Abstract
Cryptochromes (CRYs) are blue-light receptors in plants that harbor FAD as a cofactor and regulate various physiological responses. Photoactivated CRYs undergo oligomerization, which increases the binding affinity to downstream signaling partners. Despite decades of research on the activation of CRYs, little is known about how they are inactivated. Binding of blue-light inhibitors of cryptochromes (BICs) to CRY2 suppresses its photoactivation, but the underlying mechanism remains unknown. Here, we report crystal structures of CRY2N (CRY2 PHR domain) and the BIC2–CRY2N complex with resolutions of 2.7 and 2.5 Å, respectively. In the BIC2–CRY2N complex, BIC2 exhibits an extremely extended structure that sinuously winds around CRY2N. In this way, BIC2 not only restrains the transfer of electrons and protons from CRY2 to FAD during photoreduction but also interacts with the CRY2 oligomer to return it to the monomer form. Uncovering the mechanism of CRY2 inactivation lays a solid foundation for the investigation of cryptochrome protein function.
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Acknowledgements
We thank the staff of the BL17U1/BL19U1/BL19U2 beamline of NCPSS at the Shanghai Synchrotron Radiation Facility for assistance during data collection and research associates at the Center for Protein Research, Huazhong Agricultural University for technical support. This work was supported by funds from the Ministry of Science and Technology of China (2018YFA0507700), the National Natural Science Foundation of China (31722017 and 31870753), the Fok Ying-Tong Education Foundation (151021) and the Fundamental Research Funds for the Central Universities (2662017PY031).
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P.Y. conceived the project. L.M., X.W. and P.Y. designed all experiments. L.M., X.W., L.W. and L.Z. performed protein purification and crystallization. Z. Guan determined all of the structures. L.M., X.W., Y.W., Z. Gong, C.S., J.W., D.Z., Z.L. and P.Y. performed the biochemical assays. All authors analyzed the data and contributed to manuscript preparation. L.M., X.W. and P.Y. wrote the manuscript.
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Extended data
Extended Data Fig. 1 Biochemical analysis of the BIC2-CRY2 complex.
a, The interactions between two BICs (BIC1 and BIC2) and two CRYs (CRY1 and CRY2) in Arabidopsis thaliana were assessed by pull-down assays. b, Characterization of the purified full-length BIC2-CRY2 complex. Upper panel is the gel filtration chromatogram for BIC2-CRY2 purified using a SuperdexTM 200 increase 10/300 GL column. Elution volume of BIC2-CRY2 = 13.7 ml. The calibration standard for gel filtration chromatography is a mixture of thyroglobulin, γ-globulin, and ovalbumin proteins with approximate molecular weights of 669 kDa (10.3 ml), 158 kDa (13.0 ml), and 44 kDa (15.7 ml), respectively. The elution volume of BIC2-CRY2 is different to that of the 158 kDa marker, indicating BIC2-CRY2 complex is a heterodimer with a 1:1 stoichiometry. Lower panel: an SDS-PAGE gel showing the proteins present in the peak fractions of BIC2-CRY2 from gel filtration chromatography visualized by Coomassie blue staining. c-d, SV-AUC and SLS analyses of the molecular weight of the full-length BIC2-CRY2 complex in solution under dark conditions. SLS analysis was performed in a SuperdexTM 200 increase 10/300 GL column. e, The domain of CRY2 interacting with BICs was characterized by pull-down assays. FL: full length; PHR: photolyase-homology region; CCE: Cryptochrome C-terminal Extension. Uncropped gel images of a, b and e are available as source data.
Extended Data Fig. 2 Sequence alignment of CRY1 and CRY2.
The alignment was performed using MultAlin and ENDscript programs. Secondary structural elements are shown above the sequence. Sequence identity is shown in white letters on a red background, and sequence similarity is shown in red letters. The red and cyan dots indicate the residues interacting with FAD and AMP (in the BIC2-CRY2N structure, Extended Data Fig. 5), respectively. The yellow and dark blue dots below the sequence indicate residues involved in BIC2 hydrophobic and hydrophilic interactions, respectively (in the BIC2-CRY2N structure, Extended Data Fig. 7). The magenta dots indicate the residues in CRY2 that were mutated to verify the interactions observed in the BIC2-CRY2N structure.
