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The oligomeric structures of plant cryptochromes

A Publisher Correction to this article was published on 18 May 2020

This article has been updated

Abstract

Cryptochromes (CRYs) are a group of evolutionarily conserved flavoproteins found in many organisms. In plants, the well-studied CRY photoreceptor, activated by blue light, plays essential roles in plant growth and development. However, the mechanism of activation remains largely unknown. Here, we determined the oligomeric structures of the blue-light-perceiving PHR domain of Zea mays CRY1 and an Arabidopsis CRY2 constitutively active mutant. The structures form dimers and tetramers whose functional importance is examined in vitro and in vivo with Arabidopsis CRY2. Structure-based analysis suggests that blue light may be perceived by CRY to cause conformational changes, whose precise nature remains to be determined, leading to oligomerization that is essential for downstream signaling. This photoactivation mechanism may be widely used by plant CRYs. Our study reveals a molecular mechanism of plant CRY activation and also paves the way for design of CRY as a more efficient optical switch.

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Fig. 1: Reconstruction of plant CRY activation in vitro.
Fig. 2: 3D structures of ZmCRY1cW368A-PHR and ZmCRY1a-PHR.
Fig. 3: CRY active dimer formation through INT1.
Fig. 4: In vivo functional analysis of AtCRY2W349A and AtCRY2R439A.
Fig. 5: Blue light-induced conformational changes during CRY activation.
Fig. 6: Proposed model of blue-light-induced plant cryptochrome activation.

Data availability

The cryo-EM maps of ZmCRY1cW368A-PHR and AtCRY2W374A-PHR tetramers (3.2 Å and 4.2 Å) and dimers (7.2 Å and 5.6 Å) have been deposited in the EMDB with accession codes EMD-30022, EMD-30023, EMD-30024 and EMD-30025. The atomic coordinates of ZmCRY1cW368A-PHR and ZmCRY1a-PHR structures have been deposited in the Protein Data Bank with accession codes 6LZ3 and 6LZ7, respectively. Source data for Figs. 1a and 4e,f are available with the paper online.

Change history

  • 18 May 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

We thank the staff members at the cryo-EM centers of Tsinghua University (L. Zhao & H. Wang), Zhejiang University and the National Facility for Protein Science in Shanghai Zhangjiang Lab for their technical assistance on cryo-EM image and SEC-MALLS data collection and analysis. We also thank the staff members at BL19U1 for their technical assistance in X-ray diffraction data collection, and the core facility center of the Institute of Plant Physiology and Ecology for X-ray diffraction testing. This work was supported by grants from the National Key R&D Program of China (SQ2018YFA090071 to P.Z. and F.Y.), the Chinese Academy of Sciences (CAS) (XDB27020103 and QYZDB-SSW-SMC006 to P.Z., and XDB27030000 to H.L.), the National Natural Science Foundation of China (31870727 to M.Z., 31861130356 to P.Z. and 31825004 to H.L.) and the Shanghai Science and technology Commission (19XD1424500 to P.Z.). Dr. X.L. is supported by the foundation of Youth Innovation Promotion Association of CAS.

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Contributions

K.S. carried out protein expression, purification, ITC, SEC-MALLS analysis, crystallization, and cryo-EM sample preparation; X.Z. carried out cryo-EM data collection and structure determination; X.L. carried out the physiological analysis of AtCRY2 and mutants; M.M., Y.H. and F.Y. contributed to protein expression, purification, crystallization and ITC analysis; M.Z., and X.H. contributed to X-ray and cryo-EM data collection and structure determination. P.Z. and H.L. analyzed the data and wrote the manuscript with inputs from other authors. P.Z. conceived the project.

Corresponding authors

Correspondence to Hongtao Liu or Peng Zhang.

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The authors declare no competing interests.

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Editor recognition statement: Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Characterization of AtCRY2 and constitutively active/inactive mutants AtCRY2W374A/AtCRY2D387A.

a, Purified protein samples were resolved by SDS-PAGE and visualized by Coomassie blue staining. b, Color view of samples from A. c, The absorption spectra of AtCRY2 and mutants under daylight (daylight was not sufficient to induce light-activation). a.u., arbitrary units.

Extended Data Fig. 2 Size Exclusion Chromatography-Multi Angle Laser Light Scattering (SEC-MALLS) analysis of wild-type and mutant CRYs.

Representative SEC-MALLS analysis of AtCRY2, AtCRY2W374A, ZmCRY1c, ZmCRY1cW368A, AtCRY2W374AW349A, AtCRY2W374AW439A, AtCRY2D387A, respectively. Aggregates were observed in AtCRY2, and double mutants AtCRY2W374AW349A and AtCRY2W374AW439A (eluting between 8–10 ml). The chromatogram shows readings of the UV and RI detectors in blue and red, respectively. The green/yellow/black dotted lines indicate the calculated molecular mass of the protein samples eluted from a Superdex-200 10/300 column. The theoretical molecular weight of each protein is shown in parentheses.

Extended Data Fig. 3 Isothermal calorimetry titration curves of AtCRY and mutants with AtBIC116–43 peptide.

