Structural insight into light harvesting for photosystem II in green algae

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Abstract

Green algae and plants rely on light-harvesting complex II (LHCII) to collect photon energy for oxygenic photosynthesis. In Chlamydomonas reinhardtii, LHCII molecules associate with photosystem II (PSII) to form various supercomplexes, including the C2S2M2L2 type, which is the largest PSII–LHCII supercomplex in algae and plants that is presently known. Here, we report high-resolution cryo-electron microscopy (cryo-EM) maps and structural models of the C2S2M2L2 and C2S2 supercomplexes from C. reinhardtii. The C2S2 supercomplex contains an LhcbM1–LhcbM2/7–LhcbM3 heterotrimer in the strongly associated LHCII, and the LhcbM1 subunit assembles with CP43 through two interfacial galactolipid molecules. The loosely and moderately associated LHCII trimers interact closely with the minor antenna complex CP29 to form an intricate subcomplex bound to CP47 in the C2S2M2L2 supercomplex. A notable direct pathway is established for energy transfer from the loosely associated LHCII to the PSII reaction centre, as well as several indirect routes. Structure-based computational analysis on the excitation energy transfer within the two supercomplexes provides detailed mechanistic insights into the light-harvesting process in green algae.

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Fig. 1: The overall architecture of the C2S2M2L2 and C2S2 PSII–LHCII supercomplexes from C. reinhardtii.
Fig. 2: Specific interactions between CP29 and M-LHCII or L-LHCII as well as arrangement of three types of LHCII trimers around CP29.
Fig. 3: The interfaces between the light-harvesting complexes (CP29, LHCII and CP26) and the PSII core.
Fig. 4: Specific interactions between CP26 and S-LHCII.
Fig. 5: Structure-based analysis of FRET networks within the C2S2M2L2 and C2S2 supercomplexes from C. reinhardtii.

Data availability

The cryo-EM maps of C. reinhardtii PSII–LHCII supercomplexes have been deposited in the Electron Microscopy Data Bank with accession codes EMD-9956 (C2S2M2L2) and EMD-9955 (C2S2). The structure models of C2S2M2L2 and C2S2 supercomplexes are deposited in the PDB under accession codes 6KAD and 6KAC, respectively.

Code availability

The python script used for FRET rate calculation is available at https://doi.org/10.5281/zenodo.3250649.

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Acknowledgements

The cryo-EM data were collected at the Center for Biological Imaging (CBI), Core Facilities for Protein Science at the Institute of Biophysics, Chinese Academy of Sciences. The Talos Arctica beamline was sponsored by the National Laboratory of Biomacromolecules and CBI. We thank X. J. Huang, B. L. Zhu and F. Sun at the Center for Biological Imaging (IBP, CAS); X. B. Liang for support in sample preparation, data collection and storage; X. W. Pan and P. Cao for assistance with biochemical analysis; L. L. Niu and M. M. Zhang at IBP, CAS for mass spectrometry; X. Z. Zhang and M. Li for discussion; R. Tokutsu for reading of the manuscript. The project is funded by the National Key R&D Program of China (2017YFA0503702 to Z.L.), the Strategic Priority Research Program of CAS (XDB27020106 and XDB08020302 to Z.L.), the Key Research Program of Frontier Sciences of CAS (QYZDB-SSW-SMC005 to Z.L.) and the National Natural Science Foundation of China (31670749 to Z.L.), the collaborative study program of the National Institute for Physiological Sciences (to J.M.) and Grant-in-Aid for Scientific Research on Innovative Areas by Japan Society for the Promotion of Science (16H06553 to J.M.).

Author information

X.S. and A.L. prepared the cryo-EM grids for the C2S2 supercomplex. X.S. collected and processed the cryo-EM data and built and refined the structural models for both supercomplexes. A.W. prepared the C2S2M2L2 sample and characterized the sample using biochemical methods. E.K. performed computational analysis on energy transfer. K.M. and C.S. prepared cryo-EM grids for the C2S2M2L2 sample. A.L. prepared the C2S2 supercomplex sample, characterized the sample using biochemical methods and performed bioinformatics analysis. D.S. was involved in cryo-EM data processing. Z.L. participated in model building. X.S., A.L., J.M. and Z.L. analysed the structure. Z.L. and J.M. conceived and coordinated the project. The manuscript was written by X.S., A.L., J.M. and Z.L.

Correspondence to Jun Minagawa or Zhenfeng Liu.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Jean-David Rochaix, Jian-Ren Shen 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

The overall scheme for single-particle cryo-EM data processing of the C2S2M2L2.

