, which repair UV-damaged DNA in a blue light dependent manner (2Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar). Cyclobutane pyrimidine dimer (CPD) photolyases repair UV light-induced pyrimidine-dimer (Pyr<>Pyr) DNA lesions by intermolecular redox reactions between the catalytically active fully reduced flavin chromophore (FADH-) and the Pyr<>Pyr substrate (2Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar). A similar reaction mechanism is presumed for the (6-4)-photolyases, which repair pyrimidine-pyrimidone (6Lin C. Todo T. Genome Biol. 2005; 6: 220Crossref PubMed Scopus (274) Google Scholar, 4Zhao X. Liu J. Hsu D.S. Zhao S. Taylor J.S. Sancar A. J. Biol. Chem. 1997; 272: 32580-32590Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) photoproducts (Pyr (6Lin C. Todo T. Genome Biol. 2005; 6: 220Crossref PubMed Scopus (274) Google Scholar, 4Zhao X. Liu J. Hsu D.S. Zhao S. Taylor J.S. Sancar A. J. Biol. Chem. 1997; 272: 32580-32590Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) Pyr), a second class of UV light-induced DNA lesions (3Hitomi K. Kim S.T. Iwai S. Harima N. Otoshi E. Ikenaga M. Todo T. J. Biol. Chem. 1997; 272: 32591-32598Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 4Zhao X. Liu J. Hsu D.S. Zhao S. Taylor J.S. Sancar A. J. Biol. Chem. 1997; 272: 32580-32590Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Cryptochromes do not exhibit any DNA repair activities, despite significant sequence homology of plant cryptochromes to CPD-photolyases and animal cryptochromes to (6-4)-photolyases (5Partch C.L. Sancar A. Photochem. Photobiol. 2005; 81: 1291-1304Crossref PubMed Scopus (105) Google Scholar, 6Lin C. Todo T. Genome Biol. 2005; 6: 220Crossref PubMed Scopus (274) Google Scholar). Blue light absorption, phosphorylation, and effector interactions of plant cryptochromes control fundamental biological processes such as de-etiolation and flowering onset (7Sullivan J.A. Deng X.W. Dev. Biol. 2003; 260: 289-297Crossref PubMed Scopus (172) Google Scholar). Action spectra (8Ahmad M. Grancher N. Heil M. Black R.C. Giovani B. Galland P. Lardemer D. Plant Physiol. 2002; 129: 774-785Crossref PubMed Scopus (172) Google Scholar) and spectroscopic studies (9Zeugner A. Byrdin M. Bouly J.P. Bakrim N. Giovani B. Brettel K. Ahmad M. J. Biol. Chem. 2005; 280: 19437-19440Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 10Kottke T. Batschauer A. Ahmad M. Heberle J. Biochemistry (Mosc.). 2006; 45: 2472-2479Crossref Scopus (99) Google Scholar) on Arabidopsis thaliana cryptochrome 1 (AtCRY1) suggest that its native and functionally active chromophore is oxidized FAD (FADox), in contrast to photolyases, where the active chromophore is the two electron-reduced FADH-. However, in both AtCRY1 (11Giovani B. Byrdin M. Ahmad M. Brettel K. Nat. Struct. Biol. 2003; 10: 489-490Crossref PubMed Scopus (242) Google Scholar, 12Lin C. Robertson D.E. Ahmad M. Raibekas A.A. Jorns M.S. Dutton P.L. Cashmore A.R. Science. 1995; 269: 968-970Crossref PubMed Scopus (377) Google Scholar) and photolyases (2Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar), blue light activation leads to the formation of a neutral blue FADH·radical. Whereas in AtCRY1 FADox is photoreduced to FADH·upon blue light illumination, the FADH·radical in photolyases is produced after a blue light-activated electron transfer from FADH- to the Pyr<>Pyr substrate. Animal cryptochromes, either as photoreceptors or integral components of biological clocks, play crucial roles in the generation of 24-h (circadian) rhythms or their synchronization with the environmental light-dark cycle (6Lin C. Todo T. Genome Biol. 2005; 6: 220Crossref PubMed Scopus (274) Google Scholar, 13Cashmore A.R. Cell. 2003; 114: 537-543Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Drosophila cryptochrome (dCRY) is a blue light photoreceptor mediating light synchronization of the circadian clock (14Stanewsky R. Kaneko M. Emery P. Beretta B. Wager-Smith