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A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes

Abstract

Phytochromes are bimodal photoswitches composed of a photosensor and an output module. Photoactivation of the sensor is initiated by a double bond isomerization of the tetrapyrrole chromophore and eventually leads to protein conformational changes. Recently determined structural models of phytochromes identify differences between the inactive and the signalling state but do not reveal the mechanism of photosensor activation or deactivation. Here, we report a vibrational spectroscopic study on bathy phytochromes that demonstrates that the formation of the photoactivated state and thus (de)activation of the output module is based on proton translocations in the chromophore pocket coupling chromophore and protein structural changes. These proton transfer steps, involving the tetrapyrrole and a nearby histidine, also enable thermal back-isomerization of the chromophore via keto–enol tautomerization to afford the initial dark state. Thus, the same proton re-arrangements inducing the (de)activation of the output module simultaneously initiate the reversal of this process, corresponding to a negative feedback mechanism.

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Figure 1: Photocycle and structural models of the Pfr state of PaBphP and Agp2.
Figure 2: Experimental and calculated Raman spectra of the Pfr state of Agp2 in the C=C stretching region with different protonation/deuteration patterns of the chromophore.
Figure 3: Infrared difference spectra of Agp2 showing the C=O stretching region for different protonation/deuteration states of the chromophore.
Figure 4: Resonance Raman and infrared difference spectra of Agp2 obtained from samples in H2O.
Figure 5: Effect of pH on the structure and dark reversion kinetics of the Pr state of Agp2.
Figure 6: Keto–enol equilibrium of the Pr chromophore of bathy phytochromes and its role for the Pr to Pfr dark reversion.

References

  1. 1

    Briggs, W. R. & Spudich, J. L. Handbook of Photosensory Receptors (Wiley, 2005).

    Google Scholar 

  2. 2

    Rockwell, N., Su, Y. & Lagarias, J. C. Phytochrome structure and signaling mechanisms. Annu. Rev. Plant Biol. 57, 837–858 (2006).

    CAS  Article  Google Scholar 

  3. 3

    Rockwell, N. C. & Lagarias, J. C. A brief history of phytochromes. ChemPhysChem 11, 1172–1180 (2010).

    CAS  Article  Google Scholar 

  4. 4

    Karniol, B. & Vierstra, R. D. The pair of bacteriophytochromes from Agrobacterium tumefaciens are histidine kinases with opposing photobiological properties. Proc. Natl Acad. Sci. USA 100, 2807–2812 (2003).

    CAS  Article  Google Scholar 

  5. 5

    Wagner, J. R., Brunzelle, J. S., Forest, K. T. & Vierstra, R. D. A light-sensing knot revealed by the structure of the chromophore-binding domain of phytochrome. Nature 438, 325–331 (2005).

    CAS  Article  Google Scholar 

  6. 6

    Wagner, J. R., Zhang, J. R., Brunzelle, J. S., Vierstra, R. D. & Forest, K. T. High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution. J. Biol. Chem. 282, 12298–12309 (2007).

    CAS  Article  Google Scholar 

  7. 7

    Yang, X., Stojkovic, E. A., Kuk, J. & Moffatt, K. Crystal structure of the chromophore binding domain of an unusual bacteriophytochrome, RpBphP3, reveals residues that modulate photoconversion. Proc. Natl Acad. Sci. USA 104, 12571–12576 (2007).

    CAS  Article  Google Scholar 

  8. 8

    Essen, L. O., Hughes, J. & Mailliet, J. The structure of a complete phytochrome sensory module in the Pr ground state. Proc. Natl Acad. Sci. USA 105, 14709–14714 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Bellini, D. & Papiz, M. Z. Dimerization properties of the RpBphP2 chromophore-binding domain crystallized by homologue-directed mutagenesis. Acta Crystallogr. D 68, 1058–1066 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Yang, X. J., Kuk, J. & Moffat, K. Crystal structure of Pseudomonas aeruginosa bacteriophyrochrome: photoconversion and signal transduction. Proc. Natl Acad. Sci. USA 105, 14715–14720 (2008).

    CAS  Article  Google Scholar 

  11. 11

    Yang, X. J., Kuk, J. & Moffat, K. Conformational differences between the Pfr and Pr states in Pseudomonas aeruginosa bacteriophytochrome. Proc. Natl Acad. Sci. USA 106, 15639–15644 (2009).

