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Structural insights into cyanobacterial photosystem II intermediates associated with Psb28 and Tsl0063

Nature Plants volume 7, pages 1132–1142 (2021)Cite this article

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Abstract

Photosystem II (PSII) is a multisubunit pigment–protein complex and catalyses light-induced water oxidation, leading to the conversion of light energy into chemical energy and the release of dioxygen. We analysed the structures of two Psb28-bound PSII intermediates, Psb28–RC47 and Psb28–PSII, purified from a psbV-deletion strain of the thermophilic cyanobacterium Thermosynechococcus vulcanus, using cryo-electron microscopy. Both Psb28–RC47 and Psb28–PSII bind one Psb28, one Tsl0063 and an unknown subunit. Psb28 is located at the cytoplasmic surface of PSII and interacts with D1, D2 and CP47, whereas Tsl0063 is a transmembrane subunit and binds at the side of CP47/PsbH. Substantial structural perturbations are observed at the acceptor side, which result in conformational changes of the quinone (QB) and non-haem iron binding sites and thus may protect PSII from photodamage during assembly. These results provide a solid structural basis for understanding the assembly process of native PSII.

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Fig. 1: Overall structures of the Psb28–RC47 and Psb28–PSII complexes from T. vulcanus.
Fig. 2: Interactions between Psb28 and the PSII core in Psb28–RC47.
Fig. 3: Structural comparison of the protein subunits between Psb28–PSII and native monomeric PSII.
Fig. 4: Interactions between Tsl0063 and the PSII core in Psb28–RC47.
Fig. 5: Structural changes around the non-haem ion and the Mn4CaO5 cluster between Psb28–PSII and native PSII.
Fig. 6: A model proposed for the assembly of PSII.

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Data availability

The cryo-EM density maps and atomic models of the Psb28–RC47 and Psb28–PSII complexes at 3.14 Å resolution have been deposited in the Electron Microscopy Data Bank and the Protein Data Bank (EMD ID 30902 and PDB ID 7DXA for Psb28–RC47, EMD ID 30909 and PDB ID 7DXH for Psb28–PSII). The data that support the findings of this study are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank J. Lei and the staff at the Tsinghua University Branch of the National Center for Protein Sciences Beijing for providing facility support, the Explorer 100 cluster system of the Tsinghua National Laboratory for Information Science and Technology for providing computation resources, and H. Deng and X. Meng in the Proteinomics Facility at the Technology Center for Protein Sciences, Tsinghua University, for protein MS analysis. This work was supported by the National Key R&D Program of China (grant nos. 2017YFA0503700, 2016YFA0501101, 2017YFA0504600, 2020YFA0907600 and 2019YFA0906300), the National Natural Science Foundation of China (grant no. 31470339), the Strategic Priority Research Program of CAS (grant nos. XDA27050402 and XDB17000000), a CAS Key Research programme for Frontier Science (grant no. QYZDY-SSW-SMC003), Youth Innovation Promotion Association of CAS (grant no. 2020081) and CAS Interdisciplinary Innovation Team (grant no. JCTD-2020-06).

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Author notes

  1. These authors contributed equally to this work: Yanan Xiao, Guoqiang Huang.

Authors and Affiliations

  1. Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Yanan Xiao, Qingjun Zhu, Wenda Wang, Tingyun Kuang, Guangye Han & Jian-Ren Shen

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

    Yanan Xiao & Qingjun Zhu

  3. State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, China

    Guoqiang Huang, Xin You & Sen-Fang Sui

  4. Department of Biology, Southern University of Science and Technology, Shenzhen, China

    Sen-Fang Sui

  5. Research Institute for Interdisciplinary Science and Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan

    Jian-Ren Shen

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Contributions

G. Han, J.-R.S. and S.-F.S. conceived the project. Y.X., G. Han and Q.Z. performed the sample isolation and characterization. G. Huang took the cryo-EM images, processed the cryo-EM data and built the structure model. Y.X., G. Huang, X.Y., W.W., G. Han, S.-F.S. and J.-R.S. analysed the structure. Y.X., G. Huang, G. Han, J.-R.S. and S.-F.S. jointly wrote the manuscript, and all the authors contributed to the discussions of the results.

Corresponding authors

Correspondence to Guangye Han, Sen-Fang Sui or Jian-Ren Shen.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Rob Burnap, Nathan Nelson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Purification and characterization of Psb28-PSII and Psb28-RC47 complexes from the HisPsb28-ΔPsbV and HisPsb28-WT strain of T. vulcanus.

a Elution pattern of the Psb28-PSII and Psb28-RC47 complexes from the HisPsb28-ΔPsbV strain by Ni affinity chromatography column. b SDS-PAGE analysis of the purified samples shown in panel a. Lane 1: fraction I; lane 2: fraction II; lane 3: fraction III; lane M: Molecular weight marker. The band of Psb28 was indicated by a red star. Western-blotting analysis of His-Psb28 was shown in the bottom of the SDS-PAGE. c BN-PAGE analysis of the purified samples after the Ni affinity chromatography in panel a. Lane 1: fraction I; lane 2: fraction II; lane 3: fraction III. d Western-blotting analysis of native PSII and fraction III of panel a using the antibody against D1, Tsl0063 and PsbJ subunits. Lane 1: native PSII; lane 2: fraction III of panel a; lane M: Molecular weight marker. e Elution pattern of the Psb28-PSII and Psb28-RC47 complexes from the HisPsb28-WT strain by Ni affinity column. f BN-PAGE analysis of the purified samples shown in panel e. Lane 1: fraction I; lane 2: fraction II; lane 3: fraction III; lane 4: native PSII dimer with some contaminations of the PSII monomer. g Two-dimensional SDS-PAGE analysis of band 1 to band 4 shown in panel c and f. Lanes 1-2: band 1-2 from lane 3 of panel c; lanes 3-4: band 3-4 from lane 3 of panel f; lane M: Molecular weight marker. Western-blotting analyses with the antibodies against D1, His-tag and Psb27 were shown in the bottom of the SDS-PAGE. The primary antibody against D1(AS05084) was purchased from Agrisera. The primary antibody against His-tag (A02051) was purchased from Abbkine. The primary antibody against PsbJ and Tsl0063 were custom-made by Genscript, respectively. The horseradish peroxidase (HRP)-conjugated secondary antibody (Goat Anti Rabbit IgG) (AS09602) was purchased from Agrisera. Data shown in this figure is repeated more than three times, and all resulted in the same results.

