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Structural basis of LhcbM5-mediated state transitions in green algae

Abstract

In green algae and plants, state transitions serve as a short-term light-acclimation process in the regulation of the light-harvesting capacity of photosystems I and II (PSI and PSII, respectively). During the process, a portion of light-harvesting complex II (LHCII) is phosphorylated, dissociated from PSII and binds with PSI to form the supercomplex PSI–LHCI–LHCII. Here, we report high-resolution structures of PSI–LHCI–LHCII from Chlamydomonas reinhardtii, revealing the mechanism of assembly between the PSI–LHCI complex and two phosphorylated LHCII trimers containing all four types of LhcbM protein. Two specific LhcbM isoforms, namely LhcbM1 and LhcbM5, directly interact with the PSI core through their phosphorylated amino terminal regions. Furthermore, biochemical and functional studies on mutant strains lacking either LhcbM1 or LhcbM5 indicate that only LhcbM5 is indispensable in supercomplex formation. The results unravel the specific interactions and potential excitation energy transfer routes between green algal PSI and two phosphorylated LHCIIs.

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Fig. 1: Overall structure of the native CrPSI–LHCI–LHCII supercomplex.
Fig. 2: Assembly of two LHCII trimers with PSI–LHCI.
Fig. 3: Interaction between two LHCII trimers and between LHCII and Lhca2.
Fig. 4: Purification and characterization of supercomplex samples from ΔLhcbM1 and ΔLhcbM5 mutants.
Fig. 5: Overall structure of the side-layer antennae Lhca2 and Lhca9 in the CrPSI–LHCI–LHCII supercomplex from the pph1;pbcp mutant strain.
Fig. 6: Potential energy transfer pathways within the native CrPSI–LHCI–LHCII supercomplex from the pph1:pbcp mutant strain.

Data availability

The atomic coordinates of the CrPSI–LHCI–LHCII supercomplex have been deposited in the Protein Data Bank with accession codes 7DZ7 (native supercomplex from mutant pph1;pbcp) and 7DZ8 (supercomplex from mutant ΔLhcbM1). The cryo-EM maps of the native and ΔLhcbM1 mutant supercomplexes have been deposited in the Electron Microscopy Data Bank with accession codes EMD-30925 and EMD-30926, respectively. In addition, the atomic coordinates and locally refined cryo-EM maps of LHCII trimers have been deposited in the Protein Data Bank and the Electron Microscopy Data Bank with accession codes 7E0J and EMD-30934 for LHCII-1 in the native supercomplex, 7E0K and EMD-30935 for LHCII-2 in the native supercomplex, 7E0H and EMD-30932 for LHCII-1 in the supercomplex from the ΔLhcbM1 mutant and 7E0I and EMD-30933 for LHCII-2 in the supercomplex from the ΔLhcbM1 mutant. Source data are provided with this paper. All other data generated or analysed are available from the corresponding authors on reasonable request.

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Acknowledgements

We thank M. Goldschmidt-Clermont at the University of Geneva for the kind gift of the pph1;pbcp strain and insightful discussions; L. H. Chen, X. J. Huang, B. L. Zhu and F. Sun at the Centre for Biological Imaging (CAS) for support with cryo-EM data collection; X. Sheng, D. F. Song and X. Z. Zhang for discussions on cryo-EM data-processing methods; and X. B. Liang for assistance with sample preparation, data collection and storage. We thank K. Fujimura-Kamada and T. Kadowaki for conducting genetical crossing and PCR analysis, and for valuable discussion. We thank T. Mori and Y. Makino for providing technical assistance with LC–MS/MS analysis. We thank A. Watanabe and A. Ishi for technical assistance with supercomplex isolations, and E. Kim for valuable discussions. The project is funded by the National Key R&D Programme of China (no. 2017YFA0503702 to Z.L. and M.L.), the Strategic Priority Research Programme of CAS (nos. XDB27020106 to M.L. and XDB37020101 to Z.L.), the National Natural Science Foundation of China (nos. 31925024 to Z.L. and 31930064 to M.L.), the Basic Frontier Science Research Programme of CAS (no. ZDBS-LY-SM003 to Z.L.) and Grant-in-Aid from Japan Society for the Promotion of Science KAKENHI (nos. 16H06553 to J.M. and JP15H05599 to R.T.). This work was also supported by Functional Genomics Facility, NIBB Core Research Facilities and by Model Plant Research Facility, NIBB Bioresource Centre and the Cooperative Study Programme of National Institute for Physiological Sciences.