    CAS  Article  Google Scholar 

  12. 12

    Yang, X. J., Ren, Z., Kuk, J. & Moffat, K. Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome. Nature 479, 428–432 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Takala, H. et al. Signal amplification and transduction in phytochrome photosensors. Nature 509, 245–248 (2014).

    CAS  Article  Google Scholar 

  14. 14

    Anders, K., Daminelli-Widany, G., Mroginski, M. A., von Stetten, D. & Essen, L. O. Structure of the cyanobacterial phytochrome 2 photosensor implies a tryptophan switch for phytochrome signaling. J. Biol. Chem. 288, 35714–35725 (2013).

    CAS  Article  Google Scholar 

  15. 15

    Lamparter, T., Michael, N., Mittmann, F. & Esteban, B. Phytochrome from Agrobacterium tumefaciens has unusual spectral properties and reveals an N-terminal chromophore attachment site. Proc. Natl Acad. Sci. USA 99, 11628–11633 (2002).

    CAS  Article  Google Scholar 

  16. 16

    Inomata, K. et al. Assembly of synthetic locked chromophores with Agrobacterium phytochromes AGP1 and AGP2. J. Biol. Chem. 281, 28162–28173 (2006).

    CAS  Article  Google Scholar 

  17. 17

    Rottwinkel, G., Oberpichler, I. & Lamparter, T. Bathy phytochromes in rhizobial soil bacteria. J. Bacteriol. 192, 5124–5133 (2010).

    CAS  Article  Google Scholar 

  18. 18

    Zienicke, B. et al. Unusual spectral properties of bacteriophytochrome Agp2 result from a deprotonation of the chromophore in the red-absorbing form Pr. J. Biol. Chem. 44, 31738–33175 (2013).

    Article  Google Scholar 

  19. 19

    Salewski, J. et al. The structure of the biliverdin cofactor in the Pfr state of bathy and prototypical phytochromes. J. Biol. Chem. 288, 16800–16814 (2013).

    CAS  Article  Google Scholar 

  20. 20

    Scheerer, P. et al. Light-induced conformational changes of the chromophore and the protein in phytochromes: bacterial phytochromes as model systems. ChemPhysChem 26, 1090–1105 (2010).

    Article  Google Scholar 

  21. 21

    Tasler, R., Moises, T. & Frankenberg-Dinkel, N. Biochemical and spectroscopic characterization of the bacterial phytochrome of Pseudomonas aeruginosa. FEBS J. 272, 1927–1936 (2005).

    CAS  Article  Google Scholar 

  22. 22

    Mroginski, M. A. et al. Elucidating photoinduced structural changes in phytochromes by the combined application of resonance Raman spectroscopy and theoretical methods. J. Mol. Struct. 993, 15–25 (2011).

    CAS  Article  Google Scholar 

  23. 23

    Fodor, S. P. A., Lagarias, J. C. & Mathies, R. A. Resonance Raman analysis of the Pr and Pfr forms of phytochrome. Biochemistry 29, 11141–11146 (1990).

    CAS  Article  Google Scholar 

  24. 24

    Kneip, C. et al. Protonation state and structural changes of the tetrapyrrole chromophore during the Pr → Pfr phototransformation of phytochrome. A resonance Raman spectroscopic study. Biochemistry 38, 15185–15192 (1999).

    CAS  Article  Google Scholar 

  25. 25

    Andel, F. III, Lagarias, J. C. & Mathies, R. A. Resonance Raman analysis of chromophore structure in the lumi-R photoproduct of phytochrome. Biochemistry 35, 15997–16008 (1996).

    CAS  Article  Google Scholar 

  26. 26

    Borucki, B. et al. Light-induced proton release of phytochrome is coupled to the transient deprotonation of the tetrapyrrole chromophore. J. Biol. Chem. 280, 34358–34364 (2005).

    CAS  Article  Google Scholar 

  27. 27

    von Stetten, D. et al. Highly conserved residues Asp-197 and His-250 in Agp1 phytochrome control the proton affinity of the chromophore and Pfr formation. J. Biol. Chem. 282, 2116–2123 (2007).

    CAS  Article  Google Scholar 

  28. 28

    Foerstendorf, H., Mummert, E., Schäfer, E., Scheer, H. & Siebert, F. Fourier-transform infrared spectroscopy of phytochrome: difference spectra of the intermediates of the photoreactions. Biochemistry 35, 10793–10799 (1996).

