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Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8

Nature Plants volume 1, Article number: 14006 (2015) Cite this article

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Abstract

UVB from sunlight is an important environmental signal for plants. In Arabidopsis thaliana, the UVB signal is perceived by photoreceptor AtUVR8, which undergoes light-induced dimer dissociation. Crystallographic and mutational studies have identified two tryptophan residues at the dimer interface that are crucial for UVB responses. However, the molecular events leading up to dimer dissociation remain elusive. We applied dynamic crystallography to capture light-induced structural changes in photoactive AtUVR8 crystals. Here we report two intermediate structures at 1.67 Å resolution. At the epicentre of UVB signalling, concerted motions associated with Trp 285/Trp 233 lead to ejection of a water molecule, which weakens a network of hydrogen bonds and salt bridges at the dimer interface. Partial opening of the β-propeller structure, due to thermal relaxation of conformational strains originating in the epicentre, further disrupts the dimer interface and leads to dissociation. These dynamic crystallographic observations provide structural insights into the photo-perception and signalling mechanism of UVR8.

Two AtUVR8 crystal structures, both with truncated C termini, have been determined at high resolution9,10. Although they differ in space group and crystallization conditions, both structures show nearly identical dimer arrangements. As predicted, the AtUVR8 monomer adopts a seven-blade β-propeller structure14. Deletion of ∼60 residues at the C terminus of AtUVR8 did not seem to affect dimer formation nor UV-induced dimer dissociation9,10. The dimer interface, flat in shape and symmetrical in structure, buries a significant surface area where a network of hydrogen bonds and salt bridges forms extensive inter-subunit interactions9,10. UVR8 is also unique among all known photoreceptors; it does not use any non-protein cofactors as a chromophore9,10,15. Instead, AtUVR8 is highly rich in UV-absorbing aromatic residues and comprises a total of 14 tryptophans in a monomer. Aside from the C-terminal Trp 400, seven Trp residues from each monomer are found at the dimer interface whereas others are distributed among six blades at equivalent positions with their indole rings buried between blades9,10. Trp 285 and Trp 233 at the dimer interface have been identified as potential chromophores on the basis of structural analysis and mutational studies6,9–11. However, key questions concerning the photo-perception mechanism of UVR8 remain unanswered. Specifically, what is the structural nature of the triggering event? Are there any light-induced conformational changes associated with Trp 285/Trp 233? What is the sequence of molecular events leading up to dimer dissociation? Why did AtUVR8 evolve to comprise 14 Trp residues when only two of them are essential?

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Figure 1: Epicentre of UVB signalling in AtUVR8.
Figure 2: Light-induced difference densities at 180 K.
Figure 3: Light-induced structural changes.
Figure 4: Mechanism for photoreaction and dimer dissociation of UVR8.

References

  1. Heijde, M. & Ulm, R. UV-B photoreceptor-mediated signalling in plants. Trends Plant Sci. 17, 230–237 (2012).

    Article  Google Scholar 

  2. Fehér, B. et al. Functional interaction of the circadian clock and UV RESISTANCE LOCUS 8-controlled UV-B signaling pathways in Arabidopsis thaliana. Plant J. 67, 37–48 (2011).

    Article  Google Scholar 

  3. Kolowrat, C. et al. Ultraviolet stress delays chromosome replication in light/dark synchronized cells of the marine cyanobacterium Prochlorococcus marinus PCC9511. BMC Microbiol. 10, 204 (2010).

    Article  Google Scholar 

  4. Jenkins, G. I. The UV-B photoreceptor UVR8: from structure to physiology. Plant Cell 26, 21–37 (2014).

    Article  Google Scholar 

  5. Klein, R. M. Plants and near-ultraviolet radiation. Bot. Rev. 44, 1–127 (1978).

    Article  Google Scholar 

  6. Rizzini, L. et al. Perception of UV-B by the Arabidopsis UVR8 Protein. Science 332, 103–106 (2011).

    Article  Google Scholar 

  7. Favory, J.-J. et al. Interaction of COP1 and UVR8 regulates UV-B-induced photomorphogenesis and stress acclimation in Arabidopsis. EMBO J. 28, 591–601 (2009).

    Article  Google Scholar 

  8. Brown, B. A. et al. A UV-B-specific signaling component orchestrates plant UV protection. Proc. Natl Acad. Sci. USA 102, 18225–18230 (2005).

