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Long-range allosteric signaling in red light–regulated diguanylyl cyclases

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Science Advances  03 Mar 2017:
Vol. 3, no. 3, e1602498
DOI: 10.1126/sciadv.1602498
  • Fig. 1 Overview of IsPadC.

    (A) Schematic representation of IsPadC constructs characterized in this study. Individual domains are colored in dark gray, violet, blue, green, orange, and red for the N-terminal extension (NTE), Per-ARNT-Sim (PAS), cGMP phosphodiesterase–adenylyl cyclase–FhlA (GAF), phytochrome-associated (PHY), coiled-coil (cc), and DGC domains, respectively. Coiled-coil truncations are denoted as, for example, IsPadC Δ514–520 for the variant deleted in one heptad including residues 514 to 520. (B) Spectral characteristics of dark-adapted (Pr state) IsPadC in comparison to the Pfr state obtained after red light illumination. (C) Kinetic characterization of GTP to c-di-GMP conversion. The characteristic GTP concentration dependence of initial rates of c-di-GMP formation is indicative of substrate inhibition, which is observed for both light- and dark-state activities. Product formation was quantified in triplicate for several reaction times, and the SD of individual points contributed to the error estimation of the linear fit that is used to calculate the initial rate of product formation. The SE of the estimate from the linear regression is shown as an error bar for each GTP concentration. The inset shows representative traces of the high-performance liquid chromatography (HPLC) assay used for quantifying product formation. (D) Overall structure of IsPadC with individual domains in cartoon representation colored according to (A), with one protomer highlighted in pale colors. The biliverdin cofactor, in slate color, and its attachment site Cys17 from the NTE are shown as stick models.

  • Fig. 2 Light-induced changes in conformational dynamics of full-length IsPadC observed by HDX-MS.

    (A) IsPadC structure colored according to changes in relative deuterium incorporation (ΔDrel) between light-state and dark-adapted IsPadC after 15 min of deuteration (Drel of IsPadClightDrel of IsPadCdark). The scale bar in the top left corner indicates the changes in ΔDrel, with blue corresponding to reduced deuterium incorporation and red reflecting increased exchange of amide protons upon red light illumination. The biliverdin cofactor is shown as gray stick model. (B) Close-up view of the biliverdin-binding pocket and the PHY tongue region. The 2FoFc electron density map contoured at 1σ around the cofactor is shown as a light blue mesh. Important residues are shown as stick models, and the coloration corresponds to ΔDrel of the 45-s exchange time point. Residues 200 to 207 were removed for clarity. However, the coloration of residues Phe195 and Asp199 reflect the changes in Drel of the entire region 191 to 207. This region shows a higher deuterium incorporation of peptides at the interface of the PHY tongue and the biliverdin D-ring in the light state, highlighting the importance of the conformational dynamics of this region upon isomerization of the biliverdin cofactor. (C) Close-up view of the coiled-coil linker and the DGC domain colored according to ΔDrel after 10 s of deuteration. Several structural elements of the GGDEF domain, including substrate binding elements, show a reduction in conformational dynamics upon illumination. (D to F) Deuterium uptake curves of selected IsPadC peptides, with Drel plotted against the deuteration time for light- and dark-state HDX-MS experiments. The lower parts show software-estimated abundance distributions of individual deuterated species on a scale from undeuterated to all exchangeable amides deuterated. Deuteration plots for a peptide corresponding to the PHY tongue element (D), the PSM-DGC linker (E), and the internal helical spine linking the GAF and PHY domains (F). Drel values are shown as the mean of three independent measurements, and error bars correspond to the SD. An overview of all analyzed peptides is provided in figs. S6 and S7.

  • Fig. 3 Coiled-coil architecture of the sensor-effector linker.

    (A) Heptad register observed in the crystal structure of IsPadC (register 1). (B) A rotation of heptad positions e to a within the coiled-coil populates the “register 2” architecture. (C and D) Heptad units of the sensor-effector linker in registers 1 and 2, respectively, rainbow-colored according to the heptad repeats of register 1. Coiled-coil destabilizing residues are boxed. The highly conserved DXLT motif of GGDEF domains is underlined. (E) The assignment of active and inhibited states to registers 2 and 1, respectively, is confirmed by the observed DGC activity in a cell-based screening system. Wild-type IsPadC shows the expected increase in DGC activity upon red light illumination, as seen by the red coloration of the cells. In contrast, an IsPadC variant stabilizing register 1 can no longer be activated upon illumination, whereas the register 2 stabilizing variant is constitutively active. (F) For comparison, the heptad units of a superactive, artificial GCN4-GGDEF fusion (14) are also shown.