The constitutively active mutant AtCRY2W374A shows strong binding with AtCIB116–43, while wild type AtCRY2, inactive mutant AtCRY2 D387A and dimer interface mutants AtCRY2W374A/W349A and AtCRY2W374A/R439L show no significant binding with AtCIB116–43. AtCRY2W374A/W349A and AtCRY2W374A/R439L were selected for ITC binding affinity measurements because either of them could lead to the promote dissociation of AtCRYW374A dimer to a monomer.

Extended Data Fig. 4 Cryo-EM images, 2D averages and 3D reconstruction of AtCRY2W374A-PHR.

a, Representative cryo-EM micrograph of AtCRY2W374A. b-c, Representative images of the 2D class averages of tetramer and dimer, respectively. d, FSC curves at 0.5 and 0.143 of the final reconstruction of AtCRY2W374A-PHR tetramer and dimer. e, Local resolution estimation of the final sharpened cryo-EM density map of AtCRY2W374A-PHR tetramer.

Extended Data Fig. 5 Purification and structure reconstruction of ZmCRY1cW368A.

a, Purification profiles of ZmCRY1cW368A and ZmCRY1c. Protein samples of ZmCRY1c and ZmCRY1cW368A were applied to Superdex-200 10/300, and the peak fractions of ZmCRY1cW368A were visualized by SDS-PAGE and Coomassie blue staining. Molecular weights are labelled according to the SEC-MALLS results. b, Flowchart of cryo-EM data processing and 3D reconstruction of ZmCRY1cW368A. c, FSC curves at 0.5 and 0.143 of the final reconstruction of ZmCRY1cW368A-PHR. d, Local resolution estimation of the final sharpened cryo-EM density map of ZmCRY1cW368A-PHR tetramer.

Extended Data Fig. 6 Superimposition of the cryo-EM maps and structures.

a, Different views of cryo-EM map of ZmCRY1cW368A-PHR dimer (blue) superimposed to that of ZmCRY1cW368A-PHR tetramer (light gray). b, Side views of the cryo-EM density of AtCRY2W374A-PHR tetramer fitted with cryo-EM structure of ZmCRY1cW368A-PHR tetramer. c, Side and front views of the cryo-EM density of AtCRY2W374A-PHR dimer fitted with cryo-EM structure of ZmCRY1cW368A-PHR dimer. d, Superimposition of the tetrameric structures of ZmCRY1cW368A-PHR and ZmCRY1a-PHR. ZmCRY1cW368A-PHR tetramer structure molecules are colored in gray and ZmCRY1a-PHR crystal structure molecules are colored in yellow, magenta, orange and cyan.

Extended Data Fig. 7 Ribbon cartoon of ZmCRY1cW368A shows the residues interacting with FAD.

FAD is shown as a yellow stick model. Residues interacting with FAD are shown with side chains. Those residues that are conserved among CRYs from different organisms and with photolyases are colored in magenta, while those that are conserved only within plants are colored green.

Extended Data Fig. 8 Protein sequence alignment of cryptochromes-PHR and photolyases.

Protein sequences of cryptochromes-PHR and photolyases from different species are aligned. The secondary structure elements of ZmCRY1cW368A-PHR and mCRY1-PHR are indicated at the top and bottom, respectively. Protein sequences are separated by dashed lines. Regions constituting INT1 and INT2 are indicated with blue and green solid lines respectively. Highly conserved residues are shaded red, while less conserved residues are red colored and boxed. Note that specific residues from helices α6, α7, α13 and α18-α19 loop at INT1 are colored blue. Residues interacting with FAD are indicated with yellow and green triangles; the latter indicate those residues that are conserved only within plants. Species are: Zm, Zea mays; At, Arabidopsis thaliana; Os, Oryza sativa; Gm, Glycine max; mCRY, mouse CRY; dCRY, Drosophila CRY; photolyase, Escherichia coli photolyase.

Extended Data Fig. 9 Dimer interface 2 and locations of CRY mutations in ZmCRY1cW368A-PHR structure.

a, dimer interface formed by molecules A/C (or B/D). Mol A is shown as a surface model, while C is shown as a ribbon model. FAD is depicted as yellow sticks, and the “trp-triad” residues are shown as orange spheres. b, The reported CRY mutations were located in the ZmCRY1cW368A-PHR structure. The early-flowering mutation AtCRY2V367M in the Cvi nature allele and the constitutive COP-phenotype mutation AtCRY1G380R corresponds to residues Val361 and Gly371 in ZmCRY1cW368A-PHR, respectively. Their locations near the “trp-triad”residues are shown in the structure.

Extended Data Fig. 10 Sequence alignment of ZmCRY1c, ZmCRY1a, AtCRY1, and AtCRY2 at “trp-triad” residues and dimer interaction residues.

The “trp-triad” residues and dimer interaction residues are shaded in blue and magenta, respectively. The numbers of corresponding residues are indicated.

Supplementary information

Source data

Source Data Fig. 1

Unprocessed gel image for Fig.1a, lower panel.

Source Data Fig. 2

Unprocessed western blot image for Fig.4e.

Source Data Fig. 3

Unprocessed western blot image for Fig.4f.

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Shao, K., Zhang, X., Li, X. et al. The oligomeric structures of plant cryptochromes. Nat Struct Mol Biol 27, 480–488 (2020). https://doi.org/10.1038/s41594-020-0420-x

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