Source data

Extended Data Fig. 2 Further purification and characterization of Psb28-PSII and Psb28-RC47 complexes from the HisPsb28-ΔPsbV strain of T. vulcanus.

a Elution pattern of the Psb28-PSII and Psb28-RC47 complexes (fraction III in Extended Data Fig. 1a) from a Mono Q anion-exchange column. b SDS-PAGE analysis of the purified samples shown in panel a. Lane 1: fraction 1; lane 2: fraction 2; lane 3: fraction 3; lane 4: fraction 4; lane M: Molecular weight marker. c BN-PAGE analysis of the purified samples after the Mono Q anion-exchange column. Lane 1: fraction 1; lane 2: fraction 2; lane 3: fraction 3; lane 4: fraction 4. Data shown in this figure is conducted more than three times, and all resulted in the same results.

Source data

Extended Data Fig. 3 Cryo-EM densities and structural models of the PSII core intrinsic, extrinsic subunit and various cofactors of the Psb28-RC47 complex (a) and Psb28-PSII complex (b).

The intrinsic subunits and extrinsic subunit are shown as mixed cartoon/stick model. All cofactors are shown as stick models. Fe and Mg are shown as magenta and green spheres respectively. The cryo-EM density map of each subunit is depicted in gray meshes.

Extended Data Fig. 4 Sequence comparison of Tsl0063 with other subunits that have a sequence similarity above 90%.

V5V6Y3: CAB/ELIP/HLIP family protein from Thermosynechococcus sp. NK55a; A0A3M2FSJ2: light-harvesting-like protein (Ssl1498 family) from Cyanobacteria bacterium J003; Q8DMP8: Tsl0063 protein from Thermosynechococcus elongatus strain BP-1; A0A5C2M567: light-harvesting-like protein (Ssl1498 family) from Thermosynechococcus sp. CL-1; A0A3B7MFR6: light-harvesting-like protein (Ssl1498 family) from Thermosynechococcus elongatus PKUAC-SCTE542. (These information is obtained by searching the database of https://www.uniprot.org/).

Extended Data Fig. 5 Structural comparison of the Psb28-RC47 and Psb28-PSII complexes of T. vulcanus.

a, b Structural comparison of the Psb28-RC47 and Psb28-PSII complexes of T. vulcanus, viewed along the membrane plane (a) and from the stromal side (b). All subunits of Psb28-RC47 are shown in grey and the subunits of Psb28-PSII are colored differently as those in Fig. 1.

Extended Data Fig. 6 Structural comparison of the Psb28 subunit from the Psb28-RC47 complex of T. vulcanus with its crystal structure and NMR structure.

a, b Structural comparison of the Psb28 subunit from the Psb28-RC47 complex of T. vulcanus with its crystal structure from T. elongates (PDB ID code: 3ZPN) (a) and NMR structure from Synechocystis sp. strain PCC 6803 (PDB ID code: 2KVO) (b). Psb28 from Psb28-RC47 is shown in green, and its crystal structure or NMR structure is shown in grey.

Extended Data Fig. 7 Structural comparison of the subunits between Psb28-RC47, Psb28-PSII and native PSII (PDB: 3WU2).

Subunits in Psb28-RC47, Psb28-PSII and native PSII are shown in cyan, green and grey, respectively.

Extended Data Fig. 8 Structural conflicts between D2 and Psb28, D1 and Tsl0063.

a Overall structure of D1, D2, Psb28 and Tsl0063 in Psb28-PSII and native PSII (PDB: 3WU2). b Structural conflicts between D2 and Psb28. c N-terminal region (E7-F14) of Tsl0063 (Psb28-PSII) and the D-E region (E226-G236) of D1 from native PSII. Enlarged view of the boxed area shows the amino acid residues involved in the structural conflicts. D1 subunits in Psb28-PSII and native PSII are shown in yellow and grey, respectively; D2 subunits in Psb28-PSII and native PSII are shown in marine and grey, respectively; Psb28 and Tsl0063 subunits in Psb28-PSII is shown in green and cyan, respectively.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Tables 1–3.

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Source data

Source Data Extended Data Fig. 1

Unprocessed gels and western blots for Extended Data Fig. 1.

Source Data Extended Data Fig. 2

Unprocessed gels for Extended Data Fig. 2.

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Xiao, Y., Huang, G., You, X. et al. Structural insights into cyanobacterial photosystem II intermediates associated with Psb28 and Tsl0063. Nat. Plants 7, 1132–1142 (2021). https://doi.org/10.1038/s41477-021-00961-7

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