Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light-sensing kinases that control diverse cellular functions in plants, bacteria and fungi1,2,3,4,5,6,7,8,9. Bacterial phytochromes consist of a photosensory core and a carboxy-terminal regulatory domain10,11. Structures of photosensory cores are reported in the resting state12,13,14,15,16,17,18 and conformational responses to light activation have been proposed in the vicinity of the chromophore19,20,21,22,23. However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here we report crystal and solution structures of the resting and activated states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures show an open and closed form of the dimeric protein for the activated and resting states, respectively. This nanometre-scale rearrangement is controlled by refolding of an evolutionarily conserved ‘tongue’, which is in contact with the chromophore. The findings reveal an unusual mechanism in which atomic-scale conformational changes around the chromophore are first amplified into an ångstrom-scale distance change in the tongue, and further grow into a nanometre-scale conformational signal. The structural mechanism is a blueprint for understanding how phytochromes connect to the cellular signalling network.
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Protein Data Bank
Coordinates and structure factor amplitudes have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) under accession codes 4O01 and 4O0P. The solution structures are available as Source Data.
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We acknowledge beamline access at BM29 and ID23-1 at the ESRF, cSAXS at the Swiss Light Source (funded by the European Community’s FP7 grant agreement no. 312284 CALIPSO), and BioCARS at the Advanced Photon Source, Argonne National Laboratory (supported by National Institutes of Health, National Institute of General Medical Sciences grant 1R24GM111072 and in part through collaboration with P. Anfinrud). K. Forest and R. Vierstra are acknowledged for the donation of the plasmids. J. Ylänne and his group are acknowledged for advice on crystallography, and H. Häkkänen and A. Liukkonen for their assistance. We thank M.-A. Mroginski for providing force-field parameter files for the biliverdin. We acknowledge V. Chukharev and N. V. Tkachenko for the facilities for transient absorption spectroscopy measurements. We also acknowledge grants to S.W. by the Foundation of Strategic Research, Sweden, and the Swedish and European Research Councils, agreement number 279944, support to J.A.I. by Finnish Academy grant 138063, and to H.T. by the Finnish Cultural Foundation grant 0131067.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Difference scattering and kinetics.
a–c, Singular value decomposition (ΔS (q, t) = USVT , where U, S and V are matrix factors) of time-resolved solution scattering data from the PAS-GAF-PHY fragment. Two components (n) suffice to describe the data, the final product (n = 1) and a transient low-q depression (n = 2). a, The first three basis spectra (first, second and third columns of US), and original X-ray scattering data (black) with reconstruction based on the first two singular values (all columns of USVT , red) are shown. b, Relative amplitudes of the two first basis spectra (first and second columns of V). Lines are guides for the eye. c, Singular values (diagonal elements of S). d, The rise of the Pfr product state as measured by direct integration of difference scattering curves () and by optical transient absorption at 754 nm after excitation with a 680 nm laser flash. Solid curve is the function (1 − e−t/τ), where t is time and and τ a free fitting parameter. These data establish that the structural change occurs just after the Pfr state is formed in the chromophore. Note the positive signal in the absorption curve with very small amplitude at >3 ms, which appears to decay while the structural signal rises. This could be because the absorption properties depend weakly on the large-scale rearrangement. e, Direct static difference data from Fig. 1, amplified by q2 to reveal wide-angle oscillations.
Extended Data Figure 2 Light-induced changes in the secondary structure of the evolutionally conserved PHY tongue.