    CAS  Article  Google Scholar 

  29. 29

    Van Thor, J. J., Fisher, N. & Rich, P. R. Assignments of the Pfr–Pr FTIR difference spectrum of cyanobacterial phytochrome Cph1 using 15N and 13C isotopically labeled phycocyanobilin chromophore. J. Phys. Chem. B 109, 20597–20604 (2005).

    CAS  Article  Google Scholar 

  30. 30

    Schwinté, P. et al. FTIR study of the photoinduced processes of plant phytochrome phyA using isotope-labeled bilins and density functional theory calculations. Biophys. J. 95, 1256–1267 (2008).

    Article  Google Scholar 

  31. 31

    Piwowarski, P. et al. Light induced activation of bacterial phytochrome Agp1 monitored by static and time resolved FTIR spectroscopy. Chem Phys Chem 11, 1207–1214 (2010).

    CAS  Article  Google Scholar 

  32. 32

    Lightner, D. A., Holmes, D. L. & McDonagh, A. F. On the acid dissociation constants of bilirubin and biliverdin. pKa values from 13C NMR spectroscopy. J. Biol. Chem. 271, 2397–2405 (1996).

    CAS  Article  Google Scholar 

  33. 33

    Mao, J., Hauser, K. & Gunner, M. R. How cytochromes with different folds control redox potentials. Biochemistry 42, 9829–9840 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Barth, A. Infrared spectroscopy of proteins. Biochim. Biophys. Acta 1767, 1073–1101 (2007).

    CAS  Article  Google Scholar 

  35. 35

    Popp, A., Wu, L., Keiderling, T. A. & Hauser, K. Impact of β-turn sequence on β-hairpin dynamics studied with infrared-detected temperature jump. Spectrosc. Int. J. 27, 557–564 (2012).

    CAS  Article  Google Scholar 

  36. 36

    Lagarias, J. C. & Rapoport, H. Chromopeptides from phytochrome. The structure and linkage of the Pr form of the phytochrome chromophore. J. Am. Chem. Soc. 102, 4821–4828 (1980).

    CAS  Article  Google Scholar 

  37. 37

    Song, C. et al. Solid-state NMR of a canonical phytochrome reveals two Pr isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes. Proc. Natl Acad. Sci. USA 108, 3842–3847 (2011).

    CAS  Article  Google Scholar 

  38. 38

    Song, C. et al. Solid-state NMR spectroscopy to probe photoactivation in canonical phytochromes. Photochem. Photobiol. 89, 259–273 (2013).

    CAS  Article  Google Scholar 

  39. 39

    Haupts, U., Tittor, J. & Oesterhelt, D. Closing in on bacteriorhodopsin: progress in understanding the molecule. Annu. Rev. Biophys. Biomol. Struct. 28, 367–399 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Lamparter, T. & Michael, N. Agrobacterium phytochrome as an enzyme for the production of ZZE bilins. Biochemistry 44, 8461–8469 (2005).

    CAS  Article  Google Scholar 

  41. 41

    Peng, C. S., Baiz, C. R. & Tokmakoff, A. Direct observation of ground state lactam–lactim tautomerization using temperature-jump transient 2D IR spectroscopy. Proc. Natl Acad. Sci. USA 110, 9243–9248 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft, Sfb1078 (B5, B6, C3). The authors thank the ‘Norddeutscher Verbund für Hoch- und Höchstleistungsrechnen’ (HLRN) for providing computer power.

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F.V.E., P.P., M.F.L. and A.R. carried out the RR, infrared and ultraviolet–vis spectroscopic measurements. J.S. and M.A.M. performed and analysed the QM/MM calculations. P.S. provided the homology model for Agp2 and analysed the structural data. B.M.Q. and P.S. provided initial activation assays. F.B. and F.S. analysed the spectroscopic data. P.H. wrote the manuscript with contributions from all authors. The project and experiments were planned and designed by all team members.

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Correspondence to Peter Hildebrandt.

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Velazquez Escobar, F., Piwowarski, P., Salewski, J. et al. A protonation-coupled feedback mechanism controls the signalling process in bathy phytochromes. Nature Chem 7, 423–430 (2015). https://doi.org/10.1038/nchem.2225

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