  • Fig. 4 Structural plasticity of phytochrome dimerization.

    (A) Superposition of PSM modules from different published phytochrome structures featuring a parallel dimeric assembly to IsPadC (cyan) revealed almost identical arrangements of the PAS-GAF bidomain [root mean square deviation (RMSD) of 1.5, 1.2, 1.0, 0.9, and 0.9 Å for Protein Data Bank (PDB) 4OUR_B (blue) (60), 3G6O_A (violet) (16), 4Q0J_A (orange) (61), 5AKP_B (green) (26), and 5C5K_B (red) (20), respectively]. Irrespective of the β-hairpin or α-helical character of the tongue region, the PHY domains cluster in similar positions relative to the PAS-GAF domains. However, a characteristic flexibility of the central helix connecting the GAF and PHY domains is apparent, which ultimately results in altered positioning of the terminal PHY domain helices that link to the respective output modules. (B) The structural plasticity of the central helical spine and, consequently, the overall parallel dimeric phytochrome assembly are even more pronounced when comparing the nonsuperposed monomers. In this case, monomers of PSM modules have been aligned to the PAS-GAF-PHY monomer of IsPadC chain A, and the respective other monomers are displayed (RMSD of 3.2, 2.3, 2.2, 2.2, and 1.2 Å for PDB 3G6O_A, 4Q0J_A, 5AKP_B, 5C5K_B, and 4OUR_B, respectively). Again, no clustering of the PHY domain orientation with respect to the Pr- or Pfr-state character of the tongue can be observed. However, the structural differences among various phytochrome dimers are nonrandom and occur along a specific trajectory that corresponds to rotation at the dimer interface. The two extremes of this rotation correspond to structures obtained for Pfr-state crystals (violet and red). In contrast, the only parallel phytochrome structures with adjacent C-terminal domains (green and cyan) cluster in the middle of the overall trajectory. Although this suggests that the more pronounced dimer rotation of other PSM assemblies might be due to missing interactions of their output modules and linker regions, the characteristic structural transition reflecting the plasticity of the PHY domain dimerization is very likely functionally relevant for phytochromes in general.

  • Fig. 5 Schematic model of signal integration pathways in phytochrome-linked enzymatic effectors and in-solution structure of IsPadC.

    (A) The characteristic structure of IsPadC and regulatory properties of closely related homologs suggest a model of signal transduction corresponding to the violin model (28). Instead of a linear cascade of structural changes resulting in enzyme activation, the conformational dynamics of the whole system define the population of functionally relevant states, leading to either activation or inactivation with similar overall architectures (37). In the case of the phytochrome-violin, the pegbox corresponds to the effector domain, whose activity is tuned by the sensory module. Hitting the right chords on the enzymatic activity clef for stimulating GTP turnover is more complex than a specific actuation of the fingerboard, which would correspond to, for example, variation of its length (yellow lines and linkers C and D), and additionally depends on properties of the strings (gray), the shape of the violin body (blue), and the effector-pegbox (red; for example, two different DGC constructs A and B). The characteristic structural changes observed for various phytochrome structures (Fig. 4), which had also been interpreted as specific light-induced rearrangements (20, 24), reflect the structural plasticity of phytochrome dimerization. The latter, in turn, enables the modulation of the conformational dynamics of the overall system and thereby allows complex, evolutionary fine-tuning of the body of phytochrome-violin to optimize the output functionality as required by each system. The absence of characteristic structural changes, such as a defined rotation or a separation of the coiled-coil linker, enables the realization of systems featuring activation or inactivation within the same molecular architecture. (B) SAXS-based structural model for the IsPadC dimer in its dark-adapted Pr state. The surface represents the conformational space sampled by the GGDEF domains in the seven best structures according to the fit between the experimental and back-calculated SAXS data (compare fig. S10). Structures are aligned to the PAS-GAF-PHY domains.

  • Table 1 Comparison of PadC kinetics of substrate conversion and dark-state recoveries.
    Dark-state recovery at 700 nm*Comparison of initial rates at 200 μM GTP (μmol product min−1 μmol−1 enzyme2)
    Constructτ1 (s)Relative A1 (%)τ2 (s)Relative A2 (%)Dark stateLight stateFold activation
    IsPadC30 ± 146 ± 2118 ± 554 ± 10.8 ± 0.134.2 ± 0.443×
    MaPadC24.9 ± 0.450.0 ± 0.5458 ± 5650 ± 32 ± 0.215.4 ± 0.7
    TsPadC18.9 ± 0.280 ± 171 ± 320 ± 153 ± 642 ± 6−1.2×
    IsPadC Δ514–51613.5 ± 0.240.0 ± 0.3308 ± 1260 ± 111 ± 110 ± 2
    IsPadC Δ514–51713.1 ± 0.711.5 ± 0.3115 ± 189 ± 225 ± 749 ± 3
    IsPadC Δ514–52011.9 ± 0.386 ± 1393 ± 914.3 ± 0.127.2 ± 0.636 ± 41.3×
    IsPadC Δ442–477::SGn.d.n.d.48 ± 630 ± 1−1.6×
    IsPadC PSMcc19 ± 123 ± 1148 ± 277.0 ± 0.3
    IsPadC ccDGC0.2 ± 0.1−4× (to IsPadC)