Extended Data Fig. 3 Structural alignment of CRY2N and full length Drosophila CRY (dCRY, PDB ID 4JZY).
Ribbon presentation of overall structural alignment is shown at center, CRY2N is colored in cyan and dCRY in gray. Alignments of different functional loops are displayed around the overall structural alignment framed by black squares. The corresponding sequence alignments are shown beneath each black square. Different functional loops and their corresponding sequences in CRY2N and dCRY are highlighted in magenta and yellow, respectively. Magenta dashed line denotes the invisible sulfur loop in CRY2N, the corresponding residues are colored in blue. Sequence identity is indicated by red boxes.
Extended Data Fig. 4 Electron density maps of BIC2 bound to CRY2N.
a, Electron densities on the surface of the groove between the α/β and α domains of CRY2N. CRY2N is presented as a cyan surface model. BIC2 is shown as a 2Fo-Fc electron density map (contoured at 1σ in blue mesh). b, Stereo view of BIC2 electron density. The 2Fo-Fc electron density map of BIC2 is contoured at 1σ and colored in blue.
Extended Data Fig. 5 Detailed environment of the FAD binding cavity in the BIC2-CRY2N complex.
a, Sequence conservation analysis and close up view of the FAD binding cavity in BIC2-CRY2N. The FAD binding cavity is mainly formed by the CRY2N α9, α10, α12, α15, α16, α17 helices, shown in the black box at right. Sequence conservation scores displayed in different colors on the surface of CRY2 were generated by the CONSURF program. The FAD and AMP molecules are presented as green and pink ball-and-stick models. The magnesium cation is colored in magenta. b, Interface between BIC2 helix α1 and the phosphate binding loop between the α9 and α10 helices of CRY2N. Yellow dashed lines denote hydrogen bonds. c, FAD and AMP molecules in the BIC2-CRY2N complex are presented as ball-and-stick models. d, FAD displays a U-shaped conformation. The electron density (blue) of FAD is contoured at 1σ. e, Schematic representation of the contacts between CRY2 and FAD. f, Stereo representation of the electron density map of AMP bound to BIC2-CRY2N complex. The 2Fo-Fc electron density (1σ level) of AMP is shown in blue. g, The binding site of AMP. AMP is recognized by CRY2 residues N356, R357, Y399, and D406. Hydrogen bonds are shown as yellow dashed lines.
Extended Data Fig. 6 Antenna pocket of BIC2-CRY2N.
Surface and ribbon representation of the BIC2-CRY2N complex. Wheat-colored ribbon denotes BIC2. Surface of CRY2N is shown in cyan. Antenna pocket of BIC2-CRY2N is shown in the black square. BIC2 residues 72-74, which were not observed in the electron density map, form a gap between the α2 and α3 helices of BIC2.
Extended Data Fig. 7 Mutational analysis of BIC2-CRY2N interface.
a, Extensive interaction networks between BIC2 and CRY2N. Residues of BIC2 and CRY2N involved in interactions are shown in yellow and cyan rectangles, respectively. Black outlines indicate residues that were mutated to verify the interactions in the BIC2-CRY2N structure. b, Analysis of the interactions between wild type BIC2 with CRY2 mutants (labeled with asterisk). c, Interactions analysis of BIC2 mutants (labeled with asterisk) with wild type CRY2. For BIC2, we mutated residues that interact with CRY2 via their side chains (residues labeled with magenta dots in Extended Data Fig. 7d). For CRY2, there are 36 residues involved in BIC interactions. PDB 6K8K was loaded to PDBePISA (https://www.ebi.ac.uk/pdbe/pisa/) to analyze the interface of BIC2 and CRY2N. We then mutated 16 residues with the highest accessible surface area (ASA) and buried surface area (BSA). Residues that have a higher buried area percentage in PDBePISA interface analysis may make greater contributions to the interaction. Residues critical for complex formation are shown on a gray background. d, Sequence alignment of CID domains17 of BICs from different plant species. Secondary structural elements of BIC2 are shown above the sequence. The alignment was performed using MultAlin and ENDscript programs. Amino acid residue numbers are indicated to the left and right of each sequence. Sequence identity is indicated in white letters on a red background. Cyan and magenta dots represent the BIC2 residues interacting with CRY2N via main chains or side chains, respectively. The latter BIC2 residues were mutated to verify the interactions. Uncropped gel images of panels b and c are available as source data.
