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Crystal structures of the PsbS protein essential for photoprotection in plants

Nature Structural & Molecular Biology volume 22, pages 729–735 (2015)Cite this article

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Abstract

The photosystem II protein PsbS has an essential role in qE-type nonphotochemical quenching, which protects plants from photodamage under excess light conditions. qE is initiated by activation of PsbS by low pH, but the mechanism of PsbS action remains elusive. Here we report the low-pH crystal structures of PsbS from spinach in its free form and in complex with the qE inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), revealing that PsbS adopts a unique folding pattern, and, unlike other members of the light-harvesting-complex superfamily, it is a noncanonical pigment-binding protein. Structural and biochemical evidence shows that both active and inactive PsbS form homodimers in the thylakoid membranes, and DCCD binding disrupts the lumenal intermolecular hydrogen bonds of the active PsbS dimer. Activation of PsbS by low pH during qE may involve a conformational change associated with altered lumenal intermolecular interactions of the PsbS dimer.

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Figure 1: Overall structure of PsbS.
Figure 2: Structural comparison between PsbS and LHCII.
Figure 3: PsbS is not an LHCII-like pigment-binding protein.
Figure 4: Structure of low-pH PsbS dimer.
Figure 5: The low-pH PsbS dimer is physiologically relevant.
Figure 6: PsbS dimer at neutral pH.
Figure 7: DCCD-modified PsbS.

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Acknowledgements

We thank R. Bassi (Dipartimento di Biotecnologie, Università di Verona) for discussion, manuscript reading and providing seeds of npq4-E122Q E226Q double-mutant Arabidopsis, and N. Isaacs, K.K. Niyogi and J. Barber for manuscript reading. We are grateful to the staff at the Shanghai Synchrotron Radiation Facility and the Photo Factory for technical support. This work was supported by grants 2011CBA00902 (to W.C.) and 2011CBA00903 (to Z.L.) from the National Key Basic Research Program of China; grant XDB08020302 (to W.C.) from the Strategic Priority Research Program of the Chinese Academy of Sciences; and grants 31021062 (to W.C.), 31270793 (to M.L.), 31170703 (to X.P.), and 31100534 (to P.C.) from the National Natural Science Foundation of China.

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  1. National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Minrui Fan, Mei Li, Zhenfeng Liu, Peng Cao, Xiaowei Pan, Hongmei Zhang, Xuelin Zhao, Jiping Zhang & Wenrui Chang

  2. University of Chinese Academy of Sciences, Beijing, China

    Minrui Fan

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Contributions

M.F., M.L. and W.C. conceived the project. M.F. purified PsbS and performed the structural determination and the biochemical experiments with PsbS. P.C., M.L. and H.Z. assisted with data collection. X.Z. and J.Z. assisted with isolation of BBY membranes. X.P. assisted with HPLC experiments. M.F., M.L., Z.L. and W.C. discussed the results and wrote the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Sequence alignment of PsbS from different plants.

The species used for alignment are Spinacia oleracea, Arabidopsis thaliana, Oryza sativa, Zea mays, Hordeum vulgare, Populus trichocarpa, Pinus sitchensis, Selaginella moellendorffii, and Physcomitrella patens. The secondary structure of PsbS is shown above the sequence. Fully conserved residues are shaded in red. The residues mediating dimerization are marked with circles (hydrogen bond interactions) and triangles (hydrophobic interactions). The two pH-sensing glutamates are marked with squares.

Supplementary Figure 2 The purified PsbS protein sample contains chlorophylls.

(a) Size exclusion chromatography result showed that the chlorophyll molecules co-elute with PsbS from a gel filtration column. PsbS and chlorophyll were monitored by the absorption at 280 nm (A280) and 663 nm (A663), respectively.

(b) HPLC analysis of the pigments in the purified PsbS protein sample. The identification of Chl a and Chl b was based on the absorption spectra of each peak fraction.

Supplementary Figure 3 The packing mode of PsbS crystal.