    *Dark-state recoveries of the constructs were fit to a second-order exponential decay. After red light illumination of 1 min, changes in absorption at 700 nm were followed over 5 min, with automatic sampling every 5 s and an integration time of 0.01 s. The contribution of each phase in the dark recovery process is represented as relative amplitude. The SE of the estimate from the nonlinear curve fit corresponding to y = A1*exp(−x1) + A2*exp(−x2) + y0 was used as error indicator.

    †Comparison of product formation between the various constructs was performed for initial reaction rates at 50 μM GTP. Initial rates are quantified from experimental triplicates for three time points, and the SD of individual points contributed to the error estimation of the linear fit that is used to calculate the initial rate of product formation. The SE of the estimate from the linear regression is used as error indicator.

    ‡The recovery of this construct could not be determined because it appears to be locked in the Pfr-enriched state after red light illumination and does not significantly change its spectrum during a 24-hour incubation at room temperature in the dark.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/3/e1602498/DC1

      Supplementary Results

      fig. S1. Multiple sequence alignment of PadC homologs generated with Jalview (64).

      fig. S2. Spectroscopic and kinetic characterization of TsPadC and MaPadC.

      fig. S3. Characterization of the IsPadC PSMcc variant.

      fig. S4. IsPadC crystal and spectral characteristics of dark-state crystallized IsPadC.

      fig. S5. Effect of substrate binding on the overall architecture of IsPadC.

      fig. S6. Summary of HDX experiments.

      fig. S7. Individual deuterium incorporation plots of all evaluated peptides.

      fig. S8. Spectroscopic and kinetic characterization of IsPadC deletion variants.

      fig. S9. Time course of tryptic digests of IsPadC, MaPadC, and TsPadC under dark and light conditions.

      fig. S10. Details of SAXS measurements.

      table S1. Data collection, phasing, and refinement statistics.

      table S2. Overview of oligonucleotides and buffers.

      movie S1. Changes in conformational dynamics upon red light illumination of IsPadC.

      movie S2. Changes in conformational dynamics upon red light illumination of the IsPadC PSMcc variant.

      movie S3. The influence of effector deletion on conformational dynamics of the dark-state IsPadC PSMcc assembly.

      movie S4. The influence of effector deletion on conformational dynamics of the light-state IsPadC PSMcc assembly.

      References (6266)

    • Supplementary Materials

      This PDF file includes:

      • Supplementary Results
      • fig. S1. Multiple sequence alignment of PadC homologs generated with
        Jalview (64).
      • fig. S2. Spectroscopic and kinetic characterization of TsPadC and MaPadC.
      • fig. S3. Characterization of the IsPadC PSMcc variant.
      • fig. S4. IsPadC crystal and spectral characteristics of dark-state
        crystallized IsPadC.
      • fig. S5. Effect of substrate binding on the overall architecture of IsPadC.
      • fig. S6. Summary of HDX experiments.
      • fig. S7. Individual deuterium incorporation plots of all evaluated peptides.
      • fig. S8. Spectroscopic and kinetic characterization of IsPadC deletion variants.
      • fig. S9. Time course of tryptic digests of IsPadC, MaPadC, and TsPadC under dark and light conditions.
      • fig. S10. Details of SAXS measurements.
      • table S1. Data collection, phasing, and refinement statistics.
      • table S2. Overview of oligonucleotides and buffers.
      • Legends for movies S1 to S4
      • References (62–66)

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      Other Supplementary Material for this manuscript includes the following:

      • movie S1(.mp4 format). Changes in conformational dynamics upon red light illumination of IsPadC.
      • movie S2 (.mp4 format). Changes in conformational dynamics upon red light illumination of the IsPadC PSMcc variant.
      • movie S3 (.mp4 format). The influence of effector deletion on conformational dynamics of the dark-state IsPadC PSMcc assembly.
      • movie S4 (.mp4 format). The influence of effector deletion on conformational dynamics of the light-state IsPadC PSMcc assembly.

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