Extended Data Fig. 8 Comparison of CRY2N and BIC2-bound CRY2N structures.
a, Ribbon presentation of superposed CRY2N and BIC2-bound CRY2N structures. CRY2N is colored in yellow and BIC2-bound CRY2N is colored in cyan. Black squares show that helices α7 and α21 undergo slight conformational changes upon BIC2 binding. Helix α7 is involved in the BIC2 interactions, but helix α21 is not; conformational changes of helix α21 may be caused by crystal contacts. b, Superimposition of CRY2N (yellow) and BIC2-bound CRY2N (cyan) structures and comparison of their electron transfer pathways. The Trp-triad and D393 residues are presented as sticks. c, Electron density maps of residues W321, W374, W397, and D393 in CRY2N (yellow) and BIC2-bound (cyan) CRY2N.
Extended Data Fig. 9 BIC2 binding increases d(OD393-N5ISO) and d(HW397-OD393) distances and inhibits blue light-induced photobleaching of CRY2 in crystals and in solution.
a, Time-dependent distance d(OD393-N5ISO) in Step 2 obtained from MD simulations of CRY2N and the BIC2-CRY2N complex. b, Time-dependent distance d(HW397-OD393) in Step 3. c, Left panel shows the photoreduction activity of FAD in full-length CRY2D393A at 5 mg/ml (the absorbance at 450 nm is about 0.1). Middle panel shows the absorbances of CRY2WT and CRY2D393A at 450 nm at increasing concentrations. Wild-type CRY2 (CRY2WT) was used to generate the standard curve (blue line). When the concentration of CRY2WT is 8 mg/ml, the absorbance at 450 nm is 0.59. In contrast, the absorbance of CRY2D393A at 450 nm is only 0.14 when the concentration is 8 mg/ml. This indicates that CRY2D393A diminishes FAD binding. Right panel shows that CRY2D393A at a concentration of 53 mg/ml exhibits the same yellow color (the absorbance at 450 nm is 0.63) as CRY2WT at only 8 mg/ml. d, Photoreduction of CRY2N and BIC2-CRY2N crystals viewed by light microscopy. e, Photoreduction of the corresponding solutions. FAD is in oxidized form. Unbound and BIC2-bound CRY2 are full-length proteins.
Extended Data Fig. 10 Blue light induced CRY2 oligomer is able to interact with CIB1 by SEC.
a, SLS analyses of the molecular weight of full-length CRY2 in darkness and under blue light, respectively. All the runs were performed in a SuperoseTM 6 increase 10/300 GL column. b, Blue light induced full-length CRY2 tetramer interacts with truncated CIB1 (M16-N131) in SEC. All runs were performed in a SuperdexTM 200 increase 10/300 GL column. Under blue light treatment, the activated CRY2 is eluted at 11.5 ml (pink line), indicating CRY2 forms a blue light-induced tetramer. CIB1 alone is eluted at 16.2 ml under blue light (brown lines). After co-incubation, the blue light-induced tetramer co-elutes with CIB1 at ~11 ml (blue line), indicating a CRY2 tetramer interacts with CIB1. Right panel shows SDS–PAGE gels of peak fractions shown at left. The last three lanes are from separate gels. Uncropped gel images are available as source data.
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Ma, L., Wang, X., Guan, Z. et al. Structural insights into BIC-mediated inactivation of Arabidopsis cryptochrome 2. Nat Struct Mol Biol 27, 472–479 (2020). https://doi.org/10.1038/s41594-020-0410-z
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DOI: https://doi.org/10.1038/s41594-020-0410-z
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