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Cryo-EM structures of LHCII in photo-active and photo-protecting states reveal allosteric regulation of light harvesting and excess energy dissipation

Nature Plants volume 9, pages 1547–1557 (2023)Cite this article

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Abstract

The major light-harvesting complex of photosystem II (LHCII) has a dual regulatory function in a process called non-photochemical quenching to avoid the formation of reactive oxygen. LHCII undergoes reversible conformation transitions to switch between a light-harvesting state for excited-state energy transfer and an energy-quenching state for dissipating excess energy under full sunshine. Here we report cryo-electron microscopy structures of LHCII in membrane nanodiscs, which mimic in vivo LHCII, and in detergent solution at pH 7.8 and 5.4, respectively. We found that, under low pH conditions, the salt bridges at the lumenal side of LHCII are broken, accompanied by the formation of two local α-helices on the lumen side. The formation of α-helices in turn triggers allosterically global protein conformational change, resulting in a smaller crossing angle between transmembrane helices. The fluorescence decay rates corresponding to different conformational states follow the Dexter energy transfer mechanism with a characteristic transition distance of 5.6 Å between Lut1 and Chl612. The experimental observations are consistent with the computed electronic coupling strengths using multistate density function theory.

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Fig. 1: Cryo-EM structures of LHCII in nanodisc and in detergent solution.
Fig. 2: Protein secondary structures and pigments in different conformations.
Fig. 3: Relationships of key structural factors related to state transitions.
Fig. 4: Key structural factors that drive state transition.
Fig. 5: Fluorescence spectra indicate the fluorescence reversibility.

Data availability

The cryo-EM maps of spinach LHCII in nanodisc and in detergent solution at pH 7.8 or pH 5.4 have been deposited in the Electron Microscopy Data Bank under the accession codes EMD-35785, EMD-35786, EMD-35787, EMD-35782, EMD-35783 and EMD-35784. The corresponding structure models are deposited in the Protein Data Bank (PDB) under accession codes 8IX0, 8IX1, 8IX2, 8IWX, 8IWY and 8IWZ. The LHCII crystal structures used in this article can be accessed in the PDB using the accession codes 1RWT and 2BHW.

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Acknowledgements

We thank the Beijing National Laboratory for Condensed Matter Physics, Institute of Physics, Chinese Academy of Science and Beijing Branch of Songshan Lake Laboratory for Materials Science for our cryo-EM work. We thank the Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Science for our cryo-EM work, and we thank B. Zhu, X. Huang and L. Chen for their help taking EM images. We thank the cryo-EM centre of the Southern University of Science and Technology for our cryo-EM work and we thank L. Fu, J. Wu and S. Xu for their help taking EM images. We thank T. Kuang for encouragement and M. Li for in-depth discussion. We thank H. Yan for sending us the crystal structure data of LHCII from cucumber. This work was supported by the Chinese Academy of Sciences (grant nos. QYZDJ-SSW-SYS017, XDB33000000 and YJKYYQ20170046 to Y. Weng), the National Natural Science Foundation of China (grant no. 11721404 to Y. Weng) and the Shenzhen Municipal Science and Technology Innovation Commission (grant no. KQTD2017-0330155106581 to J.G.).

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Author notes

  1. These authors contributed equally: Meixia Ruan, Hao Li.

Authors and Affiliations

  1. Laboratory of Soft Matter Physics, Institute of Physics, Chinese Academy of Sciences, Beijing, China

    Meixia Ruan, Ying Zhang, Zhuan Wang, Yumei Wang, Dapeng Sun, Wei Ding & Yuxiang Weng

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

    Meixia Ruan, Ying Zhang, Wei Ding & Yuxiang Weng

  3. School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

    Hao Li & Jiali Gao

  4. Institute of Systems and Physical Biology, Shenzhen Bay Laboratory, Shenzhen, China

    Hao Li, Ruoqi Zhao, Jun Zhang, Yingjie Wang & Jiali Gao

  5. Department of Chemistry and Supercomputing Institute, University of Minnesota, Minneapolis, MN, USA

    Jiali Gao

  6. Songshan Lake Materials Laboratory, Dongguan, China

    Yuxiang Weng

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  1. Meixia Ruan
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Contributions

M.R. and H.L. purified samples and collected cryo-EM data. Y.Z., Z.W., W.D., Yumei Wang and D.S. assisted with data collection. W.D. processed cryo-EM data and reconstructed the density map. M.R., H.L. and Y. Weng analysed the structures. R.Z. and J.Z. wrote the software. R.Z., Yingjie Wang and J.G. calculated and analysed the electronic coupling. H.L. characterized and analysed the fluorescence spectra and lifetime measurement. The article was written by M.R., W.D., J.G. and Y. Weng with contributions by all authors. M.R., H.L., W.D. and Y. Weng prepared all figures. Y. Weng conceived of and coordinated the whole project.

