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Comparative Study
. 2013 Jul;20(7):859-67.
doi: 10.1038/nsmb.2597. Epub 2013 Jun 2.

A Ternary AppA-PpsR-DNA Complex Mediates Light Regulation of Photosynthesis-Related Gene Expression

Free PMC article
Comparative Study

A Ternary AppA-PpsR-DNA Complex Mediates Light Regulation of Photosynthesis-Related Gene Expression

Andreas Winkler et al. Nat Struct Mol Biol. .
Free PMC article


The anoxygenic phototrophic bacterium Rhodobacter sphaeroides uses different energy sources, depending on environmental conditions including aerobic respiration or, in the absence of oxygen, photosynthesis. Photosynthetic genes are repressed at high oxygen tension, but at intermediate levels their partial expression prepares the bacterium for using light energy. Illumination, however, enhances repression under semiaerobic conditions. Here, we describe molecular details of two proteins mediating oxygen and light control of photosynthesis-gene expression: the light-sensing antirepressor AppA and the transcriptional repressor PpsR. Our crystal structures of both proteins and their complex and hydrogen/deuterium-exchange data show that light activation of AppA-PpsR2 affects the PpsR effector region within the complex. DNA binding studies demonstrate the formation of a light-sensitive ternary AppA-PpsR-DNA complex. We discuss implications of these results for regulation by light and oxygen, highlighting new insights into blue light-mediated signal transduction.


