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Structural basis for the photoconversion of a phytochrome to the activated Pfr form

Nature volume 463, pages 250–254 (2010)Cite this article

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Abstract

Phytochromes are a collection of bilin-containing photoreceptors that regulate numerous photoresponses in plants and microorganisms through their ability to photointerconvert between a red-light-absorbing, ground state (Pr) and a far-red-light-absorbing, photoactivated state (Pfr)1,2. Although the structures of several phytochromes as Pr have been determined3,4,5,6,7, little is known about the structure of Pfr and how it initiates signalling. Here we describe the three-dimensional solution structure of the bilin-binding domain as Pfr, using the cyanobacterial phytochrome from Synechococcus OSB′. Contrary to predictions, light-induced rotation of the A pyrrole ring but not the D ring is the primary motion of the chromophore during photoconversion. Subsequent rearrangements within the protein then affect intradomain and interdomain contact sites within the phytochrome dimer. On the basis of our models, we propose that phytochromes act by propagating reversible light-driven conformational changes in the bilin to altered contacts between the adjacent output domains, which in most phytochromes direct differential phosphotransfer.

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Figure 1: Three-dimensional overlay of SyB -Cph1(GAF) Pr and Pfr solution structures.
Figure 2: Rotation of the A ring of the PCB chromophore during Pr to Pfr photoconversion.
Figure 3: Light-driven conformational changes for amino acids surrounding the chromophore.
Figure 4: Conformational rearrangement of Asp 86, Tyr 142 and Phe 82 during photoconversion from Pr to Pfr.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structures for Pr and Pfr are deposited in the Protein Data Bank under accession codes 2KOI and 2KLI, respectively.

References

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

    Article  CAS  Google Scholar 

  2. Vierstra, R. D. & Karniol, B. in Handbook of Photosensory Receptors (eds Briggs, W. R. & Spudich, J. L.) 171–196 (Wiley, 2005)

    Book  Google Scholar 

  3. 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)

    Article  ADS  CAS  Google Scholar 

  4. 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)

    Article  CAS  Google Scholar 

  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)

    Article  ADS  CAS  Google Scholar 

  6. 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)

    Article  CAS  Google Scholar 

  7. 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)

    Article  ADS  CAS  Google Scholar 

  8. Quail, P. H. Phytochrome photosensory signalling networks. Nature Rev. Mol. Cell Biol. 3, 85–93 (2002)

    Article  CAS  Google Scholar 

  9. Rudiger, W., Thummler, F., Cmiel, E. & Schneider, S. Chromophore structure of the physiologically active form (Pfr) of phytochrome. Proc. Natl Acad. Sci. USA 80, 6244–6248 (1983)

    Article  ADS  CAS  Google Scholar 

  10. 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)

    Article  CAS  Google Scholar 

  11. Inomata, K. et al. Sterically locked synthetic bilin derivatives and phytochrome Agp1 from Agrobacterium tumefaciens form photosensitive Pr- and Pfr-like adducts. J. Biol. Chem. 280, 24491–24497 (2005)

    Article  CAS  Google Scholar 

  12. Inomata, K. et al. Assembly of synthetic locked chromophores with Agrobacterium phytochromes Agp1 and Agp2. J. Biol. Chem. 281, 28162–28173 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  15. Ulijasz, A. T. et al. Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion. J. Biol. Chem. 283, 21251–21266 (2008)

    Article  CAS  Google Scholar 

  16. Wagner, J. R. et al. Mutational analysis of Deinococcus radiodurans bacteriophytochrome reveals key amino acids necessary for the photochromicity and proton exchange cycle of phytochromes. J. Biol. Chem. 283, 12212–12226 (2008)

    Article  CAS  Google Scholar 

  17. 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)

    Article  CAS  Google Scholar 

  18. van Thor, J. J. et al. Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1. Biochemistry 40, 11460–11471 (2001)

    Article  CAS  Google Scholar 

  19. 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)

    Article  CAS  Google Scholar 

  20. Fischer, A. J. et al. Multiple roles of a conserved GAF domain tyrosine residue in cyanobacterial and plant phytochromes. Biochemistry 44, 15203–15215 (2005)

    Article  CAS  Google Scholar 

  21. Oka, Y., Matsushita, T., Mochizuki, N., Quail, P. H. & Nagatani, A. Mutant screen distinguishes between residues necessary for light-signal perception and signal transfer by phytochrome B. PLoS Genet. 4, e1000158 (2008)

    Article  Google Scholar 

  22. Karniol, B., Wagner, J. R., Walker, J. M. & Vierstra, R. D. Phylogenetic analysis of the phytochrome superfamily reveals distinct microbial subfamilies of photoreceptors. Biochem. J. 392, 103–116 (2005)

    Article  CAS  Google Scholar 

  23. Rohmer, T. et al. Light-induced chromophore activity and signal transduction in phytochromes observed by 13C and 15N magic-angle spinning NMR. Proc. Natl Acad. Sci. USA 105, 15229–15234 (2008)

    Article  ADS  CAS  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. Inomata, K. et al. Assembly of Agrobacterium phytochromes Agp1 and Agp2 with doubly locked bilin chromophores. Biochemistry 48, 2817–2827 (2009)

    Article  CAS  Google Scholar 

  26. van Thor, J. J., Mackeen, M., Kuprov, I., Dwek, R. A. & Wormald, M. R. Chromophore structure in the photocycle of the cyanobacterial phytochrome Cph1. Biophys. J. 91, 1811–1822 (2006)