K. Kay S.A. Rosbash M. Hall J.C. Cell. 1998; 95: 681-692Abstract Full Text Full Text PDF PubMed Scopus (778) Google Scholar, 15Emery P. So W.V. Kaneko M. Hall J.C. Rosbash M. Cell. 1998; 95: 669-679Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). The Drosophila clock is operated by a transcriptional and translational feedback loop in which the clock proteins Period and Timeless (dTIM) inhibit their own transcription by negatively regulating the transcription factors dClock and dCycle (16Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Crossref PubMed Scopus (932) Google Scholar). dCRY resets the Drosophila clock by sequestering dTIM from the feedback loop through light-dependent dCRY-dTIM interactions (17Ceriani M.F. Darlington T.K. Staknis D. Mas P. Petti A.A. Weitz C.J. Kay S.A. Science. 1999; 285: 553-556Crossref PubMed Scopus (450) Google Scholar), which trigger the of dTIM N. M. A. Science. 1999; 285: PubMed Scopus Google Scholar) and dCRY E. N. A. Cell. Biol. 2001; PubMed Scopus Google Scholar). The dCRY a photolyase homology and a to as to its effector dTIM and light to the clock is by the of The the dCRY-dTIM and dCRY in the E. A. M. R. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. M. Rosbash M. Emery P. Science. PubMed Scopus Google Scholar). A light-induced change, by redox to to the and to for dCRY-dTIM interactions and dTIM and dCRY spectroscopic on the blue light of dCRY or any other animal cryptochrome are the of dCRY as a circadian blue light we have established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. The chromophore of the is by UV-visible absorption and spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass and reversed phase high pressure liquid We show that insect cell dCRY contains FAD in its oxidized state (FADox) and residual amounts of dCRY undergoes a reversible absorption blue light irradiation, which is assigned to the conversion of FADox to an anionic FAD⋅¯ radical. findings lead us to propose a novel photoreaction mechanism for dCRY, which is to have crucial for the resetting mechanism of the Drosophila circadian clock. Drosophila cryptochrome in Sf21 insect the A dCRY an by a for of the the the and a recombinant dCRY with a the to a mass of DNA after a in the and to the recombinant 1 Sf21 The after two the high with and used for and as in with and expression in a 1 of with the an of and in expression established in The and the with in of A and in liquid to of purification of dCRY, from of expression on and in A with and 1 The a with a and the for The a with A. The by a from to A to and on a dCRY in a of dCRY to to an with a mass The to on a a and a used with a and and monomeric dCRY to and in liquid The UV-visible absorption spectra of dCRY an with of The dCRY a of spectra on a with a of a for the spectra and a for the The of the chromophore a by spectra an of and spectra an of dCRY in a with a in and 1 of this on a and to The a with and a laser in the mass the with and in a with a in mass with the of dCRY for and for The the chromophores to the in A. T. M. Wu M. S. A. 2005; PubMed Scopus Google with a and a reversed phase chromophores and by absorption the chromophore a dCRY and to The of FAD by the with an FAD and which do not for by of UV-visible absorption spectra a UV with a of The of the to by a A with an of blue light the dCRY photoreduced by blue light for The of formation by the of the from that after of The of an in the Biochemistry (Mosc.). Scopus Google Scholar). of dCRY after a with blue a to not that not to the a used that in an of the The two the but of the to the dCRY in and produced by a of the for by through a from in and of chromophore by a with a and photoreduced with blue light for a of of dCRY by the A. J. Biol. PubMed Scopus Google Scholar) the of E. photolyase Kim S.T. Sancar A. J. Science. 