Figure 1
Figure 1. AppA and PpsR domains involved in AppA–PpsR2 formation and PpsR tetramerization
(a) The upper part shows protein constructs of AppA used in this study color-coded according to domains described previously or identified in the crystal structure presented here: BLUF – orange; linker region including the BLUF capping helix – bluish green; 4HB – red; SCHIC – blue and cysteine-rich – purple. The lower part depicts PpsR constructs of this study colored according to: N-domain – orange; αQ – red; PAS1 – blue; PAS2 – bluish green and HTH – purple. (b) Normalized MALS detection of PpsR fractionated by size exclusion chromatography (solid – UV absorbance, dashed – scattering signal). The calculated molar mass signal is plotted in green. The inset shows the quantification of the oligomer equilibrium using MST resulting in a Kd of 0.9 μM based on dimer concentration. Error bars represent the standard deviation of three individual experiments. (c) Quantification of the AppAΔC–PpsR2 interaction using MST resulted in a Kd of 1.3 μM calculated for a PpsR dimer as binding partner. MST measurements were performed at 25 °C in triplicate and error bars correspond to the standard deviation.
Figure 2
Figure 2. AppAΔC–PpsR2 formation allows light-control of PpsR-binding to DNA
(a) Quantification of native PAGE experiments addressing AppAΔC–PpsR2 formation under dark (solid) and light (dashed) conditions. PpsR (3 μM) was titrated with AppAΔC (Supplementary Fig. 2b,c) (b) DNA-binding curve of PpsR obtained from two complementary titrations of puc I (Supplementary Fig. 3a). The inset shows an active site titration of PpsR (2.5 μM) with puc II. The circled 4 and 8 reflect theoretical transition points for tetramer or octamer binding, respectively. (c) EMSA showing AppAΔC–PpsR2–DNA complex formation upon PpsR titration of puc I with 1.5 μM AppAΔC present. Lanes 2–12 – PpsR concentrations from 0 to 4 μM (details in Supplementary Fig. 3d), lane 13 – 2 μM PpsR without AppAΔC and lane 14 – 2 μM PpsR with dark-recovered AppAΔC. (d) Analogous EMSA to panel c with illumination. The ternary complex is shifted towards the well (Coomassie stain in Supplementary Fig. 3e). (e) EMSAs under dark- and light-conditions using puc II and AppAΔC concentrations enabling saturation of AppAΔC–PpsR2. Lane 1 – 3 μM PpsR, lane 2 – 5 μM AppAΔC + 3 μM PpsR and lane 3 – 5 μM AppAΔC + 13 μM PpsR (protein stains in Supplementary Figure 3f). (f) Illuminated EMSA addressing light-stability of the ternary complex with puc II in the presence of 20 μM AppAΔC enabling saturation of AppAΔC–PpsR2 throughout the PpsR titration. Lanes 1–15 – 0, 0.1, 0.25, 0.5, 1, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 10, 20, 30 μM PpsR.
Figure 3
Figure 3. Domain organization of AppA supports the dual sensor function
(a) Secondary structure representation of AppA with individual domains color-coded according to Fig 1a. (b) Overall AppAΔC structure with domains colored according to panel a. (c) Stereo-view of conserved BLUF residues around the flavin cofactor (yellow) showing the positioning of Trp104. Residues contacting Trp104 from the core β-sheet and the capping helix are shown as stick models. (d) AppAΔC structure colored with respect to deuterium incorporation in the dark after 15 s labeling. Implications of the color-coding and details for the normalization procedure (Dnorm) are described in the Supplementary Note. (e) Changes in relative deuterium incorporation between dark and blue-light conditions after 15 s labeling. Details of the color-coding are described in the main text.
Figure 4
Figure 4. Structural characterization of PpsR with implications for DNA-binding and AppAΔC–PpsR2 complex formation
(a) Secondary structure representation of PpsR color-coded according to Fig 1a. Helices αN and αP2 (grey) correspond to N-terminal helices of the N- and the PAS2-domains, respectively. (b) Domain architecture of a single PpsRΔHTH protomer colored according to panel a. Parallel dimerization is shown in the lower part in surface representation. An anti-parallel dimer colored in light-blue and dark-red according to individual protomers of chains C and D completes the tetrameric assembly. (c) Model for PpsR-binding to DNA based on the crystallographic octamer. The αQ (black helices) mediated interaction between symmetry-related tetramers enables close positioning of two HTH dimers. HTH models (transparent surface) together with the DNA double helix are based on the Fis structure (pdb 3JRE). HTH dimers extend from the PAS2 C-termini and are rotated to account for DNA-binding and tilted to prevent clashes. Colors correspond to protomers (panel b) with light-orange and grey for chains A and B, respectively. (d,e) Changes in Drel upon complex formation for AppAΔC and PpsR mapped on the structures of AppAΔC and PpsRΔHTH, respectively. (f,g) Changes in Drel upon illumination of AppAΔC–PpsR2 mapped on both structures. The 15 s time points are shown in panels d, e and g for visualization of regions protected through complexation (blue), whereas the 60 s time point in panel f was chosen to additionally demonstrate the increased exchange dynamics (red) of a 4HB peptide.
Figure 5
Figure 5. HDX details of PpsR-HTH and the AppA-BLUF capping-helix
(a,b) Deuterium uptake plots of a HTH-peptide in free PpsR (brown) and after complex formation with dark-state AppAΔC (blue, panel a), as well as for AppAΔC–PpsR2 under light-conditions (red, panel b). Main sub-panels show Drel plotted against labeling time for the peptide specified at the top. The estimated abundance distribution of individual deuterated species is presented in the lower sub-panels on a scale from undeuterated to all exchangeable amides deuterated. The observation of a bimodal distribution points to the presence of an AppAΔC–PpsR2 species with the HTH motif in a conformation different to dark-adapted complex or free PpsR. (c,d,e) To assess the quality of evaluated data selected raw-spectra are shown for the 15 s time points of one HTH-peptide of free PpsR, dark-adapted AppAΔC–PpsR2 and light-state AppAΔC–PpsR2, respectively. The seven most intense isotope peaks of the series of interest are marked with an asterisk. (f,g) Comparison of deuterium uptake of an AppA-BLUF capping-helix peptide between AppAΔC–PpsR2 under dark- (green) and light-conditions (orange, panel f), as well as in free AppA (purple, panel g). Note the broad distribution of deuterated species in free AppA indicating again EX1-like exchange kinetics. Complex formation shifts the equilibrium of the two conformations responsible for bimodal deuteration to one state, which can be partially reverted by illumination. (h,i,j) Raw data of 15 s time points of dark-adapted AppAΔC–PpsR2, light-state AppAΔC–PpsR2 and free AppA, respectively.
Figure 6
Figure 6. Molecular details of the AppA–PpsR2 core interface
(a) Overall structure of the core complex color-coded according to domains of AppA (Fig. 3b) and protomers A and B of PpsR (Fig. 4c). Details of the interface are described in the text. (b) Superposition of HDX data with the observed complex interface. Data of the 15 s time points, as shown in Figure 4d and f, are mapped on the core complex with N–Q–PAS1 shown in transparent surface and the AppA domains in cartoon representation. The full time course can be seen in Supplementary Movie 6. (c) The model of the AppAΔC–PpsR2 complex is based on the individual structures after aligning the PAS1 dimers of PpsRΔHTH and 4HB and SCHIC of AppAΔC, respectively. The HTH domains are placed in analogy to Figure 4c.
Figure 7
Figure 7. A new model for controlling photosynthetic gene expression by AppA and PpsR in a light- and oxygen-dependent manner
(i.) PpsR exists in a dimer-tetramer equilibrium and (ii.) binds to promoter regions of regulated genes as an octamer (colored according to Fig. 4c). (iii.) Addition of AppA leads to the formation of AppA–PpsR2. (iv.) This species also interacts with the promoter region forming a ternary protein-DNA complex. (v.) Illumination of this complex does not lead to dissociation of AppA and PpsR but reduces the affinity for DNA. Depending on the relative concentrations of AppA and PpsR, (vi.) excess PpsR competes successfully with AppA–PpsR2 for promoter regions under light conditions but is not able to replace the ternary complex in the dark. (vii.) The levels of AppA and PpsR are inversely regulated in response to oxygen availability. An increase in oxygen concentration favors the formation of the PpsR8–DNA species leading to enhanced repression of photosynthesis gene transcription. The model depicted accomodates in vivo results presented for anaerobic,, semi-aerobic,, and higher oxygen conditions,,.

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