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Structural insights into the assembly and energy transfer of the Lhcb9-dependent photosystem I from moss Physcomitrium patens

Nature Plants volume 9, pages 1347–1358 (2023)Cite this article

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Abstract

In plants and green algae, light-harvesting complexes I and II (LHCI and LHCII) constitute the antennae of photosystem I (PSI), thus effectively increasing the cross-section of the PSI core. The moss Physcomitrium patens (P. patens) represents a well-studied primary land-dwelling photosynthetic autotroph branching from the common ancestor of green algae and land plants at the early stage of evolution. P. patens possesses at least three types of PSI with different antenna sizes. The largest PSI form (PpPSI-L) exhibits a unique organization found neither in flowering plants nor in algae. Its formation is mediated by the P. patens-specific LHC protein, Lhcb9. While previous studies have revealed the overall architecture of PpPSI-L, its assembly details and the relationship between different PpPSI types remain unclear. Here we report the high-resolution structure of PpPSI-L. We identified 14 PSI core subunits, one Lhcb9, one phosphorylated LHCII trimer and eight LHCI monomers arranged as two belts. Our structural analysis established the essential role of Lhcb9 and the phosphorylated LHCII in stabilizing the complex. In addition, our results suggest that PpPSI switches between different types, which share identical modules. This feature may contribute to the dynamic adjustment of the light-harvesting capability of PSI under different light conditions.

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Fig. 1: Overall structure of the PpPSI-L complex.
Fig. 2: Interactions between the LHCII trimer and the PSI core.
Fig. 3: Interactions of the outer LHCI belt with the inner LHCI belt and LHCII.
Fig. 4: Structure of Lhcb9 and its interactions with surrounding subunits.
Fig. 5: Structure-based calculation of FRET rates presenting potential energy transfer pathways in the PpPSI-L complex.

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Data availability

The atomic coordinate of the PpPSI-L complex has been deposited in the Protein Data Bank with the accession code 8HTU. The composite overall and overall cryo-EM maps of the complex have been deposited in the Electron Microscopy Data Bank with accession codes EMDB-35018 and EMDB-35026. In addition, locally refined cryo-EM maps of PSI-LHCI, LHCII trimers plus PsaH-PsaL-PsaO, outer LHCIs plus Lhcb9 and outer LHCIs plus Lhcb9 processed with deepEMhancer have been deposited in the Electron Microscopy Data Bank with accession codes EMDB-35027, EMDB-35028, EMDB-35033 and EMDB-35034, respectively. All other data generated or analysed are available from the corresponding authors on reasonable request. Source data are provided with this paper.

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

We thank Y. K. He, F. Bao and C. L. Ju from the College of Life Science, Capital Normal University, for providing the P. patens strain; L. H. Chen, X. J. Huang, B. L. Zhu and F. Sun at the Center for Biological Imaging (IBP, CAS) for support in cryo-EM data collection; C. Y. Zhang and Y. Yin from the Institute of Botany, CAS, for technical assistance in sample characterization; T. Juelich (University of Chinese Academy of Sciences) for linguistic assistance during the preparation of the article. The project was funded by the Strategic Priority Research Program of CAS (XDB37020101, XDB27020106), the National Natural Science Foundation of China (31930064 and 31970264) and the National Key R&D Program of China (2022YFC2804400) and was supported by the National Laboratory of Biomacromolecules (2022kf07).

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Author notes

  1. These authors contributed equally: Haiyu Sun, Hui Shang.

Authors and Affiliations

  1. National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

    Haiyu Sun & Mei Li

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

    Haiyu Sun

  3. Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement, College of Life Science, Capital Normal University, Beijing, China

    Hui Shang & Xiaowei Pan

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  1. Haiyu Sun
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Contributions

X.P. and M.L. conceived and coordinated the project. H. Sun and H. Shang performed the purification and characterization of the PpPSI-L sample; H. Sun and X.P. processed the cryo-EM data, built and refined the structural model. H. Sun performed the multi-body refinement. H. Sun, X.P. and M.L. analysed the data and wrote the manuscript; all authors discussed and commented on the results and the manuscript.

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Correspondence to Xiaowei Pan or Mei Li.

