Crystallographic and electron microscopic analyses of a bacterial phytochrome reveal local and global rearrangements during photoconversion

Abstract

Phytochromes are multidomain photoswitches that drive light perception in plants and microorganisms by coupling photoreversible isomerization of their bilin chromophore to various signaling cascades. How changes in bilin conformation affect output by these photoreceptors remains poorly resolved and might include several species-specific routes. Here, we present detailed three-dimensional models of the photosensing module and a picture of an entire dimeric photoreceptor through structural analysis of the Deinococcus radiodurans phytochrome BphP assembled with biliverdin (BV). A 1.16-Å resolution crystal structure of the bilin-binding pocket in the dark-adapted red light-absorbing state illuminated the intricate network of bilin/protein/water interactions and confirmed the protonation and ZZZssa conformation of BV. Structural and spectroscopic comparisons with the photochemically compromised D207A mutant revealed that substitutions of Asp-207 allow inclusion of cyclic porphyrins in addition to BV. A crystal structure of the entire photosensing module showed a head-to-head, twisted dimeric arrangement with bowed helical spines and a hairpin protrusion connecting the cGMP phosphodiesterase/adenylyl cyclase/FhlA (GAF) and phytochrome-specific (PHY) domains. A key conserved hairpin feature is its anti-parallel, two β-strand stem, which we show by mutagenesis to be critical for BphP photochemistry. Comparisons of single particle electron microscopic images of the full-length BphP dimer in the red light-absorbing state and the photoactivated far-red light-absorbing state revealed a large scale reorientation of the PHY domain relative to the GAF domain, which alters the position of the downstream histidine kinase output module. Together, our data support a toggle model whereby bilin photoisomerization alters GAF/PHY domain interactions through conformational modification of the hairpin, which regulates signaling by impacting the relationship between sister output modules.

Keywords: Bilin; Electron Microscopy (EM); Photoconversion; Photomorphogenesis; Photoreceptor; Phototransduction; Phytochrome; Plant Biochemistry; Protein Structure; X-ray Crystallography.

FIGURE 1.

BV binding and photochemical properties of full-length (FL) DrBphP and its PSM and PAS-GAF fragments. A, SDS-PAGE of recombinant full-length, PSM, PAS-GAF, and D207A chromoproteins either stained for protein with Coomassie Blue (Protein) or assayed for the bound bilin by zinc-induced fluorescence (Zn). B, UV-visible absorption spectra as Pr or following saturating red-light irradiation (RL, mostly Pfr) and red light-minus-darkness difference spectra (upper dashed lines). The scales for the difference spectra were reduced by 0.7 relative to the absorption spectra. Difference absorption maxima and minima are indicated. C, Pfr → Pr thermal reversion kinetics at 45 °C monitored at 750 nm following excitation with saturating red light.

FIGURE 2.

Crystal structure of the PAS-GAF fragment from D. radiodurans BphP as Pr at 1.16 Å resolution. A, ZZZssa conformation of BV in the PAS-GAF structure (PDB code 4Q0H) displayed in front and side orientations superimposed on the F_o − F_c electron density map generated by omitting BV and contouring to 3 σ. The sulfur atom in Cys-24 (yellow) that participates in the thioether linkage to BV is included, and pyrrole rings A–D are labeled. Carbons, nitrogens, oxygens, and hydrogens are colored cyan, blue, red, and white, respectively. B, architecture of the bilin-binding pocket illustrating relevant hydrophobic and electrostatic interactions that fix BV in the Pr state. Alternative conformations of Tyr-176, Phe-203, Arg-254, and Met-259, and the positions of several well ordered waters (red spheres) are included. The pyrrole water (pw) is labeled. The coloring scheme is as in A, except that the GAF domain and Cys-24 carbons are colored in green and blue, respectively. All atoms for Asp-207 are shown to illustrate its interaction with the pyrrole water. For all other residues, only the side-chain atoms are displayed. Dashed lines locate hydrogen bond contacts.

FIGURE 3.

Crystal structure of the DrBphP PAS-GAF fragment as Pr and bearing the D207A mutation. A, structure of BV at 1.75 Å resolution (PDB code 4Q0I) displayed in front and side orientations superimposed on the F_o − F_c electron density map generated by omitting BV and Tyr-263 and contouring to 3 σ. The final refined position of Tyr-263 is also shown. The sulfur atom in Cys-24 (yellow) that participates in the thioether linkage to BV is included, and pyrrole rings A–D are labeled. Carbons, nitrogens, and oxygens are colored cyan, blue, and red, respectively. B, BV is shown as in A, superposed with the final 2F_o − F_c (white) and F_o − F_c (positive, green; negative, red) electron density maps that were contoured at 1 σ and 3 σ, respectively. Atoms of the A and D pyrroles were refined at 70% occupancy, and atoms of the B and C pyrroles were refined at 100% occupancy. C, refined positions of alternate conformations of BV having the ZZZssa and ZZZsss configurations were superposed on the electron density maps from B. D, positions of Tyr-263 for wild-type PAS-GAF fragment and the D207A mutant were shown after superposition of the GAF domains. ZZZssa BV configuration, pyrrole water (pw), and residue (res) 207 positions are shown. Coloring scheme is the same as in A–C, except that wild-type carbons are white. Dashed line locates a hydrogen bond contact.

