Structure of full-length Drosophila cryptochrome

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The cryptochrome/photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to ultraviolet and blue light exposure in all kingdoms of life1, 2, 3, 4, 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs) and 6-4 photolesions caused by ultraviolet 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 catalysed 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 carboxy-terminal tail that appends the conserved PL homology domain (PHD) and is important for function7, 8, 9, 10, 11, 12. Here, 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 centre 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.

At a glance


  1. dCRY resembles 6-4 PL with the C-terminal tail replacing the DNA substrate.
    Figure 1: dCRY resembles 6-4 PL with the C-terminal tail replacing the DNA substrate.

    a, Comparison of dCRY and 6-4 dPL. N-terminal α/β domain (blue) is coupled to the C-terminal helical domain (yellow) through a long linker (grey). In dCRY a C-terminal helix (red) docks to the PL DNA binding cleft besides the flavin (black). b, Close-up of C-terminal helix recognition groove. Trp536 of the C-terminal helix (red) juts into what would be the 6-4 PL catalytic centre adjacent to FAD. c, Superposition of 6-4 photolyase and dCRY active site with bound pyrimidine dimer ‘Dewar’ lesion, which binds in a similar position relative to FAD and His378 as Trp536 in dCRY. d, Surface and chemical complementarity between the C-terminal helix (white) and the PHD (yellow) flavin pocket (blue).

  2. Structural motifs that define Type 1 CRYs.
    Figure 2: Structural motifs that define Type 1 CRYs.

    Binding of the C-terminal tail into the dCRY active centre couples to structures of three loop regions unique to Type I CRYs. The protrusion motif, phosphate binding loop and C-terminal lid create a cavity to bind the CTT. Compared to 6-4 PL (blue), a β-strand shift in dCRY (yellow), displaces the YLP motif (Supplementary Fig. 9) 20Å from the adenosine-interacting Lys of 6-4 PL. A six-residue insertion (magenta) in the phosphate loop leaves the C-term binding groove in an open conformation. dCRY has a two-turn extension of the α8 helix leading to the protrusion motif. The C-terminal linker (red) makes close contacts with an inserted Ser-rich loop: the C-terminal lid (residues 424–432).

  3. Cofactor binding regions of dCRY.
    Figure 3: Cofactor binding regions of dCRY.

    a, E. coli CPD PL binds MTHF as an antenna cofactor. b, In dCRY few of the residues that interact with MTFH in CPD PL are present, including the Glu residue that recognizes the pterin ring or otherwise contact MTHF (E. coli PL Glu109right arrowdCRY Trp114, Asn108right arrowIle113, His44right arrowGly50). Loop regions surrounding the cofactor in dCRY and 6-4 PL also have different structures and compositions (for example, 6-4 PL Ile50right arrowdCRY Glu45, Leu51right arrowSer46, Leu40right arrowPhe42, Met54right arrowGly50). c, Flavin centre of dCRY (yellow) compared to 6-4 PL (orange). dCRY Cys416 replaces 6-4 PL Asn403 and is in hydrogen bonding distance (dotted lines) of both N5 and O4 of FAD. The linkage between the ring and ribose is altered in dCRY by semiquinone formation (black arrow).

  4. Redox active groups and conformations in dCRY.
    Figure 4: Redox active groups and conformations in dCRY.

    a, Trp triad photoreduction pathway (Trp420, Trp397 and Trp342) in dCRY (yellow) and Drosophila 6-4 photolyase (cyan). Trp536 resides as close to FADox as Trp420. 6-4 dPL conserves all three Trp residues, and also contains surface residue Trp394. Alternative electron transport pathways involving Trp314, Trp422 and Trp536 are possible in dCRY. The Trp422 indole flips relative to 6-4 dPL, but similar to CPD PLs. b, Conformational variation in the C-terminal peptide orientations of the two subunits (yellow and white) in the dCRY active centre. Asp539 hydrogen bonds back to the CTT in one molecule (white), but displaces from the pocket in the other (inset).

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Protein Data Bank


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  1. Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA

    • Brian D. Zoltowski,
    • Anand T. Vaidya,
    • Joanne Widom &
    • Brian R. Crane
  2. Laboratory of Genetics, The Rockefeller University, New York, New York 10065, USA

    • Deniz Top &
    • Michael W. Young


B.R.C., B.D.Z., A.T.V., D.T. and M.W.Y. designed the project. J.W. cloned, expressed and purified dCRY, B.D.Z. and A.T.V. purified and crystallized dCRY and collected diffraction data. B.D.Z. and B.R.C. determined the structure. D.T. and M.W.Y. performed CRY stability studies. A.T.V. performed ITC experiments. B.D.Z. and B.R.C. wrote the manuscript and all authors provided editorial input.

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Atomic coordinates for the reported crystal structures have been deposited with the Protein Data Bank under accession code 3TVS.

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  1. Supplementary Information (1.6M)

    This file contains Supplementary Text and Data, Supplementary Figures 1-12 with legends, Supplementary Tables 1-2, Supplementary Materials and Methods and additional references (see contents for details).

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