    Article  Google Scholar 

  9. Wu, D. et al. Structural basis of ultraviolet-B perception by UVR8. Nature 484, 214–219 (2012).

    Article  Google Scholar 

  10. Christie, J. M. et al. Plant UVR8 photoreceptor senses UV-B by tryptophan-mediated disruption of cross-dimer salt bridges. Science 335, 1492–1496 (2012).

    Article  Google Scholar 

  11. O'Hara, A. & Jenkins, G. I. In vivo function of tryptophans in the Arabidopsis UV-B photoreceptor UVR8. Plant Cell 24, 3755–3766 (2012).

    Article  Google Scholar 

  12. Wu, M., Strid, Å. & Eriksson, L. A. Interactions and stabilities of the UV RESISTANCE LOCUS8 (UVR8) protein dimer and its key mutants. J. Chem. Inf. Model. 53, 1736–1746 (2013).

    Article  Google Scholar 

  13. Cloix, C. et al. C-terminal region of the UV-B photoreceptor UVR8 initiates signaling through interaction with the COP1 protein. Proc. Natl Acad. Sci. USA 109, 16366–16370 (2012).

    Article  Google Scholar 

  14. Kliebenstein, D. J., Lim, J. E., Landry, L. G. & Last, R. L. Arabidopsis UVR8 regulates ultraviolet-B signal transduction and tolerance and contains sequence similarity to human regulator of chromatin condensation 1. Plant Physiol. 130, 234–243 (2002).

    Article  Google Scholar 

  15. Hofmann, N. R. The molecular mechanism of the UVR8 UV-B photoreceptor. Plant Cell 24, 3485–3485 (2012).

    Article  Google Scholar 

  16. Wu, M., Strid, Å. & Eriksson, L. A. Photochemical reaction mechanism of UV-B-induced monomerization of UVR8 dimers as the first signaling event in UV-B-regulated gene expression in plants. J. Phys. Chem. B 118, 951–965 (2014).

    Article  Google Scholar 

  17. Voityuk, A. A., Marcus, R. A. & Michel-Beyerle, M.-E. On the mechanism of photoinduced dimer dissociation in the plant UVR8 photoreceptor. Proc. Natl Acad. Sci. USA 111, 5219–5224 (2014).

    Article  Google Scholar 

  18. Liu, Z. et al. Quenching dynamics of ultraviolet-light perception by UVR8 photoreceptor. J. Phys. Chem. Lett. 5, 69–72 (2014).

    Article  Google Scholar 

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

    Article  Google Scholar 

  20. Ren, Z. et al. A molecular movie at 1.8 Å resolution displays the photocycle of photoactive yellow protein, a eubacterial blue-light receptor, from nanoseconds to seconds. Biochemistry (Mosc.) 40, 13788–13801 (2001).

    Article  Google Scholar 

  21. Ren, Z. et al. Resolution of structural heterogeneity in dynamic crystallography. Acta Crystallogr. Sect. D 69, 946–959 (2013).

    Article  Google Scholar 

  22. Huang, X., Yang, P., Ouyang, X., Chen, L. & Deng, X. W. Photoactivated UVR8-COP1 Module determines photomorphogenic UV-B signaling output in Arabidopsis. PLoS Genet. 10, e1004218 (2014).

    Article  Google Scholar 

  23. Heilmann, M., Christie, J. M., Kennis, J. T. M., Jenkins, G. I. & Mathes, T. Photoinduced transformation of UVR8 monitored by vibrational and fluorescence spectroscopy. Photochem. Photobiol. Sci. http://dx.doi.org/10.1039/C4PP00246F (2014).

  24. McGuinness, C. D., Sagoo, K., McLoskey, D. & Birch, D. J. S. Selective excitation of tryptophan fluorescence decay in proteins using a subnanosecond 295 nm light-emitting diode and time-correlated single-photon counting. Appl. Phys. Lett. 86, 261911 (2005).

    Article  Google Scholar 

  25. Peng, H. et al. Ultraviolet light-emitting diodes operating in the 340 nm wavelength range and application to time-resolved fluorescence spectroscopy. Appl. Phys. Lett. 85, 1436–1438 (2004).

    Article  Google Scholar 

  26. Heijde, M. et al. Constitutively active UVR8 photoreceptor variant in Arabidopsis. Proc. Natl Acad. Sci. USA 110, 20326–20331 (2013).