FIGURE 4.

UV-visible absorption and fluorescent properties of the D207A mutation assembled with BV and PPIX. A, absorption spectra before and after acidic denaturation of the PSM of DrBphP assembled with BV. Absorption and difference spectra were recorded for the Pr state or after saturating red-light (RL) irradiation (mostly Pfr) in nondenaturing buffer or after dissolution in 8 m urea (pH 2.0). Absorbance maxima are indicated. Difference spectra (dashed lines) were scaled to 70% of absorption spectra. B, covalent binding of BV and PPIX to the wild-type PAS-GAF fragment and the D207A mutant. Following in vitro assembly, the samples were subjected to SDS-PAGE and either stained for protein with Coomassie Blue (Protein) or assayed for the bound BV/PPIX by zinc-induced fluorescence (Zn). C and D, absorption spectra before and after acidic denaturation of the PAS-GAF fragment from wild-type DrBphP (C) and the D207A mutant (D) assembled with BV or PPIX. Absorption and difference spectra were recorded as in A. Apo, apoprotein before bilin assembly. The scales of the absorption spectra for the denatured PPIX and Apo samples from the D207A mutant were reduced 2-fold for clarity.

FIGURE 5.

Crystal structure of the PSM dimer from DrBphP as Pr at 2.75 Å resolution. A, ribbon diagram of the PSM dimer (PDB code 4Q0J) in front and side views. For one subunit, the PAS, GAF, and PHY domains and the knot lasso are colored in blue, green, orange, and yellow, respectively. BV is located by the arrow and colored in cyan with the nitrogens and oxygens colored in blue and red, respectively. The hairpin (HP) extending from the PHY domain is located by the bracket. The lines track the helical spine of one monomer. Ala-326 at the apogee of the bowed helical spine is marked by a red arrowhead. HK, histidine kinase domain; N, N terminus; C, C terminus. B, ZZZssa conformation of BV displayed in front and side orientations superposed on the F_o − F_c electron density map generated by omitting BV and contouring to 3 σ. The sulfur atom in Cys-24 (yellow) that participates in the thioether linkage to BV is included, and pyrrole rings A–D are labeled. Carbons, nitrogens, and oxygens are colored cyan, blue, and red, respectively. C, superposition of the high resolution PAS-GAF structure (PDB code 4Q0H, gray) onto the PSM structure (blue). D, close-up view of the hairpin region (orange) extending toward and contacting the GAF domain close to the chromophore. The side chains of key amino acids are included, some of which are analyzed in Figs. 9 and 10. Dashed lines locate hydrogen bond contacts. The PSM structure is colored as in A. β_ent and β_exit label the entrance (N-terminal) and exit (C-terminal) β-strands in the hairpin.

FIGURE 6.

Conformation of the PHY domain hairpin from representative Phys and its interaction with the GAF domain. PSM structures were extracted from D. radiodurans BphP as Pr (PDB code 4Q0J; this report) and a mixed Pr/Pfr state (4O01 (30)), Synechocystis Cph1 as Pr (2VEA (10)), and Cph2 as Pr (4BWI (21)), A. thaliana PhyB as Pr (4OUR (11)), P. aeruginosa BphP as Pfr (3C2W (29)), and R. palustris BphP1 (4GW9 (19)) as Pfr. The GAF and PHY domains are colored in green and orange, respectively, and the bilin is colored in cyan (arrow), the type of which is indicated. PCB, phycocyanobilin. PΦB, phytochromobilin. Side chains are shown for relevant amino acids. Dashed lines indicate hydrogen bond contacts between the DIP (Asp-Ile-Pro) motif aspartate in the GAF domain and either the conserved arginine or serine in the hairpin stem. The distance separating the GAF and PHY domain globular regions is indicated. The distances were measured from the loop separating the β1 and β2 strands of the GAF domain and the α-carbon of a conserved tryptophan (Trp-483 in DrBphP) just proximal to the exiting α-helix of the PHY domain. HP, hairpin. β_ent and β_exit label the entrance (N-terminal) and exit (C-terminal) β-strands in the hairpin.

FIGURE 7.

