Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana

Abstract

Signals generated by cryptochrome (CRY) blue-light photoreceptors are responsible for a variety of developmental and circadian responses in plants. The CRYs are also identified as circadian blue-light photoreceptors in Drosophila and components of the mammalian circadian clock. These flavoproteins all have an N-terminal domain that is similar to photolyase, and most have an additional C-terminal domain of variable length. We present here the crystal structure of the photolyase-like domain of CRY-1 from Arabidopsis thaliana. The structure reveals a fold that is very similar to photolyase, with a single molecule of FAD noncovalently bound to the protein. The surface features of the protein and the dissimilarity of a surface cavity to that of photolyase account for its lack of DNA-repair activity. Previous in vitro experiments established that the photolyase-like domain of CRY-1 can bind Mg.ATP, and we observe a single molecule of an ATP analog bound in the aforementioned surface cavity, near the bound FAD cofactor. The structure has implications for the signaling mechanism of CRY blue-light photoreceptors.

Fig. 1.

Structure of CRY1-PHR and its disulfide bond. (A) The structure of CRY1-PHR. Green, helices; purple, β-strands; dark blue, loop regions; orange, FAD cofactor; light blue, AMP-PNP, which is not bound in the native structure. (B) The disulfide bond in CRY1-PHR. The side chains of Cys-80 and Cys-190 are shown, with the carbons in dark blue and the sulfurs in yellow. Superimposed is a simulated-annealing omit map (F_o - F_c, contoured at 3 σ) (28). Figs. 1 and 3 were generated by using pymol.

Fig. 2.

Surface features near to the FAD-access cavity. Shown are the surfaces of CRY1-PHR (A) and photolyase (B). The electrostatic potential is color-coded on the surface, with red and blue representing areas of negative and positive electrostatic potential, respectively. White line, boundary of the FAD-access cavities in both parts. Figs. 2 and 5C were generated by using grasp (29).

Fig. 3.

Comparison of the aligned FAD-access cavities of CRY1-PHR and E. coli photolyase. Residues that are different between the solvent-exposed linings of the FAD-access cavities of the two proteins are shown, except for CRY1-PHR residue Ser-293 (photolyase residue Glu-274). Green carbons and labels, residues from CRY1-PHR; brown carbons and labels, residues from photolyase; red, oxygen atoms; blue, nitrogen atoms; yellow, sulfur atom. The secondary structure and the FAD cofactors are shown faded for clarity.

Fig. 4.

In vitro AMP-PNP binding to CRY1-PHR. Circles, individual data points; black line, a nonlinear least-squares fit to these data by using the equation in Materials and Methods. The K_A for the binding is 13300 ± 4700 M^-1, and n (the stoichiometry of binding) is 1.01 ± 0.15.

Fig. 5.

AMP-PNP binding to CRY1-PHR. (A) Stereo representation of the electron density of the AMP-PNP bound to CRY1-PHR. The final refined coordinates for AMP-PNP are shown, colored as in Fig. 3 with the following changes: yellow, carbon atoms; pink, phosphorus atoms. Superimposed on these coordinates is a simulated-annealing omit map (F_o - F_c, contoured at 3 σ) (28). This part of the figure was generated by using xtalview (25) and rendered with povray (www.povray.org). (B) The AMP-PNP-binding site. Shown is a ball-and-stick representation of the final refined coordinates of AMP-PNP and nearby protein residues. Dashed lines, hydrogen bonds. Atoms are colored as in A, with the following exceptions: green, carbons belonging to the protein; silver, carbons from the FAD. Distances are given in Ångströms. Both hydrogen bonds from Arg-360 to the β-phosphate of the AMP-PNP measure 3.4 Å. This part of the figure was generated in vmd (33) and rendered with povray. (C) AMP-PNP binding in the FAD-access cavity. The surface is the same as in Fig. 2. The AMP-PNP, shown as spheres, is colored as in A. A bound Mg²⁺ cation is not shown.