The structure of Arabidopsis phytochrome A reveals topological and functional diversification among the plant photoreceptor isoforms

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Plants employ a divergent cohort of phytochrome (Phy) photoreceptors to govern many aspects of morphogenesis through reversible photointerconversion between inactive Pr and active Pfr conformers. The two most influential are PhyA whose retention of Pfr enables sensation of dim light, while the relative instability of Pfr for PhyB makes it better suited for detecting full sun and temperature. To better understand these contrasts, we solved, by cryo-electron microscopy, the three-dimensional structure of full-length PhyA as Pr. Like PhyB, PhyA dimerizes through head-to-head assembly of its C-terminal histidine kinase-related domains (HKRDs), while the remainder assembles as a head-to-tail light-responsive platform. Whereas the platform and HKRDs associate asymmetrically in PhyB dimers, these lopsided connections are absent in PhyA. Analysis of truncation and site-directed mutants revealed that this decoupling and altered platform assembly have functional consequences for Pfr stability of PhyA and highlights how plant Phy structural diversification has extended light and temperature perception.

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Source data for all Pfr→Pr thermal reversion graphs in Figs. 5–7 are provided in Supplementary Table 2. Images of full SDS–PAGE gels are provided in Supplementary Fig. 3. The 3D cryo-EM consensus map of the full-length Arabidopsis PhyA dimer at 3.2 Å resolution has been deposited in the EMDB database under accession code EMD-28870. The focus-refined platform and HKRD maps at 3.1 Å and 3.4 Å average resolution, respectively, have been deposited in the EMDB database under accession codes EMD-28871 and EMD-28872. The composite 3D map has been deposited in the EMDB database under accession code EMD-28869, and its corresponding atomic model is available in the RCSB database under PDB code 8F5Z. This study made use of several publicly available protein structures for Phys and transmitter histidine kinases that were obtained from the RCSB database (http://www.rcsb.org) under accession codes 2VEA, 6TC5, 6TC7, 6TL4, 4U7O and 7RZW. Source data are provided with this paper.

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Cryo-EM data were collected on a Titan Krios microscope at the David Van Andel Cryo-Electron Microscopy Suite at Van Andel Institute. We thank G. Zhao and X. Meng for help with EM data collection, H. Zaher for help with the kinase assays and C. Sherman for technical assistance. This work was funded by the US National Institutes of Health R01 grants GM127892 and GM127892-05 (to R.D.V.) and GM131754 (to Huilin Li), and by funds provided by Washington University in St. Louis (to R.D.V.) and the Van Andel Institute (to Huilin Li).

Katrice E. McLoughlin

Present address: Burning Rock Dx, Irvine, CA, USA

These authors contributed equally: E. Sethe Burgie, Hua Li, Zachary T. K. Gannam.

Department of Biology, Washington University in St. Louis, St. Louis, MO, USA

E. Sethe Burgie, Zachary T. K. Gannam, Katrice E. McLoughlin & Richard D. Vierstra

Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA

Hua Li & Huilin Li

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E.S.B., Hua Li, Z.T.K.G., R.D.V. and Huilin Li designed the experiments. Hua Li performed the cryo-EM and 3D reconstruction. E.S.B. and Hua Li built and refined the atomic models. E.S.B., Z.T.K.G. and K.E.M. expressed and purified the assembled Phy samples and performed the mutagenesis and spectroscopic assays. E.S.B., Hua Li, Z.T.K.G., Huilin Li and R.D.V. wrote the manuscript.

Correspondence to Richard D. Vierstra or Huilin Li.

The authors declare no competing interests.