Extended Data Fig. 2 Single-particle cryo- EM data processing of the stacked C2S2 supercomplex.

a, Overall scheme of the data processing. b, Cryo-EM densities of representative cofactors and four newly found small proteins associated with the C2S2-type PSII-LHCII supercomplex from C. reinhardtii. USP, unidentified stromal protein (the side chains for some residues are assigned tentatively according to the density features); SLP, small luminal protein tentatively assigned as a small domain of Psb27.

Extended Data Fig. 3 Purification and characterization of the supercomplex samples as well as evaluation of the cryo-EM maps of stacked-C2S2 and C2S2M2L2 supercomplexes.

a, Preparation of C2S2M2L2-type PSII-LHCII supercomplex through the sucrose density gradient (SDG) ultracentrifugation and amphipol method. Three major bands were identified as LHCII trimers, PSI-LHCI supercomplexes, and C2S2M2L2 PSII-LHCII supercomplexes. b, SDS-PAGE analysis of C2S2M2L2- type PSII-LHCII supercomplex (1 μg Chl/mL) stained with Coomassie Brilliant Blue R-250. M, molecular weight marker; Type I, LHCII type I (LhcbM3/4/6/8/9); Type III, LHCII type III (LhcbM2/7); Type IV, LHCII type IV (LhcbM1). Protein labels in parentheses represent co-migrated proteins. The experiment was repeated five times independently with similar results. c, Preparation of C2S2-type PSII-LHCII supercomplex through the SDG ultracentrifugation method. The components in four major bands of SDG tube (B1-B4) are labeled nearby the bands. d, SDS-PAGE analysis of the B4 fraction used for Cryo-EM data collection. The experiment was repeated five times independently with similar results. The identities of proteins labeled nearby the bands were revealed through mass spectroscopy analysis on the in-gel digestion products of the proteins. e, The Fourier Shell Correlation (FSC) curves for the maps and structural models of stacked-C2S2(left) and C2S2M2L2 (right). Black, the gold standard FSC curves between two independent half maps. Red, the FSC curve calculated between the maps and structural models. The dotted lines indicate the criterion at a FSC value of 0.143. f, Local resolution estimation of the maps of stacked-C2S2 and C2S2M2L2supercomplexes. The rainbow bar at the bottom indicate the color codes for regions with resolutions between 2.4 (blue) − 5.6 (red) Å.

Extended Data Fig. 4 Characteristic features of Lhcb4, LhcbM1/2/3, Lhcb5 from C. reinhardtii and the galactolipids bound to LhcbM1/3.

a, Primary sequence alignment of CrLhcb4 with the homologues from plants. The regions framed in the box indicate the motifs specific to CrLhcb4/Lhcb5 but absent in plant homologues. CHLRE, C. reinhardtii; SPIOL, Spinacia oleracea; PISSA, Pisum sativum; ARATH, Arabidopsis thaliana. Color codes: dark, identical residues; light red, similar residues; cyan, missing residues. b, Sequence alignment of LhcbM1, LhcbM2 and LhcbM3 proteins. The amino acid residues labeled with green stars are those exhibiting distinct characteristic features in the cryo-EM map of the S-LHCII trimer in the C2S2 supercomplex. The region underscored by a magenta line contains additional unique features distinguishing the LhcbM3 subunit from LhcbM1/2, LhcbM4/6/8/9 and LhcbM5. c–e, Cryo-EM densities for the characteristic N-terminal regions of LhcbM1 (c), LhcbM2 (d) and LhcbM3 (e) proteins, respectively. f, Two DGDG molecules (DGD523 and DGD524) sandwiched between LhcbM1 and CP43/PsbW. g, A MGDG molecule (LMG2631) located between LhcbM3 and CP26. The cryo-EM map of C2S2 supercomplex is shown at a contour level of 4.0 rmsd (c–e) or 2.6 rmsd (f), while that of C2S2M2L2 supercomplex is shown at a contour level of 4.0 rmsd (g). h, Alignment of CrLhcb5 primary sequence with those of plant homologues.