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Extended data

Extended Data Fig. 1 Sample preparation and protein composition analysis of PpPSI-L complex.

a, Sucrose density gradient of solubilized thylakoid membranes of P. patens cultured in the media with and without glucose, respectively. The green bands corresponding to PpPSI-L and PpPSI-S are indicated and labeled. The negative stained image of PpPSI-L and PpPSI-S are also shown. b, SDS-PAGE analysis of the purified PpPSI-S and PpPSI-L complexes. The protein composition of each Coomassie band was indicated based on the mass spectrometry and proteomics data analysis. c, Sucrose density gradient of solubilized thylakoid membranes isolated from P. patens treated with high light (HL; 1000 µmol photons m−2 s−1 for 1 h) or low light (LL; 17 µmol photons m−2 s−1 for 1 h). The green bands corresponding to PpPSI-L and PpPSI-S are indicated and labeled. d, Immunoblot analysis of subunits of thylakoid membranes, PpPSI-S and PpPSI-L samples. PsaA was used as control to show that PpPSI-S and PpPSI-L samples contain PsaA with similar amount. Both pThr and Lhcb9 are present in the PpPSI-L complex, but are absent in the PpPSI-S sample. The band detected by Anti-Thr-P is identified as LhcbM proteins based on its molecular weight. Data presented in this figure were repeated at least three times, and the same results were obtained.

Source data

Extended Data Fig. 2 Characterization of PpPSI-S and PpPSI-L samples.

a, Light-induced P700 oxidation kinetics of PpPSI-S and PpPSI-L samples. The mean values and standard deviations (represented by shaded areas) were calculated from two independent measurements. b, HPLC analysis of pigment content in PpPSI-S and PpPSI-L samples. Based on the characteristic absorption spectrum of each peak fraction, the six major pigment peaks separated from the sample are identified as neoxanthin (Neo), violaxanthin (Vio), lutein (Lut), chlorophyll b (Chl b), chlorophyll a (Chl a) and β-carotene (BCR). c, Room-temperature absorption spectra of PpPSI-S and PpPSI-L samples. The PpPSI-L sample showed higher peaks around 470 and 660 nm (indicated by arrows), demonstrating that the Chl b (from LHCII) content of this fraction is higher than that of PpPSI-S complex. The spectra were normalized to the maximum in the red region. d, 77K steady-state fluorescence spectrum of PpPSI-S (black line) and PpPSI-L (red line). The blue shift of PpPSI-L compared with PpPSI-S indicates that PpPSI-L contains more Chl b than PpPSI-S. Data in this figure (b,c,d) were repeated more than three times, and all resulted in the same results.

Extended Data Fig. 3 Single particle cryo-EM analysis and evaluation of PpPSI-L complex.

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

Extended Data Fig. 4 Structure of LHCII in PpPSI-L.

a, Cartoon representation of LHCII-a monomer. Transmembrane helices A-C and two short amphiphilic helices D-E are labelled. Chlorophylls are shown as sticks with the central-Mg atoms shown as spheres. Chls a (green) and Chls b (blue) are assigned according to the conserved sites in spinach LHCII (PDB code 1RWT). Carotenoids at sites L1, L2, V1 and N1 are denoted by sticks. b, Stromal side view of the LHCII trimer. The phosphorylated Thr in LHCII-a is highlighted in ball-and-stick mode, and pigment molecules are shown as sticks. For clarity, the phytol chains of chlorophylls are omitted. c, d, Map features of characteristic residues around the N-terminal tail (c) and Y57 (d) in LhcbM2 and the corresponding sequence alignment result.

Extended Data Fig. 5 LHCI belts from P. patens and C. reinhardtii.

a, Superposition of inner LHCI belt (yellow) and outer LHCI belt (magenta) of PpPSI-L complex aligned on Lhca1. b,c, Stromal (b) and lumenal (c) side view of the outer LHC belts from PpPSI-L and CrPSI-LHCI-LHCII (PDB code 7DZ7). Two structures are superposed on the inner LHCIs, and the outer LHCIs of PpPSI-L are further aligned on the outer LHCIs of CrPSI-LHCI-LHCII. The inner and outer LHCIs are shown in cartoon mode. Lhca proteins in PpPSI-L are shown as the same colour as in Fig. 1a. Lhca proteins in CrPSI-LHCI-LHCII are coloured yellow. In (b), the PSI core and the inner LHCI belt of PpPSI-L are shown in surface mode, and coloured differently. The clash regions between Lhca1-o and Lhca2.1-i, and between Lhca3-o and Lhca3-i in the stromal side are highlighted by red boxes in (b). The long C-terminal regions of CrLhca5 and CrLhca6 in the lumenal side are shown in ribbon mode and highlighted by elliptical circles in (c).