1995; PubMed Scopus Google Scholar) and of from A. thaliana R. C.L. C.L. J. S. A. 2006; 103: PubMed Scopus Google Scholar) used as of the of of dCRY and with the R.C. PubMed Scopus Google Scholar) and to the of on the 1998; PubMed Scopus Google Scholar). FAD and MTHF, in the between the and by a of the the with between the and the of the The by a of with of the with the S. M. J. Chem. Scopus Google Scholar) the for and of dCRY from Sf21 Drosophila cryptochrome to an and from insect a expression cell and the from of insect cell expression on a purification dCRY by to other that to of which of the of and to and The purification scheme in of mg of of cell and The of the by mass not with proteins that dCRY as a of of the dCRY that the photoreceptor contains FAD and MTHF, as in other family (2Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar, C.L. Sancar A. Photochem. Photobiol. 2005; 81: 1291-1304Crossref PubMed Scopus (105) Google Scholar). We have the chromophore of dCRY by absorption and as as MALDI-TOF mass and spectra of dCRY show and with the of oxidized FAD (2Sancar A. Chem. 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B. 2006; PubMed Scopus Google Scholar). of MTHF, spectra for and spectra for The spectra a and a Whereas the the of FAD, the is of residual amounts of MTHF, as for Sancar A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). However, this not in other dCRY and significant in spectra for We that a of the insect cell-purified dCRY contains light-induced absorption of dCRY are assigned to the conversion of oxidized FAD to the red anionic FAD radical. light activation of dCRY and in of as electron for with blue light are in the and show the formation of the red anionic FAD radical from the oxidized state spectra for oxidized and anionic radical of flavin in with reaction scheme for the conversion of oxidized FAD to the anionic FAD radical blue light of dCRY and its spectra spectra and in the and of β-ME, are of the reaction FADox is to the anionic FAD radical of FADox as of The with blue light for Whereas the of FAD radical generation on the of and red and blue the of dCRY on the of and red and blue for are for or or of the with of the other with and of the do not spectra of dCRY its chromophore flavin of dCRY The an of and the an of The of FAD in the is for The spectra an of The the of which is to oxidized FAD and Sancar A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google additional for the of MTHF, we MALDI-TOF on the dCRY The mass spectra show a for FAD mass and for mass a for is mass and a is for mass that is to The with dCRY is therefore to with the of its K. J.C. 6: PubMed Scopus Google Scholar). that of the to of or its and mass is However, on we the of mass of is to that of The is assigned to the The not the and of the cofactors that are noncovalently bound to dCRY, we have the and the chromophore after a to The that dCRY contains FAD, but or that the is to the of of the of the FAD from dCRY with an FAD that of the dCRY are with The not or other that are in Blue of FAD to an FAD oxidized FAD a blue light with an and an of Blue light in a absorption with and and a but absorption between and the FADox absorption and reduced with Biochemistry (Mosc.). Scopus Google Scholar) and with spectra of photoreduced the blue light-induced as an anionic red FAD radical contrast to photolyases (2Sancar A. Chem. Rev. 2003; 103: 2203-2237Crossref PubMed Scopus (1042) Google Scholar) and AtCRY1 T. Batschauer A. Ahmad M. Heberle J. Biochemistry (Mosc.). 