Extended Data Fig. 5 The pathways for excitation energy transfer from S- LHCII/CP26/CP29 to PSII core antennae.

a–c, Comparison of the CP29−CP47 (a), S-LHCII−CP43 (b) and CP26−CP43 interfaces in the C2S2 and C2S2M2L2 structures. Color codes: cyan, C2S2; magenta, C2S2M2L2. The two structures are superposed on CP47 or CP43 for detection of relative movement of CP29 or S-LHCII and CP26. The backbones of apoproteins are shown as ribbon model, while the Chls are shown as stick models with the phytyl chain omitted for clarity. Carotenoids and other cofactors are not shown. The arrows indicate the predicted movement of CP29, S-LHCII and CP26 with respect to CP47/CP43 during the transition from C2S2M2L2 to C2S2. d, Overview of the Chl arrangement pattern in C2S2 supercomplex. For clarity, only half of the supercomplex is shown with a top view from stromal side. The red arrows indicate the potential energy transfer pathways connecting the peripheral antenna with the core antenna. e–g, The interfaces between LhcbM1 in S-LHCII and CP43 (e), between CP26 and CP43 (f) and between CP29 and CP47 (g). As Chl a616 CP29 is absent in the C2S2 supercomplex, the CP29-CP47 pathways on the stromal side are mainly contributed by Chl a603/Chl a609 CP29 −Chl a610/a616 CP47. h, The interface between CP29 and CP47 in the C2S2M2L2 supercomplex for comparison with the one in C2S2 supercomplex (g). The numbers labeled nearby the yellow dash lines are the Mg−Mg distances (Å) between two adjacent Chl molecules. Note the presence of Chl a616 CP29 in C2S2M2L2 supercomplex and it is located closer to Chl a616 CP47 than Chl a603 CP29. In dh, the Chl molecules are presented as stick models with their phytol chains omitted for clarity. The NB, ND and Mg atoms are shown as spheres to highlight the approximate orientation of Qy dipole moment of the Chl molecule. The dash lines indicate the potential energy transfer pathways between two adjacent Chl molecules at the interfaces and the Mg-Mg distances (Å) are labeled nearby the dash lines.

Extended Data Fig. 6 Potential excitation energy transfer pathways from L- LHCII to PSII core.

a, Overview of the Chl arrangement and energy transfer from L-LHCII to D2, M-LHCII and CP29. The view is from stromal side. b–e, The side view of the interfacial Chl connections between L-LHCII and D2 (b), between L-LHCII and CP29 (c), between L-LHCII and M-LHCII. (d) and between M-LHCII and CP29 (e). f, g, Potential energy transfer pathways from M-LHCII to S- LHCII’. f, top view; g, side view. The Mg-Mg distances (Å) between two adjacent interfacial Chls are labeled nearby the dash lines. The red, magenta and orange block arrows indicate the three major routes for the transfer of excitation energy from L-LHCII to the reaction center.

Extended Data Fig. 7 Cryo-EM data collection, refinement and validation statistics for the structural models of the stacked-C2S2 and C2S2M2L2 supercomplexes.

*The final particle images of C2S2 supercomplex are from particle substraction treatment of 258,242 (C2S2)2 particles (from the first round of 3D classification) followed by further 3D classification. As the final particle images combine two halves of the (C2S2)2 particles, the number becomes larger than the initial particle images.

Extended Data Fig. 8 Numbers of amino acid residues and cofactors modeled in each subunit of the stacked- C2S2/C2S2M2L2 supercomplex.

*The assignment of apoproteins (LhcbM1-3) in M-LHCII or L-LHCII is tentative due to insufficient resolution of the local map features. The assignment of Chl a/b and carotenoids in M/L-LHCIIs is deduced from those in S-LHCII of the C2S2 complex. BCR, β-carotene; Lut, lutein; Neo, neoxanthin; Vio, violaxanthin; MGDG, monogalactosyl diacylglycerol; SQDG, sulfoquinovosyl diacylglycerol; PG, phosphatidylglycerol; DGDG, digalactosyl diacylglycerol; BCT, bicarbonate ion; LMU, dodecyl-α- D-maltoside; PL9, plastoquinone 9. When the contents are different in two structures, the numbers are separated by ‘/' (left, stacked C2S2; right, C2S2M2L2). The dash symbols indicate that the subunits or cofactors are not observed in the structure.

Extended Data Fig. 9 Calculated FRET rates for the excitation transfer from Chls in S- LHCII to those in CP26 and further to CP43.

The lifetime (τ) and half-life (t1/2) were defined as τ = 1/kFRET and t1/2 = 0.693/kFRET, respectively. The numbers separated by ‘/’ represent those for stacked C2S2 (left) and C2S2M2L2 (right).

Extended Data Fig. 10 Calculated FRET rates for the excitation transfer from Chls in M- or L-LHCII to those in CP29, from Chls in L-LHCII to ChlZD2, and from ChlZD2 to Chls in CP47 or those in D1/D2.

The lifetime (τ) and half-life (t1/2) were defined as τ = 1/kFRET and t1/2 = 0.693/kFRET, respectively.

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Sheng, X., Watanabe, A., Li, A. et al. Structural insight into light harvesting for photosystem II in green algae. Nat. Plants (2019) doi:10.1038/s41477-019-0543-4

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