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Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein

Nature Plants volume 4, pages 920–929 (2018)Cite this article

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Abstract

We altered the chlorophyll (Chl) binding sites in various versions of water-soluble chlorophyll protein (WSCP) by amino acid exchanges to alter their preferences for either Chl a or Chl b. WSCP is ideally suited for this mutational analysis since it forms a tetrameric complex with only four identical Chl binding sites. A loop of 4–6 amino acids is responsible for Chl a versus Chl b selectivity. We show that a single amino acid exchange within this loop changes the relative Chl a/b affinities by a factor of 40. We obtained crystal structures of this WSCP variant binding either Chl a or Chl b. The Chl binding sites in these structures were compared with those in the major light-harvesting complex (LHCII) of the photosynthetic apparatus in plants to search for similar structural features involved in Chl a/b binding specificity.

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Fig. 1: Comparison of investigated Chl a/b binding proteins.
Fig. 2: Chl a/b affinity motifs in WSCP.
Fig. 3: Qualitative determination of relative Chl a/b binding affinities.
Fig. 4: Quantitative determination of relative Chl a/b binding affinities.
Fig. 5: Comparison of the aligned crystallographic structures of Lv-wt (grey)27 and LvPCPS (with Chl a in blue, PDB-code: 6GIW; with Chl b in green, PDB-code: 6GIX).
Fig. 6: Interaction between the different Chl-selectivity motifs with bound Chl b.
Fig. 7: Surrounding of the C-71 groups of LHCII-bound Chls b molecules.
Fig. 8: Surrounding of the C-71 group of LHCII-bound Chls a.

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Data availability

The authors declare that the data supporting the findings of this study are available from the corresponding authors upon request. Crystal structures determined in this study have been deposited in the Protein Data Bank (http://www.rcsb.org), with accession code 6GIW (LvPCPS Chl a) and 6GIX (LvPCPS Chl b).

References

  1. Mirkovic, T. et al. Light absorption and energy transfer in the antenna complexes of photosynthetic organisms. Chem. Rev. 117, 249–293 (2017).

    Article  CAS  Google Scholar 

  2. Remelli, R., Varotto, C., Sandonà, D., Croce, R. & Bassi, R. Chlorophyll binding to monomeric light-harvesting complex. A mutation analysis of chromophore-binding residues. J. Biol. Chem. 274, 33510–33521 (1999).

    Article  CAS  Google Scholar 

  3. Formaggio, E., Cinque, G. & Bassi, R. Functional architecture of the major light-harvesting complex from higher plants. J. Mol. Biol. 314, 1157–1166 (2001).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Standfuss, J., Terwisscha van Scheltinga, A. C., Lamborghini, M. & Kühlbrandt, W. Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at 2.5 Å resolution. EMBO J. 24, 919–928 (2005).

    Article  CAS  Google Scholar 

  6. Hobe, S., Fey, H., Rogl, H. & Paulsen, H. Determination of relative chlorophyll binding affinities in the major light-harvesting chlorophyll a/b complex. J. Biol. Chem. 278, 5912–5919 (2003).

    Article  CAS  Google Scholar 

  7. Kleima, F. J. et al. Decreasing the chlorophyll a/b ratio in reconstituted LHCII: structural and functional consequences. Biochemistry 38, 6587–6596 (1999).

    Article  CAS  Google Scholar 

  8. Ikegami, I., Satoh, S. & Aoki, M. Binding affinity of Chl b for the Chl a-binding sites in PSI core complexes. Plant Cell Physiol. 48, 1092–1097 (2007).

    Article  CAS  Google Scholar 

  9. Satoh, S., Ikeuchi, M., Mimuro, M. & Tanaka, A. Chlorophyll b expressed in cyanobacteria functions as a light-harvesting antenna in photosystem I through flexibility of the proteins. J. Biol. Chem. 276, 4293–4297 (2001).

    Article  CAS  Google Scholar 

  10. Xu, H., Vavilin, D. & Vermaas, W. Chlorophyll b can serve as the major pigment in functional photosystem II complexes of cyanobacteria. Proc. Natl Acad. Sci. USA 98, 14168–14173 (2001).