2006; 45: 2472-2479Crossref Scopus (99) Google Scholar, B. Byrdin M. Ahmad M. Brettel K. Nat. Struct. Biol. 2003; 10: 489-490Crossref PubMed Scopus (242) Google Scholar), neutral blue FADH·radical is The neutral FADH·radical to significant absorption or and as for E. DNA photolyase M.S. Biochemistry (Mosc.). Scopus Google Scholar). The and absorption of blue light dCRY between and is not to residual amounts of the neutral FADH·radical but represents a of anionic FAD⋅¯ Biochemistry (Mosc.). Scopus Google Scholar). is in the of the which the anionic the of Biochemistry (Mosc.). Scopus Google Scholar). with the of other flavoproteins in two either neutral or anionic flavin of Biochemistry (Mosc.). Scopus Google Scholar). for dCRY by the of the from to and the light-induced spectra of dCRY a in between and not The of and that the reaction of dCRY between the two FADox and fully reduced FAD is in the reaction cycle. the light activation mechanism of dCRY an electron transfer to the oxidized FAD the reaction in the and of as an electron as as of the absorption spectra that reaction the is The and and the and are assigned to the generation of the anionic red FAD whereas the and are to the of oxidized and in the of β-ME, the conversion of oxidized FAD to the anionic FAD radical is to after The reaction not fully after in of leads to a conversion of oxidized FAD to the radical. in of β-ME, the anionic FAD⋅¯ radical is FADox is to the anionic FAD radical in the of and and the from the of Biochemistry (Mosc.). Scopus Google Scholar). The of leads to a conversion of FADox to FAD⋅¯ that the reaction in or of contrast to the the reaction not on the of but on the of of the to FADox with a of the other with a of and of the with the of for blue light activation of dCRY is by the that as electron the reaction of FADox to FAD⋅¯ electron whereas the reaction of light the of the circadian blue light photoreceptor dCRY, we have established the expression and purification of dCRY from Sf21 insect which are related to the native Drosophila dCRY as a in that as a in A. thaliana (AtCRY1) is to in which are by the J. Deng X.W. Plant Cell. 2005; PubMed Scopus Google Scholar). Our dCRY contains oxidized FAD and residual amounts of the chromophore and dCRY in E. to oxidized FAD, whereas not established S. S. M. K. A. Photochem. Photobiol. 1999; PubMed Scopus Google Scholar, Sancar A. Photochem. 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Biochemistry (Mosc.). 2006; 45: 2472-2479Crossref Scopus (99) Google Scholar). the similar spectra and the FADox ground for both the activation mechanism of dCRY is from that of FADox is the red anionic FAD⋅¯ radical in dCRY as to the blue neutral FADH·radical in is that a photoreaction in dCRY from the anionic radical state, which the residual to light this with is We from that the two electron-reduced state of flavin (FADH-) is the of the reaction as in the of DNA The dCRY spectra are from of the DNA photolyases, where the DNA repair is in the of to of the absorption Sancar A. Biochemistry (Mosc.). Scopus Google Scholar). a light of by of in the leads to a of but not dCRY in Drosophila and in A. M. Rosbash M. Emery P. Science. PubMed Scopus Google Scholar). both proteins in the light with similar of dCRY to to a in and therefore light for We propose that the blue light-induced conversion of FADox to the anionic FAD⋅¯ radical leads to the dCRY the that trigger dCRY-dTIM dTIM is by a mechanism that and N. M. A. Science. 1999; 285: PubMed Scopus Google Scholar). its light-activated state, dCRY is of the of the which dCRY in the in and A. M. Rosbash M. Emery P. Science. PubMed Scopus Google Scholar). to the oxidized FAD state in the its UV-visible spectroscopic on dCRY have two dCRY which to the FADox with of and this we the of two dCRY of dCRY or of the However, both with the of dCRY in and A. M. Rosbash M. Emery P. Science. PubMed Scopus Google Scholar), that additional factors the in The reaction of dCRY with DNA photolyases (2Sancar A. Chem. 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Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). the of a of dCRY, we additional for electron or of blue light to the and effector However, suggest a photoreaction mechanism for dCRY that is from class and to the dCRY photoreaction mechanism is for other animal We A. B. J. A. R. and for with spectroscopic and We are to A. and for and We are to T. and A. for and in insect cell expression. We A. and P. for on the The a from R.