Corresponding authors

Correspondence to Jiali Gao, Wei Ding or Yuxiang Weng.

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Nature Plants thanks Mei Li and Nicoletta Liguor for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Sample purification of LHCII and LHCII nanodisc.

a, Sucrose density gradient ultra-centrifugation separation of LHCII trimer. b, SDS-PAGE of LHCII nanodisc, LHCII in detergent solution and membrane scaffold protein MSP1E3D1. The experiment was repeated three times independently with similar results. c, Absorption trace at 280 nm and 672 nm during the Ni-NTA column purification of LHCII nanodisc. d, Absorption trace at 280 nm and 672 nm during size exclusion chromatography column purification of LHCII nanodisc.

Extended Data Fig. 2 Fluorescence decay kinetics and UV-vis and FTIR absorption spectra of LHCII nanodisc and LHCII in detergent solution.

All spectra are the averaged results of three measurements. a, Fluorescence decay kinetics of LHCII in 0.03% β-DDM at pH 7.8 and 5.4 and LHCII nanodisc at pH 7.8 and 5.4 respectively, excited at 480 nm laser with a repetition frequency of 100 kHz, an average power density of 1.5 mW/cm2, an instrumental response factor (IRF) of 0.115 ns. b, UV-vis absorption spectra of LHCII in detergent solution and LHCII nanodisc. c, Secondary derivative FTIR spectra of LHCII trimer in 0.03% DDM at pH 7.8 and 5.4 respectively. d, Secondary derivative FTIR spectra of LHCII nanodisc at pH 7.8 and 5.4 respectively. e, Lifetime constants and the associated amplitudes of LHCII in different environments based on biexponential fitting.

Extended Data Fig. 3 Structural analysis flow chart of LHCII nanodisc at pH 7.8 (a) and 5.4 (b).

a, I, A representative cryo-EM image of 8,894 collected for LHCII nanodisc at pH 7.8. II, 2D class averages of characteristic projection views of cryo-EM particles selected for further processing. III, Gold-standard Fourier Shell Correlation (FSC) curves of unprotonated conformation at pH 7.8, the 0.143 cut-off value is indicated by a horizontal blue line. IV, Flowchart for cryo-EM data processing. V, Angular distribution plot of particles used for final 3D refinement. The distribution was calculated with CryoSPARC 4.0. The different colors indicate the different number of particles that have such orientations according to the bar shown on the right. VI, Local resolution map analyzed by the local resolution estimation tool in CryoSPARC. b, Protonated (left) and unprotonated (right) conformation at pH 5.4; the detailed illustrations of I, II, III, IV, V and VI are the same as those in a.

Extended Data Fig. 4 Structural analysis flow chart of LHCII in detergent solution at pH 7.8 (a) and 5.4 (b).

a, I, A representative cryo-EM image of 7,282 collected for LHCII in detergent solution at pH 7.8. II, 2D class averages of characteristic projection views of cryo-EM particles selected for further processing. III, Gold-standard Fourier Shell Correlation (FSC) curves of unprotonated conformation at pH 7.8, the 0.143 cut-off value is indicated by a horizontal blue line. IV, Flowchart for cryo-EM data processing. V, Angular distribution plot of particles used for final 3D refinement. The distribution was calculated with CryoSPARC 4.0. The different colors indicate the different number of particles that have such orientations according to the bar shown on the right. VI, Local resolution map analyzed by the local resolution estimation tool in CryoSPARC. b, Protonated (left) and unprotonated (right) conformation at pH 5.4; the detailed illustrations of I, II, III, IV, V and VI are the same as those in a.

Extended Data Fig. 5 Comparison of protein secondary structures and pigments in different conformations.