Extended Data Fig. 6 Structure and location of Lhcb9.

a, Superposition of the Lhcb9, LhcbM2 and Lhca1 structures. The conserved Chls are shown as spheres at their central-Mg positions. Chl 614 and carotenoid at V1 site found in LhcbM2 are shown as lines. Carotenoids located at L1, L2 and N1 binding sites are shown as sticks. The unique carotenoid located at L3 site in Lhcb9 is shown in stick-ball mode. The N- and C-terminal tails of Lhcb9 are labelled. b. Stromal side view of the monomeric Lhcb9 and LHCIs in PpPSI-L. The inner belt, outer belt and Lhcb9 are displayed as ribbon, and their helix C and N-terminal region (Nter) of Lhcb9 are highlighted in cartoon mode. Other subunits are shown in surface mode. The red arrow indicates that helix C of Lhcb9. Chl pairs 603-609(−617) are shown as sticks.

Extended Data Fig. 7 Chlorophyll arrangement in the PpPSI-L complex.

a,b, Stromal-side view of chlorophylls within the PpPSI-L complex at the stromal layer (a) and lumenal layer (b). Chlorophylls located in the interface of neighbouring LHCs and between the core and LHCs are shown as stick-ball mode and labelled, other chlorophylls are shown as lines. Red Chls from Lhcb9 and Lhca3 are shown as spheres. The pigment cluster containing two pairs of red Chls in Lhcb9 and Lhca3-o are highlight with red dashed circle. c, The detailed arrangement of the pigment cluster encircled in (a). The red Chls and three closely associated carotenoid molecules are shown as stick-ball mode. The closest Mg-to-Mg distance between the two red Chl pairs is indicated by black line and the distance is labelled. For clarity, the phytol chains of chlorophylls are omitted.

Extended Data Fig. 8 Multi-body refinement of the PpPSI-L.

a, The three bodies corresponding to PpPSI-S moiety, LHCII and outer LHCIs plus Lhcb9 are defined by the transparent masks in yellow, cyan and magenta, respectively. b, The contributions of all 18 eigenvectors to the variance. c-e, The flexibility of LHCII and Lhcb9-outLHCIs relative to the PpPSI-S moiety in the principal components along the top three eigenvectors (#1-3). The models are fitted into the bin 1 and bin 10 maps in each component and then superposed on the PpPSI-S moiety. In (c-e), the left and right panels are viewed from the stromal side and from the membrane plane. The eye symbols in the left panels in (d, e) indicate the viewing angles for the side views shown on the right panels. The Lhcb9-outLHCIs-LHCII moiety is shown in magenta and yellow for the two states (bin 1 and bin 10) in (c,d). In (e), The Lhcb9-outLHCIs and LHCII are shown in pink and cyan, respectively, for one state (bin 1) and shown in limon in another state (bin 10). The dashed lines and dotted line indicate the lumenal layer of the membrane spanning regions of these rigid bodies.

Extended Data Fig. 9 Comparison of four (inner) Lhca proteins from P. patens, Z. mays and C. reinhardtii.

a, Comparison of the inner LHCI belts from PpPSI-L, CrPSI-LHCI-LHCII and LHCI belt from ZmPSI-LHCI-LHCII. Each Lhca proteins in CrPSI-LHCI-LHCII and ZmPSI-LHCI-LHCII structures are separately aligned on the corresponding Lhca proteins in PpPSI-L structure. The AC loops are highlighted as cartoon. b-e, Structural comparison of the corresponding Lhca proteins (Lhcas located at the same positions in LHCI belt) in PpPSI-L, CrPSI-LHCI-LHCII and ZmPSI-LHCI-LHCII. The AC loop region is highlighted by red arrows. The conserved Chls are show as spheres at their central-Mg position. The specific Chls and carotenoids are shown as sticks and labelled. The conserved carotenoids are shown as lines in (b-e).

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics of PpPSI-L structure
Full size table

Supplementary information

Supplementary Information

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

Reporting Summary

Supplementary Video 1

Supplementary Video 1.

Supplementary Video 2

Supplementary Video 2.

Supplementary Video 3

Supplementary Video 3.

Source data

Source Data Extended Data Fig. 1

Unprocessed gel (ED_Fig. 1b) and western blots (ED_Fig. 1d).

Source Data Extended Data Fig. 1

MS data of PpPSI-L (ED_Fig. 1b) and MS data of PpPSI-S (ED_Fig. 1b).

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Sun, H., Shang, H., Pan, X. et al. Structural insights into the assembly and energy transfer of the Lhcb9-dependent photosystem I from moss Physcomitrium patens. Nat. Plants 9, 1347–1358 (2023). https://doi.org/10.1038/s41477-023-01463-4

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