    Article  CAS  Google Scholar 

  11. Hirashima, M., Satoh, S., Tanaka, R. & Tanaka, A. Pigment shuffling in antenna systems achieved by expressing prokaryotic chlorophyllide a oxygenase in Arabidopsis. J. Biol. Chem. 281, 15385–15393 (2006).

    Article  CAS  Google Scholar 

  12. Tanaka, A. et al. Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from chlorophyll a. Proc. Natl Acad. Sci. USA 95, 12719–12723 (1998).

    Article  CAS  Google Scholar 

  13. Wang, P. & Grimm, B. Organization of chlorophyll biosynthesis and insertion of chlorophyll into the chlorophyll-binding proteins in chloroplasts. Photosynth. Res. 126, 189–202 (2015).

    Article  CAS  Google Scholar 

  14. Dall’Osto, L., Bressan, M. & Bassi, R. Biogenesis of light harvesting proteins. Biochim. Biophys. Acta 1847, 861–871 (2015).

    Article  Google Scholar 

  15. Ros, F., Bassi, R. & Paulsen, H. Pigment-binding properties of the recombinant photosystem II subunit CP26 reconstituted in vitro. Eur. J. Biochem. 253, 653–658 (1998).

    Article  CAS  Google Scholar 

  16. Pagano, A., Cinque, G. & Bassi, R. In vitro reconstitution of the recombinant photosystem II light-harvesting complex CP24 and its spectroscopic characterization. J. Biol. Chem. 273, 17154–17165 (1998).

    Article  CAS  Google Scholar 

  17. Croce, R. & Weiss, S. & Bassi, R. Carotenoid-binding sites of the major light-harvesting complex II of higher plants. J. Biol. Chem. 274, 29613–29623 (1999).

    Article  CAS  Google Scholar 

  18. Bassi, R., Croce, R., Cugini, D. & Sandonà, D. Mutational analysis of a higher plant antenna protein provides identification of chromophores bound into multiple sites. Proc. Natl Acad. Sci. USA 96, 10056–10061 (1999).

    Article  CAS  Google Scholar 

  19. Passarini, F., Xu, P., Caffarri, S., Hille, J. & Croce, R. Towards in vivo mutation analysis: knock-out of specific chlorophylls bound to the light-harvesting complexes of Arabidopsis thaliana - the case of CP24 (Lhcb6). Biochim. Biophys. Acta - Bioenerg. 1837, 1500–1506 (2014).

    Article  CAS  Google Scholar 

  20. Xu, P., Roy, L. M. & Croce, R. Functional organization of photosystem II antenna complexes: CP29 under the spotlight. Biochim. Biophys. Acta - Bioenerg. 1858, 815–822 (2017).

    Article  CAS  Google Scholar 

  21. Mahboobe, J. et al. Structure-based exciton Hamiltonian and dynamics for the reconstituted wild-type CP29 protein antenna complex of the photosystem II. J. Phys. Chem. B 122, 4611–4624 (2018).

    Article  Google Scholar 

  22. Eggink, L. L., Park, H. & Hoober, J. K. The role of chlorophyll in photosynthesis: hypothesis. BMC Plant Biol. 1, 1471 (2001).

    Article  Google Scholar 

  23. Chen, M. & Cai, Z.-L. Theoretical study on the thermodynamic properties of chlorophyll d-peptides coordinating ligand. Biochim. Biophys. Acta - Bioenerg. 1767, 603–609 (2007).

    Article  CAS  Google Scholar 

  24. Chen, M., Eggink, L. L., Hoober, J. K. & Larkum, A. W. D. Influence of structure on binding of chlorophylls to peptide ligands. J. Am. Chem. Soc. 127, 2052–2053 (2005).

    Article  CAS  Google Scholar 

  25. Hoober, J. K., Eggink, L. L. & Chen, M. Chlorophylls, ligands and assembly of light-harvesting complexes in chloroplasts. Photosynth. Res. 94, 387–400 (2007).