a, b, Formation or disruption of salt bridge between K203 and E207 (a) at lumenal side and hydrogen bonds network among D54 (b) at stromal side of each monomer in the unprotonated (green) and protonated (magenta) conformations of LHCII in detergent solution, suggesting the protonation of E207 and D54 in LHCII after acidification. c, Average distance for K203-E207 and D54-D54 in different conformations. &: D54-D54 between three monomers. #: Unprotonated conformation at low pH (5.4) condition. &: Protonated conformation at low pH (5.4) condition. Data in bracket are the standard deviations of the average values. d, T57 and N61 in unprotonated (pink) and protonated (teal) conformations for LHCII nanodisc, the black arrow indicates the conformational transitions associated with protonation. e, Alignment of unprotonated structures at pH 7.8 (pink, green) and pH 5.4 (light blue, yellow) of LHCII in nanodisc (left) and in detergent solution (right). f, g, Structural comparison for helix E (f) and C-terminal (g) of LHCII in detergent solution without (pH 7.8, left) and with acidification (pH 5.4, right), a change from 310-helix or C-terminal random coil to α-helix is observed, along with C-terminal retraction towards helix D. h, Nex alignment of unprotonated structure at pH 7.8 (pink; green) and corresponding protonated structure at pH 5.4 (teal; magenta) of LHCII nanodisc (left) and LHCII in detergent solution (right), respectively, and a twist of the hexyl ring at stromal side occurs upon acidification for LHCII in nanodisc (expanded view). i, Lut1 and adjacent Chl610 pigment alignments of unprotonated structure at pH 7.8 (pink) and corresponding protonated structure at pH 5.4 (teal) of LHCII nanodisc, Lut1-Chl610 distance is 6.15 Å and 5.58 Å respectively, characterized by the Mg atom of Chl610 and the C27 atom in the conjugated π-system of Lut1. j, Vio alignment of unprotonated structure at pH 7.8 (pink; green) and corresponding protonated structure at pH 5.4 (teal; magenta) of LHCII in nanodisc (left) and in detergent solution (right).

Extended Data Fig. 6 Electron-density map and resolution of local structures and pigments of unprotonated structures at pH 7.8 and protonated structures at pH 5.4 for LHCII in nanodisc and in detergent solution respectively.

Local structure of pigments is double checked in COOT with best real space refinement statistics, such as Bonds, Angles, Torsions, Planes, Chirals, Non-bonded and Rama Plot. a, Local structural density map that involved D54-D54 and K203-E207 for LHCII in nanodisc (upper panel) and in detergent solution (lower panel), unprotonated structures are to the left of the dashed line (the key residues are shown in green (pH 7.8) or yellow (pH 5.4)) and protonated structures are at right (key residues are shown in blue). b, Density map of local structures and pigments for the unprotonated conformation at pH 7.8 (left) and protonated conformation at pH 5.4 (right) of LHCII in nanodisc. c, Density map of local structures and pigments for the unprotonated conformation at pH 7.8 (left) and protonated conformation at pH 5.4 (right) of LHCII in detergent solution. d, Local resolution and local correlation coefficients (in bracket, model vs map) for significant structures in different LHCII conformations, analyzed by phenix.validation_cryoem. #: Protonated conformation at pH 5.4.

Extended Data Fig. 7 Structural factors related to state transition at different conditions and their relationships.

a, Plot of Lut1-Chl610 electronic coupling strength \({\left.\ \right|V}_{{Q}_{y}^{{Chl}610},{S}_{1}^{{Lut}1}}{\left.\ \right|}^{2}/10000\) against Lut1-Chl610 separation distance in different LHCII structures. b, Plots of the fluorescence decay rate (k = 1/fluorescence lifetime, black solid circles), the summed coupling strength\({\left.\ \right|V}_{{Q}_{y}^{{Chl}612},{S}_{1}^{{Lut}1}}+{V}_{{Q}_{y}^{{Chl}610},{S}_{1}^{{Lut}1}}{\left.\ \right|}^{2}/10000\) (purple solid circles) of Lut1–Chl612 and Lut1-Chl610 pairs against the Lut1-Chl612 separation distance (R), and the fitting equation is \({k}=0.31+0.31{{\rm{e}}}^{-25\left({\rm{R}}-5.6\right)}\). c, Plot of available fluorescence lifetime (black star represents the data from the current work, blue solid circles and triangles represent data from the literatures38,39,40) and flexibility (orange solid circles, data from the literature44) of LHCII in nanodisc against the corresponding nanodisc size. d, Plot of helix D-E distance against Lut1-Chl612 separation distance from different LHCII structures, red dotted line marks the critical separation distance of 5.6 Å, green solid circles represent the data from the crystal structures (PDB code: 1RWT, 2BHW).

Extended Data Fig. 8 Configurations and excitation energies for the first singlet excited states of the chlorophyll monomer and lutein monomer.

a, Depiction of the minimal number of configurations necessary to model first singlet excited states of the chlorophyll monomer and lutein monomer. The ground-state configuration (Ψ0) is shown along with eight spin-contaminated configurations (1–8). b, Excitation energies of chlorophyll and lutein and the reference values.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics of LHCII structures at different conditions
Full size table
Extended Data Table 2 Structural parameters for LHCII at different conditions. &: Protonated structures at pH 5.4. *: Data not available. #: Salt bridge separation distance, which are slightly affected by acidification. Data in bracket are the standard deviations of the average values
Full size table

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Ruan, M., Li, H., Zhang, Y. et al. Cryo-EM structures of LHCII in photo-active and photo-protecting states reveal allosteric regulation of light harvesting and excess energy dissipation. Nat. Plants 9, 1547–1557 (2023). https://doi.org/10.1038/s41477-023-01500-2

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