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Authors

Contributions

R.T., Z.L., J.M. and M.L. conceived and coordinated the project. X.P. and A.L. collected and processed cryo-EM data. X.P. built and refined the structural models. R.T. performed biochemical and spectroscopic characterization of the PSI–LHCI(–LHCII) supercomplexes from WT and LhcbM mutants. A.L. prepared cryo-EM grids for the supercomplex sample from the pph1;pbcp mutant. C.S. and K.M. prepared cryo-EM grids for the supercomplex samples from WT and the ΔLhcbM1 mutant. T.Y. generated the ΔLhcbM5 mutant. K.T. performed qT quenching measurements. X.P., R.T., A.L., Z.L., J.M. and M.L. analysed the data and wrote the manuscript. All authors discussed and commented on the results and the manuscript.

Corresponding authors

Correspondence to Zhenfeng Liu or Jun Minagawa or Mei Li.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Plants thanks Alexey Amunts, Jean-David Rochaix and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Sample preparation and characterization of native CrPSI-LHCI-LHCII supercomplex from the pph1;pbcp mutant strain.

a, Sucrose density gradient of solubilized thylakoid membranes. Each band shown is labeled. b, SDS-PAGE analysis of thylakoids (Lane 1) and the purified PSI-LHCI-LHCII supercomplex (Lane 2). The protein composition of each Coomassie band was indicated based on the mass spectrometry and proteomics data analysis. The bands corresponding to PSII subunits are indicated by asterisk. Representative image selected from 3 biological repeats was shown. c, Room-temperature absorption spectra of PSI-LHCI and PSI-LHCI-LHCII samples. The PSI-LHCI-LHCII sample showed higher peaks around 470 and 650 nm (indicated by arrows), demonstrating that the Chl b (from LHCII) content of this fraction is higher than that of PSI-LHCI complex. The spectra were normalized to the maximum in the red region. d, HPLC analysis of pigment content in PSI-LHCI and PSI-LHCI-LHCII samples. Based on the characteristic absorption spectrum of each peak fraction, the six major pigment peaks separated from the sample are identified as loroxanthin/neoxanthin (Lor/Neo), violaxanthin (Vio), lutein (Lut), chlorophyll b (Chl b), chlorophyll a (Chl a) and β-carotene (BCR).

Source data

Extended Data Fig. 2 Single particle cryo-EM analysis and evaluation of CrPSI-LHCI-LHCII supercomplex from the pph1;pbcp mutant strain.

a, Single particle cryo-EM data processing procedure. The class corresponding to PSII-LHCII complex is framed by black box and indicated by asterisk. For the second dataset (shown in right), the PSII-LHCII complexes were excluded during particle picking. b, The gold standard Fourier shell correlation (FSC) curves of the final density map with criterion of 0.143. c, The gold standard Fourier shell correlation (FSC) curves of the refined models versus final maps with criterion of 0.5. d, Angular distribution of particles included in the final 3D reconstruction. e, Local resolution of the cryo-EM map estimated by ResMap.

Extended Data Fig. 3

Cryo-EM data collection, refinement and validation statistics of CrPSI-LHCI-LHCII structures.