SPEM images of the DrBphP dimer in the Pr and Pfr states. A and B, selected reference-free two-dimensional class averages (bottom row, Avg)) generated after negative staining of Pr (A) and of samples subjected to saturating red-light irradiation (mostly Pfr) (B) in comparisons with selected two-dimensional projections (top row, Prj) of the respective three-dimensional EM maps. Left panel B shows Pfr particles with visible HK domains, and the right panel shows particles with HK domains largely invisible. C, SPEM map of DrBphP in the Pr state shown in the surface-rendered front and side views. D and E, SPEM maps of DrBphP in the Pfr (D) and Pfr′ conformers (E). The crystal structure of the PSM dimer as Pr (PDB code 4Q0J, this report) docked as a rigid body was included in C–E. One monomer is in gray, and the other monomer is shown in blue, green, and orange for the PAS, GAF, and PHY domains, respectively. PHY domain fits the density well when it is isolated from the crystal structure and allowed to rotate by 35° clockwise and move outward by ∼9 Å for Pfr (D) or by 5 Å for Pfr′ (E). F, superposition of the SPEM maps of Pr (gray) with Pfr (magenta, left) or Pfr′ (magenta, right). The downward shift of the sister HK domains, and the rotation and outward movement of the PHY domains in the Pfr or Pfr′ models are indicated by the black arrows.

FIGURE 8.

Docking of the crystal structure of the PSM as Pfr with the EM models of the BphP dimer as Pfr. A, alignment of the crystal structure of the PSM from a Pr/Pfr mixture with the SPEM models of the Pfr and Pfr′ states shown in Fig. 7. The low resolution Pr/Pfr crystal structure (PDB code 4O01 (30)) was placed into the EM density by first fitting the PAS-GAF regions alone and then docking as a rigid body the entire PSM via its alignment with the fitted PAS-GAF region. Best fit was achieved when the PHY domain crystal structure was allowed to rotate independently of the PAS-GAF domains. One monomer is in gray, and the other monomer is shown in blue, green, and orange for the PAS, GAF, and PHY domains, respectively. B, rotation of the PHY domain relative to the PAS-GAF region for improved fit of the PSM crystal structure into the EM models of the full-length DrBphP dimer. PHY domains colored in green, cyan, and magenta were isolated from the docked PSM dimer as Pr/Pfr (PDB code 4O01). The viewing direction is from the OPM toward the PSM along the 2-fold axis of the DrBphP dimer. The gray oval marks the 2-fold axis position in each panel. The PHY domain (magenta) was rotated counter-clockwise by 25 and 34° to better fit the crystal structure into the Pfr and Pfr′ EM maps, respectively.

FIGURE 9.

Assembly and photochemical properties of amino acid substitutions affecting the hairpin/GAF domain contact. A, amino acid sequence alignment of hairpin region from the representative Phys illustrated in Fig. 6. The hairpin and bracketing PHY domain sequences are located by the orange and red lines, respectively. The WGG, PRXSF, and HbXE motifs in the hairpin β-strand stem are highlighted. Specific amino acids analyzed by site-directed mutagenesis are indicated by the red arrowheads. Identical and similar amino acids are shown by the black and gray boxes, respectively. The numbering is for D. radiodurans BphP. B, SDS-PAGE of the recombinant chromoproteins either stained for protein with Coomassie Blue (Prot) or assayed for the bound bilin by zinc-induced fluorescence (Zn). C, UV-visible absorption spectra as Pr or following saturating red-light irradiation (RL, mostly Pfr), and red light-minus-darkness difference spectra (upper lines). The scales for the difference spectra were reduced by 0.7 relative to the absorption spectra. Difference absorption maxima and minima are indicated.

FIGURE 10.

Thermal reversion rates for DrBphP PSM mutants affecting the hairpin/GAF domain contact. A, kinetics of Pfr → Pr thermal reversion for the mutant chromoproteins at 25 and 45 °C. B, half-life of Pfr for each mutant at 45 °C. Gray boxes represent the full-length (FL) chromoprotein and the PSM and PAS-GAF fragments. Green box represents the R202P mutant. Orange boxes represent various PHY-domain hairpin mutations. The dashed lines indicate the reversion rates for the PSM and PAS-GAF truncations. See Fig. 9 for the BV assembly and absorption spectra of each mutant.

FIGURE 11.

Model for DrBphP photoconversion. Model involves the reorientation of the sister PHY domains relative to the GAF domains through light-induced conformational changes in the PHY domain hairpin from β-strand to helical and altered contacts with the GAF domain. These modifications reorient and/or increase the mobility of the sister HK domains through alterations of the bowed helical spines to ultimately impact phosphotransferase activity of the dimer. HP, hairpin. Pfr and Pfr′ represent the two photoactivated states observed by SPEM.