(a) The packing of PsbS molecules within one layer in the crystal is mediated by hydrophobic interactions between the transmembrane helices of PsbS.

(b) The packing of PsbS molecules between layers in the crystal is mediated by hydrophilic interactions between the lumenal part and the N-terminal part of PsbS. One of the contact sites is indicated by a red ellipse.

Supplementary Figure 4 Structural explanation of previous mutation studies on PsbS.

(a) In Arabidopsis, the two ethylmethane sulfonate (EMS)-induced mutations G84D and G150E (equivalent to Gly31 and Gly97 in spinach) were reported to significantly affect the stability of PsbS (Li, X.-P. et al., Funct. Plant Biol. 29, 1131–1139, 2002). In the structure, we find that both Gly31 and Gly97 are located in the region where extensive hydrogen bonds are formed and stabilize the stromal conformation of PsbS. Mutations of them to large and charged residues will disrupt these interactions.

(b,c) In Arabidopsis, the mutations of the two salt bridges connecting TM1 and TM3 were reported to significantly affect the function of PsbS in qE but not its expression without reasonable explanation (Schultes, N.P. & Peterson, R.B. Biochem. Biophys. Res. Comm. 355, 464–470, 2007). Our structural analysis have revealed that PsbS cannot bind chlorophyll molecules here; therefore, the most probable explanation is that these mutations (E to V and R to L) disrupt surrounding hydrogen bonds and affect local conformations. The more hydrogen bonds around the second salt bridge (Arg42–Glu141) are consistent with the bigger effect of its mutation. The above-mentioned residues are shown as yellow sticks, and the other involved residues are shown in white.

Supplementary Figure 5 The potential chlorophyll molecule bound to PsbS.

(a) A picture of green PsbS crystals.

(b) HPLC analysis of the pigments in the PsbS crystals.

(c) The Chl a molecule is bound at the lumenal dimerization interface of PsbS dimer. The 2Fo – Fc (0.8σ level) electron density of the Chl a molecule is shown. The two PsbS monomers are shown in limon and palecyan, respectively. The surrounding non-polar residues interacting with the Chl a molecule are shown in stick. For clarity, the phytyl chain of the Chl a molecule is not shown.

(d) Comparison of the absorption spectra of the purified PsbS protein and free Chl a.

(e) Comparison of the circular dichroism (CD) spectra of the purified PsbS and LHCII proteins.

Supplementary Figure 6 PsbS is dimeric in both its active and inactive states in Arabidopsis.

Crosslinking of PsbS using the thylakoids of wild-type (a) and pH-insensitive npq4-E122Q E226Q double mutant (b) of Arabidopsis at pH 5.0 with EDC. Under low pH or high light conditions, wild-type PsbS is in its active state, but pH-insensitive PsbS mutant is still in its inactive state.

Supplementary Figure 7 The DCCD-binding property of the purified PsbS protein.

(a) Mass spectrometric determination of the molecular weight of native PsbS. The subtilisin-treated PsbS (SU-PsbS) lacking N-terminal five residues (for crystallization) was used in mass spectrometric analysis. The experimental molecular weight (21923.36) of SU-PsbS is very close to the sequence-based molecular weight (21922.5) of SU-PsbS.

(b) Mass spectrometric analysis of DCCD-incubated SU-PsbS. Considering that DCCD (206.33) is covalently bound to SU-PsbS without molecular weight loss, the three peaks (22129.94, 22336.32, 22542.57) presented in the mass spectrometric result should correspond to SU-PsbS plus one DCCD, SU-PsbS plus two DCCD, and SU-PsbS plus three DCCD, respectively. The last peak is very small and may be because of the binding of DCCD to Glu69, Glu173 and another lumenal acidic residue of PsbS.

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Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1352 kb)

Supplementary Data Set 1

Original gel and immunoblot images (PDF 347 kb)

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Fan, M., Li, M., Liu, Z. et al. Crystal structures of the PsbS protein essential for photoprotection in plants. Nat Struct Mol Biol 22, 729–735 (2015). https://doi.org/10.1038/nsmb.3068

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