Extended Data Fig. 4 Identification of each LhcbM protein in the LHCII-1 structure of CrPSI-LHCI-LHCII supercomplex from the pph1;pbcp mutant strain.

ac, Map features of characteristic residues and the corresponding sequence (residues are numbered according to LhcbM1) of LhcbM1 (a), LhcbM2/7 (b) and LhcbM3/M4 (c). The resolution and contour level are 3.13 Å and 1.42 rmsd for the masked map of LHCII-1. Unique residues used for identification are indicated by red arrows. a, LhcbM1 possesses unique N-terminal residues (RRt, underlined by a red line) with well-defined density. Moreover, Phe106 shows clear density for its side-chain, which excludes the possibility of all other LhcbM proteins as they have a Thr at the corresponding position. b, LhcbM2/7 contain unique residues Trp32 and Thr98 (residues 41 and 106 in LhcbM1), whereas the corresponding residue of Trp32 is a Phe in Type I and Type II isoforms, and the corresponding residue of Thr98 is a Phe in Type IV isoform, therefore excluding the possibility of all other LhcbM proteins. The density of the Phe147 (residue 155 in LhcbM1) side chain was used to further verify the assignment of LhcbM2/7. c, Type I LhcbM proteins have Phe40 and Trp48 (residues 41 and 49 in LhcbM1) in the N-terminal regions, whereas Type III and Type IV have Trp and Phe in the corresponding positions, and can therefore be excluded. In addition, Phe117 shows clear density for its side chain, which excludes the possibility of Type II LhcbM protein, as LhcbM5 has an Ile at the corresponding position. The well-defined densities of Met213, Met216 and Thr242 further exclude LhcbM6, LhcbM9, LhcbM8 and LhcbM9.

Extended Data Fig. 5 Identification of each LhcbM protein in the LHCII-2 structure of CrPSI-LHCI-LHCII supercomplex from the pph1;pbcp mutant strain.

ac, Map features of characteristic residues and the corresponding sequence (residues are numbered according to LhcbM1) of LhcbM5 (a), LhcbM2/7 (b) and LhcbM3/M4 (c). The resolution and contour level are 3.09 Å and 1.11 rmsd for the masked map of LHCII-2. Unique residues used for identification are indicated by red arrows. a, LhcbM5 possesses the longest N-terminal region among all LhcbM proteins (underlined by a red line), which shows clear densities in the map. The assignment of LhcbM5 was further verified by the densities of specific residues Phe197 and Phe254 (residue 242 in LhcbM1). The former is absent, while the latter is a residue with small side-chain (Gly or Thr) in all other LhcbM proteins. b, LhcbM2/7 contain unique residues Trp32 and Thr98 (residues 41 and 106 in LhcbM1), whereas the corresponding residue of Trp32 is a Phe in Type I and Type II isoforms, the corresponding residue of Thr98 is a Phe in Type IV isoform, therefore exclude the possibility of all other LhcbM proteins. The density of Phe147 (residues 155 in LhcbM1) side-chain was used to further verify the assignment of LhcbM2/7. c, Type I LhcbM proteins have Phe40 and Trp48 (residues 41 and 49 in LhcbM1) in the N-terminal regions, whereas Type III and Type IV have Trp and Phe in the corresponding positions, therefore can be excluded. In addition, Phe117 shows clear density for its side-chain, which excludes the possibility of Type II LhcbM protein as LhcbM5 has a Ile at the corresponding position. The well-defined densities of Met213, Met216 and Thr242 further exclude LhcbM6, LhcbM9, and LhcbM8 and LhcbM9.

Extended Data Fig. 6 Multi-body refinement of the CrPSI-LHCI-LHCII supercomplex from the pph1:pbcp mutant strain.

a, The three bodies corresponding to LHCII-1, LHCII-2 and PSI-10LHCI are defined by the transparent masks in magenta, cyan and yellow, respectively. b, The contributions of all 18 eigenvectors to the variance. c-e, The flexibility of LHCII-1 and LHCII-2 relative to PSI-LHCI complex in the principal components along the top three eigenvectors (#1-3). The models shown are fitted into the bin 1 and bin 10 maps in each component and then superposed on the PSI-LHCI complex region. The transparent red circles are the estimated anchor points for LHCII-1 and LHCII-2 during the rotational or pivoting movement. In (c) and (e), the views on the left are from stromal side. In (d), the view on the left is from luminal side to exhibit the anchor point. The eye symbols in the left parts of (c-e) indicate the viewing angles for the side views shown on the right.