Nature Plants thanks Andreas Möglich, Xiaojing Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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a, SDS-PAGE analysis of the recombinant full-length PhyA. Gels were either stained for protein with Coomassie blue (left) or assayed for bound PΦB by zinc-induced fluorescence (right). MM, molecular mass standards. Samples were indistinguishable to those described by Burgie et al.6 b, UV-vis absorbance spectra of PhyA. The spectra were collected from dark-adapted samples (Pr) or after saturating irradiation with 630-nm red light (RL, mostly Pfr). Absorption maxima were determined from the difference spectrum shown at 70% amplitude. The SCR at 664/723 nm is indicated in parenthesis. Spectra were the average of three technical replicates. c, Workflow used for data processing of the cryo-EM images of PhyA. In the first refined overall map at 3.5-Å resolution based on 421,969 particles, almost all PhyA domains were well seen except for the regions encompassing the PAS1 domains, which were poorly resolved. Focused refinements, excluding the PAS1 domains and using signal subtraction for the PSM, PAS2 and HKRD regions, generated a 3.2-Å map with some ambiguity in the HKRDs. Subsequent use of separate masks for the HKRDs and the platform led to improved 3.1-Å and 3.4-Å EM maps for the platform and HKRDs, respectively, which were combined to generate the final composite map of the dimer. 3DVA of particle images down-sampled to 4.14 Å per pixel resolved one of the flexible PAS1 domains (purple) at ~15-Å resolution. d, A representative cryo-EM micrograph after motion correction is shown. In total, data from 6,195 independent micrographs were utilized for map construction. e, Selected 2D class averages showing multiple views of the particles.

a, The 3D EM consensus map and focus-refined individual platform and HKRD maps color coded by local resolution. b, Eulerian angle distribution of raw particle images used in the final 3D reconstruction. c, Gold-standard Fourier shell correlation (FSC) of two half maps (orange curve) and the correlation of the atomic model and the final composite 3D map (purple curve).

The map is shown in grey mesh whereas the 3D models of the motifs/residues are shown in cartoons/sticks and colored as in Fig. 1e. Panels a-c include PΦB (red sticks) to highlight proximity to the chromophore. Key residues are indicated. a, Knot lasso extending from the GAF domain to encircle the N-terminal extension (NTE) just upstream of the nPAS domain. b, Residues 69–81 comprising part of the NTE near the knot lasso that reaches near the chromophore. c, Orthogonal views of the hairpin extending from the PHY domain to contact the GAF domain near the chromophore. d, The modulator loop extending from between the PAS1 and PAS2 domains to interact with the PHY domain of its own protomer. e, The paired DHp domains within the HKRD. Helices α1 and α2 are indicated. The cruciate feature within helix α1 is located by the brackets. Residue 905 (R905), which is normally occupied by a histidine in transmitter histidine kinases, is highlighted by the red oval. f, Closeup views of the connections between the PAS2 domain of protomer B and the nPAS and GAF domains of protomer A within the dimer. g, Structural prediction of the PAS1 fold by TrRosetta (left) and congruence of this prediction (grey) with the cryo-EM models of the PAS2 (center) and nPAS domains from PhyA (right) shown in color. The N- and C-terminal ends are indicated. For panels (e) and (f), the peptide backbone is shown in cartoon, whereas the amino acid side chains are in sticks. The prime designations identify residues from the B protomer.

The intricate interconnectivities of domains within the PSM are shown in the top three views. The fourth and fifth views highlight the modulator loop (Mod)/PHY domain connection, which provides an intra-protomer structural link with the PSM, and their connectivity to the hairpin, helical spine, and PAS2 domain features. The sixth view shows one half of the dimeric interface of the HKRD involving the DHp and CA domain α-helices. Key residues are indicated; prime designations indicate those from protomer B.

a, Diagrams of the contacts. Shown are the interacting regions in context of their α-helical, β-strand, or loop configurations. Left diagrams highlight the HKRD interprotomer interactions among the DHp and CA domains. Right diagrams highlight interactions between the HKRD (cyan/teal) and GAF (green) and PHY (orange) domains within the platform. The lines locate the specific residues involved in the predicted contacts; shown are connections found within PhyA, PhyB, or both. (A) and (B) refer to the A and B protomers within the dimer, respectively. Amino acid numberings correspond that those found within PhyA or PhyB. Some strands or helices are included for structural context but not found to participate in the interactions. b, Sequence alignment of the regions within AtPhyA and AtPhyB participating in the contacts shown in (a). Identical and similar amino acids are colored in black and grey boxes, respectively. The bars above demarcate the α-helical (red) and β-stranded (blue) features. The green circles below identify specific amino acids that participate in the contacts shown in (a).