    Article  CAS  Google Scholar 

  26. Lutz, M. Antenna chlorophyll in photosynthetic membranes. A study by resonance Raman spectroscopy. Biochim. Biophys. Acta 460, 408–430 (1977).

    Article  CAS  Google Scholar 

  27. Horigome, D. et al. Structural mechanism and photoprotective function of water-soluble chlorophyll-binding protein. J. Biol. Chem. 282, 6525–6531 (2007).

    Article  CAS  Google Scholar 

  28. Bednarczyk, D. et al. Chlorophyll fine tuning of chlorophyll spectra by protein-induced ring deformation. Angew. Chemie Int. Ed. 55, 1–6 (2016).

    Article  Google Scholar 

  29. Satoh, H., Uchida, A., Nakayama, K. & Okada, M. Water-soluble chlorophyll protein in Brassicaceae plants is a stress-induced chlorophyll-binding protein. Plant Cell Physiol. 42, 906–911 (2001).

    Article  CAS  Google Scholar 

  30. Murata, T., Toda, F., Uchino, K. & Yakushiji, E. Water-soluble chlorophyll protein of Brassica oleracea var. Botrys (cauliflower). Biochim. Biophys. Acta 245, 208–215 (1971).

    Article  CAS  Google Scholar 

  31. Kamimura, Y., Mori, T., Yamasaki, T. & Katoh, S. Isolation, properties and a possible function of a water-soluble chlorophyll a/b-protein from Brussels sprouts. Plant Cell Physiol. 38, 133–138 (1997).

    Article  CAS  Google Scholar 

  32. Shinashi, K. et al. Molecular characterization of a water-soluble chlorophyll protein from main veins of Japanese radish. J. Plant. Physiol. 157, 255–262 (2000).

    Article  CAS  Google Scholar 

  33. Bektas, I., Fellenberg, C. & Paulsen, H. Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique. Planta 236, 251–259 (2012).

    Article  CAS  Google Scholar 

  34. Murata, T. & Murata, N. Water-soluble chlorophyll-proteins from Brassica nigra and Lepidium virginicum. Carnegie Inst. Wash. Yearb. 70, 504–507 (1971).

    Google Scholar 

  35. Takahashi, S. et al. Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera). Plant Cell Physiol. 53, 879–891 (2012).

    Article  CAS  Google Scholar 

  36. Takahashi, S. et al. Molecular cloning, characterization and analysis of the intracellular localization of a water-soluble chlorophyll-binding protein (WSCP) from Virginia pepperweed (Lepidium virginicum), a unique WSCP that preferentially binds chlorophyll b in vitro. Planta 238, 1065–1080 (2013).

    Article  CAS  Google Scholar 

  37. Satoh, H., Nakayama, K. & Okada, M. Molecular cloning and functional expression of a water-soluble chlorophyll protein, a putative carrier of chlorophyll molecules in cauliflower. J. Biol. Chem. 273, 30568–30575 (1998).

    Article  CAS  Google Scholar 

  38. Palm, D. M. et al. Water-Soluble Chlorophyll Protein (WSCP) stably binds two or four chlorophylls. Biochemistry 56, 1726–1736 (2017).

    Article  CAS  Google Scholar 

  39. Schmidt, K., Fufezan, C., Krieger-Liszkay, A., Satoh, H. & Paulsen, H. Recombinant water-soluble chlorophyll protein from Brassica oleracea var. Botrys binds various chlorophyll derivatives. Biochemistry 42, 7427–7433 (2003).

    Article  CAS  Google Scholar 

  40. Bednarczyk, D., Takahashi, S., Satoh, H. & Noy, D. Assembly of water-soluble chlorophyll-binding proteins with native hydrophobic chlorophylls in water-in-oil emulsions. BBA - Bioenerg. 1847, 307–313 (2015).

    Article  CAS  Google Scholar 

  41. Takahashi, S., Ono, M., Uchida, A., Nakayama, K. & Satoh, H. Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish. J. Plant. Physiol. 170, 406–412 (2013).