    Article  Google Scholar 

  27. Neer, E. J., Schmidt, C. J., Nambudripad, R. & Smith, T. F. The ancient regulatory-protein family of WD-repeat proteins. Nature 371, 297–300 (1994).

    Article  Google Scholar 

  28. Glazer, A. N. Light guides. Directional energy transfer in a photosynthetic antenna. J. Biol. Chem. 264, 1–4 (1989).

    Google Scholar 

  29. Liu, Z. et al. Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution. Nature 428, 287–292 (2004).

    Article  Google Scholar 

  30. Porezag, D., Frauenheim, T., Köhler, T., Seifert, G. & Kaschner, R. Construction of tight-binding-like potentials on the basis of density-functional theory: Application to carbon. Phys. Rev. B 51, 12947–12957 (1995).

    Article  Google Scholar 

  31. Elstner, M. et al. Self-consistent-charge density-functional tight-binding method for simulations of complex materials properties. Phys. Rev. B 58, 7260–7268 (1998).

    Article  Google Scholar 

  32. Niehaus, T. A. et al. Tight-binding approach to time-dependent density-functional response theory. Phys. Rev. B 63, 085108 (2001).

    Article  Google Scholar 

  33. Niehaus, T. A. Approximate time-dependent density functional theory. J. Mol. Struct. THEOCHEM 914, 38–49 (2009).

    Article  Google Scholar 

  34. Rogers, D. M., Besley, N. A., O'Shea, P. & Hirst, J. D. Modeling the absorption spectrum of tryptophan in proteins. J. Phys. Chem. B 109, 23061–23069 (2005).

    Article  Google Scholar 

  35. Reshetnyak, Y. K., Koshevnik, Y. & Burstein, E. A. Decomposition of protein tryptophan fluorescence spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues. Biophys. J. 81, 1735–1758 (2001).

    Article  Google Scholar 

  36. Wöhri, A. B. et al. Light-induced structural changes in a photosynthetic reaction center caught by Laue diffraction. Science 328, 630–633 (2010).

    Article  Google Scholar 

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Acknowledgements

We thank L.C. Peng for providing the Arabidopsis thaliana cDNA library. We thank the LS-CAT staff at Advanced Photon Source, Argonne National Laboratory for beamline support. X.Y. acknowledges support from National Institute of Health grants GM036452 and EY024363. K.H.Z. acknowledges support from National Natural Science Foundation of China (Grants 31270893 and 31110103912) and State Key Laboratory of Agricultural Microbiology of China. The TD-DFTB calculation work was supported by the Research Grants Council of Hong Kong SAR grant (project No. CityU 103812 to R.Z.) and grants from City University of Hong Kong (project No. 7200350 and 9610291 to J.F.). Use of the Advanced Photon Source was supported by the US Department of Energy (Office of Science and Office of Basic Energy Sciences) under Contract No. DE-AC02-06CH11357.

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  1. Xiaoli Zeng and Zhong Ren: These authors contributed equally to this work

Authors and Affiliations

  1. Key State Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, 430070, Hubei, P.R. China

    Xiaoli Zeng, Pan-Pan Peng, Kun Tang & Kai-Hong Zhao

  2. Renz Research Inc., Westmont, 60559, Illinois, USA

    Zhong Ren

  3. Department of Physics and Materials Science, City University of Hong Kong, Kowloon, Hong Kong

    Qi Wu, Jun Fan & Ruiqin Zhang

  4. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, 60637, Illinois, USA

    Xiaojing Yang

  5. Department of Chemistry, The University of Illinois at Chicago, Chicago, 60607, Illinois, USA

    Xiaojing Yang

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Contributions

X.Y. conceived and designed the research, and initiated and coordinated collaborations; X.Z. cloned, purified and crystallized wild type and mutant proteins, and also conducted mutagenesis and spectroscopic work; Z.R. carried out data analysis, and refined and interpreted structures; Q.W., J.F. and R.Z. carried out TD-DFTB calculations; P.P.P. and K.T. contributed to mutagenesis and protein purification; K.H.Z. supervised and designed research at HZAU; X.Y. carried out X-ray experiments and data analysis; X.Y. and Z.R. wrote the manuscript.

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Correspondence to Kai-Hong Zhao or Xiaojing Yang.

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Zeng, X., Ren, Z., Wu, Q. et al. Dynamic crystallography reveals early signalling events in ultraviolet photoreceptor UVR8. Nature Plants 1, 14006 (2015). https://doi.org/10.1038/nplants.2014.6

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