Identification of a New Cryptochrome Class Ronald Brudler, Kenichi Hitomi, Hiromi Daiyasu, Hiroyuki Toh, Ken-ichi Kucho, Masahiro Ishiura, Minoru Kanehisa, Victoria A. Roberts, Takeshi Todo, John A. Tainer, Elizabeth D. Getzoff  Molecular Cell  Volume 11, Issue 1, Pages 59-67 (January 2003) DOI: 10.1016/S1097-2765(03)00008-X

Figure 1 Unrooted Phylogenetic Tree of 12 Representative Members of the Cryptochrome/Photolyase Protein Family The tree was constructed by the maximum likelihood method. The difference in AIC (Akaike information criterion) between the maximum likelihood tree and the second best tree is >1.0. Therefore, the maximum likelihood tree can be distinguished from other possible trees with statistical significance. The proteins were selected to reflect the clustering in the neighbor-joining tree (see Supplemental Data at http://www.molecule.org/cgi/content/full/11/1/59/DC1) constructed with 54 family members. The new cluster cryptochrome DASH is highlighted in red. The numbers at the nodes indicate the bootstrap probability for the clustering of sequences at the node. The scale bar corresponds to 0.1 amino acid substitutions per site. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)

Figure 2 Overall Fold of Synechocystis sp. PCC6803 Cryptochrome α helices and 310 helices, blue; β strands, green; loops, gray. The N-terminal α/β domain (upper right) and C-terminal helical domain (lower left) are connected by a long interdomain loop. The FAD cofactor (oxygen, red; nitrogen, blue; phosphorus, yellow balls) is shown with yellow bonds and binds in the cavity between the two lobes of the helical domain. The N- and C termini are labeled. From this viewpoint, the protein is about 85 Å high, 47 Å wide, and 45 Å deep. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)

Figure 3 Cryptochrome DASH Sequence Conservation, Fold, and Residue Function The sequences of cryptochrome DASH from Synechocystis sp. PCC6803 and A. thaliana were aligned to the CPD photolyase from E. coli on the basis of the crystallographic structures. Boxes around the sequences indicate the α/β (green) and helical domains (blue). Secondary structure elements are shown above the sequence (dark blue, α helices; light blue, 310 helices; green, β strands; black dots every tenth residue). Highlighted amino acids act in binding FAD (green) and MTHF (5,10-methenyltetrahydrofolate) (yellow). The regions where Synechocystis cryptochrome and E. coli photolyase differ most (deviation of Cα positions > 2.8 Å) are shown by dashed red lines. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)

Figure 4 FAD Binding Site of Cryptochrome DASH (A) Electrostatic potential surface of Synechocystis sp. cryptochrome surrounding the FAD (yellow) binding cavity. The surface was calculated with the program UHBD (Gilson et al., 1993) and colored from −4 to +4 kcal/mol·e according to the color bar. This view, looking into the FAD binding cavity, is generated by counterclockwise rotation of the model in Figure 2 by 60° around the long z axis of the protein, and then 50° clockwise rotation around the x axis. (B) Conserved electron transfer chain. Green dots indicate shortest edge-to-edge distances between FAD, Trp396, Trp373, and Trp320. The FAD cofactor and Trp residues are shown together with the 1.9 Å resolution 3Fobs - 2Fcalc omit electron density map contoured at 1σ. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)

Figure 5 Cryptochrome DASH Specificity (A) Stereo view of the superimposed active site cavities of Synechocystis cryptochrome and E. coli photolyase with docked thymine dimer. FAD is colored in black (cryptochrome) and gray (photolyase), the thymine dimer model in orange, and functionally important amino acids of the photolyase and cryptochrome in yellow and green, respectively. The proteins were superimposed on the Cα atoms of these residues. The cryptochrome protein backbone is shown in blue tubes. The thymine dimer model is taken from Medvedev and Stuchebrukhov (2001) with kind permission of the authors. (B) Cryptochrome DASH-specific amino acids (Phe148, Leu153, Arg161, Tyr226, Phe227, Thr239, Gly242, Tyr248, Ser249, Lys251, Phe252, Trp285, Phe288, Phe308, Gln359, Gly370) are shown in red and clustered around the FAD (yellow) binding site. Orientation as in Figure 2. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)

Figure 6 Functional Analysis of Cryptochrome DASH from Synechocystis sp. PCC6803 (A) DNA binding shown with gel shift assay. Lane 1, 32P-labeled DNA; 2, 32P-labeled DNA + 2.5 μM cryptochrome; 3 (control), 32P-labeled DNA + 2.5 μM bovine serum albumin; 4 (control), 32P-labeled DNA + 2.5 μM cryptochrome + 10 times unlabeled DNA. Bands corresponding to free DNA are indicated by an open arrow head, DNA-protein complexes by a filled arrow head. (B) Effects of cryptochrome DASH gene disruption on whole genome expression profiles. The horizontal axis represents the expression level of each gene in the wild-type as normalized signal intensity. The vertical axis represents the ratio between the expression level of each gene in the Δcry mutant and the wild-type. Dashed lines correspond to 2- or 0.5-fold differences between mutant and wild-type. Open circles indicate genes whose expression levels were more than 2-fold higher in the Δcry mutant than in wild-type (Table 2). (C) Northern blot analysis of Synechocystis gene slr0364. 10 μg of total RNA isolated from wild-type (WT) and the cryptochrome DASH-deficient mutant (Δcry) were loaded. Equal loading of RNA was confirmed by bands of the 23S and 16S rRNA stained with ethidium bromide (bottom). Expression of a 16 kb slr0364-containing transcript was 3.7-fold higher in the Δcry mutant than in wild-type as indicated below the blot. Molecular Cell 2003 11, 59-67DOI: (10.1016/S1097-2765(03)00008-X)