title: Mechanism of photosignaling by Drosophila cryptochrome: role of the redox status of the flavin chromophore.
abstract: Cryptochrome (CRY) is the primary circadian photoreceptor in Drosophila. Upon light absorption, dCRY undergoes a conformational change that enables it to bind to Timeless (dTIM), as well as to two different E3 ligases that ubiquitylate dTIM and dCRY, respectively, resulting in their proteolysis and resetting the phase of the circadian rhythm. Purified dCRY contains oxidized flavin (FADox), which is readily photoreduced to the anionic semiquinone through a set of 3 highly conserved Trp residues (Trp triad). The crystal structure of dCRY has revealed a fourth Trp (Trp-536) as a potential electron donor. Previously, we reported that the Trp triad played no role in photoinduced proteolysis of dCRY in Drosophila cells. Here we investigated the role of the Trp triad and Trp-536, and the redox status of the flavin on light-induced proteolysis of both dCRY and dTIM and resetting of the clock. We found that both oxidized (FADox) and reduced (FAD) forms of dCRY undergo light-induced conformational change in vitro that enable dCRY to bind JET and that Trp triad and Trp-536 mutations that block known or presumed intraprotein electron transfer reactions do not affect dCRY phototransduction under bright or dim light in vivo as measured by light-induced proteolysis of dCRY and dTIM in Drosophila S2R+ cells. We conclude that both oxidized and reduced forms of dCRY are capable of photosignaling.
authors.affiliation: From the Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 and.
authors.forename: Nuri
authors.initials: N
authors.lastname: Ozturk
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Selby
authors.forename: Dongping
authors.initials: D
authors.lastname: Zhong
authors.forename: Aziz
authors.initials: A
authors.lastname: Sancar
mesh_terms: D000818:Animals; D056931:Cryptochromes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D004583:Electrons; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D008027:Light; D055537:Light Signal Transduction / Q000528:radiation effects; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction / Q000528:radiation effects; D011487:Protein Conformation; D059748:Proteolysis / Q000528:radiation effects; D061987:Sf9 Cells; D014364:Tryptophan / Q000235:genetics
chemical_list: D056931:Cryptochromes; D029721:Drosophila Proteins; D005136:Eye Proteins; D005415:Flavins; D050505:Mutant Proteins; C119828:cry protein, Drosophila; C096445:tim protein, Drosophila; D005182:Flavin-Adenine Dinucleotide; D014364:Tryptophan
keywords: Circadian Clock; Circadian Clock Resetting; Cryptochrome; Drosophila; Flavoproteins; Photoreceptor; Protein Conformation; Signal Transduction; Timeless Degradation