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Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome

Abstract

Light is a fundamental signal that regulates important physiological processes such as development and circadian rhythm in living organisms. Phytochromes form a major family of photoreceptors responsible for red light perception in plants, fungi and bacteria1. They undergo reversible photoconversion between red-absorbing (Pr) and far-red-absorbing (Pfr) states, thereby ultimately converting a light signal into a distinct biological signal that mediates subsequent cellular responses2. Several structures of microbial phytochromes have been determined in their dark-adapted Pr or Pfr states3,4,5,6,7. However, the structural nature of initial photochemical events has not been characterized by crystallography. Here we report the crystal structures of three intermediates in the photoreaction of Pseudomonas aeruginosa bacteriophytochrome (PaBphP). We used cryotrapping crystallography to capture intermediates, and followed structural changes by scanning the temperature at which the photoreaction proceeded. Light-induced conformational changes in PaBphP originate in ring D of the biliverdin (BV) chromophore, and E-to-Z isomerization about the C15 = C16 double bond between rings C and D is the initial photochemical event. As the chromophore relaxes, the twist of the C15 methine bridge about its two dihedral angles is reversed. Structural changes extend further to rings B and A, and to the surrounding protein regions. These data indicate that absorption of a photon by the Pfr state of PaBphP converts a light signal into a structural signal via twisting and untwisting of the methine bridges in the linear tetrapyrrole within the confined protein cavity.

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Figure 1: Trap–pump–trap–probe experiment.
Figure 2: Difference maps ( F light  −  F dark ) at pump temperatures between 100 and 180 K.
Figure 3: Light-induced structural changes.
Figure 4: Light-induced molecular events in PaBphP.

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Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession codes 3NHQ, 3NOP, 3NOT and 3NOU.

References

  1. Montgomery, B. L. & Lagarias, J. C. Phytochrome ancestry: sensors of bilins and light. Trends Plant Sci. 7, 357–366 (2002)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  3. 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)

    ADS  CAS  Article  Google Scholar 

  4. Yang, X., Stojkovic, E. A., Kuk, J. & Moffat, 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)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  7. Cornilescu, G., Ulijasz, A. T., Cornilescu, C. C., Markley, J. L. & Vierstra, R. D. Solution structure of a cyanobacterial phytochrome GAF domain in the red-light-absorbing ground state. J. Mol. Biol. 383, 403–413 (2008)

    CAS  Article  Google Scholar 

  8. Moffat, K. & Henderson, R. Freeze trapping of reaction intermediates. Curr. Opin. Struct. Biol. 5, 656–663 (1995)

    CAS  Article  Google Scholar 

  9. Rajagopal, S., Schmidt, M., Anderson, S., Ihee, H. & Moffat, K. Analysis of experimental time-resolved crystallographic data by singular value decomposition. Acta Crystallogr. D 60, 860–871 (2004)

    Article  Google Scholar 

  10. Wagner, J. R., Zhang, J., 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 

  11. Foerstendorf, H., Mummert, E., Schafer, 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 

  12. Song, C. et al. Two ground state isoforms and a chromophore D-ring photoflip triggering extensive intramolecular changes in a canonical phytochrome. Proc. Natl Acad. Sci. USA 108, 3842–3847 (2011)

    ADS  CAS  Article  Google Scholar 

  13. Ulijasz, A. T. et al. Structural basis for the photoconversion of a phytochrome to the activated Pfr form. Nature 463, 250–254 (2010)

    ADS  CAS  Article  Google Scholar 

  14. Schumann, C. et al. Subpicosecond midinfrared spectroscopy of the Pfr reaction of phytochrome Agp1 from Agrobacterium tumefaciens . Biophys. J. 94, 3189–3197 (2008)

    ADS  CAS  Article  Google Scholar 

  15. van Thor, J. J., Ronayne, K. L. & Towrie, M. Formation of the early photoproduct lumi-R of cyanobacterial phytochrome Cph1 observed by ultrafast mid-infrared spectroscopy. J. Am. Chem. Soc. 129, 126–132 (2007)

    CAS  Article  Google Scholar 

  16. van Wilderen, L. J., Clark, I. P., Towrie, M. & van Thor, J. J. Mid-infrared picosecond pump–dump–probe and pump–repump–probe experiments to resolve a ground-state intermediate in cyanobacterial phytochrome Cph1. J. Phys. Chem. B 113, 16354–16364 (2009)

    CAS  Article  Google Scholar 

  17. Muller, M. G., Lindner, I., Martin, I., Gartner, W. & Holzwarth, A. R. Femtosecond kinetics of photoconversion of the higher plant photoreceptor phytochrome carrying native and modified chromophores. Biophys. J. 94, 4370–4382 (2008)