a, Secondary structure and topology of the D. radiodurans PAS-GAF-PHY fragment. The structural elements in our crystal structures are very similar to other published phytochrome structures12,13. The PHY tongue region (box), however, was found to refold upon illumination. The five-stranded β-sheet core of the GAF domain is extended by a small sixth β-strand (called 2′) that interacts with the PHY tongue (see Fig. 2 and Extended Data Fig. 4). The mini-sheet structure at the knot region14 is not included in the graph. The figure is adapted from ref. 12. b, Omit map of the PHY tongue in the dark crystal form (top panel) and the illuminated crystal form (bottom panel). In the dark crystal form, the omit map density (blue) supports the built β-turn secondary structure (orange sticks), even though most of the side chains are poorly resolved. In the illuminated crystal form, the omit map (blue) clearly reveals the density of a helix with its bulky side chains (orange). The omit maps were calculated by repeating molecular replacement and a refinement step (see Supplementary Information) with a structure in which the PHY tongue was removed. All electron density maps are contoured at 3.0σ. c, Sequence alignment of part of the GAF domain and of PHY loop region. The conserved 207D-I-P209 motif in the GAF domain and 465P-R-X-S-F469 motif in the PHY tongue are marked by asterisks. Five representatives from eubacterial (BphP), cyanobacterial (Cph), higher plant, fungi (Fph) and PAS-less phytochromes are shown. The full alignment is supplied as source data. Colour coding: dark grey, identical residues; light grey, similar residues. The amino acid sequences were aligned with Clustal Omega using the default settings31. Sequences include Deinococcus radiodurans BphP, Pseudomonas syringae pv. tomato T1 BphP, Rhodopseudomonas palustris TIE-1 BphP3, Pseudomonas aeruginosa PAO1 BphP, Agrobacterium fabrum str. C58 BphP1 (Agp1), Synechocystis sp. PCC6803 Syn-Cph1, Microcystis aeruginosa NIES-843, Nodularia spumigena CCY9414, Cyanothece sp. PCC 7822, Anabaena variabilis ATCC 29413, Physcomitrella patens Phy1, Zea mays PhyB1, Populus trichocarpa PhyA, Selaginella martensii Phy1, Arabidopsis thaliana PhyA, Synechococcus OSB' SyB-Cph1, Synechococcus OSA SyA-Cph1, Nostoc punctiforme PCC73102, Lyngbya sp. PCC 8106 and Anabaena variabilis ATCC 29413.
Extended Data Figure 3 Biliverdin structure and spectra in crystals.
a, Photographs of the crystals under cryogenic conditions at the beamline ID23-1. b, Biliverdin omit maps of the dark (top left) and illuminated (top right) form support the existence of the modelled biliverdin conformations (yellow and orange). Comparison of the electron density around the biliverdin with published structures (bottom panels). In the dark form, the electron density indicates a conformation similar to the published Pr structures13,14,32,33, including a D. radiodurans structure (PDB accession 2O9C, cyan)32. Therefore we modelled the biliverdin as a 15Za isomer. In the illuminated form, however, the electron density supports neither the biliverdin as determined in the Pfr structure of PaBphP (PDB 3NHQ, red)20, nor as determined in the Pr structure of D. radiodurans (2O9C, cyan)32. Therefore the rotation of the biliverdin D-ring cannot be reliably determined and is modelled with both possibilities (15Za, and 15Ea, orange and yellow in top right panel). Omit maps were calculated as in Extended Data Fig. 2 and contoured at 3.0σ. c, Representative absorption spectra of the dark (black) and illuminated (grey) crystals, recorded at 123 K. Note that the terms ‘illuminated’ and ‘dark’ refer here to the crystallization conditions (see Supplementary Information for details). The dark crystal spectrum resembles the Pr spectrum in solution (Fig. 1a). Illumination with red light in the crystallization drops at ambient temperature led to a slight increase of far-red absorption and disintegration of the crystals (data not shown). Dark crystals were unaffected by far-red illumination. The spectrum of the illuminated crystals shows that a substantial proportion (>50%) of the proteins reside in Pfr state. A similar absorption was detected from solubilized crystals. The illuminated crystals could be switched to Pr-like absorption with far-red illumination. Reversely, the Pfr-like features could also be increased with red light (data not shown) with illumination at ambient temperature. Exposure with light increased the scattering background in the absorption measurements. The crystals seemed unaffected by the illumination when illuminated with red light in the crystallization drops. Although the spectral analyses of the illuminated crystals do not indicate a pure Pfr spectrum, and the biliverdin conformation cannot be fit unambiguously to the electron density, the remainder of the electron density is homogeneous (Extended Data Fig. 2b). Most importantly, the tongue region of the PHY domain adopts the conformation resembling the Pfr state of PaBphP (Extended Data Fig. 4b)12,19,20. The conformations of the four monomers in an asymmetric unit are practically identical and hence we conclude that biliverdin can co-exist in both Pr and Pfr states inside this crystal form and still the protein part represents the structural aspects of the Pfr state only.
Extended Data Figure 4 Comparison of PHY tongue interactions with published structures.
a, Comparison of the dark crystal form (green/dark grey) to cyanobacterial cph1 in Pr state (PDB 2VEA, orange/light grey)13. b, Comparison of the illuminated crystal form (green/dark grey) to PaBphP in Pfr state (PDB code 3NHQ20, orange/light grey). In both the Pr and Pfr forms, key interactions are conserved between the phytochromes (black dashes), as well as the positions of three conserved tongue motives (see Extended Data Fig. 2c). The residues of these three motives are indicated as: 451W-(G/A)-G453, 465P-R-X-S-F469 and 472(W/F,Y)-X-E474,13 with numbering from the D. radiodurans sequence. Trp 451 was not modelled in our illuminated crystal structure, and part of the PHY tongue has been removed for clarity. Small changes in relative orientations between the difference crystal structures are observed, for example, a slight tilt of helix of the Pfr tongue.