a, Domain organization of full-length (FL) PhyA and NTE truncations starting at residue 25 (NΔ24) and 66 (NΔ65). The length of the NTE was extended for clarity. Dashed red lines represent the interaction of PΦB with NTE residues Tyr-70 and Ile-74. b, Thermal reversion rates of FL PhyA and its PSM bearing the NTE truncations NΔ24 and NΔ65. Data points and fit lines representative of three technical replicates are shown (see Extended Data Table 2 for statistical analyses). Also included are Pfr→Pr thermal reversion data for PSM fragments of Arabidopsis PhyA with comparable NTE truncations, remeasured here for completeness, see also Burgie et al.6. Because the reaction rates differ by orders of magnitude, rates in two time scales are shown: left panel, 120 min; right panel, 1200 min. SDS-PAGE gels and absorption and Pr-Pfr difference spectra for the preparations are shown in Supplementary Figs. 1 and 2.

a, Orthogonal views of the HKRD CA region from At PhyA superposed with the same region from At PhyB (PDB ID code 7RZW22) and the prokaryotic Walk transmitter HK from Lactobacillis plantarum (Lp) (PDB ID code 4U7O32). b and c, Models showing the predicted position of ADP (red) in the At PhyA CA domain based on the binding pocket described in Lp WalK32. Residues expected to participate in binding are indicated in (b). ADP clashes with multiple residues in the pocket of this predicted PhyA-ATP model, indicating that conformational shifts in At PhyA induced by ATP or photoactivation would be necessary for binding. Sites with substantial clash are circled. d, Amino acid sequence alignment of the possible ATP-binding pocket of At PhyA and At PhyB with comparable CA domains from bona fide histidine kinases from Bacillus subtilis (Bs YFI), Thermotoga maritima (Tm HK853), L. plantarum (Lp Walk) and Streptococcus mutans (Sm Vick), and with those from bacterial Phys (BphPs) with HK/phosphatase activities from Pseudomonas syringae (Ps BphP)18 and Deinococcus radiodurans (Dr BphP)16. Identical and similar amino acids are colored in black and grey boxes, respectively. The signature N, G1/D, F and G2 boxes and ATP lid for histidine kinases are indicated32. Arrowheads locate key residues within the ATP-binding pocket that are critical for catalysis32. e-g, At PhyA is a poor kinase as compared to Ps BphP based on autophosphorylation assays. Equimolar amounts of recombinant biliproteins were incubated for 1 min to 2 hr at ambient temperature (~24 °C) with 150 μM ATP supplemented with 10 μCi of [γ-32P]-ATP, quenched with SDS-PAGE sample buffer, and measured for 32P incorporation by autoradiography of SDS-PAGE gels. e, Time course for autophosphorylation of Ps BphP as Pfr. f, Comparisons of autophosphorylation activities of At PhyB as Pr and Pfr with those of Ps BphP after 2 hr incubations. Arrowheads locate Ps BphP. The phosphorimager scans are representative of three independent experiments. g, Images of the SDS-PAGE gels stained for protein with Coomassie blue or for the bound bilin by zinc-induced fluorescence.

Supplementary Table 1 and Figs. 1–3.

Movie of the full-length PhyA dimer resolved to 3.2 Å average resolution. The movie first shows a rotating EM map followed by a rotating cartoon view of the resulting model. The NTE, nPAS, GAF, PHY with hairpin, PAS2 with modulator and HKRD domains are in grey/black, light/dark blue, light/dark green, yellow/orange, pink/magenta and light cyan/teal, respectively. PΦB shown in red sticks.

The HKRD of Arabidopsis PhyA does not form strong non-covalent contacts with the platform. 3DVA was used to help describe the motion of the HKRD with respect to the platform in PhyA. Using this procedure, 20 EM maps were generated by averaging frames of like structures as indicated in the graph. We show four orthogonal views of the EM map of PhyA to track the movement of domains. Domain positions are indicated, including those for the PAS1 domain, which appears proximal to the PHY and PAS2 domains within the platform. The map numbers are indicated at the top left.

Source data for size-exclusion chromatography, UV–vis spectra and thermal reversion kinetics plots in Figs. 5–7.

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Burgie, E.S., Li, H., Gannam, Z.T.K. et al. The structure of Arabidopsis phytochrome A reveals topological and functional diversification among the plant photoreceptor isoforms. Nat. Plants (2023). https://doi.org/10.1038/s41477-023-01435-8

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Received: 14 December 2022

Accepted: 10 May 2023

Published: 08 June 2023

DOI: https://doi.org/10.1038/s41477-023-01435-8

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