Extended Data Fig. 7 Single particle cryo-EM data processing of CrPSI-LHCI-LHCII supercomplex from the ΔLhcbM1 mutant strain.

a, Single particle cryo-EM data processing procedure. b, The gold standard Fourier shell correlation (FSC) curves of the final density map with criterion of 0.143. c, The gold standard Fourier shell correlation (FSC) curves of the refined models versus final maps with criterion of 0.5. d, Angular distribution of particles included in the final 3D reconstruction. e, Local resolution of the cryo-EM map estimated by ResMap.

Extended Data Fig. 8 Identification of a Type I isoform replacing LhcbM1 in the structure of PSI-LHCI-LHCII supercomplex from the ΔLhcbM1 mutant strain.

Map features of characteristic residues and the corresponding sequence (residues are numbered according to LhcbM1). The density of the LHCII-1 region of PSI-LHCI-LHCII map is shown, with local resolution of approximately 3.75 Å and contour level of 1.31 rmsd. Type I LhcbM proteins have a Trp48 (residue 49 in LhcbM1) in the N-terminal regions, whereas Type III and Type IV have a Phe in the corresponding position, therefore can be excluded. Residue Tyr52 (residue 53 in LhcbM1) shows clear density for its side-chain, which excludes the possibility of Type II LhcbM protein as LhcbM5 has a Leu at the corresponding position. The map features of Tyr181 further verify the assignment of Type I isoform. Unique residues used for identification are indicated by red arrows.

Extended Data Fig. 9 Comparison of PSI-LHCI-LHCII supercomplex from pph1;pbcp and ΔLhcbM1 mutant strains.

a, Structural comparison of PSI-LHCI-LHCII supercomplex from ΔLhcbM1 mutant and in the native form (from the pph1;pbcp mutant strain), superposed on PsaA. The native supercomplex is shown in white, with LhcbM1 of LHCII-1 highlighted in lime-green. The supercomplex from ΔLhcbM1 mutant is colored orange for the PSI-LHCI moiety and the newly incorporated LhcbM3 of LHCII-1, while other LhcbM proteins are colored the same as in Fig. 1a. The N-terminal tails of LhcbM1 (lime-green) and the newly incorporated LhcbM3 (orange) in the two structures are indicated by arrows in a red square. b,c, Side view of structural comparison of two LHCII trimers in the CrPSI-LHCI-LHCII supercomplex from ΔLhcbM1 mutant and in native form, viewed from the periphery of two trimers (b) and from the PSI-LHCII interface (c). The color codes are the same as in (a). The outmost monomer in LHCII-1 shows the largest shift towards lumen as shown by the arrow in (b).

Extended Data Fig. 10 The variation of chlorophyll-chlorophyll distances at the interface between LHCII-1/LHCII-2 and PSI-LHCI in the first component of the rigid body motion.

The models correspond to the bin1 and bin10 maps of the principal component along eigenvector #1 of the multi-body refinement result. a,b, The interfacial chlorophyll pairs at the stromal layer in bin1 (a) and bin10 (b) states. c,d, The interfacial chlorophyll pairs at the luminal layer in bin1 (c) and bin10 (d) states. The numbers labeled nearby the dash lines indicate the Mg-Mg distances (Å) between two adjacent chlorophylls at the interfaces between LHCII-1/LHCII-2 and PSI-LHCI. The variation of the interfacial chlorophyll-chlorophyll distances in the top three motion components (Eigenvectors #1-3) are also summarized in Supplementary Table 2.

Supplementary information

Supplementary Information

Supplementary Figs. 1–4, Tables 1 and 2, Video 1 and additional Figs. 1 and 2 (source data for Supplementary Fig. 3b,c).

Reporting Summary

Supplementary Video 1

Analysis of the multi-body refinement result reveals the motion of two LHCII trimers relative to PSI–LHCI.

Source data

Source Data Fig. 4

Unprocessed gel in Fig. 4b.

Source Data Extended Data Fig. 1

Unprocessed gel in Extended Data Fig. 1b.

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Pan, X., Tokutsu, R., Li, A. et al. Structural basis of LhcbM5-mediated state transitions in green algae. Nat. Plants 7, 1119–1131 (2021). https://doi.org/10.1038/s41477-021-00960-8

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