    Article  ADS  CAS  Google Scholar 

  27. Strauss, H. M., Hughes, J. & Schmieder, P. Heteronuclear solution-state NMR studies of the chromophore in cyanobacterial phytochrome Cph1. Biochemistry 44, 8244–8250 (2005)

    Article  CAS  Google Scholar 

  28. 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)

    Article  ADS  CAS  Google Scholar 

  29. Harper, S. M., Neil, L. C. & Gardner, K. H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003)

    Article  ADS  CAS  Google Scholar 

  30. Vreede, J., Crielaard, W., Hellingwerf, K. J. & Bolhuis, P. G. Predicting the signaling state of photoactive yellow protein. Biophys. J. 88, 3525–3535 (2005)

    Article  CAS  Google Scholar 

  31. 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)

    Article  CAS  Google Scholar 

  32. Ulijasz, A. T. et al. Characterization of two thermostable cyanobacterial phytochromes reveals global movements in the chromophore-binding domain during photoconversion. J. Biol. Chem. 283, 21251–21266 (2008)

    Article  CAS  Google Scholar 

  33. Ulmer, T. S., Ramirez, B. E., Delaglio, F. & Bax, A. Evaluation of backbone proton positions and dynamics in a small protein by liquid crystal NMR spectroscopy. J. Am. Chem. Soc. 125, 9179–9191 (2003)

    Article  CAS  Google Scholar 

  34. Cornilescu, G. & Bax, A. Measurement of proton, nitrogen, and carbonyl chemical shielding anisotropies in a protein dissolved in a dilute liquid crystalline phase. J. Am. Chem. Soc. 122, 10143–10154 (2000)

    Article  CAS  Google Scholar 

  35. Ottiger, M., Delaglio, F., Marquardt, J. L., Tjandra, N. & Bax, A. Measurement of dipolar couplings for methylene and methyl sites in weakly oriented macromolecules and their use in structure determination. J. Magn. Reson. 134, 365–369 (1998)

    Article  ADS  CAS  Google Scholar 

  36. Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999)

    Article  CAS  Google Scholar 

  37. Wüthrich, K. NMR of Proteins and Nucleic Acids (Wiley Interscience, 1986)

    Book  Google Scholar 

  38. Bishop, J. E. et al. Phycobiliprotein-bilin linkage diversity. I. Structural studies on A- and D-ring-linked phycocyanobilins. J. Biol. Chem. 261, 6790–6796 (1986)

    CAS  PubMed  Google Scholar 

  39. Lagarias, J. C., Glazer, A. N. & Rapoport, H. Chromopeptides from C-phycocyanin. Structure and linkage of a phycocyanobilin bound to the β subunit. J. Am. Chem. Soc. 101, 5030–5037 (1979)

    Article  CAS  Google Scholar 

  40. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

    Article  ADS  CAS  Google Scholar 

  41. Schuttelkopf, A. W. & van Aalten, D. M. PRODRG: a tool for high-throughput crystallography of protein–ligand complexes. Acta Crystallogr. D 60, 1355–1363 (2004)

    Article  Google Scholar 

  42. Garrett, D. S., Powers, R., Gronenborn, A. M. & Clore, G. M. A common sense approach to peak picking two-, three- and four-dimensional spectra using automatic computer analysis of contour diagrams. J. Magn. Reson. 95, 214–220 (1991)

    ADS  CAS  Google Scholar 

  43. Ottiger, M. & Bax, A. Bicelle-based liquid crystals for NMR-measurement of dipolar couplings at acidic and basic pH values. J. Biomol. NMR 13, 187–191 (1999)

    Article  CAS  Google Scholar 

  44. Tjandra, N. & Bax, A. Direct measurement of distances and angles in biomolecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114 (1997)

    Article  ADS  CAS  Google Scholar 

  45. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Cryst. 26, 283–291 (1993)

    Article  CAS  Google Scholar 

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Acknowledgements

We thank K. T. Forest and W. M. Westler for technical advice. This work was supported by a grant from the US National Science Foundation (R.D.V.) and a postdoctoral fellowship from the American Heart Association (A.T.U.). C.C.C. was supported by the U.S. National Institutes of Health. This study was a collaboration with the National Magnetic Resonance Facility at Madison, which is supported by the US National Institute of Health.

Author Contributions R.D.V. and A.T.U. initiated the collaboration. A.T.U. and J.Z. purified the chromoproteins and A.T.U. characterized the samples. M.R. provided the isotopically labelled ALA. G.C. collected the NMR spectra and solved the NMR structures with C.C.C. and J.L.M. All authors interpreted the three-dimensional structures. R.D.V., A.T.U., C.C.C. and G.C. prepared the manuscript, tables and figures.

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Author notes

  1. Andrew T. Ulijasz and Gabriel Cornilescu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics and,,

    Andrew T. Ulijasz, Junrui Zhang & Richard D. Vierstra

  2. National Magnetic Resonance Facility at Madison, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA ,

    Gabriel Cornilescu, Claudia C. Cornilescu & John L. Markley

  3. Multidisciplinary Research Building, University of Kansas, Lawrence, Kansas 66057-1620, USA ,

    Mario Rivera

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  1. Andrew T. Ulijasz
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Correspondence to Richard D. Vierstra.

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This file contains Supplementary Table 1, Supplementary Figures 1-7 with Legends and Supplementary References. The y-axis of Supplementary Fig. 4e was corrected on January 25 2010. (PDF 2162 kb)

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Ulijasz, A., Cornilescu, G., Cornilescu, C. et al. Structural basis for the photoconversion of a phytochrome to the activated Pfr form. Nature 463, 250–254 (2010). https://doi.org/10.1038/nature08671

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