    Article  CAS  Google Scholar 

  42. Agostini, A. et al. An unusual role for the phytyl chains in the photoprotection of the chlorophylls bound to water-soluble chlorophyll-binding proteins. Sci. Rep. 7, 7504 (2017).

    Article  Google Scholar 

  43. Ramachandran, G. N., Lakshminarayanan, A. V., Balasubramanian, R. & Tegoni, G. Studies on the conformation of amino acids XII. Energy calculations on prolyl residue. Biochim. Biophys. Acta 221, 165–181 (1970).

    Article  CAS  Google Scholar 

  44. Horn, R. & Paulsen, H. Early steps in the assembly of light-harvesting chlorophyll a/b complex. J. Biol. Chem. 279, 44400–44406 (2004).

    Article  CAS  Google Scholar 

  45. Di Valentin, M. et al. Triplet-triplet energy transfer in the major intrinsic light-harvesting complex of Amphidinium carterae as revealed by ODMR and EPR spectroscopies. Biochim. Biophys. Acta - Bioenerg. 1797, 1759–1767 (2010).

    Article  Google Scholar 

  46. Iriyama, K., Ogura, N. & Takamiya, A. A simple method for extraction and partial purification of chlorophyll from plant material, using dioxane. J. Biochem. 76, 901–904 (1974).

    CAS  Google Scholar 

  47. Booth, P. J. & Paulsen, H. Assembly of light-harvesting chlorophyll a/b complex in vitro. Time-resolved fluorescence measurements. Biochemistry 35, 5103–5108 (1996).

    Article  CAS  Google Scholar 

  48. Wintermans, J. F. G. M. & De Mots, A. Spectoscophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol. Biochim. Biophys. Acta 109, 448–453 (1965).

    Article  CAS  Google Scholar 

  49. Hobe, S., Niemeier, H., Bender, A. & Paulsen, H. Carotenoid binding sites in LHCIIB: relative affinities towards major xanthophylls of higher plants. Eur. J. Biochem. 267, 616–624 (2000).

    Article  CAS  Google Scholar 

  50. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry land cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  51. CCP4. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. D50, 760–763 (1994).

    Google Scholar 

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

    Article  Google Scholar 

  53. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).

    Article  CAS  Google Scholar 

  54. Kühlbrandt, W., Wang, D. N. & Fujiyoshi, Y. Atomic model of plant light-harvesting complex by electron crystallography. Nature 367, 614–621 (1994).

    Article  Google Scholar 

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Acknowledgements

We thank T. Hares for WSCP mutant preparation and S. Müller for preliminary experiments. Moreover, we thank E. Hofmann for providing definition files for Chl ligands and helpful discussion. We are grateful to D. Noy for valuable discussion. This work has been funded by a grant from the Deutsche Forschungsgemeinschaft to H.P. (Pa 324/10-1). P.G. thanks the Studienstiftung des deutschen Volkes for support.

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Author notes

  1. These authors contributed equally to this work: Daniel M. Palm, Alessandro Agostini.

Authors and Affiliations

  1. Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany

    Daniel M. Palm, Alessandro Agostini, Vivien Averesch, Philipp Girr, Mara Werwie & Harald Paulsen

  2. Faculty of Life Sciences, Toyo University, Itakura, Gunma, Japan

    Shigekazu Takahashi

  3. Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba, Japan

    Hiroyuki Satoh

  4. Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany

    Elmar Jaenicke

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Contributions

H.P. designed the research. S.T. and H.S. designed the WSCP mutants. P.G. developed the reconstitution method. D.M.P., A.A. and V.A. prepared the WSCP samples and performed and analysed the spectroscopic measurements. E.J. resolved the crystal structures. A.A. analysed the crystal structures. D.M.P., A.A., M.W. and H.P. wrote the paper.

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Correspondence to Elmar Jaenicke or Harald Paulsen.

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Palm, D.M., Agostini, A., Averesch, V. et al. Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein. Nature Plants 4, 920–929 (2018). https://doi.org/10.1038/s41477-018-0273-z

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