Extended Data Figure 5 SAXS data and calculations.
a, Experimental SAXS data of dark (‘Pr’) and pre-illuminated (‘Pfr’) samples. Here, I is scattered intensity, 2θ is the scattering angle and q is the magnitude of the scattering vector. The data are merged from the concentration series (Extended Data Table 2b) and are normalized on 0.4 nm−1 < q < 0.6 nm−1. b, Guinier plot of the low-q region, shown for all concentrations. The curves converge at low concentration (C). Inset shows the radii of gyration (Rg) calculated from the curves in a according to the Guinier approximation. Dotted lines show the q-range used for linear regression (0.15 nm−2 < q2 < 0.120 nm−2), which is such that qRg < 1.3 as required34. Arrows indicate increasing concentration. c, Average difference scattering signals calculated from the solution-structural models using three methods: Crysol (default settings), SASTBX with spherical harmonic expansion (SHE, default settings), and SASTBX with Zernike polynomial expansion as described in Supplementary Information30. Different calculation methods result in very similar predicted difference X-ray scattering patterns. d, Determination of the relative Pr/Pfr populations represented by the BM29 data as described in Supplementary Information. SSEabs is the sum-squared error against absolute scattering data, SST is the sum of squares about the mean, and v is the Pfr fraction. We find that our Pr sample contained only Pr (top) whereas in the Pfr sample 64% of the protein molecules adopted the Pfr conformation (bottom). Notably, the static difference scattering patterns (Fig. 1) represent, up to a scaling factor, the relation between pure Pr and Pfr populations. This is in contrast to traditional SAXS which report on population mixtures, because the Pfr state cannot be easily produced with 100% population in solution.
Extended Data Figure 6 Packing interactions of the crystal forms.
a, b, Crystal packing interactions of the dark (a) and illuminated (b) crystal forms. The dimer of an asymmetric unit is shown in red and the symmetry mates in grey. For the illuminated form, only chains A and B are shown in red. Interactions are shown from two orientations for clarity. In the dark form, crystal contacts are seen in the top regions of the PHY domains and therefore may cause artefacts in the long scaffolding helix and in the opening of the PHY domains. In the illuminated form contacts are such that the PHY domains may be pushed closer together, which is consistent with the larger separation of the PHY domains as refined from the solution X-ray scattering data. It is noteworthy that the relative orientation of the monomers in the dimer is different between all three known structures for PAS-GAF-PHY phytochromes. For P. aeruginosa the dimer is parallel with variations in between different copies of the dimer in the crystallographic unit cell12, in two cyanobacterial phytochrome an antiparallel dimer is observed13,35, and in our Pr structure, the monomer have an angle of approximately 45°. The rings highlight crystal contacts.
Extended Data Figure 7 Solution-structural refinement.
a, The distribution of PHY domain separations (RPP) obtained from unbiased molecular dynamics simulations (production runs 1–3). The best-matching sets A and B of solution structures found from these conformations are indicated by crosses (with N = 100 best pairs chosen from M ≈ 6 × 106 possible combinations). Whereas the Pr structures cluster in a region of high sampling, the Pfr structures lie at the edges of the PHY-PHY distribution, suggesting inadequate sampling. To remedy this, we artificially scanned the PHY domain separation in separate simulations (production runs 4 and 5) to improve sampling. b, The new distribution of pulled PHY domain separations in the Pfr state. The final analysis and all solution-structural conclusions drawn in this study are based on the trajectories described in b. c–e, Consistency test of the structural refinement procedure. c, A cutoff parameter Rcut was introduced to reject all molecular dynamics frames RPP > Rcut. The resulting average over RPP of the best N = 100 pairs is plotted as a function of Rcut. It is found that RPP ≈ Rcut, which indicates that the best fit to the difference X-ray scattering data are always at the highest separations available in sampling range. d and e show the dependence of the total and average error (SSEtot and SSEav) as functions of Rcut. SST represents the overall sum of square about the mean. It is observed that the error decreases steeply for Rcut <∼ 5 nm, and only marginally for Rcut >∼ 5 nm. We therefore consider optimization in the latter range overfitting, and applied Rcut = 5.0 nm in the refinement for the solution structures. The refined structures (Fig. 4) should therefore be considered to represent lower limits on RPP. f, Solution-structural refinement parameters and results.
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Takala, H., Björling, A., Berntsson, O. et al. Signal amplification and transduction in phytochrome photosensors. Nature 509, 245–248 (2014). https://doi.org/10.1038/nature13310
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