    ADS  Article  Google Scholar 

  18. Schwinté, P. et al. The photoreactions of recombinant phytochrome CphA from the cyanobacterium Calothrix PCC7601: a low-temperature UV–Vis and FTIR study. Photochem. Photobiol. 85, 239–249 (2009)

    Article  Google Scholar 

  19. Rohmer, T. et al. Phytochrome as molecular machine: revealing chromophore action during the Pfr → Pr photoconversion by magic-angle spinning NMR spectroscopy. J. Am. Chem. Soc. 132, 4431–4437 (2010)

    CAS  Article  Google Scholar 

  20. Dasgupta, J., Frontiera, R. R., Taylor, K. C., Lagarias, J. C. & Mathies, R. A. Ultrafast excited-state isomerization in phytochrome revealed by femtosecond stimulated Raman spectroscopy. Proc. Natl Acad. Sci. USA 106, 1784–1789 (2009)

    ADS  CAS  Article  Google Scholar 

  21. Rockwell, N. C., Shang, L., Martin, S. S. & Lagarias, J. C. Distinct classes of red/far-red photochemistry within the phytochrome superfamily. Proc. Natl Acad. Sci. USA 106, 6123–6127 (2009)

    ADS  CAS  Article  Google Scholar 

  22. Seibeck, S. et al. Locked 5Zs-biliverdin blocks the Meta-RA to Meta-RC transition in the functional cycle of bacteriophytochrome Agp1. FEBS Lett. 581, 5425–5429 (2007)

    CAS  Article  Google Scholar 

  23. Yang, X., 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)

    ADS  CAS  Article  Google Scholar 

  24. 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 

  25. Giraud, E. et al. A new type of bacteriophytochrome acts in tandem with a classical bacteriophytochrome to control the antennae synthesis in Rhodopseudomonas palustris . J. Biol. Chem. 280, 32389–32397 (2005)

    CAS  Article  Google Scholar 

  26. Yeh, K. C., Wu, S. H., Murphy, J. T. & Lagarias, J. C. A cyanobacterial phytochrome two-component light sensory system. Science 277, 1505–1508 (1997)

    CAS  Article  Google Scholar 

  27. Li, H., Zhang, J., Vierstra, R. D. & Li, H. Quaternary organization of a phytochrome dimer as revealed by cryoelectron microscopy. Proc. Natl Acad. Sci. USA 107, 10872–10877 (2010)

    ADS  CAS  Article  Google Scholar 

  28. Anderson, S., Srajer, V. & Moffat, K. Structural heterogeneity of cryotrapped intermediates in the bacterial blue light photoreceptor, photoactive yellow protein. Photochem. Photobiol. 80, 7–14 (2004)

    CAS  Article  Google Scholar 

  29. Schmidt, M. et al. Protein kinetics: structures of intermediates and reaction mechanism from time-resolved X-ray data. Proc. Natl Acad. Sci. USA 101, 4799–4804 (2004)

    ADS  CAS  Article  Google Scholar 

  30. Rockwell, N. C. et al. A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus . Biochemistry 47, 7304–7316 (2008)

    CAS  Article  Google Scholar 

  31. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  32. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  Article  Google Scholar 

  33. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  34. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  35. Giraud, E., Lavergne, J. & Vermeglio, A. Characterization of bacteriophytochrome from photosynthetic bacteria: histidine kinase signaling triggered by light and redox sensing. Methods Enzymol. 471, 135–159 (2010)

    CAS  Article  Google Scholar 

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Acknowledgements

We thank A. Möglich for comments and reading of the manuscript, and V. Sˇrajer of BioCARS for assistance in microspectrometer experiments on crystals. We also thank the staff of LSCAT and BioCARS at the Advanced Photon Source, Argonne National Laboratory for beamline access. Supported by National Institutes of Health grant GM036452 to K.M. BioCARS is supported by National Institutes of Health grant RR07707 to K.M.

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Authors

Contributions

X.Y. initiated and designed research, collected X-ray and microspectroscopic data; carried out mutagenesis and HK assays; X.Y. and Z.R. analysed and interpreted structures; Z.R. developed data analysis methods and analysed data; J.K. purified proteins and grew crystals; K.M. initiated photoreceptor projects; X.Y., Z.R. and K.M. wrote the manuscript.

Corresponding authors

Correspondence to Xiaojing Yang or Keith Moffat.

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The authors declare no competing financial interests.

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Yang, X., Ren, Z., Kuk, J. et al. Temperature-scan cryocrystallography reveals reaction intermediates in bacteriophytochrome. Nature 479, 428–432 (2011). https://doi.org/10.1038/nature10506

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